volume 5 - 2011 | https://doi.org/10.3389/fnana.2011.00069 This article is part of the Research TopicWiring Principles of Cerebral CortexView all 11 articles Further examination showed more intricate components of this modular structure the postnatal development of this structure and potential molecular players for its formation will be reviewed I will discuss how this modular organization is transformed in mutant rodents with a disorganized layer structure in the cerebral cortex (i.e. the potential significance of this type of module will be discussed the cell column is visualized by conventional Nissl staining Dendritic bundles were first discovered using Golgi methods but these structures can today be more easily seen using microtubule-associated protein 2 (MAP2) immunohistochemistry Myelinated axon bundles can be visualized using Tau immunohistochemistry or conventional myelin staining and axon bundles of double bouquet cell can be seen using calbindin immunohistochemistry The spatial interrelationship between these differently identified morphologic minicolumns is more complex than originally expected and is neither clear nor unanimously defined In this review, I will focus on a well-examined small module exiting in layers 1 and 2 of the rat granular retrosplenial (GRS) cortex. This modular structure was first described as a module consisting of a combination of prominent apical dendritic bundles from layer 2 pyramidal neurons and thalamocortical patchy inputs, which are spatially matched with the dendritic bundles (Wyss et al., 1990) which will be reviewed first in this paper the postnatal development of this structure and potential molecular mechanisms underlying the formation of this module will be reviewed I will describe the way in which this modular organization in wild-type rodents is transformed in mutant rodents which have a disorganized layer structure in the cerebral cortex (i.e. Schematic drawing of the modular organization of the layer 1 granular retrosplenial cortex (GRS) (A) Dendritic bundles in layer 1 [postnatal day (P) 10] visualized by electroporation of enhanced green fluorescence protein (EGFP) at embryonic day (E) 18 (B–D) Confocal micrographs of layer 1 of the rat GRS stained by double-immunofluorescence for microtubule-associated protein 2 (MAP2; green) and parvalbumin (PV; red) these PV-positive neurons can inhibit intricate combinations of pyramidal neurons within different modules which receive thalamic inputs different from inputs to these PV-positive interneurons This potential organization may work as a substrate for lateral inhibition between modules OCAM may be also involved in the maintenance of compartmental organization (i.e. segregation of MAP2-positive dendritic bundles from layer 2 pyramidal neurons and the complementary aggregation of OCAM-positive apical dendrites from layer 5 pyramidal neurons) Dendritic modules in layer 1 are visualized by OCAM-immunoreactive (ir) and MAP2-ir in adults The right column shows merged images where patches of OCAM-ir and MAP2-ir are seen to interdigitate Arrowheads point to corresponding locations in two sets consisting of three figures (A–F) visualized by immunohistochemistry for OCAM and vesicular glutamate transporter 1 (VGLUT2; presumptive thalamocortical terminations) or VGLUT1 (presumptive corticocortical terminations) (A–C) Coronal sections reacted for OCAM and VGLUT2 OCAM-ir in the wild-type rat (A) is bistratified with one superficial band in layers 1b and c and another but these correspond to layer 1a and layers 3 and 4 (B) Double-immunofluorescence for OCAM and VGLUT2 directly demonstrates this complementary relationship (C) Layer 2 has low levels of both OCAM and VGLUT2 (D–F) Coronal sections reacted for OCAM and VGLUT2 The developmental time-course of the dendritic bundles in the GRS has been investigated using immunohistochemistry for MAP2 and glutamate receptor subunits 2/3 (GluR2/3; Ichinohe et al., 2003b) Bundles in layer 1 are apparent as early as postnatal day (P) 5 first using GluR2/3 immunohistochemistry and then As a step toward understanding the mechanisms underlying dendritic aggregation we further investigated the ontogeny of expression of the cell adhesion molecule OCAM OCAM exhibits a patchy distribution in layer 1 from P3 to adulthood and the regions of weak OCAM immunoreactivity selectively correspond to the dendritic bundles (using GluR2/3 and MAP2 immunohistochemistry) The periodic geometry of OCAM-positive regions support the possibility that OCAM significantly contributes to the establishment and maintenance of dendritic modules the interdigitating relationship between regions of high OCAM immunoreactivity and the dendritic bundles in layer 1 suggests that OCAM may have a repellent influence on the formation of these bundles (see also above section) The controlled ectopic induction of dendritic bundles identifies a new role for NT-3 and a new in vivo model for investigating dendritic bundles and their formation Ectopically expressed neurotrophin-3 (NT-3) induces dendritic bundles in neocortical layer 1 (A) EGFP-expressing layer 2 pyramidal neurons at P20 after electroporation of EGFP alone at E18 Note the uniform distribution of cells and dendrites (B) Electroporation of EGFP and NT-3 at E18 induces distinct dendritic bundles in the barrel cortex visualized by immunohistochemistry for OCAM and VGLUT2 (presumptive thalamocortical terminations) or VGLUT1 (presumptive corticocortical terminations) (A–C) Coronal sections reacted for OCAM and VGLUT2 from shaking rat Kawasaki (G–I) Coronal sections reacted for OCAM and VGLUT1 Pyramidal cell dendrites preferentially arborize in either patch or matrix compartments in reeler GRS (A) Coronal section reacted for VGLUT2 (red) with a Lucifer yellow (LY)-filled neuron (green) This LY-filled neuron has a soma located in a zone of low VGLUT2-ir (presumably equivalent to the OCAM-positive matrix) and many dendrite branches within a VGLUT2-dense patch (presumably equivalent to an OCAM-negative patch) These dendritic portions within VGLUT2-dense patches have spines [arrowheads in (A″)] but not the portions outside the VGLUT2-dense region (A′) (A′,A″)] show a higher magnification of the images in the white boxes [(A′) from left box and (A″) from right box] (B–E) Serial sections of a LY-filled neuron (green) The cell body is located within the OCAM-positive matrix (C) An apical dendrite-like process gives off an oblique dendrite proximally An apical tuft branches just at the border between patch and matrix compartments (C) Modular aggregations consisting of appropriate types of recipients and inputs may help to achieve efficient and quick synaptic wiring changes such as occur in the context of learning and memory The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Face perception and recognition,” by the Ministry of Education Understanding retrosplenial amnesia: insights from animal studies Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Loss of olfactory cell adhesion molecule reduces the synchrony of mitral cell activity in olfactory glomeruli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Finding your way in the dark: the retrosplenial cortex contributes to spatial memory and navigation without visual cues Pubmed Abstract | Pubmed Full Text | CrossRef Full Text NMDA receptor-dependent pattern transfer from afferents to postsynaptic cells and dendritic differentiation in the barrel cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Microstructure of the neocortex: comparative aspects Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Anterior thalamic lesions stop synaptic plasticity in retrosplenial cortex slices: expanding the pathology of diencephalic amnesia Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Honeycomb-like mosaic at the border of layers 1 and 2 in the cerebral cortex Developmental study of dendritic bundles in layer 1 of the rat granular retrosplenial cortex with special reference to a cell adhesion molecule CrossRef Full Text Unusual patch-matrix organization in the retrosplenial cortex of the reeler mouse and shaking rat Kawasaki Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Parvalbumin positive dendrites co-localize with apical dendritic bundles in rat retrosplenial cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Region specific micromodularity in the uppermost layers in primate cerebral cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Missplicing resulting from a short deletion in the reelin gene causes reeler-like neuronal disorders in the mutant shaking rat Kawasaki Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Kisvárday One axon-multiple functions: specificity of lateral inhibitory connections by large basket cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Lambert de Rouvroit The reeler mouse as a model of brain development Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Miró-Bernié Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Neurotrophin-3 is involved in the formation of apical dendritic bundles in cortical layer 2 of the rat Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Single axon branching analysis in rat thalamocortical projection from the anteroventral thalamus to the granular retrosplenial cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Receptive-field properties of transcallosal visual cortical neurons in the normal and reeler mouse Pubmed Abstract | Pubmed Full Text A unique mosaic in the visual cortex of the reeler mutant mouse Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The influence of single VB thalamocortical impulses on barrel columns of rabbit somatosensory cortex Pubmed Abstract | Pubmed Full Text Projections from the anterodorsal and anteroventral nucleus of the thalamus to the limbic cortex in the rat Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Connections of the retrosplenial granular b cortex in the rat Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Dendritic bundling in layer I of granular retrosplenial cortex: intracellular labeling and selectivity of innervation Pubmed Abstract | Pubmed Full Text | CrossRef Full Text OCAM: a new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomero-nasal axons Pubmed Abstract | Pubmed Full Text Early postnatal migration and development of layer II pyramidal neurons in the rodent cingulate/retrosplenial cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish One might call Hikari extraordinarily ordinary she takes up a new hobby--imagining what a romance would be like between her pretty friend Mari and the cute guy in their class until the roles start to get tangled in Hikari's mind Does she really have to be just the best friend in this love story And just who is on Otani's mind when his eyes drift Ichinohe launched the manga in Bessatsu Friend in April 2022 Kodansha published the manga's first volume in September 2022 The manga was nominated in Kodansha's 48th annual Manga Awards this year Source: Bessatsu Friend magazine's website Volume 2 - 2023 | https://doi.org/10.3389/fnimg.2023.1345643 This article is part of the Research TopicGlobal Excellence in Neuroimaging for Cognitive NeuroscienceView all 4 articles In recent years the common marmoset homolog of the human default mode network (DMN) has been a hot topic of discussion in the marmoset research field the posterior cingulate cortex regions (PGM A19M) and posterior parietal cortex regions (LIP but some studies claim that these form the frontoparietal network (FPN) We restarted from a neuroanatomical point of view and identified two DMN candidates: Comp-A (which has been called both the DMN and FPN) and Comp-B We performed GLM analysis on auditory task-fMRI and found Comp-B to be more appropriate as the DMN a DMN and FPN in the tasking human was closer to the resting common marmoset The human DMN appears to have an advanced function that may be underdeveloped in the common marmoset brain Default mode network regions under investigation and their areas We made comparisons of marmoset fMRI not only with human resting-state fMRI network components but also with human task-fMRI [working memory (wm)-task and motor-task] network components Through these analysis results we propose that Comp-A is the FPN and Comp-B is the DMN of the common marmoset We also found that the resting marmoset's Comp-A and Comp-B were closer to the wm-task human components than the resting human components This suggests that the marmoset may not be resting like humans do during fMRI experiments based on the combination of this result and multiseed-based connectivity analysis between mPFC and PCC regions the resting-state DMN may be underdeveloped in the common marmoset brain Human default mode network component and awake marmoset ICA components (A) Right cortical surface of the marmoset Awake resting-state marmoset ICA component-A (B) Right cortical surface of the human brain Human resting-state default mode network is mapped onto the surface (C) Right cortical surface of the marmoset brain Awake resting-state marmoset ICA component-B right and bottom left) and a sagittal view (bottom right) of awake marmoset ICA component-A right and bottom left) and a sagittal view (bottom right) of human resting-state default mode network component right and bottom left) and a sagittal view (bottom right) of awake marmoset ICA component-B Awake resting-state fMRI data of the common marmoset (Callithrix jacchus) were acquired as part of the Brain/MINDS project (Okano et al., 2015; Muta et al., 2023) A Bruker BioSpec 9.4T MRI machine (Biospin GmbH The experimental settings of the gradient recalled echo planar imaging (EPI) sequence were as follows: flip angle = 65 matrix size = 60 × 42 × 52 After preprocessing, independent component analysis (ICA) was applied to the marmoset rs-fMRI data to acquire 30 components. The number of components was chosen for compatibility with (Liu et al., 2019). MELODIC (Beckmann and Smith, 2004) was used to obtain group ICA from 48 sessions (140 frames) multi-session temporal concatenation was performed and a spatial map was obtained the two components that were used in our study were manually selected from the 30 components Fingerprint analysis (Passingham et al., 2002) was used to analyze the correspondence between marmoset and human ICA components. To apply fingerprint analysis, we used 14 sub-cortical regions as fingerprints (Supplementary Figure 1) from the Brain/MINDS 3D Marmoset Reference Brain Atlas 2019 (Woodward, 2019) The correlation between component time-series (resting marmoset FPN/DMN) and voxel time-series in sub-cortical regions was calculated for all marmoset sessions A mixed-effects model was applied for group analysis and the t-value of each voxel was calculated by one-sample t-test Mean t-values were used to quantify the fingerprints of the 14 sub-cortical ROIs the Manhattan distance between resting marmoset FPN/DMN and resting/task human FPN/DMN components was calculated using all 14 fingerprints Gilbert et al. (2023) performed an auditory task-fMRI experiment with the common marmoset We used their auditory task-fMRI data to investigate task-induced deactivation Three functional time courses were acquired from two awake marmosets (named M3 and M4) Details of the data are orientation: axial T2WI and task-fMRI NIfTI files (N = 6) were used for registration. Preprocessing and registration were performed using Statistical Parametric Mapping (SPM12) (Penny et al., 2011). SPM12 registered NIfTI images to the Marmoset MRI Standard Brain (Iriki, 2017) and task-fMRI data was smoothed using a FWHM of 1.7 mm (3.4 voxels) The preprocessed task-fMRI data was then used for GLM analysis and a cluster-extent threshold (k > 69 voxels and FWE corrected p < 0.049) was applied to acquire significant clusters under multiple comparisons CONN registered NIfTI images to the standard Montreal Neurological Institute (MNI) brain space Data were smoothed using a FWHM of 4 mm (2 voxels) for group ICA and a FWHM of 6.8 mm (3.4 voxels for compatibility with the marmoset data) for multiseed-based connectivity and fingerprint analysis A high-pass filter (1/128Hz) was applied for subsequent analyses After preprocessing, group ICA was applied to acquire 15 components from the human rs-fMRI data. We systematically checked several different numbers of components - 5/10/15/20/30 - and decided that 15 components were appropriate. For example, the default mode network became separated into two components if 30 components were chosen. MELODIC (Beckmann and Smith, 2004) was used to obtain group ICA from 200 sessions FPN and SMN components used in our study were manually selected from the 15 resting/task fMRI data components For surface mappings of human data, the command “wb_command -volume-to-surface-mapping” of the Connectome Workbench visualization software (Marcus et al., 2011) was used to map NIfTI image data onto the human cortical surface and the Brodmann label mapping (included in the HCP data) were overlaid to produce our visualizations The procedure of multiseed-based connectivity analysis of HCP resting/task fMRI data was the same as for the marmoset. A t-value threshold (t > 5.96 in Figures 3, 5) was applied to acquire significantly correlated voxels The permutation test was applied to test the significance of the Manhattan distance t-values of 14 sub-cortical ROIs were permutated in each species A two-sided rank test was performed and Bonferroni correction was applied to the results and a cluster-extent threshold (k > 55 voxels and FWE corrected p < 0.049) was applied to acquire significant clusters under multiple comparisons a mixed-effects model was used for group analysis A one-sample t-test for each voxel was performed as a 2nd-level (group) analysis Statistical significance was set at p < 0.05 Bonferroni correction was then applied to correct for the familywise error (FWE) rate and a t-value threshold was applied to acquire significantly correlated voxels a mixed-effects model was used for group analysis and the t-value of each voxel was calculated by 2nd-level analysis of OLS regression with a Tukey taper We applied a voxel-wise primary threshold (uncorrected p < 0.001 and t > 4.14) to obtain significantly activated or deactivated voxels and a cluster-extent threshold (k > 69 voxels and FWE corrected p < 0.049) was applied to acquire significant clusters for the HCP task-fMRI data a voxel-wise primary threshold (uncorrected p < 0.001 and t > 3.10) and a cluster-extent threshold (k > 55 voxels and FWE corrected p < 0.049) were applied Comp-A has a positive group ICA result in A23V Although both components showed several overlapping areas between component and task-induced deactivation A31 and PE appear as consistent areas between common marmoset and macaque monkeys therefore we prefer Comp-B as a more suitable DMN component GLM analysis results of awake marmoset passive auditory task-fMRI (A) Canonical hemodynamic response function (HRF) for the marmoset and human (B) Example design matrix for GLM analysis (TR = 3 seconds) (C) GLM analysis result (auditory stimuli > rest) of sub-cortical regions (i) Horizontal plane (z = 61) of marmoset brain shows several activated regions (ii) Sagittal plane (x = 85) shows activated region of inferior colliculus (iii) Sagittal plane (x = 98) shows activated region of medial geniculate nucleus (D) GLM analysis result (auditory stimuli > rest) mapped to the marmoset cortical surface White arrow shows activated region of auditory cortex Yellow arrow shows deactivated regions of A23b Cyan arrow shows the deactivated region of PFG This result also supports Comp-B as a DMN candidate This result suggests that the marmoset resting state may be closer to the human wm-task state and the marmoset may not be “resting” like a human does during fMRI experiments Fingerprint analysis result between awake resting marmoset and resting/tasking human ICA components (A) Example fingerprint result of three-dimensional maximum projection of t-values Correlation between Comp-A time-series and voxel time-series in sub-cortical regions was calculated The closest two ICA components are shown at the top row (awake resting marmoset) and bottom row (wm-task human) The middle row shows resting human for reference (B) Example fingerprint result of three-dimensional maximum projection of t-values (Comp-B) (C) Radar chart of fingerprint result of 14 sub-cortex regions (resting marmoset Comp-A Blue and orange asterisks show the significantly different t-values between resting marmoset vs wm-task human FPN (p < 0.05) in each ROI by the non-parametric Steel-test and Bonferroni correction (D) Radar chart of DMN fingerprint result of 14 sub-cortex regions (resting marmoset Comp-B wm-task human DMN (p < 0.05) in each ROI by non-parametric Steel-test and Bonferroni correction (E) Fingerprint distance results between awake resting marmoset components and resting/task human components White asterisks show the significantly close distance (p < 0.05) by permutation test and Bonferroni correction is not close to the resting/task human SMN components the sub-cortical activity of the resting marmoset Comp-B may be closer to that of the deactivated human DMN or that a human-like activated DMN may not really be the default mode for the marmoset Analysis results of human working memory-task fMRI data (A) Left cortical surface of the human brain The t-value range is 5 to 9.1 for positive (B) Left cortical surface of the human brain Wm-task human FPN component is mapped onto the surface (C) Left cortical surface of the human brain Wm-task human DMN component is mapped onto the surface (D) Multiseed-based connectivity analysis result of human wm-task fMRI using mPFC seeds (part of A9 Three-dimensional maximum projection of t-values from multiple seeds are shown and fingerprint analysis between the resting human DMN and resting marmoset Comp-A being closer than with Comp-B Although we propose that Comp-B is more suitable for the DMN component through several lines of evidence further investigation will be required to definitively determine the DMN in the common marmoset brain the marmoset cortex is smooth and relatively small and Comp-B showed ambiguity in regional boundaries around PE The temporal resolution of fMRI scans of the marmoset brain is low compared with the human Current marmoset data has TR = 2.0 s and group ICA was obtained from 140 frames × 48 sessions This small dataset may result in insufficient component decomposition A higher temporal resolution (or larger frame number) and larger session data would be required to correct this ambiguity for the marmoset no judgment can be made regarding this network and further research is required Publicly available datasets were analyzed in this study. This data can be found at: Pre-processed auditory task fMRI data of the common marmoset is available at https://doi.org/10.5281/zenodo.7827225 Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements Written informed consent to participate in this study was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and the institutional requirements The animal study was approved by the Experimental Animal Committee of RIKEN The study was conducted in accordance with the local legislation and institutional requirements The author(s) declare financial support was received for the research This research was supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from the Japan Agency for Medical Research and Development Cirong Liu for the valuable discussions on the default mode network Data were provided (in part) by the Human Connectome Project WU-Minn Consortium (Principal Investigators: David Van Essen and Kamil Ugurbil; 1U54MH091657) funded by the 16 NIH Institutes and Centers that support the NIH Blueprint for Neuroscience Research and by the McDonnell Center for Systems Neuroscience at Washington University The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnimg.2023.1345643/full#supplementary-material The default mode network in chimpanzees (Pan 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cortical layers in awake marmosets User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability Ichinohe N and Woodward A (2024) A reappraisal of the default mode and frontoparietal networks in the common marmoset brain Received: 28 November 2023; Accepted: 20 December 2023; Published: 09 January 2024 Copyright © 2024 Okuno, Ichinohe and Woodward. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Takuto Okuno, dGFrdXRvLm9rdW5vQHJpa2VuLmpw To investigate the effect of an excessive tibial plateau angle (TPA) and change in compressive load on tensile forces experienced by the cranial cruciate 16 cadaveric stifle joints from 16 orthopedically normal Beagles Stifle joints were categorized into unchanged (mean TPA 30.4°) and excessive (mean TPA before and after modification The excessive TPA group underwent a TPA-increasing procedure (curvilinear osteotomy of the proximal aspect of the tibia) to achieve the desired TPA A robotic system was used to apply a 30- and 60-N compressive load to specimens and LCL were sequentially transected; load application was repeated after each transection Orthogonal force components were measured in situ Forces on ligaments were calculated after repeated output force measurements as the contribution of each component was eliminated Increasing the compressive load increased tensile forces on the craniomedial and caudolateral bands of the CCL was greater for the excessive TPA group than for the unchanged TPA group Results indicated that stress on the CCL may increase when the compressive load increases The TPA-increasing procedure resulted in increased tensile force on the CCL at a 60-N compressive load without affecting forces on the MCL or LCL excessive TPA) were younger than dogs with a TPA ≤ 30° (deemed a normal TPA) This finding suggests that dogs with an excessive TPA are at a higher risk of early CCL rupture than dogs with a TPA ≤ 30° In a previous study,8 our group showed that an excessive TPA (approx 40°) experimentally created by means of a TPA-increasing procedure may promote degenerative changes in the extracellular matrix of the CCL in dogs not many studies have investigated the effects of excessive TPA on tensile forces experienced by the ligaments of canine stifle joints The objective of the study reported here was to investigate the effects of excessive TPA on these forces by comparing the tensile forces on the CCL and LCL in stifle joints from orthopedically normal Beagles with and without experimentally created excessive TPAs during in situ application of a compressive load selected to simulate weight-bearing conditions A secondary aim was to compare the tensile forces on these ligaments between 2 loads (the estimate of weight-bearing force vs half of that force) and sex of the dog from which the specimen was obtained were recorded where r is the radius of the 30-mm TPLO saw blade Figure 1Radiographic images (mediolateral views) of 2 cadaveric stifle joints collected from skeletally mature orthopedically normal Beagles in a study to investigate the effect of an excessive TPA on tensile forces experienced by the CCL B—Specimen from the excessive TPA group after the TPA-increasing procedure was performed Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.459 The upper mechanism could move in 2 translational and 3 rotational axes and the lower mechanism could move in 1 translational axis The tibial clamp was fixed to the upper mechanism via a UFS and the femoral clamp was fixed to the lower mechanism Figure 2Photograph (A) and schematic view (B) of the testing system used in the present study A—The testing system consisted of a 6-DOF manipulator with a 6-DOF UFS The upper and lower arrows indicate the tibial and femoral clamps B—The 6-DOF robotic simulator was used to test the biomechanical behavior of the stifle joint The upper mechanism is indicated with a bracket on the right side of the image and the lower mechanism is indicated with an arrow on the left side The UFS (blue square in panel B) was used to measure load and torque and define the load applied to the joint Horizontal and vertical straight dotted lines depict the X- and Z-axes and the dotted circle depicts the site of the tibial clamp The joint coordinate system was used to define the positions of the femur and tibia and motions of the joint in each stifle joint specimen. The motions that could be performed with the stifle joint were as follows: flexion-extension rotation, medial-lateral translation, valgus-varus rotation, cranial-caudal translation, internal-external rotation, and proximal-distal translation about each axis (Figure 3) The flexion-extension axis was defined on the basis of insertions of the MCL and LCL on the femur and the internal-external rotation axis was defined on the basis of bone landmarks on the tibia The valgus-varus axis was defined as the line perpendicular to the flexion-extension and internal-external rotation axes The conceptual drawing shows the motions of the femur and the tibia The procedure was also performed with half of this load (30 N) applied to observe the change in the in situ force experienced by each ligament with the change in load The proximal-distal testing was performed and output recorded for each specimen under loads of 30 and 60 N as follows: first with the joint and its ligaments intact; second with the CrMB of the CCL transected; third with the CaLB of the CCL transected; fourth The outputs of the 3 forces in the UFS were recorded before (fx and fz') transection of each ligament in the assigned order and the in situ forces on each ligament were calculated as described we performed joint incision and blunt dissection at the boundary line between the CrMB and CaLB orientation during sample preparation Two nylon sutures were used as guides for transection; one was passed into the joint and around the CrMB and the other was passed into the joint and around the entire CCL Transections were performed with a scalpel Data analysis was performed with statistical software.d The Shapiro-Wilk test confirmed that continuous data were normally distributed and LCL were compared by means of 1-way ANOVA for repeated measurements and the Tukey-Kramer test was performed for post hoc analysis The paired t test was used to compare the TPA before and after osteotomy for the excessive TPA group and to compare the tensile forces on a given ligament type at compressive loads of 30 and 60 N The 2-sample t test was used to compare the age and TPA of dogs between groups prior to experimental procedures and to compare ligament tensile forces between the intact and excessive TPA groups at each load Differences were considered significant at values of P < 0.05 These analyses were performed after the F test to evaluate homogeneity of variance The mean ± SD TPA of stifle joints was 30.4 ± 2.9° (range 29° to 34°; prior to modification) for the unchanged and excessive TPA groups respectively; these results did not differ significantly (P = 0.714) between groups the mean ± SD age (19.5 ± 12.2 months; range 12 to 48 months) and body weight (9.5 ± 0.6 kg; range 8.1 to 10 kg) for the unchanged TPA group did not differ significantly from those for the excessive TPA group (15.1 ± 2.4 months [range 12 to 19 months; P = 0.348] and 10.4 ± 1.2 kg [range the mean ± SD TPA after osteotomy (41.1 ± 1.7°) was significantly (P < 0.001) greater than that prior to modification When the 30-N compressive load was applied, the in situ tensile forces on the CrMB of the CCL, CaLB of the CCL, MCL, and LCL in the unchanged TPA group were 8.0 ± 4.5 N, 8.4 ± 4.8 N, 2.8 ± 2.7 N, and 10.7 ± 11.4 N, respectively (Figure 4) No significant (P = 0.141) difference was found among the tensile forces on the 4 ligaments at this load When the 60-N compressive load was applied with a significant (P = 0.003) difference among ligaments; post hoc testing indicated these forces on the CaLB and LCL were significantly (P = 0.004 and 0.010 Figure 4Mean ± SD in situ tensile forces on the CrMB of the CCL, CaLB of the CCL, MCL, and LCL in cadaveric stifle joints of 8 skeletally mature Beagles of the unchanged TPA group at compressive loads of 30 N (white bars) and 60 N (gray bars). The force on each ligament was determined according to a previously described method.18 *Values are significantly (P < 0.05) different between the 30- and 60-N loads for a given ligament (paired t test) compared with that for the MCL under the same load (ANOVA followed by the Tukey-Kramer test) The in situ tensile forces on the CrMB and CaLB of the CCL in the unchanged TPA group were significantly (P = 0.007 and 0.010, respectively) higher at the 60-N compressive load than those at the 30-N load (Figure 4) no significant difference in these forces was observed between the 30- and 60-N loads for the MCL (P = 0.844) or LCL (P = 0.210) When the 30-N compressive load was applied, the in situ tensile forces on the CrMB of the CCL, CaLB of the CCL, MCL, and LCL in the excessive TPA group were 7.7 ± 2.9 N, 10.0 ± 6.4 N, 1.2 ± 1.5 N, and 11.9 ± 9.5 N, respectively (Figure 5) Significant (P = 0.010) differences in these forces were detected among ligaments and post hoc testing revealed that the tensile forces on the CaLB and LCL were significantly higher than that on the MCL (P = 0.030 and 0.007 with a significant (P < 0.001) difference among ligaments; on post hoc testing these forces were significantly higher in the CrMB (P = 0.012) Figure 5Mean ± SD in situ tensile forces on the CrMB of the CCL, CaLB of the CCL, MCL, and LCL ligaments in cadaveric stifle joints of 8 skeletally mature Beagles of the excessive TPA group at compressive loads of 30 and 60 N. See Figure 4 for key The in situ tensile forces on the CrMB and CaLB of the CCL in the excessive TPA group were significantly (P = 0.002 and 0.002 respectively) higher at the 60-N compressive load than those at the 30-N load no significant difference in these forces was found between the 30- and 60-N loads for the MCL (P = 0.939) or LCL (P = 0.189) No significant difference was found between the unchanged TPA and excessive TPA groups for in situ tensile forces on the CrMB of the CCL (P = 0.894) or LCL (P = 0.823) when the 30-N compressive load was applied the tensile force on the CrMB was significantly higher in the excessive TPA group than in the unchanged TPA group and the tensile force on the MCL was significantly lower in the excessive TPA group than in the unchanged TPA group (P = 0.004 our results for the unchanged TPAs were fairly similar to those reported in that investigation These findings collectively suggest that as compressive force parallel to the tibial axis increases (eg the CaLB may become taut at a stifle joint angle of 135°; therefore the stress may be more focused on the CaLB than on the CrMB the most commonly identified lesion was a rupture of the CrMB (20/25 dogs) although results of the present study suggested that the tensile force on the CrMB is somewhat smaller than that on the CaLB (without a significant difference) our results indicated that as the TPA increases a greater force that may lead to injury is generated on the CrMB the data indicated that the tensile force on the CrMB of the CCL was significantly higher in the excessive TPA group than in the unchanged TPA group This suggested that the proximal tibial shape may be important for the tensile forces experienced by the stifle joint ligaments and that an excessive TPA may magnify CCL stress and alter CCL strength On the basis of results of those previous investigations and the present study and because dogs with an excessive TPA (≥ 35°) are at risk for sustaining a CCL rupture in the future we suggest that these dogs should be monitored closely changes to the patellar ligament might influence the shear force on the CCL Although the present study did not investigate the forces of the quadriceps muscle and patellar ligament it was possible that translocation of the enthesis of the patellar ligament changed the shear force on the stifle joint The present study showed that tensile force on the CCL increased in situ with an increased compressive load while the collateral ligament tensile forces remained unchanged We showed that an excessive TPA increases stress in the CCL the TPA-increasing procedure in this study increased the tensile force on the CCL without having a major impact on the MCL and LCL No funding was received in association with this study The authors declare that there were no conflicts of interest The authors acknowledge Daichi Katori, Kotaro Kawakita, Yukino Suyama, Shoko Terashima, and Hitoshi Mukaitouge from Nippon Veterinary and Life Science University for their help with study experiments. English language editing was provided by Editage (www.editage.com) Estimate of the annual economic impact of treatment of cranial cruciate ligament injury in dogs in the United States The cruciate ligaments of the canine stifle: an anatomical and functional analysis Cranial tibial thrust: a primary force in the canine stifle and body weight as risk factors for rupture of the cranial cruciate ligament in young dogs Comparison of tibial plateau angles in normal and cranial cruciate deficient stifles of Labrador Retrievers Risk factors for excessive tibial plateau angle in large-breed dogs with cranial cruciate ligament disease Histological and immunohistological analysis of degenerative changes in the cranial cruciate ligament in a canine model of excessive tibial plateau angle Tibial plateau leveling osteotomy for cranial cruciate ligament rupture in the canine Vet Clin North Am Small Anim Pract 1993;23:777–795 Analysis of passive tibio-femoral joint movement of Beagle dogs during flexion in cadaveric hind limbs without muscle Change in tibial plateau angle after tibial plateau leveling osteotomy in dogs Combination tibial plateau leveling osteotomy and cranial closing wedge osteotomy of the tibia for the treatment of cranial cruciate ligament-deficient stifles with excessive tibial plateau angle A novel robotic system for joint biomechanical tests: application to the human knee joint Tibial displacement with stifle joint flexion and cranial cruciate ligament transection in the dog: an ex vivo study using a robotic simulator Optimization of graft fixation at the time of anterior cruciate ligament reconstruction: part I: effect of initial tension A joint coordinate system for the clinical description of three-dimensional motions: application to the knee Forces and moments in six-DOF at the human knee joint: mathematical description for control The use of a universal force-moment sensor to determine in-situ forces in ligaments: a new methodology Evaluation of a pressure walkway system for measurement of vertical limb forces in clinically normal dogs Biomechanics of tibial plateau leveling of the canine cruciate-deficient stifle joint: a theoretical model Partial rupture of the cranial cruciate ligament of the stifle in dogs: 25 cases (1982–1988) Degenerative changes of the cranial cruciate ligament harvested from dogs with cranial cruciate ligament rupture Angle between the patellar ligament and tibial plateau in dogs with partial rupture of the cranial cruciate ligament Subscribe to newsletters © 2025 American Veterinary Medical Association. All rights reserved. Powered by KGL PubFactory Metrics details Autism spectrum disorder (ASD) is a multifactorial disorder with characteristic synaptic and gene expression changes Early intervention during childhood is thought to benefit prognosis we examined the changes in cortical synaptogenesis and gene expression from birth to the juvenile stage in a marmoset model of ASD induced by valproic acid (VPA) treatment while juvenile-age VPA-treated marmosets showed increased synaptogenesis synaptic plasticity transiently increased and was associated with altered vocalization Synaptogenesis-related genes were downregulated early postnatally the differentially expressed genes were associated with circuit remodeling similar to the expression changes observed in humans we provide a functional and molecular characterization of a non-human primate model of ASD highlighting its similarity to features observed in human ASD there is an urgent need to identify biological abnormalities both to understand the pathogenesis and to develop effective pharmacological treatments that can be started during the early developmental stage Synaptic development involves two processes: genetically programmed synaptogenesis and activity-dependent remodeling Impairment of these processes is likely to adversely affect normal circuit generation with consequent ASD symptoms We also analyzed gene expression modulations at these ages The present study indicated concurrent changes in synaptic and molecular phenotypes with development Synapses of the model animal were underdeveloped in neonates which was different from synaptic overdevelopment commonly observed in the model at puberty and in human ASD Synaptic plasticity was transiently enhanced during infancy We also clarified behavioral outcomes at infancy Clustering of genes based on the temporal profile of modulation revealed genes associated with synaptogenesis and synaptic remodeling Molecular phenotypes of human idiopathic ASD were well replicated in this model after infancy in a large number of coexpression modules but only partially in various rodent models suggesting a closer molecular pathway in the primate model that is affected in ASD This study demonstrates the importance of early phenotypes in the pathogenesis of ASD and suggests novel molecular targets for early-age intervention and good outcomes throughout life a Slice preparations from UE animals at 0, 3, and 6 M, with schematic adapted from the atlas64 on the right b Photomicrograph of biocytin-stained dendritic spines in layer 3 pyramidal neurons from UE and VPA animals c Dendritic spine density in the basal dendrites of layer 3 pyramidal neurons from UE (blue) and VPA (red) animals The spine density was measured at the dendritic segment 25–50 µm (0 M) or 50–75 µm (3 M and 6 M) from the soma and n = 35 dendrites in 3 animals (6 M VPA) Two-sided t-test with Holm-Sidak correction in UE animals between 0 M and 3 M p = 0.0053; VPA animals between 3 M and 6 M ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant d Representative traces of mEPSCs recorded from layer 3 pyramidal neurons e The frequency (left) and amplitude (right) of mEPSCs in UE and VPA animals Two-sided t-test with Holm-Sidak correction for the frequency in UE animals between 0 M and 3 M ***p < 0.001; *p < 0.05; ns not significant f Representative mIPSC traces recorded from layer 3 pyramidal neurons g The frequency (left) and amplitude (right) of mIPSCs in UE and VPA animals Two-sided t-test with Holm-Sidak correction for the frequency between UE and VPA h logFC values of the spine density and the mEPSC and mIPSC frequencies *p < 0.05 (statistical tests and p values are as in the legends of c i The E/I ratio of miniature synaptic currents for UE and VPA animals Two-sided t-test with Holm-Sidak correction between UE and VPA j The E/I ratio of the evoked synaptic currents k logFC values of the E/I ratio in the frequency of miniature synaptic currents and the evoked synaptic currents **p < 0.01 and *p < 0.05 (statistical tests and p-values are as in the legends of i and j) The slightly different time courses between the spine density and mIPSC frequency may reflect differences in the efficacy of synaptic transmission and the E/I ratio a The time course of normalized field EPSP amplitudes before and after LFS onset that were recorded from slices obtained at 0 M (top) and 6 M (bottom) in UE (blue) and VPA (red) animals Insets represent the average traces of field EPSPs before (−10 to 0 min b Normalized field EPSP amplitudes in UE and VPA animals at post-LFS 30–40 min c Cumulative distribution of spine volume in UE and VPA animals at 0 Two-sided Kolmogorov–Smirnov test between UE and VPA animals d Representative electron micrograph of synapses in UE and VPA animals at 3 M These images are representative of 234 PSDs (UE) and 230 PSDs (VPA) e Cumulative distribution of the PSD length in UE and VPA animals at 3 M and 6 M Two-sided Kolmogorov–Smirnov test between UE and VPA animals at 3 M f logFC values for the normalized post-LFS field EPSP amplitudes and the spine volume plotted as a function of age **p < 0.01 (statistical tests and p values are as in the legends of b and c) prenatal VPA exposure does not affect mGluR-dependent LTD prenatal VPA exposure affects the E/I ratio and spine volume at 3 M with the effects at other ages being weaker This suggests that 3 M is another timepoint with strong synaptic phenotypes early synaptic abnormalities in the prefrontal cortex may affect vocal development a Representative vocalization spectrogram for UE (top) and VPA (top) animals at 12 weeks of age The type of call is shown below the spectrogram b The average number of calls in UE and VPA animals at 11–13 weeks n = 8 animals (UE) and n = 5 animals (VPA) c Average ratio of call types in UE and VPA animals d The average entropy of calls in UE and VPA animals We speculated that these phenotypes are accompanied by altered gene expression We searched for VPA-modulated gene expression using a custom-made microarray Among 9362 genes expressed in the three cortical regions (areas 8 there were 1037 differentially expressed genes (DEGs) with an absolute value of logFC of >0.4 and an adjusted p-value (padj) of <0.05 at either age a Distribution of the logFC values of gene expression significantly modulated by VPA exposure The logFC values at 3 M are plotted against those at 0 M (left) while the logFC values at 6 M are plotted against those at 3 M (right) The microarray data are from 10 samples from 5 animals (0 M UE) b Common upregulated and downregulated genes across ages P values are for the enrichment of common genes between ages (one-sided Fisher’s exact test) The traces in light colors represent data from individual genes in the cluster The traces in dark colors represent the average of all genes in the cluster d List of genes with the highest expression modulations in each cluster and critical period-related genes are marked e The distance between the average logFC values of gene expression for each cluster and the logFC values of structural and physiological parameters (spine density The column labeled “3r” shows the distance between the negative of the average logFC values of cluster 3 and the structural and physiological parameters to highlight parameters that are inversely correlated with cluster 3 f Enrichment of SFARI and critical period-related genes in each cluster The dotted line is the ratio among the entire gene set One-sided Fisher’s exact test for SFARI gene enrichment p = 0.68 (6 M); for critical period gene enrichment The color of the bars represents the direction of regulation of the pathway based on the logFC values at 0 M (cluster 1) and 3 M (clusters 2 and 3) The p-values of enrichment were provided by the IPA software The red dotted line represents the threshold of significance (p = 0.05) These results suggest that clusters 2 and 3 include genes associated with synaptic modulations that specifically occur at 3 M statins and an anti-inflammatory drug were predicted inflammation and calcium signaling were predicted Modulated genes with padj < 0.1 are plotted Spearman’s correlation coefficients (r) and two-sided p-values (p) are shown These data suggest that VPA-exposed marmosets replicate a broad range of human ASD components while rodent models generally replicate a part of the pathology including modulations in spine density and miniature synaptic current frequencies were correlated with modulations in the expression of cluster 1 genes The E/I balance and plasticity-related phenotypes were correlated with modulations in cluster 2 genes and inversely correlated with modulations in cluster 3 genes The entropy of calls was reduced at infancy when cluster 2 and 3 genes were maximally modulated This suggests that the phenotypes after complete maturation of the circuit may be different from the phenotypes we observed until puberty; moreover different therapeutic approaches may be needed for infantile and adult people with ASD Future research on phenotypes at later times into adulthood and in different areas or layers will provide further insight into ASD pathology Abnormal expression of the NMDA receptor subunits GRIN1 (glutamate ionotropic receptor NMDA type subunit 1 cluster 3) and GRIN2A may explain the age-dependent modulation of LTD the marmoset ASD model appeared to be a useful model for translational study the present study has the following limitations individuals with idiopathic ASD have different etiologies and exhibit different symptoms While the synaptic and molecular phenotypes in the ASD marmoset model after infancy revealed in this study were similar to the overall conditions of people with idiopathic ASD the translational validity of this model will be further enhanced by determining the subtype that this model best represents we did not address the roles of non-neuronal components such as astrocytes and microglia of which accumulating evidence suggests involvement in the pathogenesis of ASD early-age intervention proposed in this study requires early screening and diagnosis which are not fully established and medication to neonates and children generally involves the risk of adverse side effects this study indicated the critical importance of early biological phenotypes in ASD These findings could contribute to future age-dependent therapy for ASD in early life All experiments were approved by the Animal Research Committee of the National Center of Neurology and Psychiatry and the Animal Care and Use Committee of the National Institute of Radiological Sciences and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals Marmosets were bred in the National Center of Neurology and Psychiatry or the National Institute of Radiological Sciences The temperature and humidity were maintained at 27–30 °C and 40–50% Serum progesterone levels in female marmosets were measured; furthermore VPA was prepared as a 4% solution in 10% glucose solution and intragastrically administered to pregnant marmosets daily for 7 days from post-conception day 60 at 200 mg/kg/day Some UE animals received 10% glucose solution while other UE animals received no treatment Untreated animals were used because of the limited availability of treated animals The offspring of VPA-administered or UE marmosets were used at 0 M (4–11 days for electrophysiological and morphological analyses 3 M (88–110 days for electrophysiological and morphological analyses and 6 M (189–273 days for electrophysiological and morphological analyses There were no between-group differences in the pregnancy periods of the glucose-administered UE and VPA marmosets (139.5 ± 1.2 days for glucose-administered UE [mean ± SD n = 6] and 140.6 ± 1.7 days for VPA [n = 16] as well as the body weight at birth (29.0 ± 1.9 g for glucose-administered UE and 30.7 ± 2.4 g for VPA There was no significant difference in the body weight at birth between glucose-administered and untreated UE animals (29.7 ± 2.9 g for untreated animals 7–8 slices were obtained from each hemisphere The slices were placed on an interface-style chamber perfused with ACSF at 32 °C to allow recovery The slices were placed on a recording chamber that was continuously perfused with ACSF at 28 °C 1 µM tetrodotoxin (Fujifilm Wako Pure Chemical) was added to the ACSF 20 µM) or the GABAA receptor blocker picrotoxin (Cayman Chemical The internal solution contained the following (in mM): 130 Cs methanesulfonate Axon Instruments) was used for signal recording with a low-pass filter of 2 kHz The signals were recorded on a PC using a data acquisition board (PCI-6221 National Instruments) and Igor Pro software (WaveMetrics version 6.0) at a 10 kHz sampling frequency Data with unstable baseline or with a series resistance >28 MΩ (0 M) or >18 MΩ (3 and 6 M) were excluded which was close to the calculated equilibrium potential for Cl− which was close to the equilibrium potential for monovalent cations Miniature synaptic currents were analyzed using Mini Analysis (Synaptosoft version 6.03) and a custom-made program for MATLAB (Mathworks The signal was further bandpass filtered at 4–1000 Hz the standard deviation (SD) of the baseline was measured in a 50 ms segment lacking events We set the current amplitude threshold (in units of SD of the baseline) and current area threshold (in units of ms times SD of the baseline) for miniature events at 3 and 4.5 (0 M mEPSCs) Stimulus-evoked synaptic currents were induced by field stimulation of layers 4–5 using a bipolar tungsten electrode (tip separation 150 µm) connected to an isolator (BSI-950 which evoked approximately half-maximal EPSCs EPSCs and IPSCs were recorded at holding potentials of −65 and 0 mV Stimulus-evoked field potentials were recorded using an interface-style recording chamber under a binocular microscope The chamber was maintained at 35 °C and a glass recording electrode containing 0.5 M NaCl was placed in layer 3 at ~400–500 µm from the pia A bipolar tungsten stimulation electrode was placed in layers 4–5 at ~1–1.2 mm from the pia The signal was amplified using an amplifier (ER-1 Cygnus) with a low-pass filter at 1 kHz and recorded on a PC at a 10 kHz sampling frequency Test stimuli were applied at a 30 s interstimulus interval Up to two pathways were used in the same slice The stimulus intensity was adjusted to evoke a field EPSP with one phase decay (100–500 µA) The field EPSP was completely abolished after perfusion with 20 µM NBQX (Tocris) and 50 µM D-APV (Cayman Chemical) When the baseline amplitude was stable for >20 min The dependency of LFS-induced LTD on NMDA receptors was confirmed using 100 µM D-APV in 0 M slices We induced mGluR-dependent LTD by perfusion with 100 µM DHPG (Tocris) for 10 min After whole-cell recording using biocytin-containing electrodes the slices were fixed in 4% paraformaldehyde overnight and stained using the Vectastain Elite ABC Kit (Vector Laboratories) and DAB Substrate Kit (Vector Laboratories) The slices were dehydrated using a graded ethanol series the other spine volumes were estimated by their optical density measurements the fixed slices were cryosectioned at 40 µm and after defatting with chloroform/ethanol and rehydration the sections were stained with 0.1% thionin slices were immediately fixed after slicing in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) for at least 5 days The slices were then treated using 1% osmium oxide in cacodylate buffer for 60 min and stained with 2% uranyl acetate for 60 min the tissues were embedded in Epon 812 (TAAB) Ultrathin sections (70 nm thick) were prepared and examined using a Tecnai Spirit electron microscope (Thermo Fisher Scientific-FEI) Continuous sequences of multiple calls without a silent gap (multisyllabic calls) were considered a single bout of call Call type entropy was calculated using the ratio of each call type ri as −Σi ri log2 ri Differential gene expression was evaluated based on the p value from Welch’s t-test with Benjamini-Hochberg adjustment (padj) DEGs (absolute value of logFC > 0.4 and padj < 0.05 at either age) were clustered using the k-means algorithm which yielded the lowest value of Akaike’s information criterion we used data from the probe with the lowest padj To compare UE and VPA data at multiple timepoints the data were tested by Welch’s t-test with Holm-Sidak correction Two-way repeated-measures ANOVA was used to compare the spine density along the dendrites and the paired-pulse ratio Comparisons between two groups were performed using the t-test The Kolmogorov–Smirnov test was used to compare the distribution of spine volume and PSD length Enrichment of genes was analyzed using Fisher’s exact test Spearman’s correlation was used to determine the correlation of logFC values between marmosets or rats and humans The concordance of gene expression modulations between the model animals and human ASD was tested using the binomial test Tests for gene enrichment and concordance were one-sided Further information on research design is available in the Nature Research Reporting Summary linked to this article The source codes for the analysis of miniature synaptic currents and spine volume have been deposited at GitHub (https://github.com/ncnp-bisai/matlab) Insufficient evidence for “autism-specific” genes and management of children with autism spectrum disorder Dendritic spine pathology in neuropsychiatric disorders Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits Synaptopathology involved in autism spectrum disorder Regional differences in synaptogenesis in human cerebral cortex Postnatal development of dendritic structure of layer III pyramidal neurons in the medial prefrontal cortex of marmoset Pre- and postnatal development of the primary visual cortex of the common marmoset and elimination of synapses as overlapping processes The dorsal medial prefrontal cortex responds preferentially to social interactions during natural viewing Oxytocin’s neurochemical effects in the medial prefrontal cortex underlie recovery of task-specific brain activity in autism: a randomized controlled trial A comprehensive transcriptional map of primate brain development Evolutionary conservation and divergence of the human brain transcriptome Comparative hippocampal synaptic proteomes of rodents and primates: differences in neuroplasticity-related proteins Advances in nonhuman primate models of autism: Integrating neuroscience and behavior Postnatal development of layer III pyramidal cells in the primary visual Indifference of marmosets with prenatal valproate exposure to third-party non-reciprocal interactions with otherwise avoided non-reciprocal individuals Inequity aversion is observed in common marmosets but not in marmoset models of autism induced by prenatal exposure to valproic acid Prenatal valproate exposure and risk of 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subcortical development of the marmoset brain from infancy to adulthood Mapping gray matter development: Implications for typical development and vulnerability to psychopathology Comparison of androgen receptor and oestrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testost Changes in endocrine profile and reproductive organs during puberty in the male marmoset monkey (Callithrix jacchus) Hypothalamic-pituitary-gonadal relationships in man from birth to puberty Cytoarchitectonic subdivisions of the dorsolateral frontal cortex of the marmoset monkey (Callithrix jacchus) and their projections to dorsal visual areas Rearrangement of the dendritic morphology in limbic regions and altered exploratory behavior in a rat model of autism spectrum disorder Balancing plasticity/stability across brain development Excitation-inhibition balance as a framework for investigating mechanisms in neuropsychiatric disorders Plasticity of cortical excitatory-inhibitory balance Understanding intellectual disability and autism spectrum disorders from common mouse models: synapses to behaviour Induction of NMDA receptor-dependent long-term depression in visual cortex does not require metabotropic glutamate receptors Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses Cognitive control of vocalizations in the primate ventrolateral-dorsomedial frontal (VLF-DMF) brain network Gene expression patterns in visual cortex during the critical period: synaptic stabilization and reversal by visual deprivation Gene expression analysis of the critical period in the visual cortex and regional gene expression patterns in autism Transcriptome of iPSC-derived neuronal cells reveals a module of co-expressed genes consistently associated with autism spectrum disorder FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders Transcriptional and splicing dysregulation in the prefrontal cortex in valproic acid rat model of autism Development of non-phosphorylated neurofilament protein expression in neurones of the New World monkey dorsolateral frontal cortex Topographic and laminar maturation of striate cortex in early postnatal marmoset monkeys as revealed by neurofilament immunohistochemistry DNA methylation and susceptibility to autism spectrum disorder Emerging roles of synapse organizers in the regulation of critical periods The regulatory role of long-term depression in juvenile and adult mouse ocular dominance plasticity Limiting parental interaction during vocal development affects acoustic call structure in marmoset monkeys Cadm1-expressing synapses on Purkinje cell dendrites are involved in mouse ultrasonic vocalization activity Astrocytes and microglia and their potential link with autism spectrum disorders Efficacy of early interventions for infants and young children with Loss of mevalonate/cholesterol homeostasis in the brain: a focus on autism spectrum disorder and Rett syndrome Sustained correction of associative learning deficits after brief early treatment in a rat model of Fragile X Syndrome A dual effect of ursolic acid to the treatment of multiple sclerosis through both immunomodulation and direct remyelination The Marmoset Brain in Stereotaxic Coordinates (Academic Press A quantitative acoustic analysis of the vocal repertoire of the common marmoset (Callithrix jacchus) Acoustic analysis of vocal development in a New World primate Structure and usage of the vocal repertoire of Callithrix jacchus Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: Potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I) Developmental genetic profiles of glutamate receptor system protector of normal tissue and mitochondria and reelin in marmoset cortex: potential molecular mechanisms of pruning phase of spines in primate synaptic formation process Download references Tsuchiya for assisting with the marmoset experiments This work was supported by Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (29-6 JSPS KAKENHI Grant Number JP18K06497 (J.N.) and AMED Grant Number JP21dm0207066 (N.I.) National Center of Neurology and Psychiatry Department of Degenerative Neurological Diseases National Institutes for Quantum and Radiological Science and Technology planned the project and designed the experiments Sakai performed electron microscopy analyses supported electrophysiological experiments All authors discussed the results and commented on the manuscript The authors declare no competing interests Peer review information Nature Communications thanks Nafiseh Atapour and the other anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-021-25487-6 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. 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Volume 12 - 2018 | https://doi.org/10.3389/fnana.2018.00089 Neural activity in the middle temporal (MT) area is modulated by the direction and speed of motion of visual stimuli The area is buried in a sulcus in the macaque but exposed to the cortical surface in the marmoset making the marmoset an ideal animal model for studying MT function To better understand the details of the roles of this area in cognition underlying anatomical connections need to be clarified Because most anatomical tracing studies in marmosets have used retrograde tracers the axonal projections remain uncharacterized In order to examine axonal projections from MT we utilized adeno-associated viral (AAV) tracers which work as anterograde tracers by expressing either green or red fluorescent protein in infected neurons AAV tracers were injected into three sites in MT based on retinotopy maps obtained via in vivo optical intrinsic signal imaging Brains were sectioned and divided into three series one for fluorescent image scanning and two for myelin and Nissl substance staining to identify specific brain areas Overall projection patterns were similar across the injections V3 (VLP) and V4 (VLA) and surrounding areas in the temporal cortex including MTC (V4T) There were also projections to the dorsal visual pathway There was a visuotopic relationship with occipital visual areas In a marmoset in which two tracer injections were made the projection targets did not overlap in A8aV and AIP suggesting topographic projections from different parts of MT Most of these areas are known to send projections back to MT suggesting that they are reciprocally connected with it studies on MT functions have played an important role in cognitive neuroscience so it is difficult to compare results in terms of currently known areas The experiments were performed in two marmosets (Callithrix jacchus; Table 1) All experimental procedures were approved by the Experimental Animal Committee of RIKEN or by the Experimental Animal Committee of the National Center of Neurology and Psychiatry The marmosets were handled in accordance with the “Guiding Principles of the Care and Use of Animals in the Field of Physiological Science” formulated by the Japanese Physiological Society Virus injections based on retinotopy maps obtained using in vivo optical intrinsic signal imaging (A) Cortical surface images through the intact dura in regions around the middle temporal (MT) area in the left hemispheres of marmosets 1 and 2 (B,C) Retinotopy maps and tracer injection sites (B,C) Color-coded retinotopy maps of visual field locations estimated with the annulus (B) and wedge (C) apertures The green and red dots indicate virus injection sites (D) Zoomed-in images of parts of brain sections around the injection sites showing virus tracer spread which was set on a custom holder with a rotating stage on a tripod The fovea direction was back-projected on a computer monitor by rotating the stage 180° to align stimulus locations an expected value was calculated for each pixel using the following formula: USA) which also delivered trigger signals to an image acquisition PC Virus injections were made to MT regions representing near, but not in, the central visual field (green tracer, case 1; Figures 1B,C) and a peripheral and lower visual field (red tracer and a peripheral visual field around the horizontal meridian in marmoset 2 (red tracer which work as anterograde tracers by expressing fluorescent proteins in infected neurons were injected into each designated site through a glass-pipette attached to an injector (Nanoject II Two minutes after positioning of the pipette tip at a depth of 800 μm from the cortical surface 200 nl of a viral tracer was injected at a rate of 25 nl/min The tracers were a mixture of AAV1-Thy1S-tTA (1 × 109 vector genomes (vg)/μl) and AAV1-TRE-clover (5 × 109 vg/μl) for green fluorescent protein and AAV1-Thy1S-tTA (1 × 109 vg/μl) and AAV1-TRE3-tdTomato (5 × 109 vg/μl) for red fluorescent protein the bone was replaced directly over the cortex after carefully ensuring that there was no ongoing bleeding The bone was sealed with thin dental cement and the skin was sutured Each voxel of the 3D-reconstructed fluorescent signals was projected onto the mid-depth cortical surface based on the gradients of the potential The projected fluorescent signals and identified brain areas were assigned to each polygon of the surface Custom Matlab scripts were used unless otherwise indicated Based on in vivo optical intrinsic signal imaging (Figures 1A–C), the virus tracers were injected into MT regions representing near the central field (case 1), a peripheral lower visual field (case 2) and a peripheral visual field around the horizontal meridian (case 3). The spreads of all injection sites were ~1 mm in diameter when examined in histological sections (Figure 1D) which may correspond to thick stripes in V2 Figure 2. Brain section images at levels of the occipital cortex. (A) Fluorescent section image from cases 1 (green) and 2 (red). Green and red channel images taken with FITC and TRITC filters were overlaid. The + and − symbols indicate receptive field locations in the upper and lower visual fields, respectively, for each visual cortical area (Paxinos et al., 2012) Some retrogradely labeled cells in V1 were indicated by arrows (D–F) Images of brain sections from case 3 (red) slightly caudal from the sections in (A–C) shown in the same format as in (A–C) except that a far-red channel image (Cy5 filter) was used as a green channel and overlaid to enhance visibility of the projection by making the background autofluorescence appear yellowish In case 3, consistent with the above-described cases, there were projections to layer 6 of V2 and V1 (Figure 2D). Because the site of this injection was an MT region representing an area around the horizontal meridian, the injection labeled axons going to both upper and lower visual fields (Figure 2D). The seemingly weaker projection may be because the volume of infected neurons was smaller than in the other cases (Figure 1D) although the same amount of tracer was injected All injection sites had projections to V3, V3A (DA) and V4 (VLA; Figures 2–4). Overall the projections to the occipital visual areas targeted supragranular layers and layer 6, with weaker labeling in layer 4 (except layer 4B in V1), suggesting feedback-type connections (Rockland and Pandya, 1979; Maunsell and van Essen, 1983b) Figure 3. Brain section images at caudal levels from the injection sites, utilizing the same format as in Figure 2 (A) Fluorescent section image from case 1 (green) and 2 (red) (B) Image of the corresponding myelin-stained section (C) Images of the corresponding Nissl substance-stained section (D–F) Images of brain sections from case 3 Figure 4. Brain section images at levels of the injection sites showing projections to intraparietal areas, utilizing the same format as in Figure 2 Inset in (A) is a higher magnification view showing horizontal connection in gray matter In (A) the centers of the injection sites were slightly caudal from this section whereas it was slightly rostral from this section in (D) There were strong projections from MT to V6 (DM) in all the injections (Figures 2, 3). Those projections targeted all layers. In case 2, labeling was found in both upper and lower quadrant visual fields located in V6, consistent with the fact that V6 covers the entire contralateral visual fields (Galletti et al., 1999) Figure 5. Brain section images at levels of rostral from the injection sites, utilizing the same format as in Figure 2 For intraparietal areas, there were projections to LIP, MIP and AIP (Figures 4, 5). These targeted all layers (Figures 4A,D), and there was a tendency toward stronger labeling in superficial layers (Figure 5A). In the marmoset that received two tracer injections, labeling was found in the same area but separated in AIP (Figure 5A) suggesting that there is a topographic relationship between MT projections and AIP Figure 6. Brain section images at levels of the motor cortex, utilizing the same format as in Figure 2 Brain section images at levels of the prefrontal cortex (A,D,G) Fluorescent section images from cases 2 (red; A) (B,E,H) Images of the next myelin-stained sections (C,F,I) Images of the Nissl substance-stained section Flat map showing MT projection in case 1 (green) and 2 (magenta) White regions indicate overlaps of the two tracer projections The inset shows a part of the flat map in increased brightness The gray regions indicate injection sites in MT Figure 9. Flat map showing MT projection in case 3 (magenta). The same format as in Figure 8 In subcortical structures, MT projections were found in the superior colliculus (Figure 4A), caudate nucleus, lateral and inferior pulvinar nuclei and pontine nuclei (Figure 5) in all injections To visualize overall projection patterns, 3D reconstructions of the axonal projections were generated using the brain section images (Figures 10–12) MT had axon bundles in the white matter separately projecting to the prefrontal cortex temporal cortex (with horizontal connection in the gray matter) MT in the other hemisphere through the corpus callosum Lateral view of 3D reconstructions showing MT projection Dorsal view of 3D reconstructions showing MT projection Rostral view of 3D reconstruction showing MT projection MT projected to nearby temporal areas, MST, FST, FSTv (PGa/IPa), the occipital visual areas V1, V2, V3 (VLP), V4 (VLA), V4T (MTC), the dorsal visual pathway V3A (DA), parietal V6, V6A, intraparietal AIP, MIP, LIP and frontal A4ab, prefrontal A8aV and A8C in all tracer injections (Figure 13) New findings in this study are that there was MT projection to V6 (DM) A4ab and topographic MT projections to AIP and A8aV in marmosets A summary flat map showing consistency between injections the number of cases where MT projection was found was indicated by gray scale Those are the areas that MT projected to in the present study This demonstrates that these connections were well conserved during evolution those findings were consistent with the MT projections detected in marmosets we also found MT projections to the pulvinar terminal nucleus and pontine nucleus in marmosets suggesting that these connections are reciprocal TT and NI contributed to the experimental design data interpretation and writing of the manuscript SW and WS performed the marmoset experiments NK and KS performed the histological experiments This research was supported by AMED under grant number JP18dm0207001 and by MEXT KAKENHI under grant number JP 17K13274 (awarded to HA) We thank Hiromi Nito and Noriko Murayama for their help with histology and image processing. The data-sets will be available after publication at a Brain/MINDS data portal https://www.bminds.brain.riken.jp/ 3D reconstruction of brain section images for creating axonal projection maps in marmosets Direction and orientation selectivity of neurons in visual area MT of the macaque A representation of the visual field in the caudal third of the middle tempral gyrus of the owl monkey (Aotus trivirgatus) Connection from cortical area V2 to MT in macaque monkey Resolving the organization of the third tier visual cortex in primates: a hypothesis-based approach Exact geodesics and shortest paths on polyhedral surfaces Near-isometric flattening of brain surfaces Visual receptive field organization and cortico-cortical connections of the lateral intraparietal area (area LIP) in the macaque doi: 10.1146/annurev.neuro.26.041002.131052 Pathways for motion analysis: cortical connections of the medial superior temporal and fundus of the superior temporal visual areas in the macaque A relationship between behavioral choice and the visual responses of neurons in macaque MT Cytoarchitectonic subdivisions of the dorsolateral frontal cortex of the marmoset monkey (Callithrix jacchus) and their projections to dorsal visual areas Anatomical and physiological definition of the motor cortex of the marmoset monkey Sensitivity of human visual and vestibular cortical regions to egomotion-compatible visual stimulation Spatial precision of population activity in primate area MT Multiple visual areas in the caudal superior temporal sulcus of the macaque Segregation of efferent connections and receptive field properties in visual area V2 of the macaque Cortical input to the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN) in macaques: a retrograde tracing study Cortical projections to the nucleus of the optic tract and dorsal terminal nucleus and to the dorsolateral pontine nucleus in macaques: a dual retrograde tracing study Response properties and receptive fields of cells in an anatomically defined region of the superior temporal sulcus in the monkey Cortical connections of areas V3and VP of macaque monkey extrastriate visual cortex doi: 10.1002/(sici)1096-9861(19970303)379:1<21::aid-cne3>3.0.co;2-k Receptive-field properties of neurons in middle temporal visual area (MT) of owl monkeys The cortical visual area V6: brain location and visual topography The cortical connections of area V6: an occipito-parietal network processing visual information Amygdalofugal and amygdalopetal connections with modality-specific visual cortical areas in macaques (Macaca fuscata Cortical connections of MT in four species of primates: areal Inferior frontal eye field projections to the pursuit-related dorsolateral pontine nucleus and middle temporal area (MT) in the monkey Structure and function of the middle temporal visual area (MT) in the marmoset: comparisons with the macaque monkey Projections from cortical visual areas of the superior temporal sulcus to the superior colliculus A weighted and directed interareal connectivity matrix for macaque cerebral cortex Functional properties of neurons in middle temporal visual area of the macaque monkey The connections of the middle temporal visual area (MT) and their relationship to a cortical hierarchy in the macaque Representation of glossy material surface in ventral superior temporal sulcal area of common marmosets occipital and temporal lobes of the monkey A selective impairment of motion perception following lesions of the middle temporal visual area (MT) Quantitative analysis of the corticocortical projections to the middle temporal area in the marmoset monkey: evolutionary and functional implications Cortical connections of area V6Av in the macaque: a visual-input node to the eye/hand coordination system The Marmoset Brain in Stereotaxic Coordinates Google Scholar Topography of the afferent connectivity of area 17 in the macaque monkey: a double-labelling study A modified technique for high-resolution staining of myelin Parallel motion signals to the medial and lateral motion areas V6 and MT+ Contrasting patterns of cortical input to architectural subdivisions of the area 8 complex: a retrograde tracing study in marmoset monkeys Bistratified distribution of terminal arbors of individual axons projecting from area V1 to middle temporal area (MT) in the macaque monkey Morphology of individual axons projecting from area V2 to MT in the macaque Laminar origins and terminations of cortical connections of the occipital lobe in the rhesus monkey Connections of the dorsomedial visual area: pathways for early integration of dorsal and ventral streams in extrastriate cortex Cortical afferents of visual area MT in the Cebus monkey: possible homologies between New and Old Worldmonkeys Cortical microstimulation influences perceptual judgements of motion direction Intrinsic connections and architectonics of the superior temporal sulcus in the rhesus monkey Post-rolandic cortical projections of the superior temporal sulcus in the rhesus monkey A computational analysis of the relationship between neuronal and behavioral responses to visual motion The organization of connections between areas V5 and V1 in macaque monkey visual cortex Independent projection streams from macaque striate cortex to the second visual area and middle temporal area The organization of connections between the pulvinar and visual area MT in the macaque monkey Topographic patterns of V2 cortical connections in macaque monkeys doi: 10.1002/(sici)1096-9861(19960715)371:1<129::aid-cne8>3.0.co;2-5 Functional columns in superior temporal sulcus areas of the common marmoset Emergence of complex wave patterns in primate cerebral cortex Visual Motion Discrimination by Propagating Patterns in Primate Cerebral Cortex Cortical connections of visual area MT in the macaque Projections to the superior temporal sulcus from the central and peripheral field representations of V1 and V2 Subcortical projections of area MT in the macaque The projections from striate cortex (V1) to areas V2 and V3 in the macaque monkey: asymmetries “FluoRender: an application of 2D image space methods for 3D and 4D confocal microscopy data visualization in neurobiology research,” in Proceedings of the IEEE Pacific Visualization Symposium (Songdo: IEEE) Google Scholar Systematic comparison of adeno-associated virus and biotinylated dextran amine reveals equivalent sensitivity between tracers and novel projection targets in the mouse brain Retinotopic patterns of connections of area 17 with visual areas V-II and MT in macaque monkeys Rapid adaptation induces persistent biases in population codes for visual motion Correlated variability in the neurons with the strongest tuning improves direction coding Correlated neuronal discharge rate and its implications for psychophysical performance Yamamori T and Ichinohe N (2018) Axonal Projections From the Middle Temporal Area in the Common Marmoset Received: 20 June 2018; Accepted: 10 October 2018; Published: 30 October 2018 Copyright © 2018 Abe, Tani, Mashiko, Kitamura, Hayami, Watanabe, Sakai, Suzuki, Mizukami, Watakabe, Yamamori and Ichinohe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Hiroshi Abe, aGlyb3NoaS5hYmVAcmlrZW4uanA= Noritaka Ichinohe, bmljaGlub0BuY25wLmdvLmpw † These authors have contributed equally to this work The February issue of Kodansha's Bessatsu Friend magazine announced on Friday that Rumi Ichinohe's Kimi no Yokogao o Miteita (I Was Looking At You In Profile) manga is entering the climax of its "Mari Arc" in the magazine's next issue on February 13 who is vaguely rooting for his best friend Mari to start a relationship with their cheerful classmate Ōtani after their handsome classmate Asagiri introduced Ōtani to Mari Mari herself has only thought about romance vaguely but Hikari is sure that Ōtani has a crush on Mari and will publish the second volume on February 13 The April issue of Kodansha's Bessatsu Friend magazine announced last Friday that manga creator Rumi Ichinohe will launch a new manga titled Kimi no Yokogao o Miteita (I Was Looking At You In Profile) in the magazine's next issue on April 13 The manga will feature on the issue's front cover and the first chapter will have a color opening page The magazine describes the manga as an "ensemble youth romance story" centering on two girls and two boys Source: Bessatsu Friend magazine's website Volume 16 - 2022 | https://doi.org/10.3389/fnbeh.2022.943759 Individuals with autism spectrum disorder (ASD) are exposed to a variety of stressors owing to their behavioral traits Cortisol is a hormone typically associated with stress and its concentration and response to stress are higher in individuals with ASD than in controls The mechanisms underlying cortisol dysregulation in ASD have been explored in rodents Although rodent models have successfully replicated the major symptoms of autism (i.e. and restricted/repetitive patterns of behavior) evidence suggests that the hypothalamic-pituitary-adrenal (HPA) axis system differs between rodents and primates We developed an ASD model in the common marmoset (Callithrix jacchus) utilizing prenatal exposure to valproic acid (VPA) we collected the salivary cortisol levels in VPA-exposed and unexposed marmosets in the morning and afternoon Our results revealed that both VPA-exposed and unexposed marmosets showed similar diurnal changes in cortisol levels which were lower in the afternoon than in the morning heightened cortisol levels were observed throughout the day in VPA-exposed marmosets These results are consistent with those of ASD in humans Our results suggest that VPA-exposed marmosets show similarities not only in their behavioral patterns and brain pathologies validating the usefulness of VPA-exposed marmosets also as a tool for ASD stress research the primate ASD model appears to be preferable for studying the hormonal stress response in ASD VPA-exposed marmosets recapitulate human ASD well in the four major cell types of the brain consisting of neurons and three types of glia VPA-exposed marmosets appear to provide a suitable model for translational research on ASD In this study, we collected the salivary cortisol from marmosets in the morning and afternoon. Salivary measurements of cortisol have been shown to closely mirror those in the serum (Perogamvros et al., 2010; VanBruggen et al., 2011) We found that VPA-exposed marmosets had heightened basal cortisol levels Our results suggest that VPA-exposed marmosets may be useful for translational research on stress pathophysiology in ASD All experimental animal care procedures were conducted under approved protocols according to the regulations of the National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan. Ten UE marmosets (five males and five females) and nine VPA-exposed marmosets (five males and four females) were included in this study (Table 1) The ages of the experimental animals range from 2- to 8-year-old The mean age was 4.7 ± 1.62 years (UE marmosets: 4.5 ± 1.43 The subjects were born and raised in family cages they were transferred to individual stainless steel home cages (Natsume Manufacturing Co. Japan) in order to avoid the confounding effect on cortisol levels due to interactions with conspecifics of the same cage The subjects were kept at room temperature of 29 ± 2°C and maintained on a 12 h:12 h light:dark cycle with free access to food and water The lights in the breeding room were turned on at 7:00 a.m Marmosets in the facility were familiar with human contact and approached experimenters to obtain food rewards without hesitation Valproic acid marmosets were exposed to valproic acid during their fetal stage, whereas UE marmosets were not (Yasue et al., 2015) The dams of VPA-exposed marmosets were housed in their cages Their blood progesterone levels were monitored periodically to determine the timing of pregnancy The VPA group received 200 mg/kg intragastric sodium valproate via an oral catheter daily on days 60 to 66 after conception This period was determined based on the administration period (E12 of the rat fetus) used to produce VPA-exposed rodent models of ASD All VPA dams received the medication without vomiting and showed no signs of abnormal pregnancy or delivery The dams of UE marmosets were administered neither VPA nor a solvent during this period to prevent miscarriage VPA marmosets displayed no malformations or body weight differences compared with UE marmosets A 2.0-mL Costar Spin-X centrifuge tube with a nylon filter (0.22 μm) was filled with the swabs and centrifuged at 10,000 rpm for 5 min to extract the liquid portion of the sample The collected samples were stored in a −80°C freezer until further use Saliva samples were collected three times per individual with no two collections occurring in the same month The duration of the experiment was 3 months Cortisol levels (μg/dl) were measured using an AIA-360 Automated Immunoassay Analyzer with AIA-pack cortisol test cups (Tosoh Corporation Japan) and averaged per subject across all saliva collections either in the morning or the evening Two-factor repeated-measures ANOVA and student-t test were performed using JMP 16 software (SAS Institute, Cary, NC, United States) when the data were determined to be normally distributed by the Shapiro–Wilk test or by the F-test. If we found that the data showed a non-normal distribution, a mixed design ANOVA by a downloadable program (ARTool) (Wobbrock et al., 2018) was performed using R P-values less than 0.05 were considered statistically significant Salivary samples were successfully collected by experimenters while the marmosets were in their home cages They showed no signs of aggression or aversion during sampling No significant effect of sex on salivary cortisol levels was observed in both UE and VPA-exposed groups in the morning and evening we analyzed the data for male and female together Figure 1 shows group salivary cortisol levels (mean ± SD) measured in the morning (7:30 a.m.) and afternoon (6:30 p.m.) Two-factor repeated measures ANOVA showed a significant difference in the group factor [F(1,17) = 9.3153 p = 0.0072] and time factor [F(1,17) = 100.8143 We did not find any significant difference in interaction effects [F(1,17) = 4.0257 These results suggested that the VPA-exposed marmosets maintained diurnal changes with high cortisol levels in the morning that fell throughout the afternoon and that salivary cortisol levels in the VPA-exposed group were significantly higher than those in the UE group at the times examined in this study Salivary cortisol levels in the morning (7:30 a.m.) and afternoon (6:30 p.m.) in the valproic acid-exposed (VPA group) and unexposed (UE group) marmosets Values are expressed as mean ± standard deviation (n = 10 for UE group We have also examined the correlation between age and salivary cortisol level Although no statistically significant correlation between age and cortisol level was found at any time of day or in any group there was a positive correlation in UE marmosets in the afternoon (r = 0.58) and a weak positive correlation in VPA-exposed marmosets (r = 0.32) no correlation between age and salivary cortisol level was found for both groups Unfortunately, we found that among the VPA-exposed marmosets used in the current experiment, only three VPA-exposed marmosets were involved in the task presented in the previous papers (Yasue et al., 2015, 2018; Nakagami et al., 2022) one marmoset showed the lowest morning salivary cortisol level (23.1 μg/dL) This level was half that of the other two VPA-exposed marmosets and was similar to the average level of the UE marmoset This VPA-exposed marmoset had the best performance in three social tasks (social gazing and third-party reciprocity) among these three VPA-exposed marmosets These anecdotal observations suggest that there may be a relationship between cortisol levels and levels of social impairment in VPA-exposed marmosets Of the animals in this experiment, there were two pairs of siblings in UE marmosets and two pairs in VPA marmosets (Table 1) None of these pairs had cortisol levels biased in one direction relative to the mean in both morning and evening (data not shown) suggesting that the results in this study were not biased due to the inclusion of siblings Note that saliva sampling in this study was from marmosets that were in individual cages so the results of this study may include isolation-related stress We considered the possibility that the time between the animals’ transfer to the single cage and the sampling start point could affect the results of this study there was no significant difference in the time from isolation to the experiment between UE and VPA-exposed marmosets (p = 0.8659 We also examined temporal changes of cortisol level in samples at three time points taken from each animal separately in the morning and afternoon A mixed design ANOVA showed a significant difference only in the group factor [F(1,16) = 7.6731 but not in the time factor and interaction effects in the morning or afternoon The results indicate that there is no specific pattern of variation in cortisol level among three samples over 3 months in either UE or VPA suggesting the effect of isolation time before the experiment is minimal in both groups This suggests that VPA-exposed marmosets may be in a constant state of stress our results are consistent with these reports VPA-exposed marmosets replicated the abnormal endocrine function observed in people with ASD this technique is still invasive and not suitable for long-term cortisol level monitoring we established a method to collect saliva from marmosets after acclimatization by training The current procedure using marmosets will allow the repeated examination of cortisol levels in ASD models in the same individuals both at basal levels and in the stress response and will contribute to a reduction in the number of experimental animals This study revealed that VPA-exposed marmosets reproduced the variability of cortisol levels in human ASD. Marmosets are cooperative and highly social primates and are considered a suitable model animal to study stress in social life, and they also show higher social function deficits as in cases of ASD (Yasue et al., 2015, 2018; Watanabe et al., 2021; Nakagami et al., 2022) Further examination of cortisol levels in VPA-exposed marmosets would provide a new avenue for studying the biology of stress faced by individuals with ASD and for developing novel therapeutic interventions The original contributions presented in this study are included in the article/supplementary material Further inquiries can be directed to the corresponding authors This animal study was reviewed and approved by the regulations of the National Center of Neurology and Psychiatry (NCNP) KN managed the production and physical condition of the animals All authors have read and approved the final manuscript This research was supported by an Intramural Research Grant (Grant Number: 2-7 to NI) for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry by Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) the Japan Agency for Medical Research and Development (AMED) (Grant Number: 22 dm0207066h0004 to NI) and by JPSP KAKENHI (Grant Numbers: JP24600020 and JP15K01791 to AN and 16H02058 We thank Akiko Tsuchiya for providing technical support and Satoshi Watanabe for advice on statistical analysis American Psychological Association [APA] and American Psychiatric Association [APA] (2013) Diagnostic and Statistical Manual of Mental Disorders: DSM-5 Google Scholar Exaggerated responses to stress in the BTBR T+tf/J mouse: an unusual behavioral phenotype Circadian profile of peripheral hormone levels in Sprague-Dawley rats and in common marmosets (Callithrix jacchus) cocaine and ketamine: effects on the pituitary-adrenal axis in rhesus monkeys Variable cortisol circadian rhythms in children with autism and anticipatory stress and sensory sensitivity in children with autism Google Scholar The use of salivary cortisol measurements for the non-invasive assessment of adrenal cortical function in Guinea pigs In utero exposure to valproic-acid alters circadian organisation and clock-gene expression: implications for autism spectrum disorders Corticosteroid and neurosteroid dysregulation in an animal model of autism Vasopressin and oxytocin: hypothalamic modulators of the stress response: a review CrossRef Full Text | Google Scholar Do neonatal bilateral ibotenic acid lesions of the hippocampal formation or of the amygdala impair HPA axis responsiveness and regulation in infant rhesus macaques (Macaca mulatta) Stress and stress reduction in common marmosets Google Scholar Common marmosets (Callithrix jacchus) evaluate third-party social interactions of human actors but Japanese monkeys (Macaca fuscata) do not Marmoset monkeys evaluate third-party reciprocity Google Scholar Daytime secretion of salivary cortisol and alpha-amylase in preschool-aged children with autism and typically developing children Comparison of blood sampling methods for plasma corticosterone measurements in mice associated with minimal stress-related artefacts Glucocorticoids and brown adipose tissue: do glucocorticoids really inhibit thermogenesis Prevalence and characteristics of autism spectrum disorder among children aged 8 years—autism and developmental disabilities monitoring network Enhanced anxiety and stress-induced corticosterone release are associated with increased Crh expression in a mouse model of Rett syndrome Reduced childhood social attention in an autism model marmoset predicts impaired social skills and inflexible behavior in adulthood Comparison of corticosterone responses to acute stressors: chronic jugular vein versus trunk blood samples in mice Salivary cortisone is a potential biomarker for serum free cortisol Unaltered hormonal response to stress in a mouse model of fragile X syndrome Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid Low stress reactivity and neuroendocrine factors in the BTBR T+tf/J mouse model of autism Individual differences in the diurnal cycle of cortisol Google Scholar Enhanced cortisol response to stress in children in autism Altered circadian patterns of salivary cortisol in low-functioning children and adolescents with autism Early neurogenesis in the anterior hypothalamus of the rhesus monkey The relationship between serum and salivary cortisol levels in response to different intensities of exercise Functional and molecular characterization of a non-human primate model of autism spectrum disorder shows similarity with the human disease Wobbrock, J. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Nobuyuki Kawai, a2F3YWlAaXMubmFnb3lhLXUuYWMuanA=; Noritaka Ichinohe, bmljaGlub0BuY25wLmdvLmpw Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish. Volume 10 - 2019 | https://doi.org/10.3389/fpls.2019.00401 Soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) is the most destructive pest affecting soybeans [Glycine max (L.) Merr.] in the U.S identified in PI 88788 (rhg1) and Peking (rhg1/Rhg4) have been widely used to develop SCN resistant cultivars in the U.S some SCN populations have evolved to overcome the PI 88788 and Peking derived resistance making it a priority for breeders to identify new alleles and sources of SCN resistance 461 soybean accessions from various origins were screened using a greenhouse SCN bioassay and genotyped with Illumina SoySNP50K iSelect BeadChips and three KASP SNP markers developed at the Rhg1 and Rhg4 loci to perform a genome-wide association study (GWAS) and a haplotype analysis at the Rhg1 and Rhg4 loci which identified 12 SNPs at four genomic regions on Chrs 7 and 18 that were significantly associated with SCN resistance (P < 0.001) and 24 predicted genes were found near the significant SNPs on Chrs 7 and 10 KASP SNP genotyping results of the 462 accessions at the Rhg1 and Rhg4 loci identified 30 that carried PI 88788-type resistance and 58 that carried neither the Peking-type nor the PI 88788-type resistance alleles indicating they may possess novel SCN resistance alleles By using two subsets of SNPs near the Rhg1 and Rhg4 loci obtained from SoySNP iSelect BeadChips a haplotype analysis of 461 accessions grouped those 58 accessions differently from the accessions carrying Peking or PI 88788 derived resistance thereby validating the genotyping results at Rhg1 and Rhg4 and newly characterized SCN resistant accessions will be beneficial for the development of DNA markers to be used for marker-assisted breeding and developing soybean cultivars carrying novel sources of SCN resistance The continued planting of soybean varieties with PI 88788 and Peking derived SCN resistance will only increase the number of SCN populations that have overcome their defense mechanisms it is paramount importance to soybean breeders to identify new SCN resistant germplasm sources from diverse genetic backgrounds that have different defense mechanisms than Peking and PI 88788 and (3) to conduct a haplotype analysis of the 461 accessions near the Rhg1 and Rhg4 loci by using two subsets of SNPs on Chr 18 (103 SNPs) and Chr 8 (64 SNPs) obtained from the SoySNP50K iSelect BeadChips A total of 448 accessions from MG 0 to VIII were selected from the United States Department of Agriculture (USDA) Soybean Germplasm Collection and the University of Georgia (UGA) soybean breeding program for SCN greenhouse screening procedures. The accessions were assembled from 28 different countries, with 62% of them originating from China, the center of origin for the domestication of soybean (Hymowitz, 1970) The accessions were selected based on the origin diversity 289 accessions have not previously been phenotyped with SCN race 3 A total of 159 accessions with phenotypes available in Germplasm Resources Information Network (GRIN) database were also included in this panel to confirm their resistance and validate our KASP markers at Rhg1 and Rhg4 loci along with six known SCN susceptible cultivars (Hutcheson Lee 74) and one resistant line (G93-9009) were evaluated in the greenhouse and PI 548316) were also included in phenotyping for HG Type determination Based on the screening results of the three sets 106 accessions which were rated as resistant or MR were subsequently phenotyped with a new SCN race 3 population in the greenhouse during winter 2016 This new and more aggressive population of SCN race 3 (compared to HG type 0) was collected from Collins According to HG Type designation using the seven indicator lines this more aggressive SCN race 3 population was designated as HG Type 5 These accessions were sown in 10.2 cm wide clay pots filled with a fumigated sandy loam soil in December 2016 Pots were arranged in a RCBD with four replications A heat mat was placed underneath the pots to maintain temperature at 28–30°C and then thinned to a single seedling per pot after 7–9 days Twelve young leaves per line were collected, and then freeze-dried for 48 h. DNA was extracted from soybean leaves using a modified CTAB method (Keim et al., 1988) and stored at -20° C until use DNA concentration was quantified using a TECAN Infinite T1000 Pro (Tecan US United States) and diluted with water to 10–20 ng/μL for KASP assays KASP reactions were run in a 4 μL reaction The PCR fluorescent end reading was performed using a Light Cycler 480 Real Time PCR system (Roche More than 40,000 SNPs of the 461 accessions were obtained from the SoySNP50K Infinium Chip data 3 and Soybean Breeding and Genetics Lab database at the UGA SNPs were eliminated for analysis if they had no assigned physical position or a minor allele frequency (MAF) less than 0.05 35,817 SNPs met these criteria and were used to conduct GWAS The genotyping results of SNP markers GSM 381 and GSM 191 were also included in the GWAS analysis Because PI 670017 does not have SoySNP50K data cluster analysis was performed for 461 accessions Rhg1 and Rhg4 are two major effect loci that provide resistance to SCN race 3, as described above. At the Rhg1 locus on Chr 18, three genes are known to contribute to SCN resistance (Glyma.18g022400, Glyma.18g022500, and Glyma.18g022700 (corresponding to Glyma18g02580; Glyma18g02590, and Glyma18g02610, respectively, based on version 1.0 of Williams 82) (Cook et al., 2012) Based on the soybean reference genome sequence version 2.0 of Williams 82 approximately 500 kb flanking both sides of these three genes was selected for analysis resulted in the selection of 103 SNPs for the haplotype analysis Likewise, at the Rhg4 locus on Chr 8, the serine hydroxymethylytransferase (SHMT) gene was attributed with conveying SCN resistance (Liu et al., 2012) Two SNPs (ss715602757 and ss715602764) are situated close to this gene thus a 0.5 Mb region (based on the soybean reference genome sequence version 2.0 of Williams 82) flanking both sides of these SNPs were selected for analysis which resulted in the selection of 64 SNPs for the haplotype analysis at the Rhg4 locus The phenotyping data from the cyst counts were subjected to analysis of variance (ANOVA). A split-plot analysis for a mixed linear model was applied with blocks and sets being treated as random effects, while accession was considered as a fixed effect. R packages 4 and JMP software (SAS Institute No transformation of phenotype data was conducted prior to performing the GWAS analysis Both CMLM and ECMLM account for population structure and kinship while GLM with PCAs control for population structure Two criteria were used to predict candidate genes responsible for SCN resistance: (1) if a gene was implicated as a gene providing disease resistance for nematodes or other pathogens in previous studies or (2) if genes were located at genomic regions where the peak SNPs were placed as a result of the GWAS analysis The gene models without functional annotations or belonging to unknown functional families were excluded 31 were breeding lines or cultivars from the U.S. and 32 accessions were from six other countries Summary of greenhouse soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) bio-assay phenotyping results of 462 soybean accessions for SCN resistance which is very close to being classified as MR A large number (n = 311) of accessions rated moderately susceptible and susceptible in the greenhouse screening assay (including the six susceptible checks) matched the expected genotyping results by carrying susceptible alleles at both the Rhg1 and Rhg4 loci Summary of genotyping results at known major soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) resistance loci Rhg1 and Rhg4 on chromosomes 18 and 8 and phenotyping results of a greenhouse SCN bio-assay of 462 soybean accessions that were screened for SCN race 3 resistance Furthermore, 462 soybean accessions were genotyped using a functional KASP SNP marker GSM 039A (Pham et al., 2013) for southern root-knot nematode (Meloidogyne incognita Kofoid and White) resistance which is one of the most damaging pests in the Southern U.S Genotyping revealed that 135 accessions carried a previously identified allele providing resistance to this nematode 58 accessions were rated as resistant to SCN based on the greenhouse screening assay 18 appear to be carrying novel genes or alleles conveying SCN resistance These 58 accessions will be further subjected to a greenhouse phenotyping for southern root-knot nematode resistance Compared to the genotyping results generated from SNP markers (GSM 381 and GSM 383) at the Rhg1 locus 82% of the resistant accessions grouped into two clusters with Peking and PI 88788 A discrepancy was observed between our rhg1 markers and the results of the haplotype analysis for 16 accessions Dendogram of 461 soybean accessions generated using haplotype SNP markers at the Rhg1 locus (n = 103) on chromosome (Chr) 18 and Rhg4 locus (n = 64) on Chr 8 SNP data were obtained using the SoySNP50K iSelectBeadChips A total of 58 unique accessions (green) were grouped separately from PI 88788-type (pink) and Peking-type resistance (blue) at both of the Rhg1 and Rhg4 regions Cluster analysis based on the other subset of SNPs (n = 64) occupying a 997-kb region at the Rhg4 locus on Chr 8 placed 26 accessions into the Peking group, which was lower than the indicated genotyping results using KASP SNP marker GSM 191 at the Rhg1 locus (59 accessions). Of these 59 accessions, 30 accessions were placed in one cluster group and three were placed in a different cluster group, which were distinct from the Peking cluster (Figure 1B) One explanation for the difference in results between the haplotype analysis and the KASP functional markers genotyping might be that no informative SNPs at the Rhg1 and Rhg4 loci were present in the SoySNP50K Chip data LD decay distance was used to examine candidate genes within QTL regions and gene models that resided within a haplotype block or 125 kb genomic region of each significant SNP outside of haplotype block were considered Neighbor-joining tree (A,B) and principal component analysis (C,D) depicting different clusters that were formed among 461 soybean accessions grouped by country of origin (A,B) and maturity group (C,D) (MG) (A,B) Clusters are colored according to country of origin (China: red; Japan: pink; United States: green; other colors: other countries) (C,D) Clusters are colored according to MG The result of neighbor joining tree (A,B) was similar with PCA using first two components (C,D) The ss715606985 SNP on Chr 10 (resistance allele = GG) accounted for 25.6% of the phenotypic variation with an average FI index of 3.8% which was significantly lower than the average FI index of the whole panel (49.76%) A total of 55 resistant and MR accessions identified using the greenhouse screening assay carried the ss715606985 resistance allele on Chr 10 based on GWAS carried resistance alleles at the Rhg1 locus based on GSM 381 SNP marker allele calls no QTLs for SCN resistance at this region on Chr 10 have been reported using the populations derived from these known resistance sources Manhattan plot (A) and quantile-quantile (Q-Q) plot (B) generated from genome-wide analysis for soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) resistance among 461 soybean accessions using compressed mix linear model (CMLM) in GAPIT package Summary of significant SNPs that were identified for soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) resistance using multiple statistical GWAS models (GLM and ECMLM) among 462 soybean accessions phenotyped using a greenhouse SCN bio-assay for SCN resistance and genotyped using KASP SNP markers GSM 381 and GSM 383 at the Rhg1 locus KASP SNP marker GSM 191 at the Rhg4 locus and SoySNP50K iSelect BeadChips possible candidate genes were as follows: (1) a leucine rich repeat (LRR) protein kinase family protein gene (5) a transcription regulation gene and (6) others in miscellaneous groups Of these listed candidate genes and their ontologies some of them occupy domains on Chr 7 where R genes have previously been categorized such as a (LRR) receptor gene (Glyma.07g199500) Predicted genes for soybean cyst nematode (Heterodera glycines Ichinohe) (SCN) resistance located in three 100 kb haplotype block genomic regions near significant SNPs detected for SCN resistance on chromosomes 7 and 10 that were identified in a genome wide association study (GWAS) a new aggressive SCN population was found that was designated as HG Type 5 based on HG Type test and race 3 based on the previous determination test with four indicator lines Based on greenhouse phenotyping results with HG Type 0 106 R and MR lines were phenotyped again with HG Type 5 Our study was able to identify 34 accessions which are resistant or MR to the aggressive HG Type 5 Based on genes reported at Rhg1 and Rhg4 loci (Cook et al., 2012; Liu et al., 2012), three functional markers were designed to detect rhg1 and Rhg4 resistance alleles (Shi et al., 2015) to assist in identifying sources for SCN resistance other than rhg1 and Rhg4 alleles The genotyping results of the three KASP SNP markers at the Rhg1 and Rhg4 loci indicated that 58 accessions rated resistant and MR from the greenhouse screening did not carry either the rhg1 or the Rhg4 resistance alleles Haplotype analysis of these 58 accessions based upon SNPs at Rhg1 and Rhg4 regions also indicated that that these 58 accessions were not clustered with the accessions carrying the Peking-type or PI 88788-type haplotypes Based on both the genotyping results of the KASP SNP markers at the Rhg1 and Rhg4 loci and haplotype analysis using SNPs surrounding the Rhg1 and Rhg4 genomic regions on Chrs 18 and 8 these 58 accessions may possess novel resistance alleles conferring SCN resistance that are different than Peking or PI 88788 The origin for five of the 58 highest rated resistant accessions (PI 574484 and each was also characterized as being highly resistant to aggressive SCN HG Type 5 Four of them (except PI 603529) have been characterized as having desirable agronomic and consumer traits and low shattering (<2 in scale 5) (USDA-GRIN) based on genotyping results using SNP marker GSM 039A PI 561329 is also predicted to have resistance to southern root-knot nematode as were 18 out of the 58 SCN resistant or MR accessions were identified These 18 accessions appeared to be carrying novel genes or alleles conveying SCN resistance these accessions could be valuable germplasm sources for resistance to two different nematode species: SCN (Heterodera glycines Ichinohe) and southern root-knot nematode (Meloidogyne incognita Kofoid and White) The authors concluded that the N-ethylmaleimide sensitive factor (NSF) gene which was located near SNP ss715597431 on Chr 7 might be co-inherited with the NSF gene at Rhg1 locus on Chr 18 They found 855 soybean accessions from USDA germplasm which carried rhg1 resistance allele are also homozygous for SNP ss715597431 we found 87 of 89 soybean accessions carrying resistance allele at Rhg1 (both PI 88788 and Peking) had beneficial allele at ss715597431 locus two were rated as moderately and highly susceptible PI 398823 carried Peking-type resistance allele at Rhg1 locus but it did not possess resistance allele at Rhg4 five MR (FI: 10.7–28.9%) accessions that do not possess a resistance allele at Rhg1 based on our genotyping results carry resistance allele at SNP ss715597431 locus The genotyping results of SNP marker ss715606985 revealed that 55 of the resistant and MR accessions carried the same resistance allele (A); interestingly all of these 55 resistant accessions including Peking also carried the same resistance alleles as PI 567516C although no QTL controlling SCN resistance on Chr 10 was reported in bi-parental mapping populations derived from Peking the results of the greenhouse screening indicated that the average FI of accessions carrying the resistance allele at SNP marker ss715606985 on Chr 10 was significantly low (3.8%) suggesting that this SNP marker could locate in a genomic region responsible for SCN resistance This result highlights one of the advantages of using GWAS over bi-parental mapping populations by demonstrating that QTL mapping is limited by the number of recombination events occurring between two parents to map traits whereas GWAS is able to capture more allelic diversity from a larger panel of accessions with a greater number of recombination events and thus is able to significantly associate traits at genomic regions that bi-parental mapping populations are unable to detect The genomic regions containing SNPs with the FDR P-value < 0.05 were also examined to compare them with previous reported QTL. Two additional genomic regions on Chrs 6 (LG C2) and 11 (LG B1) were identified (Supplementary Table S2). The genomic region on Chr 6 (LG C2) flanked by two SNPs: ss715594958 and ss715594959 were overlapped with reported QTL on Chr 6 from PI 438489B (Yue et al., 2001) Thirty-seven resistant or MR accessions carried the same beneficial allele (A allele) at both SNPs 29 carried resistance allele at Rhg1 based on KASP markers (18 accessions carried Peking allele and 11 carried PI 88788 allele) GmSNAP11 contributed to SCN resistance as an additive effect and the FI of genotypes having both GmSNAP18 and GmSNAP11 was lower than that of genotypes containing only GmSNAP18 we examined 66 accessions carrying resistance alleles at Rhg1 locus which also carry three beneficial alleles of three closest SNPs to GmSNAP11 65 were rated as resistant or MR to HG Type 0 PI 398823 was highly susceptible since it only carries the Peking type allele at Rhg1 locus Further research is needed to confirm if these genes might be responsible for conveying SCN resistance in soybean carried out greenhouse phenotyping and population genotyping JB participated in the result interpretation and edited manuscript JN provided oversight for greenhouse phenotyping All authors read and approved the final manuscript The funding for this research was provided by the United Soybean Board and Vietnam Educational Foundation as fellowship to Dung Tran The authors wish to acknowledge the excellent technical assistance provided by Steve Finnerty and Dale Wood in executing the experiments and SCN phenotyping in the greenhouse Special thanks also go to the members in UGA’s Soybean Breeding and Genetics Lab for their assistance with planting and tissue sampling The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00401/full#supplementary-material FIGURE S1 | Distribution of female index among 461 soybean accessions using Origin 6.0 software (OriginLab Corporation FIGURE S2 | Genome-wide linkage disequilibrium (LD) decay plot for 461 soybean accessions based on 35,817 SNPs measured as r2 between pairs of marker was plotted against the genetic distance (cM) The fitting curve (blue line) illustrated the model fit to LD decay The estimated LD decay rate was about 125kb which was measured by when the r2 dropped to half of its FIGURE S3 | Manhattan plots and Q- Q plots generated from genome-wide analysis using TASSEL software and GAPIT package (A) General linear model (GLM) generated by TASSEL; 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Merr.] soybean cyst nematode (Heterodera glycines) (SCN) resistance to Heterodera glycines (Rhg) genes kompetitive allele specific polymerase chain reaction (KASP) Noe J and Li Z (2019) Genome-Wide Association Analysis Pinpoints Additional Major Genomic Regions Conferring Resistance to Soybean Cyst Nematode (Heterodera glycines Ichinohe) Copyright © 2019 Tran, Steketee, Boehm, Noe and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Zenglu Li, emxpQHVnYS5lZHU= Volume 10 - 2019 | https://doi.org/10.3389/fmicb.2019.00050 pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18 We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation These results highlight the importance of viroporins in virus-induced NLRP3 inflammasome activation Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV the underlying molecular mechanisms are not fully understood the role of the 3a protein in activating the NLRP3 inflammasome remains unknown we examined the role of the 3a protein in activating the NLRP3 inflammasome Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory All animal experiments were approved by the Animal Committees of the Institute of Medical Science (The University of Tokyo) Bone marrow-derived macrophages (BMMs) were prepared as described previously (Ichinohe et al., 2009) bone marrow was obtained from the tibia and femur by flushing with Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor 10% heat-inactivated fetal bovine serum (FBS) and L-glutamine (2 mM) at 37°C/5% CO2 HEK293FT cells (a human embryonic kidney cell line) and HeLa cells (a human epithelial carcinoma cell line) were maintained in DMEM supplemented with 10% FBS and streptomycin (100 μg/ml) (Nacalai Tesque) MDCK cells (Madin-Darby canine kidney cells) and HT-1080 cells (a human fibrosarcoma cell line) were grown in Eagle’s minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% FBS Influenza A virus strain A/PR8 (H1N1) was grown at 35°C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs (Ichinohe et al., 2009). The viral titer was quantified in a standard plaque assay using MDCK cells (Pang et al., 2013) To construct plasmids expressing the E mutant V25F the mutated E fragments were amplified by inverse PCR with wild-type E-containing plasmids and specific primer sets ligated in a ligase- and T4 kinase-containing reaction and then transformed into DH5α competent cells (TOYOBO) To construct plasmids expressing the 3a mutant 3a-CS fragments were amplified from wild-type 3a-containing plasmids using 3a-specific primer sets and transformed as described above HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 μg pLenti6-E/3a/M-V5His or pLenti-M2 using polyethylenimine (PEI) Max the cells were lysed with RIPA buffer (50 mM Tris–HCl And the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes The membranes were incubated over night with mouse anti-V5-tag (R960-25 followed by horseradish peroxide-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories) or anti-rabbit IgG (Invitrogen) After washing 3 times with washing buffer (0.05% Tween-20/PBS) the membranes were exposed using Chemi-Lumi One Super (Nacalai Tesque) and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus (GE Healthcare) To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 μg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 μg of ER-mCherry or DsRed-Golgi (Ito et al., 2012) cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS After washing with PBS and blocking with 4% BSA/PBS the cells were incubated with a mouse anti-flag antibody (M2 Sigma) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies) To observe the cellular distribution of NLRP3 in the E- or 3a-expressing cells HeLa cells were cultured on coverslips and transfected with 1 μg of pCA7-HA-E or pCA7 control vector together with 0.5 μg of pCA7-NLRP3 cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS the cells were incubated with rabbit anti-HA (561 MBL) and mouse anti-NLRP3 (Cryo-2; AdipoGen) antibodies followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (Life Technologies) Fluorescent signals were observed by confocal microscopy (A1R+ Statistical significance was tested using a two-tailed Student’s t-test P-values < 0.05 were considered statistically significant These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs The 3a protein of SARS-CoV stimulates IL-1β secretion (A–C) HEK293FT cells were transfected with pLenti6-E-V5 Samples were analyzed by immunoblot with mouse monoclonal antibodies against V5-tag (A) (D,E) LPS-primed BMM were infected with the lentivirus expressing SARS-CoV E Supernatants were collected at 24 h post-infection and analyzed for IL-1β by ELISA Data are representative of at least three independent experiments and indicate the mean ± SD (D,E); ∗∗∗P < 0.001 suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion Ion channel activity of the 3a protein is required for IL-1β secretion amino acid sequence of cysteine-rich domain (residue 127–133) of wild-type 3a and 3a-CS mutant (B) LPS-primed BMM were infected with the lentivirus expressing SARS-CoV E and indicate the mean ± SD (B); ∗∗∗P < 0.001 Subcellular localization of SARS-CoV 3a protein and 3a-CS mutant (A,B) HeLa cells were transfected with the expression plasmid encoding flag-tagged 3a or 3a-CS and that encoding either DsRed-monomer-Golgi (A) or ER-mCherry (B) and observed with a confocal microscope at 24 h post-transfection these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome NLRP3 inflammasome activation by SARS-CoV 3a HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages K+ efflux is required for activation of the NLRP3 inflammasome by SARS-CoV 3a protein (A,B) BMMs were infected with influenza virus A/PR8 (A) or lentiviruses expressing SARS-CoV 3a or E proteins (B) and cultured in the presence or absence of KCl (130 mM) Cell-free supernatants were collected at 24 h post-infection and indicate the mean ± SD; ∗∗P < 0.01 and ∗∗∗P < 0.001 Mitochondrial ROS-dependent activation of the NLRP3 inflammasome by SARS-CoV 3a protein (A,B) LPS-primed BMMs were stimulated with ATP (A) or lentiviruses expressing SARS-CoV 3a or E proteins (B) in the presence or absence of Mito-TEMPO (500 μM) Cell-free supernatants were collected at 24 h (lentiviruses) or 6 h (ATP) post-infection or stimulation we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome both K+ efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis I-YC and TI designed the study and wrote the manuscript This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (25713018 and 15H01254) and the SENSHIN Medical Research Foundation I-YC was a qualified recipient of the Postdoctoral Research Abroad Program and was supported by the research grant MOST 103-2917-I-564-028 from the Ministry of Science and Technology of the Republic of China Matsuyama (National Institute of Infectious Diseases Japan) for providing the total RNA extracted from SARS-CoV-infected Vero cells The NLRP3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral RNA The role of potassium in inflammasome activation by bacteria Inflammasomes: current understanding and open questions Castano-Rodriguez Role of severe acute respiratory syndrome Coronavirus viroporins E RNase L activates the NLRP3 inflammasome during viral infections The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function Response of host inflammasomes to viral infection HCV genomic RNA activates the NLRP3 inflammasome in human myeloid cells Identification of a novel coronavirus in patients with severe acute respiratory syndrome NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals The p7 viroporin of the hepatitis C virus contributes to liver inflammation by stimulating production of Interleukin-1beta Fernandes-Alnemri The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation Aetiology: Koch’s postulates fulfilled for SARS virus The NALP3 inflammasome is involved in the innate immune response to amyloid-beta Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization Critical functions of priming and lysosomal damage for NLRP3 activation Inflammasome recognition of influenza virus is essential for adaptive immune responses Influenza virus activates inflammasomes via its intracellular M2 ion channel Mitochondrial protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA virus infection Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome A mitochondria-targeted triphenylphosphonium-conjugated nitroxide functions as a radioprotector/mitigator Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and NLRP3 inflammasomes The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling A novel coronavirus associated with severe acute respiratory syndrome Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release Cryopyrin activates the inflammasome in response to toxins and ATP Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection The YXXPhi motif within the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is crucial for its intracellular transport The DHX33 RNA helicase senses cytosolic RNA and activates the NLRP3 inflammasome The RNA- and TRIM25-binding domains of influenza virus NS1 protein are essential for suppression of NLRP3 inflammasome-mediated IL-1beta secretion K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter Critical role for calcium mobilization in activation of the NLRP3 inflammasome Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome IL-1R signaling in dendritic cells replaces pattern-recognition receptors in promoting CD8(+) T cell responses to influenza a virus Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel Coronavirus as a possible cause of severe acute respiratory syndrome Immunopathogenesis of coronavirus infections: implications for SARS Structure and inhibition of the SARS coronavirus envelope protein ion channel Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration The NLRP3 inflammasome: a sensor for metabolic danger Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation Characterization of viral proteins encoded by the SARS-coronavirus genome Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein Rhinovirus-induced calcium flux triggers NLRP3 and NLRC5 activation in bronchial cells Antioxidant properties of MitoTEMPOL and its hydroxylamine NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids EV71 3D protein binds with NLRP3 and enhances the assembly of inflammasome complex SARS coronavirus E protein forms cation-selective ion channels Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus Subcellular localization and membrane association of SARS-CoV 3a protein SARS-Coronavirus open reading frame-3a drives multimodal necrotic cell death Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients Thioredoxin-interacting protein links oxidative stress to inflammasome activation A role for mitochondria in NLRP3 inflammasome activation Chang MF and Ichinohe T (2019) Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome Copyright © 2019 Chen, Moriyama, Chang and Ichinohe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Takeshi Ichinohe, aWNoaW5vaGVAaW1zLnUtdG9reW8uYWMuanA= Volume 7 - 2013 | https://doi.org/10.3389/fncir.2013.00031 Abnormalities in the processes of the generation and/or pruning of dendritic spines have been implicated in several mental disorders including autism and schizophrenia We have chosen to examine the common marmoset (Callithrix jacchus) as a primate model to explore the processes we studied the postnatal development of basal dendritic trees and spines of layer-III pyramidal cells in the primary visual sensory cortex (V1) a visual association cortex (inferior temporal area Basal dendrites in all three areas were longer in adulthood compared with those in the newborn rapid dendritic growth occurred in both TE and PFC around the second postnatal month This early growth spurt resulted in much larger dendritic arbors in TE and PFC than in V1 The density of the spines along the dendrites peaked at 3 months of age and declined afterwards in all three areas: the degree of spine pruning being greater in V1 than in TE and PFC The estimates of the total numbers of spines in the basal dendrites of a single pyramidal cell were larger in TE and PFC than in V1 throughout development and peaked around 3 months after birth in all three areas These developmental profiles of spines and dendrites will help in determining assay points for the screening of molecules involved in spinogenesis and pruning in the marmoset cortex An understanding of the molecular underpinnings of spine pathology may provide insights into the etiology of these diseases and lead to the discovery of new drug targets and they may not represent a model suitable for examining the relationship between spine development and neuropsychiatric disorders we studied the developmental profiles of the basal dendritic trees and spines of layer-III pyramidal cells in the primary visual sensory cortex (V1) and an executive control area (area 12 of the prefrontal cortex We found that all three areas showed an overshoot-type pattern of spine development which was similar to that seen in humans and macaques Basal dendrites in all three areas became longer after birth neurons in TE and PFC exhibited dramatic growth of the basal dendrites during the first 2 months after birth; the basal dendrites of layer-III pyramidal cells in TE and PFC thus covered a wider cortical area than those in V1 The three areas exhibited similar developmental changes in the time courses of the density of spines along dendrites with a peak in the third month after birth the estimated total number of spines on basal dendrites peaked at 3 months of age in all three areas and it was nearly three times larger in TE and in PFC than in V1 The developmental time courses of the dendrites and spines and the areal differences will help to determine assay points for the future screening of the molecules that are involved in spinogenesis and pruning in the marmoset cortex we used common marmosets (Callithrix jacchus) of either sex with the following ages: postnatal day 0 (0D; n = 1) All animal care procedures and experiments were conducted in accordance with protocols that were approved by the ethics committee for primate research at the National Center of Neurology and Psychiatry in Japan and that were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals Vital data of the animals used in the present study and sliced parallel to the cortical surface at a thickness of 250 μm Slices were incubated in 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Co Cortical sampling position (A) and labeled cells (B–D) The animals were lightly fixed with paraformaldehyde and sliced at 250 μm tangential to the cortical surface Slices were incubated in 4,6-diamidino-2-phenylindole solution to visualize neuronal nuclei Layer III pyramidal cells were individually injected with Lucifer yellow and reacted for diaminobenzidine product (B–D) Examples of injected cells from the primary visual cortex (V1) and the prefrontal cortex (PFC) of a 3-month-old monkey are shown in (B) Pyramidal cells were individually injected with 8% Lucifer Yellow (Sigma-Aldrich Co. LLC, St. Louis, MO) under the visual guidance of ultraviolet illumination (Elston et al., 1997). All cell bodies of the injected cells were located in the supragranular layer, which was immediately above the granular layer and easily identified in 0.1% DAPI-stained preparations (Elston and Rosa, 1997) Only cells located in the lower part of layer III were injected reconstructions of the basal dendritic trees of layer-III pyramidal cells (in the tangential plane) sampled from the primary visual (V1) and prefrontal (PFC) cortices at day 0 (0D) The illustrated cells represent the average of all cells sampled for each cortical area/age group Insets illustrate the spine density for cells of each cortical area/age group at 50–70 μm from the cell body Left Scale Bars = 100 μm (for all of skeletonized reconstructions of the basal dendritic trees of pyramidal cells) Right Scale Bars = 10 μm (for all of Insets illustrate the spine density for cells of each cortical area/age group at 50–70 μm from the cell body) Spine densities were calculated by drawing the horizontally projecting dendrites of randomly selected cells in their entirety while visualizing them with a Nikon 100X oil immersion objective (numerical aperture, 1.40) and then counting the number of spines per 10-μm segment (Eayrs and Goodhead, 1959; Valverde, 1967) Spine density was quantified as a function of distance from the cell body to the distal tips of the dendrites We selected horizontally projecting dendrites for our calculations of spine densities to avoid trigonometric errors Spine densities were calculated per 10-μm interval along the entire length of 20 randomly selected dendrites in each cortical area for each age group No distinction was made between the different spine types (e.g. The mean total number of spines within the basal dendritic trees of the cells in each cortical area/age was estimated by summing the products of the mean intersections of each 10 μm of Sholl annuli of the cells (see above) multiplied by the mean spine densities of each 10 μm of the corresponding distance from the cell body of 20 dendrites (for technical details, see Elston, 2001) When other statistical methods (Mann–Whitney U-Test These statistical tests were performed with StatView (SAS Institute and we described the results as p < 0.05 or p < 0.0001 with other information (e.g. F value: F-test plays an important role in the ANOVA and F value assesses whether any of the sampled groups is on average superior or inferior to the others vs the null hypothesis that all groups yield the same mean; U value: Mann–Whitney's U-test is a non-parametric statistical test for assessing whether one of two samples of independent observations have larger values than the other U value assesses how far the measured data are higher in one group than in the other.) We set the statistically significant level to p = 0.05 A total of 324 pyramidal cells in layer III were included in the analyses (Table 2) The following results were based on 50,098 individual dendritic spines that were drawn and tallied In order to examine the development of dendritic arbors, we measured basal dendritic field areas and the entire dendritic lengths that were summed across the basal dendrites. V1, TE, and PFC differed in their growth profiles (Figure 2). The dendritic field areas of the pyramidal cells in V1 gradually increased from 0D to 4.5Y, except for a small decline from 3M to 6M (Figure 3A; One-Way ANOVA The dendritic field area at 4.5Y was 167% larger than that at 0D the dendritic field areas of TE and PFC showed a marked increase from 0D to 2M (TE: 319% increase p = 4.3 × 10−7; PFC: 209% increase Dendritic field areas of TE and PFC were larger at 4.5Y than at 0D (TE: 264% larger; p = 4.3 × 10−7; PFC: 200% larger Statistical analyses (One-Way ANOVAs) revealed that the differences in the sizes of the basal dendritic trees of pyramidal cells in any given cortical area were significant across the age groups (p < 0.001; V1 Dendritic field areas (A) and total dendritic lengths (B) of layer-III pyramidal cells in V1 Because of the different developmental profiles of the dendritic field areas between the three cortical areas and of the different sizes at 0D, there were relative differences in the dendritic field areas among the cortical areas at any given age (Figure 3A) the rank order of the dendritic field areas was PFC The averaged dendritic field area of PFC neurons was 302% larger than that in V1 (p = 4.9 × 10−9) and the averaged dendritic field area in TE was 195% larger than that in V1 (p = 1.4 × 10−7) the dendritic field areas of TE and PFC neurons were 269% and 324% larger than those of V1 respectively (t-test; p = 1.3 × 10−24 for TE vs p = 1.3 × 10−24 for PFC vs Statistical analyses (One-Way ANOVAs) revealed that the sizes of the dendritic trees of cells at any given age were significantly different among the cortical areas in all animals (p < 0.001; 0D The total basal dendritic lengths that were summed across the basal dendrites exhibited developmental changes similar to those of the basal dendritic field area (Figures 3A,B) The total dendritic lengths in TE and PFC markedly increased from 0D to 2M (t-test p = 1.8 × 10−7 for TE and 0.014 for PFC) the dendritic lengths in TE and PFC stayed almost at the same level until 4.5Y the dendritic lengths of V1 neurons gradually increased from 0D to 4.5Y The dendrites in TE and PFC were markedly longer than those in V1 throughout development One-Way ANOVAs revealed that the differences in the total lengths of the basal dendritic trees of pyramidal cells in any given cortical area were significant across the age groups (p < 0.001; V1 One-Way ANOVAs revealed that the sizes of the dendritic trees of cells at any given age were significantly different among the cortical areas in all animals (p < 0.001; 0D (A) Sholl plots of the branching patterns of the basal dendritic trees of layer-III pyramidal cells from V1 The shaded areas indicate the standard deviations (B,C) Developmental changes in the peak number of Sholl intersections and the distance from the soma to the position of the peak number of Sholl intersections of layer-III pyramidal cells In addition, the distance from the soma to the Sholl annulus with the peak branching complexity increased with age (Figure 4C) the distance of the peak dendritic complexity from the soma gradually increased from 0D to 6M and then remained nearly the same until 4.5Y the distance increased from 0D to 3M and then remained at the same level from 3M to 4.5Y Lastly, the statistical analysis (One-Way repeated measures ANOVA) revealed that the numbers of dendritic intersections with Sholl annuli were significantly different among the cortical areas (p < 0.001) at each given age (0D, F2 = 39.58; 2M, F2 = 16.67; 3M, F2 = 54.85; 6M, F2 = 136.32; 4.5Y, F2 = 92.99: Figure 4A) Repeated measures ANOVA revealed that the differences in the spine densities across ages were significantly different in each cortical area (p < 0.001; V1 Repeated measures ANOVA also revealed significant differences (p < 0.05) in the spine densities among the cortical areas for a given age (0D (A) Profiles of the spine densities of the basal dendrites of layer-III pyramidal cells as a function of distance from soma The peak density (spine number/10 μm) (B) the distance from the soma to the position of peak density (C) of layer-III pyramidal cells in V1 The total number of spines in the basal dendritic trees of average layer-III pyramidal cells in V1 All the above analyses were based on samples from one animal per age group except for 2M group in which data from three animals were pooled (Table 2) One-Way ANOVAs showed that all the parameters analyzed were not different between the three 2M animals [basal dendritic field area p = 0.08 (F2 = 2.93); total basal dendrite length p = 0.30 (F2 = 1.28); estimated total spine dendrites The developmental profiles of layer-III pyramidal cells in the same three areas have recently been examined in the macaque monkey (Macaca fascicularis) (Elston et al., 2009, 2010a) These studies employed exactly the same method as that used in the present study allowing us to make a direct comparison between the two species although no clear developmental trend was observed in the three areas of both species The density then decreased toward adult values with different degrees among V1 spine density in V1 decreased most drastically While the spine density in V1 was higher in adults than in newborns in the marmoset it was lower in adults than in newborns in the macaque The ratio of the total spine number in the macaque divided by that of the marmoset tended to decrease gradually from birth to adulthood in the three areas (V1: birth, 1015%, 3M, 162%, adult, 112%; TE: birth, 728%, 3M, 168%, adult, 197%; PFC: 2D/0D, 1408%, 3M, 270%, adult, 238%). This calculation was based on Figure 6 in the present study and data from Elston et al. (2009, 2010a) The total number of spines on basal dendrites at birth was much larger in the macaque than in the marmoset The difference at birth resulted from the 5-fold difference in the dendritic fields and the 2-fold difference in the spine densities between the two species The total number of spines on the basal dendrites in the adult marmoset was consistent between this and previous studies [V1: this study, 950; Elston et al., 1996, V1, 900; TE: this study, TEr, 3100; Elston et al., 1996, 3000 in TEc, 4200 in TEa (Thus, TEr in our study was similar to TEc in Elston et al., 1996); PFC: this study, 3500; Elston et al., 2001 the measured values were not different among the three 2M animals in the present study These results warranted the validity of our sampling and analytical methods Although our data were based mostly on one animal per one age/area the density is 12 synapses/μm; in marmosets and PFC in the marmoset all exhibited an overshoot type of spine development with a peak at 3M The dendritic field area and the total number of spines differed across ages and across areas These results provide a starting point for studies of the molecular mechanisms of spinogenesis and pruning in the primate cerebral cortex Comparisons among the transcriptomes of different time epochs of spine development in selected cortical areas are a strategic beginning for this endeavor we can manipulate these genes with viral vector infection techniques or use the transgenic marmoset to assess their function in spine development We thank Yoko Oga and Tsuguhisa Okamoto for their technical help This work was supported by an Intramural Research Grant (grant number 23–7) for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry Grant-in-Aids for Scientific Research on Innovative Areas “Face perception and recognition” and “Shitsukan” by the Ministry of Education and a Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) (to Noritaka Ichinohe) by grants (23240047 Sports and Culture of Japan (to Ichiro Fujita) Tomofumi Oga was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education Agustín-Pavón Lesions of ventrolateral prefrontal or anterior orbitofrontal cortex in primates heighten negative emotion Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Synaptogenesis in the prefrontal cortex of rhesus monkeys Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Changes of synaptic density in the primary visual cortex of the macaque monkey from fetal to adult stage Pubmed Abstract | Pubmed Full Text Evidence for a decrease in basilar dendrites of pyramidal cells in schizophrenic medial prefrontal cortex Pubmed Abstract | Pubmed Full Text Vergleichende Lokalisationslehre der Großhirnrinde Architectural subdivisions of medial and orbital frontal cortices in the marmoset monkey (Callithrix jacchus) Pubmed 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of functionally related cortical visual areas Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Comparison of dendritic fields of layer III pyramidal neurons in striate and extrastriate visual areas of the marmoset: a Lucifer yellow intracellular injection Pubmed Abstract | Pubmed Full Text Visuotopic organization of striate cortex in the marmoset monkey (Callithrix jacchus) Pubmed Abstract | Pubmed Full Text | CrossRef Full Text When cortical development goes wrong: schizophrenia as a neurodevelopmental disease of microcircuits Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Morphometric study of human cerebral cortex development Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text “Theories of visual cortex organization in primates,” in Cerebral Cortex The dendritic architecture of prefrontal pyramidal neurons in schizophrenic patients Pubmed Abstract | Pubmed Full Text Dendritic anomalies in disorders associated with mental retardation Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Perturbation of dendritic protrusions in intellectual disability Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Comparison of androgen receptor and estrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testosterone concentrations Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Lifespan alterations of basal dendritic trees of pyramidal neurons in the human prefrontal cortex: a layer-specific pattern Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Generation of transgenic non-human primates with germline transmission Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Affiliative processes and vocal development Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Apical dendritic spines of the visual cortex and light deprivation in the mouse Pubmed Abstract | Pubmed Full Text Synapse pathology in psychiatric and neurologic disease Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Synaptogenesis in monkey somatosensory cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Fujita I and Ichinohe N (2013) Postnatal development of layer III pyramidal cells in the primary visual Received: 11 December 2012; Paper pending published: 31 December 2012; Accepted: 09 February 2013; Published online: 08 March 2013 Copyright © 2013 Oga, Aoi, Sasaki, Fujita and Ichinohe. This is an open-access article distributed under the terms of the Creative Commons Attribution License distribution and reproduction in other forums provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc *Correspondence: Noritaka Ichinohe, Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan. e-mail:bmljaGlub0BuY25wLmdvLmpw Volume 9 - 2015 | https://doi.org/10.3389/fnins.2015.00459 Mirror neurons respond when executing a motor act and when observing others' similar act mirror neurons have been found only in macaques To investigate the degree of phylogenetic specialization of mirror neurons during the course of their evolution we determined whether mirror neurons with similar properties to macaques occur in a New World monkey where mirror neurons have been reported in macaques since no sulcal landmarks exist in the frontal cortex We addressed this problem using “in vivo” connection imaging methods we first identified cells responsive to others' grasping action in a clear landmark and injected fluorescent tracers into the region we identified clusters of labeled cells in the ventrolateral frontal cortex which were confirmed to be within the ventrolateral frontal cortex including PMv after sacrifice We next implanted electrodes into the ventrolateral frontal cortex and STS and recorded single/multi-units under an awake condition we found neurons in the ventrolateral frontal cortex with characteristic “mirror” properties quite similar to those in macaques This finding suggests that mirror neurons occur in a common ancestor of New and Old World monkeys and its common properties are preserved during the course of primate evolution This study would provide valuable insight into our understanding of primate evolution We simultaneously recorded multiunits from a part of the STS containing cells responsive to the sight of others' action which was determined by multiunit recording beforehand and from a circumscribed area in the frontal cortex which was identified in vivo to have cells fluorescently labeled by a retrograde tracer that had been injected to the STS site after the first recording Experiments were performed with three adult common marmoset monkeys (C This study was approved by the Experimental Animal Committee of the National Institute of Neuroscience and Psychiatry and the animals were cared for in accordance with the “Guiding Principles of the Care and Use of Animals in the Field of Physiological Science” of the Japanese Physiological Society As a general guideline to the preparation of marmosets, we followed Bourne and Rosa's procedure (Bourne and Rosa, 2003) The food was withdrawn in the evening before the day of the experiment Surgery and electrophysiological recordings were conducted under anesthesia induced by an intramuscular injection of ketamine hydrochloride (Ketalar 25 mg/kg i.m.) following an intramuscular injection of atropine sulfate (0.15 μg/kg) and maintained with an intravenous infusion of remifentanil (Ultiva muscular paralysis was induced with rocronium bromide (Eslax The animal was artificially ventilated with a mixture of 70% N2O and rectal temperature were monitored continuously throughout the experiments The animals were placed in a stereotactic apparatus and the head holder and the recording chamber were implanted on the skull the pupil was fully dilated with topical tropicamide (0.5%) and phenylephrine hydrochloride (0.5%) A contact lens whose power was measured using a retinoscope was used to focus the eye contralateral to the recorded hemisphere at a distance of 57 cm electrodes were inserted with reference to two sulcal landmarks A micromanipulator lowered a linear array 32-channel multicontact electrode (Neuronexus US) perpendicular to the cortical surface of the posterior and ventral parts of STS where strong responses to others' grasping action were commonly observed under our anesthetic condition The linear array multicontact electrode contained four shanks (400 μm shank separation) and each shank had eight contacts (impedance ~1 MΩ at 1 kHz) with an intercontact spacing of 200 μm Multiunit activities were simultaneously recorded from the 32 (4 shanks × 8 contacts) contacts only 4–5 bottom contacts of each shank were inserted into the cortex to minimize the penetration damage of the cortex The timing of multiunit activity and task events (stimulus onset and offset) were recorded and stored with < 1 ms resolution using a TDT signal processing system (RZ2 The visual stimulus set consisted of 33 movies that showed reaching and grasping motor acts of an actor marmoset Actions performed by the marmosets were recorded with a video camera (HDR-CX560V Using graphics software (Adobe Premiere Pro CS4 we edited the recorded videos to generate video clips of 1 s duration (30 frames) with a resolution of 640 × 480 pixels Three different marmoset subjects were used as the actor animal with two different types of food (a piece of potato and bun) and with two different views (frontal and lateral) The size of the stimulus (video clip) was ~20° Each stimulus was presented 12 times in a pseudorandom order The tracer was diluted to 1% in 0.1 M phosphate-buffered saline (PBS) and 0.12–0.15 μL of the tracer solution was pressure-injected through a glass micropipette with a 50-μm-inner-diameter tip which was attached to a 10 μL Hamilton syringe The injection sites were immediately confirmed under a fluorescence stereomicroscope (VB-G05 Japan) with a filter for red fluorescent protein (RFP The retrograde tracer was injected into an area in STS that contained the cells strongly responsive to the video clip of others' action as determined from the electrophysiological recording under anesthesia (see above) the injection site was close to the posterior tip of STS and ventral to STS an artificial dura was placed on the cortex and the bone was put back and the wound was closed All surgical procedures were the same as in the electrophysiological experiment except that remifentanil and rocronium bromide were not administered The anesthesia was maintained using a mixture of 70% N2O One to three weeks after the injection, craniotomy and duratomy around STS and the frontal regions were performed. Fluorescently labeled spots were identified in vivo in the lateral frontal cortex and at the injection site as well under a fluorescence stereomicroscope with a filter for RFP (Ichinohe et al., 2012) The labeled spots were observed in the lateral frontal cortex including area 6V according to a histological examination Two micromanipulators were used to lower linear array multicontact electrodes (Neuronexus US) vertically perpendicular to the cortical surface in the STS injection site and in the labeled spots in the lateral frontal cortex for the chronic experiment and intercontact spacing were the same as those used for the experiment conducted under the anesthetic condition After the animal recovered from the electrode implantation surgery (about 2 days) the animal sat comfortably in a primate chair and the head was fixed Multiunit activity was recorded simultaneously from STS and the ventrolateral frontal cortex the signals from the electrode implanted in STS of the second and third animals were not detected probably owing to damage caused by several penetrations (electrodes for recordings under the anesthetic and awake conditions and a glass pipet for tracer injection) touching the food by the experimenter or the animal) occurred the experimenter pushed a button to turn on a small LED The LED was invisible to the animal but visible to a video camera (HDR-CX560V The animal behaviors recorded by the video camera and neuronal data were synchronized with reference to the LED state accompanied by each behavioral event the time resolution of this synchronization was 33.3 ms which was limited by that of the video camera We recorded visual responses of cells in STS and the ventrolateral frontal cortex while an experimenter was reaching and grasping food in front of the animal with its head fixed which was placed just in front of the primate chair The position of the food on the tray was the same across all trials in a session The experimenter performed the following grasping action types He/she reached and grasped (1) a piece of banana with his/her hand (3) a piece of banana with a pair of forceps and (4) he/she mimed to reach and grasp as if there was a piece of food These four action types were performed from either the right or left side of the animal (in total 4 action types × 2 reaching directions = 8 conditions) we recorded motor-related responses of cells while the animal took a piece of banana/bun from a tray with its head fixed Each condition was designed as a block that consisted of at least 10 trials and each block was pseudorandomly intermingled expressed as the mean firing rate (spikes per second) of multiunit activity were measured in two different time epochs: Epoch 1 corresponds to a 1-s period centered at the hand-food contact and Epoch 2 corresponds to a 1-s period starting 5 s before the hand-food contact (baseline response) The significance of the response when the animal was observing grasping action executed by the experimenter and when the animal itself was grasping was examined by comparing the responses in Epoch 1 with those in Epoch 2 by the paired t-test Individual multiunits were defined as mirror neurons when the neuronal responses were significant while observing at least one of the eight grasping action conditions [p < 0.05 after Bonferroni correction for multiple comparisons (p < 0.05/8)] and while executing grasping action (p < 0.05) To analyze the effect of the grasping action types under the observation conditions we applied Two-way factorial ANOVA (with reaching direction and grasping action type as factors) to the neuronal responses during Epoch 1 The time course of the population neuronal responses for the mirror neurons responsive during observation (execution) was calculated by aligning at the moment when the experimenter's hand touched the food under the most preferred grasping action condition (animal's hand touched the food) normalizing by the maximum magnitude of response during the grasping action We also analyzed the multiunits that responded while observing under at least one of the eight grasping action conditions [paired t-test between Epochs 1 and 2 p < 0.05 after Bonferroni correction for multiple comparison (p < 0.05/8)] but failed to respond while executing a grasping action The time course of the population activity was calculated by aligning at the moment when the experimenter's hand touched the food for each grasping action type in the preferred reaching direction for each multiunit and normalized by its maximum response Two-way factorial ANOVA (with recording area and grasping type as factors) was applied to the responses around the time the food was touched (Epoch 1) We also analyzed normalized responses by calculating z scores and obtained similar results To confirm that single units showed the mirror neuron properties single-unit data were sorted offline using the t-distribution E-M sorting algorithm provided in the Plexon Off-line Sorter software from the multiunit data (Plexon Inc. the animals were sedated with ketamine hydrochloride (Ketalar 25 mg/kg i.m.) and overdosed with sodium pentobarbital (Nembutal with 0.1 M PBS (pH 7.4) and 4% paraformaldehyde in PBS (Merck and a brain block was put in ice-cold 0.1 M PBS with 10 Coronal sections were prepared at 50 μm in a series of three sections and the third sections for Nissl substrate with thionin we failed to stain the first sections in three series by immuno-histochemistry of CTB due to unknown reason we confirmed that linear array multicontact electrodes were implanted in the ventrolateral frontal cortex from sections stained by Nissl substrate and myelin Pseudocolor represents the density of labeled cells Bottom: Coronal sections stained by Nissl substrate and myelin showing the areal boarder in the ventrolateral frontal cortex and distribution of the labeled cells The asterisks indicate recording tracks of shanks of electrodes in the ventrolateral frontal cortex Electrophysiological mapping of STS under anesthetic condition (A) Cortical surface around STS of a common marmoset Four green dots indicate the penetration sites of four shanks of a linear array multicontact electrode The red line indicates the presumed cortical surface because only 4–5 bottom contacts of each shank were inserted (B) Multiunit responses to the sight of grasping action of another marmoset which were arranged by channel configuration Rasters and peristimulus time histograms were aligned to the stimulus onset at time = 0 The bin width for the peristimulus time histogram was 20 ms To minimize damage to the cortex caused by the electrode penetration multiunits were recorded only from channels on the upper part of each shank The multiunits on the bottom two channels on the second shank strongly responded to the movie (C) Multiunit responses to the sight of human grasping action the multiunits on the bottom two channels on the second shank strongly responded to the movie To search for mirror neurons, we implanted each of two linear array multicontact (32-channels) electrodes into fluorescently bright spots in the ventrolateral frontal cortex including area 6V and in STS injection sites, which were histologically confirmed later (Figure 1C) (Burman et al., 2008; Paxinos et al., 2012) After waiting for the animal to recover (about 2 days) we conducted multiunit recording under an awake condition We trained the animal to sit in a primate chair with its head fixed the animal observed an experimenter's action The experimenter performed one of the eight grasping action types in front of the primate chair with a tray placed just in front of the chair (Materials and Methods) we let the animal grasp a piece of banana or bun on the tray Individual multiunits were considered to be mirror neurons when their responses were significantly evoked when the animal was observing the experimenter's grasping action (at least in one of eight observation conditions; t-test with Bonferroni correction p < 0.05/8) and when the animal itself was performing a grasping action (execution condition; t-test compared with the preceding baseline activity and when the animal grasped a piece of food with its peak observed at around the time the food was touched Three examples of multiunit responses (A–C) with mirror neuron properties under observation and execution conditions in the ventrolateral frontal cortex Rasters and peristimulus time histograms were aligned to the touch of food at time = 0 The bin width for the peristimulus time histogram was 100 ms The left (right) column indicated the multiunit responses when an experimenter grasped food from the left (right) side of the animal The first to fourth rows indicate the multiunit responses when an experimenter grasped (1) a piece of banana with his/her hand The bottom indicates the multiunit responses when the animal itself grasped a piece of banana or bun The responses in the shaded area were used for statistical analysis *p < 0.05/8 under observation condition p < 0.05 under execution condition (paired-t test) which was aligned at the moment when the experimenter or the animal touched the food The responses were averaged after normalizing the responses by the peak responses of the individual mirror neurons The magnitude of responses gradually increased and reached the peak at around the time the food was touched under both observation and execution conditions Time course of normalized activity of neuronal population for 27 mirror neurons in the ventrolateral frontal cortex The responses were aligned at the moment when the experimenter or animal touched the food and were averaged after normalizing the responses by the peak responses Black and gray lines indicate the responses when an experimenter grasped a piece of food under the most preferred grasping action condition and when the animal itself grasped a piece of food and those of one single unit decreased under both observation and execution conditions Among the multiunits and single units that showed either significant visual or motor responses 34 and 41% satisfied the mirror neuron criteria multiunits with mirror neuron properties we found involved single units with the mirror neuron properties Figure 5. Two examples of single-unit responses under observation and execution conditions in the ventrolateral frontal cortex. Display formats were the same as those in Figure 3 (A) Single-unit responses that satisfied the mirror neuron criteria (B) Magnitude of single-unit responses that significantly increased under the observation condition but decreased under the execution condition *p < 0.05∕8 under observation condition The peak of the response for the execution of grasping action was also unclear while that for the observation was distinctly located at around the time the food was touched we found a small number of “mirror neurons” in STS and the magnitude of their responses to the self-action was obviously smaller than those in the ventrolateral frontal cortex Figure 6. An example of a mirror neuron in STS and time course of the population activity for seven mirror neurons. (A) Rasters and peristimulus time histograms of an example of STS mirror neuron responses. Display formats are the same as in Figure 3. (B) Time course of the normalized activity of neuronal population for seven mirror neurons in STS. Display formats are the same as in Figure 4 and no significant main effect of multiunit groups was shown (p = 0.065) there was a significant interaction between multiunit groups and grasping action type (p = 0.015) The difference between no food condition and the other food conditions was largest in the visual-responsive cells in STS than in the other two groups in the ventrolateral frontal cortex (p = 0.0049; One-way ANOVA) the representation of the grasping action type was different in detail among STS and non-mirror neurons in the ventrolateral frontal cortex STS cells were more sensitive to the reaching goal Comparison of visual-responsive cells in STS visual-dominant cells in the ventrolateral frontal cortex and mirror neurons in the ventrolateral frontal cortex (A) Time course of normalized activity of neuronal population for 30 visual-responsive multiunits in STS 35 visual-dominant multiunits in the ventrolateral frontal cortex and 27 mirror neurons in the ventrolateral frontal cortex Normalized responses of the multiunits for the preferred reaching direction when an experimenter grasped a piece of banana with his/her hand (blue) a piece of banana with a pair of forceps (green) and he/she mimed to reach and grasp as if there was a piece of food (purple) The responses were aligned at the moment when the experimenter or animal touched the food (B) Response magnitudes of visual-responsive cells in STS (green) visual-dominant cells in the ventrolateral frontal cortex (red) and mirror neurons in the ventrolateral frontal cortex (blue) for each grasping action type Error bars indicate standard error of the mean we combined electrophysiology with in vivo surface connection imaging First we determined the STS region responsive to the sight of others' action we visualized the anatomical connections between the STS region and the ventrolateral frontal region using in vivo surface connection imaging method we conducted unit recordings from the two connected regions Otherwise it might be necessary to spend long time to conduct unit recording across the large frontal regions without referring landmarks in marmosets that have no sulcus in the frontal cortex Another advantage is to enable simultaneous unit recording from two anatomically connected regions with a confirmation in vivo There was a potential disadvantage with this procedure The regions in which first unit recording was conducted and the anatomical tracers were injected could be damaged owing to several penetration of electrodes and a glass pipet we failed to record from STS of the second and third animals resulting small samples in the injection site because the electrodes were chronically implanted after visualizing the anatomical connection we conducted the unit recording only in one session or in fixed recording site for each awake animal Although we used 32-channel multicontact electrodes electrode with a larger number of channels or with movable device would be more preferable to increase the sample size The anatomical input from extrastriate cortex indicates that the ventrolateral frontal cortex also contributes to visual information processing for social communication These properties were also commonly observed in the mirror neurons in the ventrolateral frontal cortex of marmoset which was inconsistent with the temporal profile difference found in mirror neurons of songbirds it was difficult to compare whether the two peaks were significantly different because the profiles of hand motion for marmosets (execution) and for human (observation) were different and the time resolution of monitor system was poor (33.3 ms) This issue might be resolved by using actor marmosets for the observation condition and a monitor system with better time resolution A combination between a grasping hand and the target to grasp is essential in activating mirror neurons in PMv and some cells in STS of macaque monkeys (Perrett et al., 1989; Gallese et al., 1996; Barraclough et al., 2009; Nelissen et al., 2011) we showed that the absence of the target to grasp decreased the magnitude of responses of the mirror neurons in the ventrolateral frontal cortex and the visual-responsive cells in STS of common marmosets by directly comparing between the responses of mirror neurons and non-mirror neurons in the ventrolateral frontal cortex and visual-responsive cells in STS we found that mirror and non-mirror neurons in the ventrolateral frontal cortex were not as sensitive to the presence of the target to grasp as STS cells This suggests that the mirror neurons in the ventrolateral frontal cortex and STS cells play different roles in understanding others' action This inhibition might arise from the ventrolateral frontal cortex (mirror neurons or visual- dominant neurons) and transmitted to STS through a direct connection Common marmosets live together as a large family, with the mother, the father, and older siblings. Almost all family members engage in parenting behaviors including carrying, grooming, protecting, and feeding infants (Ferrari, 1992; Rothe et al., 1993). Moreover, they imitate a novel action demonstrated by a conspecific (Voelkl and Huber, 2000, 2007) Future research on mirror neurons in the common marmoset and their role in unique social behaviors will further deepen our understanding of the functions of mirror neurons This work was supported by an Intramural Research Grant (Grant No 23-7) for Neurological and Psychiatric Disorders from NCNP Grant-in-Aid for Scientific Research (C) (26430031) a Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) “Shitsukan,” and “Adolescent Mind and Self-Regulation” of MEXT and the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from AMED and Keiko Nakagaki for assistance with the experiments and Tomoaki Chiba and Hiromi Mashiko for help with the data analysis Visual adaptation to goal-directed hand actions Evolution of mirror systems: a simple mechanism for complex cognitive functions Ventral premotor and inferior parietal cortices make distinct contribution to action organization and intention understanding Preparation for the in vivo recording of neuronal responses in the visual cortex of anaesthetised marmosets (Callithrix jacchus) Action observation activates premotor and parietal areas in a somatotopic manner: an fMRI study Cortical and thalamic projections to cytoarchitectural areas 6Va and 8C of the marmoset monkey: connectionally distinct subdivisions of the lateral premotor cortex Mirror neurons encode the subjective value of an observed action View-based encoding of actions in mirror neurons of area f5 in macaque premotor cortex Mirror neurons differentially encode the peripersonal and extrapersonal space of monkeys Mirror neurons in monkey area F5 do not adapt to the observation of repeated actions PubMed Abstract | CrossRef Full Text | Google Scholar ALE meta-analysis of action observation and imitation in the human brain Estimating the phylogeny and divergence times of primates using a supermatrix approach Understanding motor events: a neurophysiological study Neurons in primary motor cortex engaged during action observation From monkey mirror neurons to primate behaviours: possible ‘direct’ and ‘indirect’ pathways Mirror neurons responding to the observation of ingestive and communicative mouth actions in the monkey ventral premotor cortex CrossRef Full Text | Google Scholar Parietal lobe: from action organization to intention understanding Evidence for a causal inverse model in an avian cortico-basal ganglia circuit Toward a phylogenetic classification of Primates based on DNA evidence complemented by fossil evidence Motion sensitive cells in the macaque superior temporal polysensory area lack of response to the sight of the animal's own limb movement Distinct feedforward and intrinsic neurons in posterior inferotemporal cortex revealed by in vivo connection imaging Neural processing of auditory feedback during vocal practice in a songbird Demystifying social cognition: a Hebbian perspective understanding actions: action representation in mirror neurons Corticospinal neurons in macaque ventral premotor cortex with mirror properties: a potential mechanism for action suppression Afferent and efferent projections of the inferior area 6 in the macaque monkey Responses of primate frontal cortex neurons during natural vocal communication Single-neuron responses in humans during execution and observation of actions Action observation circuits in the macaque monkey cortex Google Scholar Frameworks of analysis for the neural representation of animate objects and actions Precise auditory-vocal mirroring in neurons for learned vocal communication doi: 10.1146/annurev.neuro.27.070203.144230 Neurophysiological mechanisms underlying the understanding and imitation of action Infant survival and number of helpers in captive groups of common marmosets (Callithrix jacchus) CrossRef Full Text | Google Scholar Frontal lobe connections of the superior temporal sulcus in the rhesus monkey Microstimulation reveals specialized subregions for different complex movements in posterior parietal cortex of prosimian galagos Congruent activity during action and action observation in motor cortex Umiltà Mapping visual cortex in monkeys and humans using surface-based atlases The conditions for tool use in primates: implications for the evolution of material culture Imitation as faithful copying of a novel technique in marmoset monkeys Goda N and Ichinohe N (2015) Mirror Neurons in a New World Monkey Received: 04 June 2015; Accepted: 18 November 2015; Published: 10 December 2015 Copyright © 2015 Suzuki, Banno, Miyakawa, Abe, Goda and Ichinohe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Wataru Suzuki, d2F0YXJ1c0BuY25wLmdvLmpw; Noritaka Ichinohe, bmljaGlub0BuY25wLmdvLmpw Metrics details Mitochondria contribute to cellular innate immunity against RNA viruses Mitochondrial-mediated innate immunity is regulated by signalling molecules that are recruited to the mitochondrial membrane and depends on the mitochondrial inner membrane potential (Δψm) Here we examine the physiological relevance of Δψm and the mitochondrial-associating influenza A viral protein PB1-F2 in innate immunity PB1-F2 completely translocates into the mitochondrial inner membrane space via Tom40 channels and its accumulation accelerates mitochondrial fragmentation due to reduced Δψm PB1-F2 variants lacking a C-terminal polypeptide which is frequently found in low pathogenic subtypes PB1-F2-mediated attenuation of Δψm suppresses the RIG-I signalling pathway and activation of NLRP3 inflammasomes PB1-F2 translocation into mitochondria strongly correlates with impaired cellular innate immunity making this translocation event a potential therapeutic target the mechanisms by which it translocates into mitochondria and affects the immune response remain unclear we investigated the mechanistic and functional roles of PB1-F2 in mitochondrial-mediated innate immunity We observed that PB1-F2 translocation into mitochondria leads to Δψm attenuation that strongly correlated with impaired cellular innate immunity (a) Cellular fractions from A/PR8 (16 HA U ml−1)-infected HEK293 cells were collected by differential centrifugation and analyzed by Western blotting using the indicated organelle markers (b) Subcellular localization of PB1-F2 variants and immunofluorescence with PB1-F2 specific antibodies was used to detect expression (middle panels) Mitochondria in the same cells were identified by stable expression of mitochondrially targeted red fluorescent protein (Mito-RFP The bottom four rows depict other organelle markers for the endoplasmic reticulum (ER-green and anti-PDI) and peroxisomes (anti-catalase) (c) Each full-length PB1-F2 variant was synthesized in vitro and incubated with isolated mitochondria (Mt) from HEK293 cells for the indicated times at 25 °C The reactants were then treated with proteinase K and analyzed by Western blotting (a) Isolated mitochondria from PB1-F2-expressing HEK293 cells were treated with either 1 M KCl or 0.1 M Na2CO3 (pH 11.5) for 30 min on ice the supernatant (S) and pellets (P) were analyzed by immunoblotting with antibodies against PB1-F2 or several mitochondrial proteins as indicated (b) Mitochondria isolated from A/PR8 (16 HA U ml−1)-infected HEK293 cells were diluted into either regular (−SW) or hypotonic swelling (+SW) buffer and maintained on ice for 30 min the supernatant (S) and pellets (P) were analyzed by Western blotting using the indicated sub-mitochondrial markers HtrA2 was used as a positive control for an unanchored IMS protein CoxIV and mtHsp70 are MIM and matrix proteins (c) Sub-mitochondrial fractionation of PB1-F2 (A/PR8) by sucrose density gradient centrifugation fractions (numbered) were analyzed by Western blotting with antibody against PB1-F2 or against indicated sub-mitochondrial markers (d) The mitochondrial fraction isolated from A/PR8 (16 HA U ml−1)-infected HEK293 cells was treated with proteinase K (100 μg ml−1) in the absence or presence of 500 μg ml−1 digitonin (pre-permeabilized condition) for 15 min on ice The reactants were developed by immunoblotting with antibodies against PB1-F2 or against several mitochondrial membrane markers as indicated (e) HEK293 cells were co-transfected with 5 ng of NLuc-PB1-F2 (88W) expression plasmid with increasing amounts (0–200 ng) of either Venus(N)-PB1-F2 (88W) (open triangle) or equal mass pairs of Venus(N)- and Venus(C)-PB1-F2 (88W) (filled square) expression plasmids and were analyzed 21 h later using a biomolecular fluorescence complementation BRET saturation assay Inset figure depicts the dose-dependent complementation (Venus(N+C)-PB1-F2 (88W)) of Venus fluorescence signal these biochemical lines of evidence indicate that PB1-F2 constitutively localizes to the mitochondrial inner membrane space (IMS) and assembles into a highly ordered oligomer (a) HeLa cells were treated with either control siRNA or siRNA directed against representative TOM components (TOM22 TOM40 and TOM70) to evaluate the effect of PB1-F2 import into mitochondria The silencing efficiency for each Tom protein was confirmed by immunoblotting with specific antibodies (b,c) Subcellular localization of (b) mitochondrially targeted enhanced GFP (Su9-eGFP) and (c) PB1-F2 (PR8) in the siRNA-treated HeLa cells (TOM40 and TOM70) and the green and red bars represent mitochondrial and cytosolic localization **P<0.01 and ***P<0.001 (by unpaired t-test) (d) Immunofluorescence images of the subcellular localization of Su9-eGFP or PB1-F2 (PR8) in HeLa cells treated with either TOM22 or TOM20 siRNAs these findings indicate that the translocation mechanism of the viral protein into mitochondria is distinct from the canonical host-protein import system that is generally mediated by Tom20/Tom22 receptors (and Tom70 for precursors with a hydrophobic presequence) which might account for the unique structural properties of the MTS in PB1-F2 (a) A549 cells were infected with either the A/PR8 (16 HA U ml−1) or A/CA/04/09 (multiplicity of infection (MOI) of 10) strains for 22 h and the mitochondrial morphology of infected cells was monitored by immunofluorescence microscopy (left panels) Mitochondria in the same cells were stained with an anti-HtrA2 polyclonal antibody (middle panels) Images on the right depict a magnified version of the boxed areas in each middle image (b) Quantification of mitochondrial morphology in a Cells were scored as one of the four morphological categories as depicted in the inset (c) HeLa cells were transfected with each PB1-F2 variant plasmid and their mitochondrial morphologies were classified Mfn2 and OPA-1) in the A/PR8-infected (I) and uninfected (U) cells were analyzed by immunoblotting (e) A549 cells were infected with A/PR8 (16 HA U ml−1) or uninfected and the cells were stained with MitoTracker Red CMXRos which detects mitochondrial membrane potential (Δψm) The two right panels are magnified images of the boxed area in the infected cells (left panel) and the circles in the images are highlighted examples of mitochondria loss of Δψm (a) HEK293 cells were infected with A/PR8 (16 HA U ml−1) for about 15 h and the cells were stained with the cationic fluorescent dye TMRM and analyzed by flow cytometry (right panel; red trace) CCCP-treated cells (40 μM) were also analyzed (left panel) Grey histograms in both panels represent a profile of unstained cells except that HEK293 cells were transfected with the expression plasmid indicated inside the panel Cells were analyzed by flow cytometry at 24 h post transfection (c) The kinetics of Δψm disruption in A/PR8-infected J774A.1 macrophages Cells infected with A/PR8 were collected at the indicated time points (0 The percentage of JC-1 reduction (y axis) is presented The immunoblot on the right represents a profile of PB1-F2 expression at each time point as well as the loading controls β-actin and OPA-1 Five bands (a–e) of OPA-1 isoforms were detected by immunoblotting with the antibody against OPA-1 and bands a and b are a mixture of L-OPA-1 isoforms (d) Δψm is dispensable for PB1-F2 translocation into mitochondria HeLa cells transfected with either mitochondrial-targeted dihydrofolate reductase (mtDHFR) or PB1-F2 were treated with (+) or without (−) CCCP (40 μM) and their translocation into mitochondria was monitored by immunofluorescence microscopy (left images) Quantification is listed in the right score panel these observations indicate that mitochondrially targeted PB1-F2 leads to the attenuation of Δψm (a) Mitochondrial morphology in HeLa cells treated with control siRNA Each siRNA-treated cell was transfected with PB1-F2 expression plasmids as indicated or treated with 20 μM CCCP (DMSO as a control) PB1-F2-expressing cells were identified by immunofluorescence and mitochondria were visualized using an antibody against CoxIV Insets depict the magnified images of each boxed area Cells were scored as one of three morphological categories as shown in the inset (c) Model of mitochondria fission induced by PB1-F2 We propose that the observed mitochondrial fragmentation occurs via a Drp-1-dependent pathway (a,b) HEK293 cells were co-transfected with 75 ng of plasmid encoding Myc-tagged MAVS 50 and 100 ng) of plasmids encoding the PB1-F2 variants and either the (a) IFN-β or the (b) NF-κB reporter plasmids Transfected cells were analyzed 24 h later for reporter gene-dependent luciferase activities Western blots reveal the abundance of Myc-MAVS proteins (c) HEK293 cells were co-transfected with 75 ng of plasmid encoding Myc-tagged RIG-I (1-250) (d) HEK293 cells were co-transfected with 30 ng of plasmid encoding NLRP3 pro-IL-1β (150 ng) and each PB1-F2 variant (300 ng) Cell-free supernatants were collected 24 h post transfection Inset: the functional role of the MOM-localized PB1-F2 chimeric mutant in NLRP3 inflammasomes (e) The reconstituted level of secreted IL-1β in d was confirmed by immunoblotting with antibodies against human IL-1β and pro-IL-1β and β-actin was used as a loading control in whole cell lysates (f) The inhibition of ASC oligomerization by mitochondrial-targeted PB1-F2 variants HEK293 cells transfected with plasmids encoding NLRP3 and each PB1-F2 variant were lysed and their lysates were cross-linked with BS3 The samples were analyzed by immunoblotting with antibody against FLAG The bottom blots confirm the expression levels of NLRP3 and ASC proteins in their lysates These findings demonstrate that mitochondrially targeted PB1-F2 acts as a negative regulator of NLRP3 inflammasomes these data demonstrate that Δψm dissipation introduced by translocation of the influenza A virus PB1-F2 into mitochondria correlates with the severity of defective mitochondrial-mediated innate immunity including the RIG-I signalling pathway and activation of NLRP3 inflammasomes which downregulates mitochondrial-mediated immune responses by attenuating Δψm as demonstrated in the present study we demonstrated that full-length versions of PB1-F2 from different viral strains specifically translocate into mitochondria and we further revealed mechanistic insight into how the viral protein is incorporated into the organelle our in vivo import analysis clearly demonstrated that PB1-F2 bypasses the general import receptors Tom20/Tom22 and is targeted directly to the Tom40 channel likely by evading host cell surveillance during import our findings provide a mechanism for PB1-F2 import into mitochondria and localization in mitochondrial sub-compartments Further studies are needed to identify other viral proteins (includes another influenza A viral protein) that have an affinity for mitochondria and to elucidate whether these proteins highjack the host import machinery and thus it we speculate that mitochondrially targeted PB1-F2 might trigger OMA-1 activation and induce such punctate mitochondria which corresponded to the lack of the C-terminal region that is important for mitochondrial translocation secondary bacterial infections are associated with increased severity and lethality and future studies aimed at evaluating the role of PB1-F2 in the differential susceptibility to pathogen infection may prove interesting Mitochondria are involved in various cellular processes and also appear to act as a platform for first-line innate immunity against RNA viruses The findings of the present study reveal a role for mitochondrial targeting of PB1-F2 in immune evasion and the inhibition of PB1-F2 translocation into the organelle might serve as a potential therapeutic target CCCP was purchased from Sigma-Aldrich (St Louis the MitoProbe JC-1 assay kit and TMRM were purchased from Molecular Probes/Invitrogen (Carlsbad and bis(sulfosuccinimidyl) suberate (BS3) was obtained from Thermo Scientific (Rockford Furimazine was supplied by Promega (Madison USA) and poly(I:C) was purchased from InvivoGen (San Diego All other reagents were biochemical research grade and the produced virus was stored at −80 °C until infection experiments The influenza virus A/CA/04/09 strain (H1N1) was a generous gift from Professor Yoshihiro Kawaoka (the University of Tokyo) The animal experiments (six- to eight-week old female C57BL/6J mice from The Jackson Laboratory) were approved by the Animal Committees of the Institute of Medical Science 1:1,000) polyclonal antibodies and anti-AIF (E-1 1:500) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz 1:1,000) rabbit monoclonal antibodies and anti-Hsp60 (D307 anti-caspase-3 (1:1,000) and anti-cleaved caspase-3 (Asp175; 1:1,000) polyclonal antibodies were purchased from Cell Signalling Technology (Danvers 1:1,000) monoclonal antibodies were supplied by Sigma-Aldrich Monoclonal antibodies against haemagglutinin (HA; HA.11 1:1,000) were obtained from Covance (Princeton and a polyclonal antibody against HtrA2 (Omi; 1:200) was purchased from R&D Systems (Minneapolis anti-cytochrome c (1:500) and anti-Drp-1 (1:1,000) monoclonal antibodies were supplied by BD Biosciences (San Jose and an anti-mitochondrial heat shock protein 70 (mtHsp70; 1:2,000) monoclonal antibody was obtained from Affinity BioReagents (Golden Anti-influenza A virus M2 protein (1:1,000) monoclonal and human MAVS (1:1,000) and IL-1β (1:1,000) polyclonal antibodies were purchased from Abcam (Cambridge and anti-human IL-1β (1:500) and anti-mouse IL-6 (1:500) antibodies used for ELISA were obtained from eBioscience (San Diego The Alexa Fluor 488 (1:500) anti-mouse monoclonal and the Alexa Fluor 488 (1:500) and Alexa Fluor 568 (1:500) anti-rabbit polyclonal antibodies were obtained from Molecular Probes/Invitrogen and the Cy3-conjugated sheep anti-mouse (1:1,000) monoclonal antibody was from Jackson ImmunoResearch Laboratories (West Grove All constructs used in the present study were confirmed by DNA sequencing (ABI 3100) either virally infected (A/PR8) or plasmid-transfected HEK293 cells (22 h post infection/transfection) were washed once with cold 1 × phosphate-buffered saline (PBS; pH 7.2) and lysed in 800 μl of homogenization buffer (20 mM HEPES (pH 7.5) 70 mM sucrose and 220 mM mannitol) by 30 strokes in a Dounce homogenizer The homogenate was centrifuged at 800 g for 5 min to precipitate the nuclei and the resulting supernatant was further centrifuged at 10,000 g for 10 min (4 °C) to precipitate the crude mitochondrial fraction The resulting supernatant was further centrifuged at 100,000 g for 30 min (4 °C) to precipitate light membrane organelles and the final supernatant was used as the cytosolic fraction the isolated mitochondria pellet was washed once with homogenization buffer the pellet was resuspended in buffer and treated with proteinase K (100 μg ml−1) in the absence or presence of 0.05% digitonin (weight per volume) The proteolytic reaction was performed on ice for 15 min and the reactants were subjected to Western blot analysis with the indicated antibodies Cell-free protein synthesis of each PB1-F2 variant (PR8 88W and 58W) was performed using TNT Coupled Wheat Germ Extract System (Promega) according to the manufacturer's protocol The synthesized proteins were incubated with mitochondria isolated from HEK293 cells in import buffer (20 mM HEPES-KOH (pH 7.4) The reactant from each time point was then treated with proteinase K (50 μg ml−1) to avoid non-specific binding of MOM and the reactant was subjected to Western blot analysis Isolated mitochondria (suspended in 100 μl of homogenization buffer) were diluted in 900 μl of hypotonic buffer (10 mM HEPES-KOH buffer (pH 7.5) containing 0.5 mM EDTA and protease inhibitor cocktail (Roche)) and maintained on ice for 30 min to cause swelling The mixture was then sonicated five times for 30 s each followed by centrifugation at 5,000g for 10 min The resulting supernatant was further spun at 200,000 g for 45 min at 4 °C and the pellet was resuspended in 100 μl of 5 mM HEPES-KOH buffer (pH 7.5) followed by layering over a linear gradient (0.8 to 1.6 M) of sucrose in hypotonic buffer and finally centrifuged at 100,000 g for 15 h at 4 °C in an Optima TL Ultracentrifuge (Beckman Coulter) 200-μl fractions were collected and subjected to Western blot analysis HEK293 cells (2.5 × 105 cells per well) were co-transfected with a constant amount (5 ng) of NLuc-tagged PB1-F2 (88W) plasmid and increasing amounts of Venus-tagged constructs using Lipofectamine 2000 reagent (Invitrogen) Cells were harvested 21 h post transfection and transferred to each well of white 96-well microplates NLuc substrate (furimazine; 5 μM) was added and the plates were analyzed via a BRET saturation assay using a Flexstation 3 Microplate Reader (Molecular Devices) at 37 °C and all cells were imaged by confocal microscopy (Carl Zeiss LSM510) An anti-influenza A virus M2 protein monoclonal antibody was used to detect A/CA/04/09-infected cells A semi-intact cell assay was performed as previously described25 PB1-F2-expressing HeLa cells were fixed with 3.7% formaldehyde for 10 min permeabilized with either 400 μg ml−1 (pre-permeabilized) or 2,000 μg ml−1 (normal condition) digitonin in PBS and then processed for immunofluorescence microscopy For the RNA interference knockdown experiments 21-nucleotide siRNA (sense strand-only shown) against human TOM20 (5′-aguuaccugaccuuaaagatt-3′) hDrp-1 (5′-gcagaagaaugggguaaautt-3′) and hOMA-1 (5′-gaucaauuggguuauucautt-3′) were purchased from QIAGEN HeLa cells were transfected with 50 nM siRNA (final concentration) twice within 24 h using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocols the siRNA-treated cells were transfected with each of the indicated expression plasmids using X-tremeGENE HP reagent followed by an in vivo import assay AllStars Negative Control siRNA (QIAGEN) was used as the control The ΔΨm was analyzed by BD FACSCalibur (BD Biosciences) with the cationic fluorescent dye TMRM (Molecular Probes/Invitrogen) Cells (~1 × 106 cells ml−1) that were infected with A/PR8 or plasmid transfected were washed once with 1 × PBS (pH 7.2) and harvested into a centrifuge tube The cells were then resuspended in 1 ml of 1 × PBS (pH 7.2) containing 2 μM TMRM and incubated at 37 °C for 30 min After washing three times with 1 × PBS (pH 7.2) Data analyses and graphs were prepared using FlowJo software (Tree Star HEK293 cells were plated in 24-well plates (2 × 105 cells per well) the cells were co-transfected with 100 ng of a luciferase reporter plasmid (p125luc or pELAM) 2.5 ng of the Renilla luciferase internal control vector phRL-TK (Promega) and each of the indicated expression plasmids using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocols Empty vector (pcDNA3.1(−)) was used to maintain equivalent amounts of DNA in each well Cells were harvested 24 h post transfection and analyzed using a dual-luciferase reporter assay on the GloMax 20/20n luminometer (Promega) Each experiment was replicated at least three times HEK293 cells were plated in 24-well plates (4 × 105 cells per well) the cells were co-transfected with 30 ng of pCA7-NLRP3 5 ng of pCA7-procaspase-1 and 150 ng of pCA-pro-IL-1β and each of the indicated expression plasmids using Lipofectamine 2000 Empty vectors (pcDNA3.1(−)) were used to maintain equivalent amounts of DNA in each well clarified medium supernatants were collected the above cells were washed once with 1 × PBS (pH 7.2) and then resuspended in 150 mM NaCl solution followed by lysis with 15 strokes through a syringe with a 21-gauge needle After centrifugation (12,000 g for 15 min) the clarified supernatant was cross-linked with 4 mM BS3 for 30 min at room temperature followed by incubation with 50 mM Tris-HCl buffer for 15 min at room temperature to completely quench the cross-linking reaction The resulting sample was resolved on 10% SDS–PAGE and immunoblotted with anti-FLAG monoclonal antibody The analysis of variance test (GraphPad QuickCalcs) was used for the statistical analyses A P-value<0.05 was considered statistically significant Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity Cytochrome c: functions beyond respiration Mitochondrial calcium signalling in cell death New insights into the role of mitochondria in aging: mitochondrial dynamics and more Identification and characterization of MAVS a mitochondrial antiviral signalling protein that activates NF-κB and IRF3 Mitoxosome: a mitochondrial platform for cross-talk between cellular stress and antiviral signalling Mitochondrial membrane potential is required for MAVS-mediated antiviral signalling A novel influenza A virus mitochondrial protein that induces cell death An insight into the PB1F2 protein and its multifunctional role in enhancing the pathogenicity of the influenza A viruses The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function Mitochondrial targeting sequence of the influenza A virus PB1-F2 protein and its function in mitochondria Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1 and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space A structural perspective of the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments Regulation of mitochondrial morphology through proteolytic cleavage of OPA1 Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics Mitofusin 2 inhibits mitochondrial antiviral signalling Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential Activation and regulation of the inflammasomes Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation Measles virus V protein inhibits NLRP3 inflammasome-mediated interleukin-1β secretion Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signalling protein off the mitochondria to evade innate immunity Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates Mitochondrial dynamics regulate the RIG-I-like receptor antiviral pathway SLP-2 is required for stress-induced mitochondrial hyperfusion Expression of the 1918 Influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens Monoclonal antibodies against the PB1-F2 protein of H1N1 influenza A virus Download references We are grateful to Koji Okamoto (the Osaka University Japan) and Thomas Langer (the University of Cologne) for their valuable comments on the study We appreciate technical support from the Research Support Centre the Kyushu University for the fluorescence measurements and Yuko Fuchigami for her technical assistance with DNA cloning and sequencing The anti-PB1-F2 (A/PR8 strain) monoclonal antibody was a kind gift from Viktor Wixler (the Münster University Hospital Medical School) We are also grateful to Yoshihiro Kawaoka (the University of Tokyo) for kindly providing the pPolI/CA04-PB1-F2-stop88W plasmid and the A/CA/04/09 (H1N1) virus Hideki Hasegawa (the National Institute of Infectious Diseases Japan) for the A/Beijing/262/95 (H1N1) virus Michael Whitt (the University of Tennessee) for the recombinant vesicular stomatitis virus and Victoria Allan (the University of Manchester) for the ER-green plasmid This work was supported by the JSPS KAKENHI Grants (No the Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (T.K.) the Kao Foundation for Arts and Sciences (T.K the Tokyo Biochemical Research Foundation (T.I.) the Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.I.) and the Sasakawa Scientific Research Grant from the Japan Science Society (T.Y.) This study was also partly supported by the Grant for Joint Research Project of the Institute of Medical Science the University of Tokyo (T.K.) and Research Fellow of the Japan Society for the Promotion of Science (O.S.) International Research Center for Infectious Diseases established the knockdown cell lines and performed the semi-intact cell assay Analyzed data and interpreted all experimental results designed the overall direction and contributed to writing the paper The authors declare no competing financial interests Supplementary Table 1 and Supplementary References (PDF 2684 kb) Download citation a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science a proton-selective ion channel essential for efficient viral replication which localizes to the mitochondria and attenuates host antiviral immunity are involved in the inflammatory response by stimulating NLRP3 inflammasome-dependent IL-1β secretion the precise mechanisms by which these viral proteins activate the NLRP3 inflammasome remain unclear a research group at The Institute of Medical Science The University of Tokyo (IMSUT) observed nucleus- and mitochondria-derived double-stranded DNA (dsDNA) in extracellular web-like structures in the cytoplasm and extracellular space around influenza virus-infected macrophages 10.1016/j.isci.2020.101270 Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system. The Institute of Medical Science, The University of Tokyo Copyright © 2025 by the American Association for the Advancement of Science (AAAS) Volume 13 - 2019 | https://doi.org/10.3389/fncel.2019.00344 Autism spectrum disorder (ASD) is one of the most widespread neurodevelopmental disorders characterized by impairment in social interactions Using immunohistochemistry and positron emission tomography (PET) several studies have provided evidence of the existence of activated microglia in ASD patients we developed an animal model of ASD using the new world monkey common marmoset (Callithrix jacchus) and demonstrated ASD-like social impairment after the in utero administration of valproic acid (VPA) To characterize microglia in this marmoset model of ASD from early toddler to adult morphological analyses of microglia in VPA marmosets and age-matched unexposed (UE) marmosets were performed using immunohistochemistry for microglia-specific markers The most robust morphological difference between VPA marmosets and UE marmosets throughout the life span evaluated were the microglia processes in VPA marmosets being frequently segmented by thin and faintly Iba1-positive structures The segmentation of microglial processes was only rarely observed in UE marmosets This feature of segmentation of microglial processes in VPA marmosets can also be observed in images from previous studies on ASD conducted in humans and animal models Apoptotic cells have been shown to have segmented processes our results might suggest that microglia in patients and animals with ASD symptoms could frequently be in the apoptotic phase with high turnover rates of microglia found in some pathological conditions All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health United States) and approved by the Animal Research Committee of the National Institute of Neuroscience (Kodaira Characteristics of the animals used in this study The perfusion solutions were delivered using a Masterflex 7553–7570 peristaltic pump (Cole-Parmer The perfused brains were removed and incubated overnight in the PFA solution followed by a graded series of sucrose solutions over 1 week at concentrations of 10 The brains were then sectioned 40 μm thick using a sliding microtome (Retratome REM-710 Endogenous peroxidase activity was blocked by a 20-min incubation of the tissue sections in 80% methanol containing 3% hydrogen peroxide sections were washed three times with PBS containing 0.3% Triton X-100 (PBST) After incubation in 1% bovine serum albumin (BSA; Sigma sections were further incubated in rabbit anti-Iba1 primary antibody (Wako Japan) diluted 1:1,000 in PBST containing 1% BSA at 4°C overnight sections were subjected to incubation for an additional 2 h with biotinylated anti-rabbit IgG secondary antibody (Vector Laboratories United States) diluted 1:200 in PBST containing 1% BSA at room temperature and were incubated with avidin-biotin-peroxidase complex solution (Vector Laboratories Iba1 positive structures were visualized using 3,3′-diaminobenzidine tetrahydrochloride as a chromogen Images were captured using an All-in-One Fluorescence Microscope (BZ-X700; KEYENCE In addition to immunohistochemistry of Iba1, a cytosolic microglial marker, we also prepared sections for immunofluorescent staining for P2Y12, a microglia surface marker (Wolf et al., 2017) After blocking non-specific binding by incubating the sections for 2 h with PBST containing 1% BSA the sections were incubated with rabbit anti-P2Y12 primary antibody (1:1000; Sigma-Aldrich United States) in PBST containing 1% BSA at 4°C overnight The sections were then washed extensively with PBST and incubated with secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000; Invitrogen United States) in PBST containing 1% BSA at 4°C for 2 h the sections were mounted and cover-slipped using VECTASHIELD® (Vector Laboratories Fluorescent images were captured using an All-in-One Fluorescence Microscope XY images acquired at 1-μm z-step intervals were merged we performed qualitative microscopic examination of microglial morphology on tissues stained for Iba1 and P2RY12 quantitative analysis of morphology of microglial processes and somata and microglial density was performed on sections stained for Iba1 For performing the quantitative analysis of morphology of microglial processes and somata 50 Iba1-positive microglia on layer 3 of area 12o were selected from each animal used in this study the number of microglia examined both in UE and VPA marmosets was 200 at 2M Selected microglial somata and corresponding processes were traced three dimensionally by adjusting focal plane through depth of each section using a Nikon ECLIPSE 80i microscope (Nikon Japan) and the computer-aided tracing system Neurolucida (MBF Bioscience We did not aim to complete reconstruction of each microglia through multiple sections in order to acquire global information of a single microglia as this was difficult due to dense and clouded Iba1-positive processes in each section which enabled us to quantify the length and width of microglial processes and several indices of somata was used for performing the quantitative analysis the number of Sholl annuli intersections was quantified The diameters of all processes were measured every 10 μm starting at 15 μm from the center of the cell body to avoid mismeasurement of cell body per se The quantified factors regarding the cell bodies were (1) the cell area and (2) the form factor. The form factor was calculated using the equation (4π × area)/circumference2, as described by Soltys et al. (2001). Thus, the maximum form factor value of perfectly circular cells was 1 and activated microglia had larger form factors than did microglia in a ramified state (Soltys et al., 2001) we performed analysis of microglial density the centers of the microglial cell bodies on layer 3 of area 12o were located under a Nikon ECLIPSE 80i microscope (Nikon Japan) in ten Iba1-immunostained sections from each animal used in this study and the number of microglia were counted in a 150 μm × 80 μm area The density value was expressed as the number of microglia cell bodies per 1000 μm2 The data were evaluated using analysis of variance (ANOVA) followed by Tukey-Kramer post hoc tests Statistical analyses were performed using JSTAT software (Tokyo Japan) and KaleidaGraph 3.6 software (Synergy Software All values were expressed as the mean ± STD and p-values less than 0.05 were considered statistically significant Segmented processes of microglia in VPA marmosets (A) Representative images of Iba1-positive microglia at 3 months (M) in UE and VPA marmosets Arrowheads indicate thin and faintly Iba1-positive structures segmenting microglial processes (B) Representative images of Iba1-positive microglia at 6 M in UE and VPA marmosets (C) Representative images of P2RY12-positive microglia at 6 M in VPA marmosets Arrows indicate segmented microglial processes Inset corresponds to the enlarged image of (C) Arrowheads in inset indicate thin delicate processes bridging P2RY12-positive microglial processes (D) Histogram shows the number of thin (less than 0.15 μm) and faintly Iba1-positive structures segmenting microglial processes in UE and VPA marmosets through the developmental stages examined Morphological changes of microglial processes in developmental VPA marmosets assayed on sections stained for Iba1 (A) Histogram shows the number of microglial processes directly deriving from a soma in UE and VPA marmosets through developmental stages examined (B) Histogram shows the total length of microglial processes of a microglia in UE and VPA marmosets through developmental stages examined (C) Line graphs show the number of intersections of microglial processes and concentric circle denotes microglia in UE and VPA marmosets in each developmental stage examined (D) Histograms show the diameter of microglia at every 10 μm distance from the center of a cell body in UE and VPA marmosets of each developmental stage examined There was no significant difference in the area of the microglia cell bodies for UE marmosets compared to that for VPA marmosets (Figure 3A). In contrast, the form factor of the cell bodies at 3 and 6 months, and adulthood was greater in VPA marmosets than in UE marmosets (Figure 3B). The solidity of the microglia was statistically lower in VPA marmosets than in UE marmosets at 3 and 6 months of age (Figure 3C) Morphological changes of microglial cell bodies and an index for microglial somata and processes (i.e. solidity) in VPA marmosets assayed on sections stained for Iba1 (A) Histogram shows the area of microglial cell body in UE and VPA marmosets through the developmental stages examined (B) Histogram shows the area of microglial cell body in UE and VPA marmosets through the developmental stages examined (C) Histogram shows the solidity of a microglia in UE and VPA marmosets through the developmental stages examined The density of microglia cell bodies in VPA marmosets at 6 months was statistically lower than that in UE marmosets (Figure 4) There were no statistical differences at any other ages evaluated Effects of prenatal exposure to VPA on microglial density in marmoset cortex assayed on sections stained for Iba1 (A) Representative images show microglial distribution in UE and VPA marmosets through the developmental stages examined (B) Histogram shows the density of microglia in UE and VPA marmosets through the developmental stages examined we performed qualitative and quantitative analysis of microglia in VPA marmosets with autistic behavior and UE marmosets at the ages from early toddler to adulthood on the sections stained for Iba1 Quantitative analysis on the tissue stained for Iba1 showed that most of the parameters evaluated demonstrated age-dependent differences the number of thin and faintly Iba1-positive structures segmenting the microglial processes was robustly greater in VPA marmosets than in UE marmosets throughout the evaluated life span This microglia segmentation seemed to be a hallmark of the VPA marmoset cortex it is possible that microglia with segmented processes found in this study might be in the apoptotic phase with high turnover rate of microglia This possibility should be tested in a future study throughout the life span evaluated in our study microglia processes in VPA marmosets tended to be thinner than those in UE marmosets with the differences being statistically significant in early adolescence and adulthood thinning microglia processes along with development seemingly paralleled the increasing number of thin and faintly Iba1-positive structures segmenting microglia processes with the passage of age since segmentation of microglia processes occurs in patients with ASD and in rodent and marmoset models of ASD this feature may serve as a good biomarker for assessing brain pathology The raw data supporting the conclusions of this manuscript will be made available by the authors All animal experiments were approved by the Animal Research Committee of the National Institute of Neuroscience (Kodaira ToS performed the histological preparation KN and TM provided the UE and VPA marmosets This work was supported by an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (Grant Number 23-7) by the Strategic Research Program for Brain Science by a Grant-in-Aid for Scientific Research on Innovative Areas “Foundation of Synapse Neurocircuit Pathology” (No and by JSPS KAKENHI grant numbers 17K18401 (Grant-in-Aid for Young Scientists) and 16J40220 (Grant-in-Aid for JSPS Research Fellow) Akiko Tsuchiya for their valuable technical support and animal care Tomoko Manabe for her technical assistance for histochemistry Disassembly of the dying: mechanisms and functions A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure Neuroglia in the autistic brain: evidence from a preclinical model Prevalence and characteristics of autism spectrum disorder among 4-year-old children in the autism and developmental disabilities monitoring network Microglia turnover with aging and in an Alzheimer’s model via long-term in vivo single-cell imaging Transcriptome analysis 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the cat Google Scholar Morphology of reactive microglia in the injured cerebral cortex fractal analysis and complementary quantitative methods doi: 10.1002/1097-4547(20010101)63:1<90::aid-jnr11>3.0.co;2-9 Functional plasticity of microglia: a review Microglial activation in young adults with autism spectrum disorder Microglia gone rogue: impacts on psychiatric disorders across the lifespan A new fate mapping system reveals context-dependent random or clonal expansion of microglia Fetal valproate syndrome and autism: additional evidence of an association A male with fetal valproate syndrome and autism doi: 10.1146/annurev-physiol-022516-034406 Kohsaka S and Ichinohe N (2019) Segmented Iba1-Positive Processes of Microglia in Autism Model Marmosets Copyright © 2019 Sanagi, Sasaki, Nakagaki, Minamimoto, Kohsaka and Ichinohe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Noritaka Ichinohe, bmljaGlub0BuY25wLmdvLmpw Volume 9 - 2015 | https://doi.org/10.3389/fnana.2015.00030 Neuronal activities recorded from the dorsal bank of the anterior cingulate sulcus have suggested that this cortical area is involved in control of search vs goal-based action selection and encoding of prediction error regarding action value to explore potential anatomical bases for these neuronal activities we injected retrograde tracers (CTB-Alexa-488 and CTB-gold) into the dorsal bank of the anterior cingulate sulcus and examined the distribution of labeled cell bodies in macaque monkey brains The Nissl staining showed that the cortex in the dorsal bank of the anterior cingulate sulcus has consistent layer 4 which means that the cortical region is a part of the granular prefrontal cortex The injection site belonged to the sulcal portion of area 9m in two cases and the sulcal portion of area 8Bm in one case In addition to the continuous distribution of labeled cells in the two areas (areas 9m and 8Bm) around the injection site the labeled cells were densely distributed in the cingulate areas (areas 24 The dense labeling of cells was also found in other prefrontal areas (areas 46 and 12) in the two cases with injection into the sulcal portion of area 9m whereas the dense labeling of cells was found in pre-motor areas (F6 and F7) in the case with injection into the sulcal portion of area 8Bm The dense labeling of cells in the prefrontal and premotor areas was more similar to those previously found after injections into dorsal parts of areas 9 and 8B Subcortical distribution of labeled cells was found in the mediodorsal nucleus of thalamus and substantia nigra pars compacta in all the cases the dorsal bank of the anterior cingulate sulcus should be the most ventral parts of areas 9m and 8Bm To examine anatomical inputs to the dorsal bank of the anterior cingulate sulcus we injected retrograde tracers (CTB-Alexa-488 and CTB-gold) into the sulcal portions of areas 9m and 8Bm and examined the distribution of labeled cell bodies in cortical and subcortical structures in macaque monkeys We used two male rhesus monkeys (Macaca mulatta) weighing 7–10 kg. The two monkeys (Monkey 1 and Monkey 2) used in this study were the same animals used in our electrophysiological studies (Matsumoto et al., 2006, 2007) We kept the animal number codes consistent between this study and the previous studies All procedures were approved by the RIKEN Animal Experiment Committee and were in accordance with the Guideline for Animal Experiments of the Japan Neuroscience Society The surgical procedures, task, training, and electrophysiological experiments have already been described with electrophysiological results (Matsumoto et al., 2006, 2007) After confirming the location of the targeted part of the dorsal bank of the anterior cingulate sulcus by anatomical MRI a head holder and two electrophysiology recording chambers (20 mm in diameter) were implanted by an aseptic surgery under phenobarbital–induced anesthesia (35 mg per kilogram body weight Retrograde tracers were injected into the dorsal bank of the anterior cingulate sulcus (Figure 1) the animals were fully awake with the head fixed in the monkey chair A 24G stainless steel needle was filled with retrograde tracers CTB-Alexa-488 (Invitrogen-Molecular Probes diluted in 0.1 M phosphate-buffered saline (PBS) The needle filled with the tracers was connected to a silicon tube filled with PBS and further connected to a 10 μl micro syringe filled with PBS (Hamilton The needle was attached to an oil hydraulic micromanipulator (Narishige Japan) and slowly advanced through the same grid as that used for single-cell recordings to the dorsal bank of the anterior cingulate sulcus The depth location of the anterior cingulate sulcus was determined during recordings with an electrode by characteristic absence of neuronal activities in the sulcus The needle tip was first advanced to 0.52 mm above the dorsal surface of the anterior cingulate sulcus and then withdrawn to 1.00 mm above the dorsal surface of the anterior cingulate sulcus to make a space for the tracers to stay in the gray matter We left the needle at that position for about 5 min and then 1 μl of tracers were injected over a period of 10 min using a syringe pump (KDS210 The needle was left at that position for another 5 min and then slowly retracted (A) Photomicrograph of coronal section showing the site of CTB-Alexa-488 injection in Case 1L (B) Photomicrograph of coronal section showing the site of CTB-Alexa-488 injection in Case 2R (C) Photomicrograph of coronal section showing the site of CTB-gold injection site in Case 2L (D) Medial and lateral views of the brain reconstructed from structural MRI images Red arrows indicate the sites of tracer injections into the dorsal bank of the anterior cingulate sulcus The animals were kept in their home cages for 1 week for the tracer transport after the tracer injections and then they were perfused first with 2 L of phosphate buffer (pH 7.3) followed by 4 L of a solution containing 4% paraformaldehyde in PB The brain was subsequently removed from the skull and cut sagittally the brain specimens were kept in 30% sucrose in PB we cut them into 50 μm-thick sections with a sliding microtome The sections were divided into five series The first series were used for visualizing either Alexa488 or Gold The second series were stained for the Nissl substance with thionin The contralateral hemispheres were not stained for the injected tracers Sections were incubated for 1 h with 0.1 M PBS (pH 7.3) containing 0.5% Triton X-100 and 5% normal goat serum (PBS-TG) at room temperature and then for 40–48 h at 4°C with PBS-TG containing a monoclonal anti-Alexa-488 antibody (Invitrogen-Molecular Probes the sections were placed in PBS-TG containing biotinylated goat anti-mouse IgG (Vector Immunoreaction was visualized by incubation with ABC (one drop of reagents in 7 ml of 0.1 M PB; ABC Elite kits followed by diaminobenzidine histochemical reaction with 0.03% nickel ammonium sulfate Sections were washed first with 0.1 M PBS, followed by 0.01 M PBS. An IntenSE M silver enhancement kit (Amersham plc, Amersham, UK) was used to visualize the CTB-gold signals (Sincich et al., 2007) A one-to-one cocktail of the IntenSE M kit solution and 33% gum Arabic solution was used as reagent Development of reaction products was monitored under a microscope and terminated by rinsing the sections in 0.01 M PBS followed by several rinses in 0.1 M PBS We determined the extent of injection site by the area in which the tracers filled the entire neuropil Total number of labeled neurons (N) and the ratio of the number of labeled neurons in superficial layers (layers 1–3) to the number of labeled neurons in deep layers (layers 5 and 6) (S/D) in each cortical area a separate functional area from SMA-proper (F3) Cytoarchitecture of the cortical areas around the injection sites (dorsal bank parts of areas 9m and 8Bm) together with that of the neighboring cortical area in the ventral bank of anterior cingulate sulcus (area 24c) The injection sites were located in the dorsal bank of the anterior cingulate sulcus in all the three cases (Figure 1) close to the dorsal lip of the sulcus in two cases (Case 1L and Case 2R) and at the middle between the dorsal lip and fundus in the third case (Case 2L) the injection site was judged to be located in area 9m in two cases (Case 1L and Case 2R) and in area 8Bm in the third case (Case 2L) Distribution of retrogradely labeled neurons on the flat map of the cerebral cortex in Case 1L The density of labeled neurons was converted into a pseudo-color-code The flat map was flipped horizontally to make the comparison between the cases easier Distribution of retrogradely labeled neurons on coronal sections in Case 1L (A–O) The abbreviations used to mark cortical areas are shown in the Abbreviations in the main text The red square indicates the injection site The coronal sections were flipped horizontally to make the comparison between the cases easier Distribution of retrogradely labeled neurons on the flat map of the cerebral cortex in Case 2R Distribution of retrogradely labeled neurons on coronal sections in Case 2R (A–P) Distribution of retrogradely labeled neurons on the flat map of the cerebral cortex in Case 2L Distribution of retrogradely labeled neurons on coronal sections in Case 2L (A–N) In most of the cortical areas where labeled cells occurred, the labeled cells were located in both the superficial layers (layers 1–3) and deep layers (layers 5 and 6). A large difference in the number of labeled cells between the superficial and deep layers with a ratio larger than 4 was found in a few cases, but the results were inconsistent between the cases (Figure 9 and Table 1) Normalized numbers of retrogradely labeled neurons in each cortical area counted separately for superficial and deep layers The number in each area was normalized by the total number of labeled neurons in the cortical area that had the maximum number of labeled neurons (a sum of the numbers in superficial and deep layers) Only the cortical areas in which labeled neurons were found in at least two of the three cases were included The distribution of labeled cells indicated that there were strong projections from the cingulate cortical areas (areas 24 and 23) to the injection sites commonly in all the three cases There were mutual connections between the sulcal parts of areas 9m and 8Bm the sulcus part of area 9m received definite projections from other prefrontal areas (areas 46 and 12) while the sulcus part of area 8Bm received minimal projections from these prefrontal areas instead received strong projections from motor related areas (F6 i.e. the pre-supplementary motor area and F7 i.e. the dorsal premotor cortex) while the sulcus part of area 9m received minimal projections from these motor related areas There were also common projections from superior temporal areas only from area TPO or from surrounding areas as well there were common projections from the mediodorsal nucleus of thalamus Projections from the ventral tegmental area were observed in two of the three cases but the projections from the ventral tegmental area were weaker than those from the substantia nigra pars compacta Minimal projection was observed in the basal nuclei of amygdala in the area 9m cases The mutual connections between the two areas in the dorsal bank of the anterior cingulate sulcus may be strong enough to convey motor-related information from area 8Bm to area 9m and cognitive-control-related information from area 9m to area 8Bm observations with new behavioral paradigms may find differences in neuronal properties between the two areas because we had only one case with tracer injection into area 8Bm further studies are necessary to definitely conclude the difference between the projections to the sulcus part of area 9m and those to the sulcus part of area 8Bm A general principle has been proposed for cortico-cortial projections within the prefrontal cortex regarding layer localization of cells-of-origin and terminals: projections from an agranular area to a granular area originate in deep layers and terminate in superficial layers while projections from a granular area to an agranular area originate in superficial layers and terminate in deep layers (Barbas and Hilgetag, 2002; Barbas and Zikopoulos, 2007) cingulate and pre-motor areas to the cingulate sulcus parts of areas 9m and 8Bm didn't take either type in the layer distribution of cells-of-origin which are consistent with the proposal as layer 4 was not fully distinguished in areas 9m and 8Bm as compared with that in the lateral prefrontal areas The diverse inputs related to different aspects of outcomes along with the motor related inputs from the premotor areas and the context information from the dorsal and dorsolateral prefrontal areas may make the dorsal bank of the anterior cingulate sulcus an optimal anatomical location to integrate goal directed action plans with various types of value and context information MKE and NI observed the sections and analyzed the data This work was supported by the Japan Society for he Promotion of Science through the Funding Program for World-Leading Innovative R and D on Science and Technology (FIRST Program) to KT and NI by Grant-in-Aid for Scientific Research on Innovative Areas “Shitsukan” and “Seisyun-no” from MEXT Hiromi Mashiko and for conducting histological procedures. Eiji Hoshi (Tokyo Metropolitan Institute of Medical Science) for generously providing a program subdivision on the medial bank of olfactory sulcus; 13l subdivision on the lateral part of middle orbital gyrus; 14c ventral subdivision near callosal sulcus; 24b dorsal subdivision in ventral bank of anterior cingulate sulcus; 29 Medial superior temporal area (visual); MT Area TAa in the sts dorsal bank (auditory); TEad Dorsal subregion of anterior TE (visual); TEav Ventral subregion of anterior TE (visual); TEm Area TEm in the sts ventral bank (visual); TEO Dorsal subregion of posterior TE (visual); TEpv Ventral subregion of posterior TE (visual); TF/TH Area TF and TH of parahippocampal cortex; TFO Visual area V2; V23a/b area V23a and V23b in posterior cingulate cortex; V3 Lamina-specific alterations in the dopamine innervation of the prefrontal cortex in Schizophrenic subjects Ipsilateral connections of the anterior cingulate cortex with the frontal and medial temporal cortices in the macaque monkeys Medial prefrontal cortices are unified by common connections with superior temporal cortices and distinguished by input from memory-related areas in the rhesus monkey Rules relating connections to cortical structure in primate prefrontal cortex CrossRef Full Text | Google Scholar Architecture and intrinsic connections of the prefrontal cortex in the rhesus monkey The prefrontal cortex and flexible behavior Beitraege zur histologischen lokalisation der grosshirnrinde Neuronal encoding of subjective value in dorsal and ventral anterior cingulate cortex Limbic connections of the orbital and medial prefrontal cortex in macaque monkeys Sensory and premotor connections of the orbital and medial prefrontal cortex of macaque monkeys Connectional networks within the orbital and medial prefrontal cortex of macaque monkeys Common regions of the human frontal lobe recruited by diverse cognitive demands and serotoninergic receptors and complementarity of their subtypes in primate prefrontal cortex Haber, S. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Noritaka Ichinohe, Department of Ultrastructural Research, National Center of Neurology and Psychiatry, National Institute of Neuroscience, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, JapanbmljaGlub0BuY25wLmdvLmpw Volume 8 - 2014 | https://doi.org/10.3389/fnsys.2014.00098 This article is part of the Research TopicThe Claustrum: charting a way forward for the brain’s most mysterious nucleusView all 14 articles The identity of the claustrum as a part of cerebral cortex and in particular of the adjacent insular cortex has been investigated by connectivity features and patterns of gene expression we mapped the cortical and claustral expression of several cortical genes in rodent and macaque monkey brains (nurr1 and netrinG2) to further assess shared features between cortex and claustrum these genes were densely expressed in the claustrum but very sparsely in the cortex and not present in the striatum claustral cell types can be distinguished by co-expression of these genes we performed a panel of double ISH in mouse and macaque brain NetrinG2 and nurr1 genes were co-expressed across entire cortex and claustrum but cux2 and nurr1 were co-expressed only in the insular cortex and claustrum The nurr1+ claustral neurons expressed VGluT1 a marker for cortical glutamatergic cells and send cortical projections our data suggest a partial commonality between claustral neurons and a subtype of cortical neurons in the monkey brain many nurr1+ neurons were scattered in the white matter between the claustrum and the insular cortex possibly representing their migratory history we injected Lucifer Yellow intracellularly in mouse and rat slices to investigate whether dendrites of insular and claustral neurons can cross the border of the two brain regions Dendrites of claustral neurons did not invade the overlying insular territory gene expression profile of the claustrum is similar to that of the neocortex but with modifications in density of expression and cellular co-localization of specific genes Given the shared features of gene expression and extrinsic connectivity the possibility arises that Latexin+/nurr1+ neurons in the cortical deep layers and the claustrum may be categorized as the same subclass of neurons To examine the evolutionarily conserved expression of these genes, we previously performed ISH analyses in the macaque brain (Watakabe et al., 2007; Watakabe, 2009) we found that nurr1+ neurons are present in both the claustrum and cortex in the macaque brain as opposed to lateral-restricted expression in the rodent brain nurr1 mRNA was expressed in layer 6 across the entire neocortex in macaques even while latexin mRNA was not detected in the neocortical regions in this species The differential gene expression in rodent and macaque led us to further investigations to clarify the evolutionary fingerprint of the claustral neurons We aimed to clarify by double ISH whether these genes exhibit similar distribution patterns as that of nurr1 in the two species Whether they are co-expressed outside claustrum would be a good measure to understand the significance of their expression in both cortex and claustrum we reexamined the latexin mRNA expression in the insular/claustral regions of the macaque brain We also examined the expression of the nurr1 gene in the embryonic monkey and postnatal mouse cortex to see if we could differentiate cortical and claustral neurons in the earlier developmental time point we examined the morphology of claustral neurons to ascertain whether their dendrites extend beyond the insular/claustral border in rodents If the claustral neurons exhibit long dendrites that cross into insular cortex this could be taken as strong support for a cortical identity Tracer injection into the adult macaque monkey was performed in RIKEN Institute in accordance with the protocol approved by the Experimental Animal Committee of the RIKEN Institute, which was also concordant with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996. Adult macaque monkey brain sections for ISH were obtained from the samples that were used in a previous study (Watakabe et al., 2007) Embryonic macaque monkey brain was obtained from Tsukuba Primate Research Center Tracer injection into rat brain and perfusion of mice and rats for ISH and Lucifer Yellow injection followed the animal care guidelines of the National Institute for Basic Biology and National Institute for Physiological Sciences The ISH probes for the mouse and monkey nurr1 and VGluT1 gene have been described previously (Watakabe et al., 2007) PCR was performed on the mouse and monkey cDNAs using the following primer sets 5′-TGGAGTGGGAGTTCTGAAAG-3′ and 5′-GGAAACTTCCTGGGTTGTGC-3′; for the netrinG2 gene 5′-ATGCCGAAGGCCTCCATGCA-3′ and 5′-CTGTCACAATTTGAGAGTCTGC-3′ 5′-TTGGTGGCACAGAACTACATCA-3′ and 5′-GTGACACTTTGGGATTATTTGG-3′ To investigate dendritic morphology, Lucifer Yellow injection was performed essentially as described previously (Oga et al., 2013) 11-week old male BL6 mice (~ 30 g) or 7-week old male Wistar rats (~ 150 g) were perfused with 30 and 150 ml of 4% paraformaldehyde/0.1 M phosphate buffer at the rate of 1 or 10 ml/min for mice and rats The fixed brain was excised and sliced at 200 μ m After counterstaining with 1 μ g/ml DAPI Lucifer Yellow was injected into individual cells under the visual guidance of ultraviolet illumination In the low-power view of the dark field image the insular/claustral regions appeared as a dark column next to the rhinal fissure which itself was sandwiched between the brighter neocortex and piriform cortices The injections were targeted from the posterior side of the slices To identify the precise border between the claustrum and the insular cortex, and to enhance the fluorescent signals of the Lucifer Yellow, the injected slices were processed for immunostaining using the biotinylated rabbit antibody to Lucifer Yellow (Invitrogen, A5751) and the mouse monoclonal antibody to latexin (Arimatsu et al., 1992 1:500) or parvalbumin (Swant235; 1:1000) in 2% bovine serum albumin and 5% sucrose in 0.1 M phosphate buffer for 5–7 days at room temperature followed by detection with streptavidin-Alexa488 and Cy3-conjugated anti-mouse antibody The distance between the “Top,” “Bottom,” “Right,” and “Left” borders and the center of the cell body was calculated based on these values To clarify whether the cux2 and netrin G2 genes are expressed in the same cell populations that express latexin and nurr1 we first performed double ISH using these gene markers in the mouse cortex and later in the macaque brain Colocalization of cux2 and nurr1 mRNAs in the claustrum and cortical deep layer of the mouse brain DIG-labeled cux2 antisense probe (detected in red) and FITC-labeled nurr1 antisense probe (detected in green) were hybridized to the coronal sections of the mouse brain (A,B) Double ISH of cux2 (red) and nurr1 (green) mRNAs in the frontal section (A) shows only the red channel (cux2 signal) while (B) shows the merged view for red and green channels of the same section (C–E) The dashed rectangles in (A,B) were magnified and shown for different channels (C,D) show the signals for cux2 and nurr1 mRNAs and (E) shows the merged image of (C,D) (F–H) Double ISH of cux2 and nurr1 in the more posterior section (F,G) show red (cux2 signal) and green (nurr1) channels while (H) shows the merged view for red and green channels (I–K) Dashed rectangles in (F–K) were magnified The arrowheads in panels (I–K) indicate the nurr1+/cux2− cells in layer 6b (L) Hoechst nuclear staining to confirm lamina boundaries in (I–K) The dashed lines indicate the lamina borders determined by the density of nuclear staining (M–O) Dashed rectangles in (I–K) were magnified Note exact colocalization of the two mRNAs in (O) and netrinG2 gene are all expressed in the same neuronal population that is enriched both in the claustrum and in layer 6 and some in layer 5 of the mouse lateral cortical areas Colocalization of netrinG2 and nurr1 mRNAs in the mouse brain (A,B) Double ISH of netrinG2 (red) and nurr1 (green) mRNAs in the mouse frontal section (C–E) Dashed rectangles in (A,B) were magnified and shown for netrinG2 (C) (F–H) Dashed rectangles in (C–E) were magnified The arrows in (F,H) indicate a netrinG2-single positive cell suggesting further differentiation of nurr1-mRNA expressing cortical neurons Colocalization of cux2 and nurr1 mRNAs in the claustrum and insular cortex of the monkey brain (A,D,G) Double ISH of cux2 (red) and nurr1 (green) mRNAs in the monkey brain section that spans the deep layers 5 (L5) and 6 (L6) of the insular cortex and the claustrum and merged (G) channels were shown for the same section to demonstrate the colocalization of cux2 and nurr1 mRNA signals (B,E,H) Dashed rectangles in (A,D,G) were magnified (C,F,I) Dashed rectangles in (B,E,H) were magnified (J,K) Double ISH of cux2 and nurr1 mRNAs in monkey TE both cux2 (red) and nurr1 (green) mRNA signals are shown as merged Note the absence of cux2 mRNA signals in the deep layers in this area In a previous study, the netrinG2 gene was reported to be expressed in the claustrum and layer 6 neurons of the insular cortex (Miyashita et al., 2005). As shown in Figures 4A–H we found by double ISH that the netrinG2 gene is co-expressed with the nurr1 gene in layer 6 of the insular cortex as well as in the claustrum we observed nurr1mRNA-positive cells in layer 6b that do not express netrinG2 mRNA The expression of netrinG2 and nurr1 mRNAs in the insular cortex generally coincided in limited subpopulation The coexpression of netrinG2 and nurr1 genes were observed in all the areas we examined including Gene expression profiling for insular and claustral neurons by netrin G2 (A,D,G) Double ISH of netrinG2 (red) and nurr1 (green) mRNAs in the monkey brain section that spans layer 6 (L6) of the insular cortex and the claustrum (Cla) and merged (G) channels were shown for the same section to demonstrate the colocalization of netrinG2 and nurr1 mRNA signals (B,E,H) Rectangles in (A,D,G) were magnified (C,F,I) Rectangles in (B,E,H) were magnified (J–L) Double ISH of latexin (red) and nurr1 (green) mRNAs in the monkey brain section that spans layer 6 (L6) of the insular cortex and the claustrum (Cla) The rectangles in the claustrum indicated by the arrows were magnified on the right side of each panel (M) A merged view for double ISH of nurr1 (red) and VGluT1 (green) mRNAs in the monkey brain section that spans the entire layers of the insular cortex (Ins) the claustrum (Cla) and the striatum (St) is shown and merged (P) channels were shown for the rectangle in the claustrum indicated by the white arrow All the nurr1+ neurons were VGluT1+ glutamatergic in this analysis while some VGluT1+ cells in the claustrum exhibited only low level expression of nurr1 mRNA (compare N–P) We conclude that claustral expression of latexin and netrinG2 genes is conserved across mice and monkeys whereas cortical expression patterns are modified in monkeys (A–D) FastBlue was injected into areas V4 and 7a and several claustral cells were retrogradely labeled (B,D) The same section processed for nurr1 ISH (A,C) exhibited nurr1 expression in all the FastBlue-labeled cells (see the insets) The rectangles within the claustrum are shown magnified in the insets for each panel (D) indicate the schematic plot for (C); the nurr1 single-positive cells were plotted in red and the nurr1/FastBlue double positive cells were plotted in blue (E,F) FluoroGold was injected into V1 and the claustral cells were retrogradely labeled The same section was processed for nurr1 ISH (G,H) The claustral regions of (A,B) were magnified but “standard” pyramidal neurons (i.e. with a discernible apical dendrites oriented toward the pia) Expression of nurr1 mRNA in the embryonic monkey and postnatal mice (A) Single ISH of nurr1 mRNA in the coronal section of the adult monkey brain hemisphere Note a dense expression of nurr1 mRNA in the claustrum and layer 6 of the insular cortex Note the scattered cell population in the white matter between the insular cortex and the claustrum (C,D) The rectangles in (B) were magnified (C) shows the shapes of the cell bodies at the bottom of the insular cortex most of which were typical pyramidal except those at the very bottom (right side of the panel) (D) shows the shapes of the cell bodies at the top of the claustrum which are elongated in horizontal direction (E) ISH of nurr1 mRNA in the coronal section of the embryonic E110 monkey brain hemisphere Note the abundant scattered cell population in the white matter between the insular cortex and the claustrum (G,H) The rectangles in (F) were magnified (G) shows the shapes of the cell bodies in the white matter many of which appear like inverted pyramidal cells (H) shows the cell bodies at the top of the claustram (I,J) ISH of nurr1 mRNA in the coronal section of the postnatal P0 and P8 mouse brain hemisphere Note similarity of the shapes of the cells to those in the monkey claustrum (G) For comparison, we investigated the distribution of nurr1 mRNA in P0 and P8 mice (Figures 6I–K). The distribution of nurr1 mRNA was almost identical to that in the adult brain at this stage. As in the embryonic monkey, the proximal dendrites were more evident in the postnatal mice (Figures 6I–K) which enabled us to determine the precise border between insular cortex and the claustrum Lucifer Yellow injection to the mouse and rat claustral neurons (A) Dark field image of the mouse slice that received Lucifer Yellow injection to the insular/claustral region The fluorescent image of the antibody-enhanced Lucifer Yellow-filled neurons is superimposed to indicate the location of clustered injection (B) Maximal projection stacks of confocal sections for magnified view of the insular/claustral region of (A) Note the meshwork of dendrites that cover the insular cortex and the claustrum (C) Another injection into the mouse slice (D) Reconstruction of the Lucifer Yellow injected neurons shown in (C) Different cells are shown by different colors for better representation of each cell (E) Merged view for latexin (red) and Lucifer Yellow (green)-stained slice of the rat brain The densely stained oval at the bottom of the insular cortex is considered as the claustrum (F) Maximal projection stacks of confocal sections for magnified view of the insular/claustral region of (E) Note that the insular/clautral border can be delineated by the dense fiber staining of latexin within the claustrum Latexin+ neurons were observed in a scattered manner outside the claustrum as well (G) Magnified view of the insular/claustral border of (F) Note the differential dendritic arborization of the claustral and insular cells The dotted line indicates the insular/claustral border identified on the basis of latexin fiber staining C-13 may have a dendrite toward the posterior side These examples suggest that lack of vertically orienting apical dendrites for at least some claustral cells may be because they are oriented along the longitudinal axis We conclude that while there does not appear to exist clear-cut border of dendritic fields for claustral and insular cells the dendrites of these cells are mostly contained within each territory Figure 8. Quantitative analysis of dendritic field extension for Lucifer-Yellow injected cells. (A) A typical example of the fluorescent image of the analyzed insular/claustral region. The axis of analysis (shown by the bar and the arrow at the bottom) was determined so that the pia is to the “Top.” This panel was taken from the same section shown in Figures 7E–G The red dots on each cell indicate the tips of the dendrites that determined the “Top,” “Bottom,” “Right,” and “Left” borders of the dendritic field of the cell of interest The distance from the center of the cell body to these borders were measured (dotted lines) to examine the polarity of dendritic extensions Because we wanted to measure the overlaps between the insular and claustral territory we measured the distance in these max projection images and not the 3D distance from the tips to the cell body (B) Rader plots for 28 claustral and 29 outer cells chosen from eight slices of two rats were superimposed The ends of each axis correspond to 300 μ m from the center The “claustral” cells were determined based on the counterstaining by latexin or parvalubumin The “outer” cells were those that were located outside the dense latexin-positive regions but within the regions containing scattered latexin+ cells (C) The averages and standard deviations for Left (L) and Bottom (B) values for claustral and outer cells are shown The difference of the “Top” values between the claustral and outer cells or the difference of the “Top” and “Bottom” values for the outer cells were statistically significant with the indicated p-value (D) Five representative examples each of the dendritic reconstruction for the claustral and outer cells by Imaris FilamentTracer All these neurons are oriented so that the top is to the pia Green dots indicate the position of the cell bodies The red arrowheads of the outer cells indicate the apical dendrites that were clearly different from other basal dendrites by morphology Most of the claustral cells lacked such apical dendrites candidates for apical dendrites which may be oriented toward the anterior or posterior directions (orthogonal to the slice plane) were present in some cells which were shown by black arrowheads with question marks Figure 9. Examples of atypically oriented pyramidal cells in the claustrum. (A) Dendritic reconstruction of a claustral cell (C-15) shown in Figure 8D The region indicated by the square was cut off for 3D reconstruction of the confocal stack images in (B,C) Note the orientation of the neuron: the top is to the pia matter of the insular cortex (B) A maximal projection stack of confocal images of the region shown by a rectangle in (A) was viewed from the top surface of the slice there does not appear to exist apical dendrites (C) The 3D reconstruction of the confocal images for C-15 was rotated so that the slice is viewed obliquely from the bottom side Note the positions of the red and blue circles The purple triangle represents the pipette position for Lucifer Yellow (LY) injection The candidate primary and secondary apical dendrites that are oriented toward the bottom of the slice were colored in red and blue The arrowheads and the question mark indicates the potential apical dendrites that may be severed Since we injected from the posterior side of the coronal slice this means that the candidate apical dendrites are oriented toward the anterior side of the mouse brain (D–F) Dendritic reconstruction of a claustral cell (C-13) The rotation of the 3D-reconstruction revealed an appendage that appears like an apical dendrite severed at the slice top surface (1) latexin mRNA was expressed only in the claustrum in macaques and (2) cux2 mRNA was expressed only in the insular cortex and the claustrum (in contrast with the widespread cortical expression of nurr1 and netrinG2 mRNAs) these genes would not be co-expressed simply by coincidence in the cortex the exact matching of distribution outside claustrum demonstrates the significant similarity of the claustral cells and a subtype of cortical deep layer neuron Summary of expression patterns for claustral enriched genes and we found by double ISH that LGR7 mRNA colocalizes with nurr1 mRNA in the mouse brain (data not shown) we observed VGluT1 mRNA-positive cells in the white matter that surrounds the monkey claustrum we found that netrinG2 mRNA is expressed in the upper subpopulation of nurr1-mRNA positive cells in all the areas examined including motor frontal and visual cortices (data not shown) we found that cux2 mRNA is not expressed in the deep layers outside the insular cortex in the macaque cortex latexin mRNA was not expressed even in the insular cortex where cux2 mRNA is co-expressed with nurr1 mRNA One possible interpretation would be that latexin and cux2 mRNA expression is downregulated as a result of cortical differentiation the nurr1+ neurons in the monkey brain may be ontogenetically independent from those of rodent cortex Further study is needed to clarify this point and to understand the functional significance of species difference observed here Tetsuya Sasaki in National Institute of Neuroscience for helping us with the Lucifer Yellow injection We thank Drs Yumiko Hatanaka and Yasuyoshi Arimatsu for providing latexin antibody Sachi Okabayashi of the Corporation for Production and Research of Laboratory Primates; Dr Yasuhiro Yasutomi of the Tsukuba Primate Research Center National Institute of Infectious Diseases for providing the embryonic monkey tissue Confocal images were acquired at Spectrography and Bioimaging Facility Supported by the grant from the JSPS to Akiya Watakabe (KAKENHI19500304 and 22500300) and Scientific Research on Innovative Areas (Neocortical Organization) (22123009 to Tetsuo Yamamori) Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Early regional specification for a molecular neuronal phenotype in the rat neocortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Molecular markers distinguishing supragranular and infragranular layers in the human prefrontal cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Chemically defined feedback connections from infragranular layers of sensory association cortices in the rat Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Development of the endopiriform nucleus and the claustrum in the rat brain Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text A serial section golgi analysis of the primate claustrum Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The rat claustrum: afferent and efferent connections with visual cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Cux1 and Cux2 regulate dendritic branching and synapses of the upper layer neurons of the cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Claustrum in the hedgehog (Erinaceus europaeus) brain: cytoarchitecture and connections with cortical and subcortical structures Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The claustrum: a historical review of its anatomy Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Fate-restricted neural progenitors in the mammalian cerebral cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The expression of vesicular glutamate transporters defines two classes of excitatory synapse Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Vglut2 afferents to the medial prefrontal and primary somatosensory cortices: a combined retrograde tracing in situ hybridization study [corrected] Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Retinol-binding protein gene is highly expressed in higher-order association areas of the primate neocortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Morphology of claustral neurons in the rat Pubmed Abstract | Pubmed Full Text Neurons of the claustrum in the cat; a Golgi study Pubmed Abstract | Pubmed Full Text Proteomic analysis illuminates a novel structural definition of the claustrum and insula Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Strong expression of NETRIN-G2 in the monkey claustrum Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Nishimura-Akiyoshi Axonal netrin-Gs transneuronally determine lamina-specific subdendritic segments Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Localization of LGR7 gene expression in adult mouse brain using LGR7 knock-out/LacZ knock-in mice: correlation with LGR7 mRNA distribution Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Topography of Gng2- and NetrinG2-expression suggests an insular origin of the human claustrum Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon traced by the expression of the genes Dlx-2 doi: 10.1002/1096-9861(20000828)424:3<409::AID-CNE3>3.0.CO;2-7 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Neuroepithelial origin of the insular and endopiriform parts of the claustrum Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The types of neurons in the claustrum of bison bonasus: nissl and golgi study Pubmed Abstract | Pubmed Full Text Electrophysiological and morphological features of rat claustral neurons: an intracellular staining study Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Functional specificity of claustrum connections in the rat: interhemispheric communication between specific parts of motor cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Cerebral hemisphere regulation of motivated behavior Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Types of neurons of the claustrum in the rabbit–Nissl Pubmed Abstract | Pubmed Full Text Comparative molecular neuroanatomy of mammalian neocortex: what can gene expression tell us about areas and layers Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Area-specific substratification of deep layer neurons in the rat cortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Comparative analysis of layer-specific genes in Mammalian neocortex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Rockland KS and Yamamori T (2014) Characterization of claustral neurons by comparative gene expression profiling and dye-injection analyses Received: 31 January 2014; Accepted: 07 May 2014; Published online: 23 May 2014 Copyright © 2014 Watakabe, Ohsawa, Ichinohe, Rockland and Yamamori. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Akiya Watakabe, Division of Brain Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585 Aichi, Japan e-mail:d2F0YWthYmVAbmliYi5hYy5qcA== UTokyo FOCUS Heat waves can reduce the body's immune response to flu according to new research in mice at the University of Tokyo The results have implications for how climate change may affect the future of vaccinations and nutrition Climate change is predicted to reduce crop yields and nutritional value as well as widen the ranges of disease-spreading insects the effects of heat waves on immunity to influenza had not been studied before University of Tokyo Associate Professor Takeshi Ichinohe and third-year doctoral student Miyu Moriyama investigated how high temperatures affect mice infected with influenza virus I think this is why no one else has studied how high temperatures affect flu," said Ichinohe The influenza virus survives better in dry so it usually infects more people in winter Ichinohe is interested in how the body responds after infection young adult female mice at either refrigerator-cold temperature (4 degrees Celsius or 39.2 degrees Fahrenheit) the immune systems of mice in hot rooms did not respond effectively Most affected by the high heat condition was a critical step between the immune system recognizing influenza virus and mounting a specific heat-exposed mice had no other significant changes to their immune system: They had normal reactions to flu vaccines injected under the skin which are increasingly becoming regarded as important for health remained normal in the mice living in hot rooms mice exposed to high temperature ate less and lost 10 percent of their body weight within 24 hours of moving to the hot rooms Their weight stabilized by day two and then mice were infected by breathing in live flu virus on their eighth day of exposure to heat Mice living in heat wave temperatures could mount a normal immune response if researchers provided supplemental nutrition before and after infection Researchers gave mice either glucose (sugar) or short-chain fatty acids chemicals naturally produced by intestinal bacteria researchers surgically connected mice so that body fluids moved freely between underfed and normally fed mice The fluids from normally fed mice prompted the immune systems of underfed mice to respond normally to the flu virus "Does the immune system not respond to influenza virus maybe because the heat changes gene expression Or maybe because the mice don't have enough nutrients We need to do more experiments to understand these details," said Moriyama The results may shed light on the unfortunate experience of getting sick again while recovering from another illness "People often lose their appetite when they feel sick If someone stops eating long enough to develop a nutritional deficit that may weaken the immune system and increase the likelihood of getting sick again," said Ichinohe University of Tokyo Associate Professor Takeshi Ichinohe (right) and third-year doctoral student Miyu Moriyama © 2019 The University of Tokyo CC-BY　 An important area of future study will be the effect of high temperature on different types of vaccinations Flu vaccines injected into the upper arm use inactivated virus but vaccines sprayed into the nose use live attenuated (weakened) virus "The route of delivery and the type of virus both may change how the immune system responds in high temperatures," said Moriyama Until more research can clarify what these findings may mean for humans Ichinohe and Moriyama cautiously recommend a proactive approach to public health "Perhaps vaccines and nutritional supplements could be given simultaneously to communities in food-insecure areas Clinical management of emerging infectious diseases may require nutritional supplements in addition to standard antiviral therapies," said Ichinohe The researchers are planning future projects to better understand the effects of temperature and nutrition on the immune system The strain of flu used in this research was A/PR8/34 influenza virus a type of H1N1 flu originally isolated in Puerto Rico in 1934 and now common in laboratory mouse studies Inquiries about the content of this page: Division for Strategic Public Relations Send inquiry Kashiwa Campus Hongo Campus Komaba Campus Access and Campus Maps Back Researchers at the University of Tokyo have found that heat waves may make people more susceptible to the flu A study in mice showed that their immune response to the influenza virus was weakened by exposure to extreme heat While it is known that rising temperatures will contribute to poor nutrition in regions where crop yields will suffer, this is the first time that experts have investigated how heat waves may impact flu immunity I think this is why no one else has studied how high temperatures affect flu,” explained study co-author Professor Takeshi Ichinohe young adult female mice to either refrigerator-cold temperatures around 39.2 degrees Fahrenheit or heat wave temperatures at around 96.8 degrees Fahrenheit The study revealed that the immune systems of mice in hot rooms did not respond effectively when they were infected with the flu The heat inhibited a critical step taken by the immune system when it recognizes the influenza virus and mounts a specific response the heat-exposed mice showed no other significant changes to their immune system They demonstrated normal reactions to flu vaccines and their gut bacteria remained normal “Does the immune system not respond to influenza virus maybe because the heat changes gene expression Or maybe because the mice don’t have enough nutrients We need to do more experiments to understand these details,” said study co-author and doctoral student Miyu Moriyama The study findings may help to explain why individuals often become sick while recovering from a different illness “People often lose their appetite when they feel sick that may weaken the immune system and increase the likelihood of getting sick again,” said Professor Ichinohe “Perhaps vaccines and nutritional supplements could be given simultaneously to communities in food-insecure areas may require nutritional supplements in addition to standard antiviral therapies.” The study is published in the Proceedings of the National Academy of Sciences By Chrissy Sexton, Earth.com Staff WriterPaid for by Earth.com There are times when you struggle with self-esteem and feel ashamed of how you look You seek love and support to build your confidence and you’re searching for something that reflects this journey that will fill the void that you didn’t get to have If you’re looking for a story that offers gentle encouragement and shows people helping each other build confidence It’s very fluffy and won’t give you headaches with heavy drama Unlike many stories with 20 chapters of misunderstandings and bad communication If you enjoy soft stories that make your heart feel warm without heavy drama then My Sweet Girl is definitely a perfect choice for you My Sweet Girl is a manga written and illustrated by Rumi Ichinohe. Originally serialized in Kodansha’s Bessatsu Friend magazine from 2015 to 2021, the series gathered generally positive reviews, with many praising the art style. Kodansha also licensed it in English and describes the plot as:“Love isn’t for me.” That is what Tsugumi Koeda a first-year high school student always told herself flat body has given Koeda a serious complex making it totally impossible for her to ever imagine falling in love whenever Tsugumi’s with the natural and frank Masamune feelings that she wants to be “a girl” like everybody else start to grow more and more… This is a pure and slow-step love story dedicated to all the girls who find things awkward when it comes to love I was looking through my plan-to-read list and remembered I had read the first two chapters of My Sweet Girl a few months ago I decided to pick it up again a few days ago and ended up binging the entire story I’ve read so much manga filled with miscommunication and dragged-out arcs that drive me crazy which I really appreciate.My Sweet Girl follows a typical shojo high school romance structure with the male lead being popular and the female lead being less so What stands out is how the story addresses the female lead’s body complex She believes no one will love her because she’s very skinny contrasting with the societal preference for a curvier body type initially starting with him teasing her but not bullying her and the two characters are together for most of the story I love seeing couples in a relationship and how they grow together Being in my early twenties and having a body type similar to that of the female lead It’s hard to find people who help build your confidence in real life Reading this story made me wish I had a friend like the male lead to help me grow but seeing stories that reflect what I wish had happened to me is comforting.The romance in My Sweet Girl is adorable The male lead encourages the female lead to see herself as more than just “bones,” and she helps him with his conflicts as well This mutual support and character development are rare in manga There are characters and moments of drama that might make you want to punch someone focusing on the cute moments between the leads While some parts of the drama don’t have a strong closure I didn’t mind because I was so invested in their cuteness the story showcases character development and mutual support and if I’m taking the time to write this recommendation it means the story genuinely touched me.My Sweet Girl – Should You Read This Manga?My Sweet Girl is a refreshing shojo high school romance that stands out for its heartwarming and genuine story The plot centers around a popular male lead and a less popular female lead who struggles with her body image Unlike many manga filled with miscommunication and dragged-out drama this story quickly develops the romance and focuses on the couple’s growth together Looking for more finished romance manga? Check out 6 Finished Silly Heartwarming Romance Manga That Will Leave You Melting.© Rumi Ichinohe / Kodansha Ltd Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser Japanese version Indonesian publisher M&C! announced last week that it has licensed Marina Umezawa's Ms. Seiyuu and Devil Producer (Seiyū-san to Do S na P-sama), and Rumi Ichinohe's My Sweet Girl (Kimi wa Kawaii Onna no Ko) manga The company will release the one volume of Ms Seiyuu and Devil Producer and the first volume of My Sweet Girl on March 21 M&C! describes the Ms Umezawa published the manga in Shogakukan's Sho-Comi magazine in 2016 Shogakukan published the manga's one compiled book volume in August 2016 M&C! previously published Umezawa's Eyes Behind the Glasses (Sonna Me de Minaide) A Mysterious Feeling Called Love (Hatsukoi Wazurai) and I Believe in You (Megane-chan no Akaiito) manga Ichinohe's My Sweet Girl manga centers around first-year high school student Tsumugi Koeda a girl who has a complex about being short and she has given up on any romantic ventures because of this runs into her while performing her school duties one day which is the first time anyone has said that to her Ichinohe launched the manga in Kodansha's Bessatsu Friend magazine in 2015 and Kodansha published the manga's sixth compiled book volume on January 12 previously published Ichinohe's Table for Two (Futari no Table) and Silent Kiss manga a prominent Japanese choreographer from New York City recently spent 10 days in Canton teaching Canton Ballet dancers a new work she created for them Titled “Whispering Wind,” the dance centers on a boy walking through the woods who feels a wind blowing The ballet’s female dancers portray the wind Male dancers dressed in black will carry the girls against a black backdrop “It’s a mood piece,” says Cassandra Crowley who has known Ichinohe for more than 20 years “It’s been fascinating to watch her transform our little American girls into little Japanese girls,” Crowley says “She’s been working with every part of their movement.” “Whispering Wind” will be part of Canton Ballet’s spring season April 3 and 4 at the Palace Theatre The Japanese-themed program is a tie-in with the Canton Museum of Art’s “Kimono As Art” exhibition The January issue of Kodansha's Bessatsu Friend magazine announced on Friday that Rumi Ichinohe's My Sweet Girl (Kimi wa Kawaii Onna no Ko) manga will end in two chapters The 12th compiled volume of the manga announced on November 13 that the manga will end in its 13th volume in April 2021 Ichinohe launched the manga in Kodansha's Bessatsu Friend magazine in 2015 Kodansha Comics published the 11th compiled volume in English on November 3 Japanese publisher Kodansha announced the 15 nominees in three categories for its 48th annual Manga Awards on Monday Kodansha will announce the winner for each category on May 14 Kodansha gave out a "Best Children's Manga" award as well but starting in 2015 Kodansha has integrated the nominees for that category into the Best Shōnen Manga and Best Shōjo Manga categories instead Source: Kodansha Japanese publisher Kodansha announced the winners on Tuesday for its 48th annual Manga Awards Other nominees included The Fragrant Flower Blooms With Dignity, Gachiakuta, Tank Chair, and Daemons of the Shadow Realm Other nominees inluded In the Clear Moonlit Dusk, Firefly Wedding, and A Sign of Affection Other nominees included Oshi no Ko, The Darwin Incident, Chiikawa, Nagatan to Ao to: Ichika no Ryōrichō, and Bōkyō Tarō Source: Kodansha Researchers develop electrogenerated chemiluminescence cells that possess unprecedented levels of luminance and current efficiency Electrogenerated chemiluminescence (ECL) cells are inherently self-emissive and hold promise for next generation displays due to their simplicity of design and production they trail behind light-emitting diodes (LEDs) and organic LEDs in luminance researchers have engineered yellow phosphorescent ECL cells with an iridium complex and a redox mediator achieving the highest reported luminance and current efficiency for iridium complex-based cells This advancement holds great promise for the future development of high-performance ECL-based displays Electrogenerated chemiluminescence (ECL) cells characterized by their self-emissive nature have gathered significant interest for prospective display applications due to their uncomplicated structure and straightforward fabrication process These cells are created by sandwiching a solution-based emitting layer between two transparent electrodes when compared to other self-emissive devices like light-emitting diodes (LED) and organic LEDs the luminescent performance of ECL cells remains subpar and is currently undergoing improvement iridium complexes are widely used in efficient organic LEDs due to their ability to produce room-temperature phosphorescent emissions from their excited states The emission colors can be easily adjusted by changing ligands and researchers have explored their properties using both theoretical and experimental methods In a recent study led by Associate Professor Takashi Kasahara and Nanami Ichinohe, a master’s student at Hosei University, and including collaborators from University of Fukui and National Cheng Kung University, researchers have successfully designed a highly luminescent ECL cell by utilizing an iridium complex and a mediator. This study has been published in the journal Electrochemistry on 15 February 2024 “ECL is a light-emitting phenomenon induced by an electron transfer reaction between the radical anion and cation of the luminescent material Although conventional ECL solutions have been typically prepared by dissolving specific amounts of a single luminescent material in an organic solvent in this study we have prepared a solution that contained two luminescent materials.” “We expect that this ECL system using the redox mediator will be actively investigated by many research groups across the world and contribute to future (in probably several decades from now) highly luminescent and efficient solution-based self-emissive display applications,” envisions Dr researchers have successfully engineered a yellow phosphorescent ECL cell incorporating an iridium complex and a redox mediator This cell exhibits the highest luminance ever reported for iridium complex-based cells and is expected to pave the way for the development of next-generation ECL-based displays Iridium complex-mediator duo elevates self-emission Title of original paper: Yellow Phosphorescent Electrogenerated Chemiluminescence Cell Based on a Cyclometalated Iridium Complex with a Redox Mediator Year/Volume/pages: 2024 Volume 92 Issue 2 Pages 027004 DOI: 10.5796/electrochemistry.23-00147 Takashi Kasahara is an Associate Professor in the Department of Electrical and Electronic Engineering Faculty of Science and Engineering at Hosei University he was associated with the Seiko Epson Corporation With over 50 research papers (29 Articles and 21 Conference papers) and 529 citations Kasahara's research centers around nanotechnology he has authored several books on organic electronic materials CAMDEN — Ten Japanese exchange students and two chaperones arrived in Camden but only after their flight from New York to Boston was canceled they boarded a bus from Boston to Portland and were picked up by an MSAD 28 school bus at 1:30 in the morning for the final leg of the trip to Camden marks the 14th year Camden and Hirakawa have exchanged students The students and the two chaperones will live with host families during their stay this week Camden-Rockport Middle School students are encouraged to discover just how similar it is to be a teenager whether you’re from a seaside town in Maine or the seaside Aomori Prefecture in Japan The CRMS band played both the national anthems from the United States and Japan Comments were made by CRMS Principal Thom Ingraham welcoming the exchange students and from Ikuko Ichinohe from Japan Piehl studied at the University of Minnesota majoring in Japanese history and minoring in Japanese language He currently is an assistant English teacher at seven schools in Aomori but the kids have had a lot of time to sleep They’ve just been really excited to finally get here and meet everybody I think we’re going to have a blast with everyone There are going to be a lot of activities.” Contact Chris Wolf at news@penbaypilot.com Thanks to our readers and especially our supporters who help to keep PenBayPilot.com an open and accessible community hub Your support is even more critical during rapidly changing times While we work hard to keep you informed about the Midcoast community We are grateful to those who already participate Join for as little as $2.99 per month and support local journalism on a community hub that serves everyone X