Student Research A farmer in Hawaii grew an avocado weighing as much as a newborn baby.KONA A farmer in Hawaii grew an avocado weighing as much as a newborn baby and his family members have been growing giant avocados in Hawaii for almost 80 years MORE: Avozilla, the 4-pound avocado as big as your face, can make 18 pieces of avocado toast The farmer said his family farm's first avocado tree was grafted by his oldest brother in 1941 and it continues to produce delicious and unusually large avocados to this day a neighbor and frequent consumer of Fukumitsu's avocados believes that Fukumitsu may have grown the world's largest avocado She said the avocado she recently received from Fukumitsu tipped the scales at over 6 pounds The Guinness Book of World Records says the record holder for the heaviest avocado is currently by a fellow Hawaiian resident Felicidad Pasalo with an avocado that weighed 5 pounds and 8 ounces McElroy said when she reached out to the Guinness Book of World Records representatives told her it would take about twelve weeks for the organization to authenticate the avocado Just off Broad Street in Falls Church, hordes of the verminous Skaven and humans of The Empire fight tooth and nail over a damned city On other nights, newcomers can be found learning Magic: The Gathering tricks from wizened masters. Since the 1980s, local adventurers have rallied to go on quests in Dungeons and Dragons and more obscure tabletop games After this weekend, the battles and revelry inside The Compleat Strategist (103 East Broad Street) will go silent for good, as the tabletop gaming store shuts down to make way for a Whole Foods-anchored mixed-use development The store is closing for good on Saturday (Jan but there are still treasures buried among the codexes and outdated rulebooks for those who know what to look for The Compleat Strategist manager Adam Fukumitsu says the store has gotten many well wishes since the closure was announced late last month A lot of patrons have asked how has business been expecting the store must have been seeing difficulties but Fukumitsu said that isn’t the case “Business has been rocking for two years,” Fukumitsu said According to Fukumitsu, after a month or two of quarantine, tabletop gaming saw a surge as locals looked for new activities to keep them sane through lockdown. Board games saw a boost in popularity, and online gaming sites like Roll20 boosted the sale of physical books for players D&D came back like a rocket starting in 2015,” Fukumitsu said The store opened as Strategy and Fantasy World in 1977 and was bought by the New York-based, family-owned The Compleat Strategist in the 1980s Fukumitsu has worked at the store since 2013 He says the store being pushed out by redevelopment wasn’t exactly a surprise “It was a train we’ve seen coming for a decade now,” Fukumitsu said but there’ve been weird delays over the years…In 2019 we heard it was going forward and there was a lot of weird push and pull.” Fukumitsu said at one point the property was eyed for development by Todd Hitt before the real estate scion was arrested and found guilty of being involved in a real estate Ponzi scheme The Compleat Strategist is commemorating its impending closure with tabletop battles As more retail moves to digital storefronts Fukumitsu says the sense of community that gamers can find at brick-and-mortar stores will be difficult to replace “The community has been figuring out where they go now,” Fukumitsu said he says local gaming groups have plans to go around to peoples’ homes and one player has talked about getting access to a company-owned warehouse to play “We’re getting hit at both ends by Amazon,” Fukumitsu joked. “They’re both eating our lunch in sales and now kicking us off the property…but less online is that play space It’ll be interesting to see what happens.” Marc Forbes started visiting the store as a gaming enthusiast before he became an employee in 2016 “My entire social life was tied up in this place,” Forbes said “We’re going to try to keep that going after it closes but it’s going to be harder… I’m really going to miss this place.” Joon has expanded its offerings in Tysons with the addition of a bar and patio Named after a poetic Farsi term for “wine,” May Bar began serving customers inside the… Owners of six Woofie’s franchises in Northern Virginia are hosting a “Paws in the Park” pet adoption event tomorrow (Saturday) at Wolf Trap National Park Participating Woofie’s include Reston/Herndon A Fairfax Connector bus to Tysons (staff photo by James Jarvis) The Fairfax County Department of Transportation (FCDOT) has the green light to apply for a federal grant to replace… The country band Delta Spur performs on the Plaza at Tysons Corner Center for the mall’s 2023 summer concert series (courtesy Tysons Corner Center) Fresh produce Manga creator Shigeyuki Fukumitsu made his debut in 1997 with his series Musume Aji in Seirin Kogeisha's Garo magazine He carved a niche for himself by telling stories about down-on-their-luck protagonists who continue to fight the odds towards something better His semi-autobiographical works include Boku no Shōkibo na Seikatsu (My Small Life) Boku no Shōkibo na Shippai (My Small Mistake) and Tsuma ni Koisuru 66 no Hōhō (66 Ways to Fall in Love with Your Wife) let readers into his life and a view of his personal relationships The first volume of Fukumitsu's Tsuma to Boku no Shōukibona Ikuji (My Wife and My Small Childcare) shows Fukumitsu's foray into fatherhood Fukumitsu and his wife decided to become parents after his manga artist career took off and was able to support his family they are saddened to learn that he has hearing loss in both ears the manga shows the family of four and their life together Fukumitsu's manga is ongoing and currently serialized in Kodansha's Young Manga Third and on the Comic Days website Source: Comic Natalie Millennial brunch aficionados may need to add a new location to the top of their bucket lists: Hōlualoa in Hawaii This is where a farmer is growing what are thought to be the world's biggest avocados which are both the size and weight of a newborn baby Kenji Fukumitsu and his family have been harvesting the avocados for around 80 years, and they all come from the same tree, according to Big Island Video News The tree was first grafted by Fukumitsu's older brother in 1941 and it continues to bear fruit to this day Read more: You can now buy avocados the size of your head in Australia for $12 each The ginormous fruits tend to weigh over 6lbs each which likely makes them the biggest in the world Kind-hearted Fukumitsu gives away many of the mammoth avocados to the nearby Urgent Care of Kona medical centre "Every month or so we would hear a big clunk and there's a bag or a box of at least 20 or 30 avocados," Dr Joy McElroy of the centre said And it occurred to us that this is not normal It was Dr McElroy who decided to look up the world record for avocado size and found that Fukumitsu's were bigger than the world record holder's Read more: A woman discovered a 5-pound avocado that's bigger than her head — and it could break a world record The current record is held by fellow Hawaiian Felicidad Pasalo, who set the record on January 3 2018 with an avocado that weighed 5lbs 8oz Dr McElroy contacted the Guinness Book of World Records but was told it would take the organisation 12 weeks to confirm whether Fukumitsu's were record-breaking the avocado in question is unlikely to last 12 weeks.  Fukumitsu is rather relaxed about the whole thing Fukumitsu is as stumped as the next person: "That This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page The last seven weeks of the semester are brimming with cultural celebrations the Beacon has outlined five of these annual events for you to add to your calendar From the annual lūʻau to first-of-their kind events take advantage of these opportunities to celebrate the student body and enjoy the hard work club leaders have put into hosting these events for our community The Hawaiʻi Club Annual Lūʻau is one of UP’s most famous cultural events on campus the theme is “'A'ohe hana nui ke alu 'ia me ke aloha,” which translates to no task is too big when done by all with aloha “We hope to really convey the message that when we come together as a community and just have a lot of mutual respect for one another that we can accomplish anything,” Michelle Fukumitsu there will be a photo booth and a country store with Hawaiian food and goods for sale Attendees will be served a traditional Hawaiian meal including poi Following the meal, the show will include a variety of dances led by Hawaiʻi Club students, UP faculty, the Vietnamese Student Association, Portland State University’s Pacific Islanders Club and other culture clubs Admission is $14 for UP affiliates. Tickets can be purchased in advance at this link South Asian Student Union Cultural Night: A Night in India South Asian Student Union (SASU) is bringing elements of their culture to campus with a first-ever annual culture night on March 30 they’ve narrowed their event this year to focus on Indian culture calling their first event A Night in India There will be a fashion show and short dance performance by SASU members to open the event showcasing South Asian traditional dress they’ve brought from home Following will be an assortment of interactive features that attendees can explore for the evening including an art gallery a mehendi — commonly known as Henna — station and a catered Indian dinner “The goal is showing a vibrant culture: vibrant colors “We just want to put that out there and help people to see it for themselves.” Admission is $7 and tickets can be purchased in-person only. Keep up with SASU’s instagram page for ticket sale times and locations Filipino American Student Association Tahanan: Pilipino Cultural Night  Filipino American Student Association’s (FASA) annual Pilipino Cultural Night (PCN) will take place on April 14 in the Quiet Side of Commons the theme is “Tahanan,” which means house or home honoring the community they’ve built within their club “We really found a home in FASA and a lot of people are not from here or don’t come from a community where they can express or celebrate their Filipino culture,” Jaedina Bayking they have a home where they can really do that.” Traditional and other popular Filipino dances will be part of the main performance. Other cultural clubs are also invited to be a part of the pre-show. Attendees will also be able to enjoy a traditional meal served with halo-halo For information on ticket sales, check FASA’s instagram page closer to the event date Vietnamese Student Association Culture Show: Hello Việt Nam 2023   the Vietnamese Student Association’s (VSA) seventh annual Culture Show is being held in the Chiles Center for the first time The theme this year is “Ý Nghĩa Của Tôi” which translates to my meaning The message emphasizes the idea that each individual has their own interpretation of what it means to be Vietnamese Attendees can expect a traditional Vietnamese meal and a student-led show that will center on Vietnamese culture one born in Vietnam and one born in America who explore their identities as Vietnamese “The whole purpose of the script is to convey that whether you were born in Vietnam Dances will also be part of the show, including a traditional Vietnamese Lion Dance Admission is $10 for UP affiliates. Tickets can be purchased in advance at this link Unity Ball — hosted by Latinx Student Union After a successful first Unity Ball last year Latinx Student Union (LSU) is preparing another cultural event unlike any other The second annual Unity Ball will take place on April 22 and the show is scheduled to begin around 7 p.m Unity Ball is more than a cultural show highlighting LSU It strives to showcase every culture on campus in an inclusive way By inviting other clubs to participate in their show Unity Ball is one of the most collaborative events on campus Last year’s event included more than ten cultural clubs on campus LSU is concentrating on Peruvian culture since their club represents a variety of countries This means Peruvian food and dance will be spotlighted by LSU However Peru won’t be the only country represented at the ball dances and dishes from a variety of cultures will be included Since Unity Ball is such a collaborative event LSU is also welcoming community members to volunteer for the day of the event to help bring it all together For information on ticket sales or volunteering, check LSU's instagram page closer to the event date Chiara Profenna is the DEI editor for The Beacon. She can be reached at profenna23@up.edu Guide: Where to have a “cafecito con chisme” in Portland Society for Mexican American Philosophy set to host first biannual Summer Institute at UP Letter from the Editor: The future of DEI at The Beacon remains bright The Student Voice Of The University of Portland Since 1935 The Student Voice of the University of Portland Since 1935 City and County of Honolulu Copyright © 2025 City and County of Honolulu Hawaii club is hosting their 44th annual Lū'au on Saturday the first Lū'au since 2019 due to COVID-19 Attendees can expect traditional Lū'au food hula performances and a fire dancer along with a shaved ice stand and local Hawaiian goodies Attendees will be required to show their UP IDs or proof of vaccination or a negative COVID-19 test to attend the Lū'au an abundance of attendees are made up of friends and family coming from Hawaii but due to the Lū'au being hosted at a later date ticket sales are low in comparison to previous years “Usually we will try to coordinate the date of the Lū'au to be when spring break is in Hawaii so that people would be able to make travel arrangements if they wanted to come up,” Chan said Chiles was not available during that time.” Despite not meeting the anticipated ticket sales Michelle Fukumitsu and Kacie Moku are using this as an opportunity to stay cautious of COVID-19 Masks will be required when not eating or drinking and tables will be taken out to allow for space The number of seats allowed at each table will also be decreased this year’s Lū'au will have new acts not seen in previous years our pre show consists of a lot more acts from different clubs and different talents around the school,” Moku said “It's normally shorter but we have many performers from Guam club and FASA We have a skit being done with a student fire dancer as well as two singers that are going to be performing.” With this being Hawaii club’s 44th Lū'au the club is excited to continue spreading their aloha spirit onto the University campus student volunteers and the parent island coordinators in Hawaii began planning over the summer to ensure everything runs smoothly in time for spring flowers and decorations are shipped from Hawaii to Portland to help the Lū'au capture the traditional elements Those interested in attending the Lū'au can purchase tickets through the ticket website up until April 2 “Lū’au and Hawaii Club are so meaningful because they provide a chance to bring a little bit of home with us to Portland as well as a chance to connect with others and help celebrate and appreciate our home and the community and aloha it all brings,” Chan Moku and Fukumitsu said in an email to The Beacon Janea Melido is a reporter for The Beacon. She can be reached at melido24@up.edu Big Island Video News video courtesy Hawaii News Now / Lynn Beittel of Visionary Video Hawaiʻi - A Kona farmer happily shares his avocados with the neighborhood Turns out they are the biggest avocados on Earth (BIVN) – Hōlualoa farmer Kenji Fukumitsu has been bringing giant avocados to Urgent Care of Kona for years “Just out of kindness,” according to Dr so I share them with some of our friends. If you eat it during November month “Every month or so we would hear a big clunk and there’s a bag or a box of at least 20 or 30 avocados,” McElroy recounted McElroy said the large avocados are usually shared in the office Fukumitsu is kind enough to just give them to us.” This year, Urgent Care of Kona staff decided to take a look at the record books. According to the Guinness World Records, the biggest was over 4 pounds, registered in Venezuela. However, the Guinness website listed the record holder for heaviest avocado as Hilo resident Felicidad Pasalo 2018 with an avocado that weighed 5 lbs 8 oz Pamela Wang of Kealakekua weighed a previous record- avocado at 5 lbs 3.6 oz “Happy to be out-Guinnessed by this worthy opponent,” Wang commented on Facebook “This baby is 6 pounds!” McElroy said The problem is they have to have someone to authentic [the record] this baby isn’t going to last 12 weeks “We didn’t think nothing of it,” Fukumitsu said Fukumitsu’s giant avocado-producing tree was grafted by his oldest brother I don’t know,” Fukumitsu answered Filed Under: Kailua-Kona Tagged With: avocado, Holualoa, Joy McElroy, Kenji Fukumitsu Copyright © 2025 · Dynamik-Gen on Genesis Framework · WordPress · Log in Volume 11 - 2017 | https://doi.org/10.3389/fncel.2017.00133 This article is part of the Research TopicThe Cerebellum Inside Out: Cells, Circuits and FunctionsView all 20 articles Thyroid hormone 3,3′,5-Triiodo-L-thyronine (T3) is essential for proper brain development Perinatal loss of T3 causes severe growth defects in neurons and glia including strong inhibition of dendrite formation in Purkinje cells in the cerebellar cortex Here we show that T3 promotes dendritic outgrowth of Purkinje cells through induction of peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α) a master regulator of mitochondrial biogenesis PGC-1α expression in Purkinje cells is upregulated during dendritic outgrowth in normal mice while it is significantly retarded in hypothyroid mice or in cultures depleted of T3 PGC-1α knockdown or molecular perturbation of PGC-1α signaling inhibits enhanced dendritic outgrowth and mitochondrial generation and activation caused by T3 treatment PGC-1α overexpression promotes dendrite extension even in the absence of T3 PGC-1α knockdown also downregulates dendrite formation in Purkinje cells in vivo Our findings suggest that the growth-promoting activity of T3 is partly mediated by PGC-1α signaling in developing Purkinje cells the downstream target of T3/TR mediating dendritic development of Purkinje cells remains to be elucidated In order to increase mitochondrial mass to fill the expanding dendritic volume not only fission/fusion dynamics but mitochondrial biogenesis must be upregulated during dendrite formation we investigated the mechanistic link between the T3-induced mitochondrial biogenesis and dendritic development in Purkinje cells We demonstrate that T3 enhances mitochondrial biogenesis and dendritic growth through induction of PGC-1α in Purkinje cells Commercial sources for reagents used for supplemental experiments were as follows: 3,3′,5-Triiodo-L-thyronine (T3; Sigma-Aldrich) 2-Mercapto-1-methylimidazole (MMI; Sigma-Aldrich) Sodium perchlorate monohydrate (PM; Sigma-Aldrich) Pregnant ICR mice and pups of either sex (Nihon-SLC) were used in this study. Mother and pups were housed individually ad libitum under a 12:12 h light-dark cycle at 23°C. The induction of developmental hypothyroidism was performed as previously described (Sawant et al., 2015) Pregnant and nursing mother mice were treated with 0.08% MMI 1.0% PM and 5.0% sucrose in drinking water from day 18 of gestation until postnatal day 14 (P14) A control group was treated with 5.0% sucrose in water All experiments were handled in agreement with guidelines of the Animal Experiment Committee of Kyoto University The full length RIP140 cDNA (GenBank: NM_173440.2) was amplified from a mouse brain cDNA library and fused with FLAG-tag to create pAAV-FLAG-RIP140 Primary cultures of cerebellar neurons were performed as described previously with slight modification (Fujishima et al., 2012) P0 mouse cerebella were dissected and dissociated in DMEM/F12 supplemented with 10% fetal bovine serum and plated on glass-based culture dishes coated with poly-D-lysine cell cultures were washed with DMEM/F12 to remove FBS and media were replaced by serum-free maintenance medium (DMEM/F12 containing 1% penicillin–streptomycin Ten nanomolar of T3 was added at 0 and 5 days in vitro (DIV) Plasmids were transfected to dissociated cells by using Amaxa Mouse Neuron Nucleofector Kit Mito-EGFP was introduced to Purkinje cells by infecting adeno-associated viruses (AAV) at 0 DIV AAV (109–1010 plaque-forming units) was purified by using AVB Sepharose High Performance In utero electroporation for Purkinje cells was performed as described previously (Nishiyama et al., 2012; Fukumitsu et al., 2015) pregnant mice on day 11.5 of gestation were anesthetized by an intra-abdominal injection of somnopentyl (Kyoritsu) The plasmid DNA solution (5 μg/μl) purified using the Qiagen Plasmid Maxi Kit was injected by using an aspirator tube (Drummond) into the fourth ventricle of embryos The positive electrode (CUY650P3; Nepa Gene) was placed on the anterior end of the fourth ventricle through the uterus 970 ms) were delivered with a square-wave electroporation generator (CUY21; Nepa Gene) Detailed protocols for immunofluorescence were described in a previous report (Kaneko et al., 2011) Antibodies used for immunofluorescence were as follows: mouse anti-Calbindin D28K (Swant): rabbit anti-Calbindin D28K (Millipore): goat anti-Calbindin (Santa Cruz): mouse anti-Pyruvate dehydrogenase (PDH; Abcam): rabbit anti- PGC-1α (Abcam): rabbit anti-DsRed (Clontech): mouse anti-Pax-6 (R&D): rabbit anti-cytochrome C oxidase IV (COX-IV; Abcam); and Alexa 488- Immunofluorescence images were taken with a confocal microscope (FV1000; Olympus) with a 40× 60× or 100× objective (NA 0.95 dendrites were traced with Neurolucida software (MBF Bioscience) The dendritic area was quantified with ImageJ For quantification of dendrite mitochondrial content (ratio of mitochondrial area to dendrite area) and area occupied by Mito-EGFP signal was divided by the entire dendritic volume by using ImageJ PDH signal was calculated as the sum of PDH absolute value in cells labeled with volume markers PGC-1α signal in the cell soma was averaged in each cell The length and number of dendritic protrusions were measured in the dendritic segment of 10 μm in length from the distal tips using ImageJ software Data were analyzed using Student’s t test for single comparisons and by one-way or two-way analysis of variance (ANOVA) with Tukey’s HSD post hoc or Tukey-Kramer HSD analysis for multiple comparisons 3,3′,5-Triiodo-L-thyronine (T3) enhances mitochondrial biogenesis and dendritic outgrowth in cerebellar Purkinje cells Primary cerebellar cell cultures prepared from P0 mice were incubated for 10 days in the absence or presence of different concentrations of T3 The morphology of Purkinje cells was visualized by immunostaining with anti-Calbindin (A,B) Dose-dependent effects of T3 on total length (A) and branch number (B) of Purkinje cell dendrites at 10 DIV (C) Purkinje cells were labeled with adeno-associated viruses (AAV)-Mito-EGFP to visualize mitochondria and cultured with or without 10 nM T3 Cells were costained with anti-Calbindin and anti-pyruvate dehydrogenase (PDH) antibodies (D,E) Quantification of mitochondrial content (D) and PDH expression (E) in Purkinje cells with or without T3 treatment Signal intensity was normalized to the value of cells in the T3-deficient (T3-) condition Data represent mean ± SEM; ***p < 0.001 T3-induced dendritic outgrowth is associated with increased generation and activity of mitochondria a close family member which may function redundantly with PGC-1α was not detected in Purkinje cells at the stages examined (data not shown) PGC-1α expression in the developing cerebellar cortex (A) Shape changes of Purkinje cell dendrites during postnatal development (B,C) Sagittal cerebellar sections were immunostained for PGC-1α and Calbindin at different ages of development and were observed at low (B) and high (C) magnification PGC-1α was predominantly detected in the Purkinje cells from P7 These results suggest that T3 induces the onset of PGC-1α expression in Purkinje cells around P8 while other factors may regulate PGC-1α expression in later stages of cerebellar development PGC-1α expression in Purkinje cells is downregulated in hypothyroid mice (A–C) PGC-1α expression was compared at P7 (A) Representative images from four mice in each condition are shown Dendritic growth of Purkinje cells is retarded in the hypothyroid condition the EGL is thicker in hypothyroid animals compared to control animals Each section was immunostained with anti-Calbindin and anti-PGC-1α DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (D) Quantitative comparison of PGC-1α expression in Purkinje cells in control (euthyroid) and hypothyroid animals N = 15 cells from four mice from two independent experiments for each points two-way analysis of variances (ANOVA) with Tukey’s HSD post hoc analysis PGC-1α expression is induced in cultured Purkinje cells by T3 treatment (A) Cerebellar cells were cultured in the presence of 10 nM T3 and then immunostained with anti-Calbindin and PGC-1α at the indicated day in culture Boxed regions in the upper panels are enlarged in lower panels PGC-1α expression is gradually increased in Purkinje cells (arrows) from 6 DIV (B) Quantitative comparison of PGC-1α expression in Purkinje cells cultured with (black dots and line) or without (gray dots and line) T3 two-way ANOVA with Tukey’s HSD post hoc analysis (C) Dissociated cerebellar cells were cultured with or without T3 treatment and immunostained for Calbindin (D) Quantitative comparison of PGC-1α expression in Purkinje cells and granule cells cultured with or without T3 (E,F) Immunofluorescent images (E) and quantitative comparison (F) of PGC-1α expression in Purkinje cells treated with or without T3 for 24 h (D,F) The average pixel intensities of PGC-1α signals in the cell soma were measured To further confirm that PGC-1α expression is induced by T3, we analyzed if PGC-1α expression is upregulated in Purkinje cells immediately after exposure to T3. To this end, we cultured cerebellar cells in the absence of T3 and treated them with T3 at 9 DIV. We detected significant increase in PGC-1α expression in Purkinje cells at 24 h after T3 treatment (Figures 4E,F) these results strongly suggest that T3 regulates the onset of PGC-1α expression in developing Purkinje cells PGC-1α knockdown inhibits T3-induced dendritic growth in Purkinje cells (A) HEK293T cells expressing mouse PGC-1α were transfected with PGC-1α-scramble (scr) or short hairpin RNA (shRNA) construct and analyzed by western blotting with anti-PGC-1α and anti-β-actin antibodies (B,C) Cultured Purkinje cells were transfected with PGC-1α-shRNA or scr-shRNA (control) constructs and stained for Calbindin and PGC-1α (B) or Calbindin and cytochrome C oxidase IV (COX-IV) (C) at 10 DIV (D) Representative images of Purkinje cells transfected with scr shRNA (control) PGC-1α shRNA (PGC-1α shRNA) or PGC-1α shRNA plus an shRNA-resistant mutant of PGC-1α (shRNA + rescue) Cells were cultured in the presence of 10 nM T3 until 10 DIV and immunostained with anti-Calbindin and anti-PDH antibodies (E–G) Quantitative analyses of the total dendritic length (E) number of dendritic branches (F) and PDH signal (G) one-way ANOVA with Tukey’s HSD post hoc analysis (H) Quantification of the effects of PGC-1α knockdown on Purkinje cells morphology in the presence or absence of T3 Molecular perturbation of PGC-1α inhibits dendritic outgrowth and mitochondrial activity in Purkinje cells (A) Representative images of Purkinje cells overexpressing EGFP (control) Cells were stained for Calbindin at 10 DIV (B–D) Quantitative analyses of the total dendritic length (B) number of dendritic branches (C) and PDH signal (D) 30 cells for NRF1DN and 30 cells for RIP140 one-way ANOVA followed by Tukey Kramer HSD tests Knockdown of PGC-1α inhibits dendritic outgrowth in vivo Purkinje cells (A) Representative images of Purkinje cells transfected with scr shRNA (control) or PGC-1α shRNA construct (B) Dual color images of GFP derived from shRNA constructs (green) and immunostaining with anti-PGC-1α (magenta) (C,D) Quantitative analyses of the total dendritic length (C) and number of dendritic branches (D) in Purkinje cells expressing scr shRNA (control) or PGC-1α shRNA constructs (E) Representative images of distal dendrites of Purkinje cells expressing scr shRNA (control) and PGC-1α shRNA (F,G) The length (F) and number (G) of dendritic protrusions in the Purkinje cells expressing scr shRNA (control) or PGC-1α shRNA (shRNA) Twelve cells from three mice were analyzed for each group These results together support that the cell-autonomous function of PGC-1α is required for the later steps of dendritic development in Purkinje cells in response to T3 These results substantiate the view that T3 promotes dendritic outgrowth in Purkinje cells at least in part by inducing PGC-1α signal PGC-1α overexpression enhances dendritic outgrowth of Purkinje cells in the absence of T3 (A,B) The morphology of Purkinje cells transfected with tdTomato (control) or PGC-1α-mCherry (+PGC-1α) Cells were cultured with (B) or without (A) T3 and stained for Calbindin at 10 DIV PGC-1α overexpression induced dendritic outgrowth in the absence of T3 (A) (C–E) Quantitative analyses of total dendritic length (C) branch numbers (D) and PDH signal (E) Purkinje cells expressing tdTomato (control) or PGC-1α were cultured with or without T3 treatment ***p < 0.001 and *p < 0.05 Mitochondria produce the majority of cellular ATP via aerobic metabolism The large expansion of growing dendrites in differentiating neurons should be accompanied by a rapid increase in mitochondrial mass and activity to handle their rising demand for energy we demonstrate that T3 enables intensive outgrowth of Purkinje cell dendrites partly by upregulating PGC-1α expression We provide several lines of evidence that PGC-1α is a downstream target of T3 and enhances mitochondrial biogenesis and activity in the Purkinje cells treated with T3 PGC-1α expression begins around P7 when cells undergo the latter step of remodeling from stellate shaped cells to young Purkinje cells knockdown of PGC-1α does not affect Purkinje cell migration nor the first remodeling but strongly inhibits dendrite extension and spine maturation after P7 It is therefore probable that RORα and PGC-1α mediate differential steps of dendrite formation and maturation downstream of T3 it is possible that T3 indirectly induces PGC-1α expression by enhancing neurotrophin production Though we do not garner evidence for it here these activity-dependent pathways may regulate PGC-1α expression during spine formation in later stages whereas T3 may regulate spinogenesis independently of PGC-1α RORα and PGC1α thus likely form intricate cross-regulatory loop rather than simple signaling cascade The mechanism by which PGC-1α enhances dendrite outgrowth remains to be fully elucidated KFj and MK conceived and designed the experiments All authors read and approved the final manuscript This work was supported by the KAKENHI (#26290005) of the Japan Society for the Promotion of Science (JSPS) and the Naito Foundation to MK 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 Galbraith for technical assistance and the members of Kengaku lab and iCeMS for helpful discussions Carlton for critical reading of the manuscript Early dendritic development of Purkinje cells in the rat cerebellum A light and electron microscopic study using axonal tracing in “in vitro” slices Thyroid hormone receptors in brain development and function Induction of early Purkinje cell dendritic differentiation by thyroid hormone requires RORα Retinoid-related orphan receptor α controls the early steps of purkinje cell dendritic differentiation Interactions between mitochondria and the transcription factor myocyte enhancer factor 2 (MEF2) regulate neuronal structural and functional plasticity and metaplasticity Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines An interaction between thyroid hormone and nerve growth factor promotes the development of hippocampus olfactory bulbs and cerebellum: a comparative biochemical study of normal and hypothyroid rats Localization of the transcriptional coactivator PGC-1α to GABAergic neurons during maturation of the rat brain Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells Control of mitochondrial transcription specificity factors (TFB1M and TFB2M) by nuclear respiratory factors (NRF-1 and NRF-2) and PGC-1 family coactivators RORα coordinates reciprocal signaling in cerebellar development through sonic hedgehog and calcium-dependent pathways Thyroid hormone induces cerebellar purkinje cell dendritic development via the thyroid hormone receptor α1 Transcriptional control of mitochondrial biogenesis and function doi: 10.1146/annurev.physiol.010908.163119 Peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) coactivates the cardiac-enriched nuclear receptors estrogen-related receptor-α and -γ Identification of novel leucine-rich interaction motif within PGC-1α Immunohistochemical characterization of the orphan nuclear receptor ROR α in the mouse nervous system Remodeling of monoplanar purkinje cell dendrites during cerebellar circuit formation The role of thyroid hormone on cerebellar development Promoter-specific regulation of the brain-derived neurotropic factor gene by thyroid hormone in the developing rat cerebellum RORα augments thyroid hormone receptor-mediated transcriptional activation “Effects of thyroid hormones on central nervous system development,” in Neurobehavioural Teratology Peroxisome proliferator-activated receptor γ coactivator-1 promotes cardiac mitochondrial biogenesis Transcriptional co-activator PGC-1α drives the formation of slow-twitch muscle fibres Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation Transcriptional coactivator PGC-1α integrates the mammalian clock and energy metabolism Deletion of the thyroid hormone receptor α 1 prevents the structural alterations of the cerebellum induced by hypothyroidism Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex The effects of early hypo- and hyperthyroidism on the development of the rat cerebellar cortex Selective and regulated gene expression in murine Purkinje cells by in utero electroporation Molecular basis of thyroid hormone-dependent brain development Thyroid hormone receptor β mutation causes severe impairment of cerebellar development A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response Light-regulated thyroid hormone signaling is required for rod photoreceptor development in the mouse retina The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor α (ERRα) Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development Bioenergetic analysis of peroxisome proliferator-activated receptor γ coactivators 1α and 1β (PGC-1α and PGC-1β) in muscle cells RORα regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo Mitochondrial biogenesis is required for axonal growth The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor α in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes Effects of thyroid hormone on synaptogenesis in the molecular layer of the developing rat cerebellum PGC-1α and PGC-1β regulate mitochondrial density in neurons Receptor interacting protein 140 as a thyroid hormone-dependent negative co-regulator for the induction of cellular retinoic acid binding protein I gene Requirement of Helix 1 and the AF-2 domain of the thyroid hormone receptor for coactivation by PGC-1 Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1 T3-mediated expression of PGC-1α via a far upstream located thyroid hormone response element Aberrant cerebellar development of transgenic mice expressing dominant-negative thyroid hormone receptor in cerebellar purkinje cells The mechanism of action of thyroid hormones Fujishima K and Kengaku M (2017) Thyroid Hormone Induces PGC-1α during Dendritic Outgrowth in Mouse Cerebellar Purkinje Cells Received: 28 December 2016; Accepted: 20 April 2017; Published: 09 May 2017 Copyright © 2017 Hatsukano, Kurisu, Fukumitsu, Fujishima and Kengaku. 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) or licensor 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: Mineko Kengaku, a2VuZ2FrdUBpY2Vtcy5reW90by11LmFjLmpw 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 (Hakipu‘u) – The Trust for Public Land (TPL) the City and County of Honolulu’s Clean Water and Natural Lands Program (CWNL) and DLNR’s Legacy Land Conservation Program (LLCP) announce the acquisition and protection of 1.5 acres known as Hakipu‘u Lo‘i Kalo located in Hakipu‘u For over a decade the Hakipu‘u community and kuleana descendants have been working to protect Hakipu‘u Lo‘i Kalo This lo‘i kalo (wetland taro patch) sits at the foot of the Ko‘olau mountains These lo‘i have been in active cultivation since ancient times and are some of the last lo‘i in Hakipu‘u; a place once overseen by Hawaiʻi’s kahu and revered to this day for its traditional navigators Threatened by development and an end to kalo farming and community access  TPL worked in partnership with the Hakipu‘u community for more than 8 years to find a conservation solution to protect these lo‘i TPL took out a loan in 2016 to purchase the land and acted as a temporary owner allowing the community time to raise the funds to purchase the property TPL led efforts to raise $1 million to purchase and protect Hakipu‘u Lo‘i Kalo and convey it to community ownership under Ho’āla ‘Āina Kūpono “We were humbled to work closely with the Hakipu‘u community and Ho’āla ‘Āina Kūpono to protect Hakipu‘u Lo‘i Kalo This community teaches all of us by example We are confident that under community stewardship Hakipu‘u Lo‘i will thrive and live on as a stronghold of Hawaiian agriculture and cultural practice,” said Reyna Ramolete Hayashi The City and County of Honolulu’s Clean Water and Natural Lands Program acquired a real property interest in the form of a conservation easement valued at $650,000 and the State of Hawai‘i’s Legacy Land Conservation Program granted $350,000 to Hoʻāla ‘Āina Kūpono to purchase and protect the land [email protected]  Metrics details Social animals actively engage in contact with conspecifics and experience stress upon isolation the neural mechanisms coordinating the sensing and seeking of social contacts are unclear Here we report that amylin-calcitonin receptor (Calcr) signaling in the medial preoptic area (MPOA) mediates affiliative social contacts among adult female mice Isolation of females from free social interactions first induces active contact-seeking concurrent with a loss of Amylin mRNA expression in the MPOA Reunion with peers induces physical contacts activates both amylin- and Calcr-expressing neurons and leads to a recovery of Amylin mRNA expression Chemogenetic activation of amylin neurons increases and molecular knockdown of either amylin or Calcr attenuates contact-seeking behavior Our data provide evidence in support of a previously postulated origin of social affiliation in mammals less is known about the early responses to social isolation in which automated scoring of proximity to a social versus a nonsocial chamber can lead to misinterpretation of the subject animals’ sociability and found that amylin-Calcr signaling in the cMPOA is required for contact seeking behavior in an affiliative context These data collectively indicated that amylin-ir MPOA neurons are activated by the presence of social stimuli and that such activation is associated with an increase amylin expression level within the MPOA and limited tactile inputs that are available during this in-cage isolation are not sufficient to maintain amylin expression in the MPOA suggesting that free social interaction is required movement (locomotion or any physical movements not otherwise classified); 2) exploration of the environment: sniffing (targeted at the partition or through the partition) digging; 3) contact-seeking: biting at the partition 3- and 4-together groups did not differ in any variables throughout this experiment suggesting that these isolation-induced behaviors were not because of separation from the nest partition-biting was increased when the subject mice were isolated and inaccessible peers were present beyond the partition suggesting that the partition-biting may represent social motivation or contact-seeking behavior with the exception of piloerection which is difficult to detect in mice With respect to a stress response, 120 min after the introduction of the real partition mice in complete and somatic isolation exhibited a significant increase in c-Fos expression in the paraventricular nucleus of the hypothalamus (PVH), the central regulator of endocrine stress responses, compared to mice in the 3- or 4-together conditions (Fig. 2m-n) These findings suggest that both complete and somatic isolation are stressful for female mice possibly because the peer separation of adult mice is not as threatening as maternal separation of infants To identify the neural mechanisms of sensing and seeking for social contacts we next focused on the initial active response to somatic isolation which induces more contact-seeking behavior and retains the visual eliminating complications in interpreting changes in behavior or neuronal activities while reunion and light-induced defensive huddle both involve increased physical contacts with cage mates these two conditions have distinct features and reunion does not include a stress response while the transfer to a dark novel cage did not induce statistically significant changes in any brain regions no overlap was found between the brain regions activated by the reunion and those activated by somatic isolation The BSTpr and MeAD were commonly activated after reunion and bright-light induced defensive huddle suggesting these regions might be activated in response to direct physical contact suggesting that these regions were associated specifically with affiliative contacts these data suggest Calcr+ neurons in the cMPOA and MePD are involved in affiliative cMPOA lesions reduced partition-biting after somatic isolation and initial contact after reunion to levels similar to that observed in the Calcr knockdown mice These data indicate that the cMPOA functions in inducing contact-seeking and the acute phase of affiliative contact behaviors were largely mediated via Calcr in the cMPOA the decrease of contact-seeking following ovariectomy may be attributed to the decrease in amylin expression in the cMPOA among the various functions of Calcr+ neurons in facilitating social contacts amylin plays a significant role in the induction of contact-seeking behaviors One distinction remains; while Agrp neurons are activated during hunger (lack of the goal) Calcr+ neurons are activated during reunion (achievement of the goal) To further elucidate the exact role of Calcr+ cMPOA neurons in social motivation future studies should examine how Calcr+ neurons exhibit social condition-dependent activities with higher temporal resolution thus it is reasonable to speculate that increased amylin by satiety could induce sociability in mice food scarcity is one of the major limiting factors of group size and individual animals increase solitary foraging to avoid intragroup food competition the limited amylin binding to Calcr in the cMPOA may downregulate contact-seeking behaviors to better adapt the environment Further direct experimental demonstration is needed to prove this appealing possibility future work examining the relevant projections from the MeA to the MPOA in higher anatomical and molecular resolutions would be of interest to the field one being about the security of self and the other is about the interest toward others and were conducted in accordance with the National Institutes of Health guide (NIH publication no Amylin-KO and Amylin-Cre mice were backcrossed to BALB/c for at least five generations Cg-Gt (ROSA)26Sor tm3(CAG-EYFP) Hze /J (JAX 007903) was from the Jackson Laboratory Mice were maintained under controlled conditions (12/12 h light/dark cycle [lights on at 08.00 a.m.]; 23±2 °C; 55±10 % humidity with food and water ad libitum) Female mice were housed in groups of four or five after weaning at 4 weeks Mice were 3-10 months old at the start of the experiments The reagents used in the experiments and their commercial sources were as follows: Amylin (mouse CNO (10 mg/ml stock) was dissolved in saline (0.9 % NaCl) containing 20% (v/v) dimethyl sulfoxide (Wako The housing condition was manipulated to examine the effect of cage mate presence on amylin expression. For the experiment in the Fig. 1e virgin female mice were group-housed (in groups of five) from weaning and were subjected to perfusion fixation and brain dissection after 0 Additional virgin females were single-housed for 6 days and then were group-housed back with their siblings or 14 days after the second round of group housing The effects of cage mates were investigated by housing virgin female mice with one or four unfamiliar females or with four castrated males mice were subjected to perfusion fixation and the brains were dissected one of the five mice was placed into the mesh cage Brain samples of in-cage isolated mice and one of the group-housed mice (in groups of four) were dissected after 6 days of social isolation Group-housed mice (in groups of four) were placed in a chamber (44 × 24 × 15 cm) containing purified paper bedding (Alpha-Dri) and two water bottles (Fig. 2a) and divided into left and right compartments by a central removable partition Mice had access to food pellets and water ad libitum in each compartment Two types of partition were prepared: a sham partition through which mice could see and sniff each other but were not in free physical contact one of the four mice was placed into the compartment without the previous nest and the sham partition was replaced with the real partition Their behavior was tracked for more than 3 h using a video camera (iVIS HF R52 in which mice tuck their feet beneath the body with their back hunched up in which mice tuck their feet beneath the body without their back hunched up were scored in a one-zero fashion using still images extracted at 1-min intervals for 3-h The affiliative nature of these behaviors was confirmed by the lack of other kinds of agonistic behaviors in this study was implanted intraperitoneally to measure the mouse locomotor activity The activity and core temperature were recorded at 1-min intervals Group-housed mice (in groups of two) were brought to the behavioral testing room and allowed to acclimate to a 12/12 h light/dark cycle (lights on at 02.00 p.m.) for 3 days mice (in groups of two) were placed into a dark novel cage their behavior was tracked for 30 min using an infrared camera (400-CAM035-2 and they were then returned to the home cage mice were placed into a dark or a brightly illuminated novel cage and their behavior was tracked for 30 min a 93.2 ± 5.5 lux fluorescent lamp was placed above the novel cage Mice were scored for whether or not they made social contacts with conspecifics for 10 min using CAPTIV-L2100 (TEA The number of fecal boli deposited in the novel cage was quantified after each behavioral test as anxiety measurement Behavioral tests were performed during the dark period (10.00 a.m.–02.00 p.m.) The predator odor-induced defensive huddle test was conducted in the home cage Group-housed C57BL/6 female mice (in groups of two) were acclimate to a 12/12 h light/dark cycle (lights on at 08.00 a.m.) for 3 days mice (group of two) were brought to the behavioral testing room equipped with a draft chamber and allowed to acclimate for 30 min A piece of filter paper (2 × 2 cm) soaked with 2MT (270.6 μmol) was placed at the edge of the home cage and the mouse behavior was tracked for 30 min The bacterial artificial chromosome (BAC) modification was carried out using BAC recombineering technology The organic anion transporting polypeptide 3 Solute carrier organic anion transporter family member 1A5 and pyridine nucleotide-disulphide oxidoreductase domain 1 genes were included upstream or downstream of Amylin gene in the RP23-438K19 clone NotI sites were inserted 5′ and 3′ of Amylin gene using the Red/ET Recombination System to cut off superfluous genes The Cre sequence was inserted in-frame into the second to third exons shared by the major Amylin isoforms (NM_010491) The Cre insertion cassette consisted of a 5′ homology arm (5′-GAAAGAGGTAGCTTGATTTCTGTGGGTTTTTTTTTTTTCTTTTCAGGGATCTTGAGAA-3′) the Ampicillin-resistant gene flanked by FRTs and a 5′ homology arm (5′-TCAGGAAATCACCAGAGCATTTACACATAAAGTATTAAGTGCTGCAGAAGTACATTGACT-3′) The purified BAC modifying cassette was electroporated into DH10α cells containing the RP23-438K19 BAC and screened by polymerase chain reaction (PCR) The ampicillin-resistant gene was removed with the FLP/FLPe recombinase encoded by the 706-Flp plasmid Fingerprinting analysis using BamHI restriction enzyme and DNA sequencing were carried out to confirm the presence of the correct modifications in the BAC clone The linearized BAC DNA was purified with Sepharose® CL-4B (Sigma-Aldrich The purified BAC insert was microinjected into fertilized eggs (C57BL/6 J) at the pronuclear stage Successfully developed two cell-stage embryos were transferred to pseudopregnant recipient Jcl:ICR females (Clea Japan) We identified one mouse line that exhibit faithful Amylin-Cre expression in neurons expressing endogenous Amylin This Amylin-Cre mouse line was then confirmed to be normal for the growth Transgenic mice were genotyped by PCR using three primers (5′-CTTTGATGGTTCCAATTTACACT-3′ and 5′-TCCGGTTATTCAACTTGCACCATGC-3′) to detect a 205-bp sequence present in the Amylin-Cre transgene and a 303-bp sequence present in the Amylin gene 293FT cell line (Invitrogen) was used in AAV production Mice were anesthetized with xylazine hydrochloride (0.24 mg Japan) and lethal amount of sodium pentobarbital (3.24 mg Japan) and then perfused with 4% PFA in phosphate-buffered saline (PBS) The brains were immersed in 4% PFA at 4 °C overnight and then in 30% sucrose in PBS for 2 days Japan) and cut into 40-μm coronal sections using a Cryostat (Leica CM1950) Every third brain section was prepared for histochemistry brain sections were washed twice with PBS containing 0.2% Triton X-100 (PBST) for 10 min treated with methanol containing 0.3% H2O2 for 5 min and treated with 0.8% Block Ace (Megmilk Snow Brand Co. Sections were then incubated with anti-c-Fos (1:5,000 USA) antibodies diluted into 0.8% Block Ace PBST overnight at 4°C sections were washed and incubated with biotin-conjugated horse anti-rabbit secondary antibody (1:2000 USA) for 2 h and then with ABC peroxidase reagent (Vector Laboratories) Signal-positive cells were detected by diaminobenzidine (DAB) reaction (Vector Laboratories) the sections were similarly incubated with an anti-NeuN (1:5000 Merck Millipore) antibody in combination with an anti-Neurophysin I (NPI) antibody (1:2,000 Santa Cruz Biotechnology) and then with an anti-mouse (1:2000 Vector Laboratories) or anti-rabbit secondary antibody in combination with an anti-goat secondary antibody (1:2000 The sections were then incubated with the ABC alkaline phosphatase reagent (Vector Laboratories) Signal-positive cells were detected with ImmPACT Vector Red (Vector Laboratories) For double labeling of Calcr and c-Fos or amylin and c-Fos c-Fos-immunoreactive neurons were detected by DAB reaction with nickel Calcr- or amylin+ cells were detected by DAB reaction without nickel brain sections were washed three times with PBST for 10 min and treated with 0.8% Block Ace in PBST for 30 min for blocking Sections were then incubated with the primary antibody diluted into 0.8% Block Ace PBST overnight at 4°C incubated with DAPI diluted in PBST for 5 min the sections were mounted between slide and coverslip using Vectashield Vibrance antifade mounting media (Vector laboratories) The signal of some primary antibodies was enhanced by biotin-Streptavidin-Alexa Fluor 568 (Invitrogen) We used the following primary and secondary antibodies: anti-NeuN (1:5,000 and Alexa 488- or Alexa 568-conjugated anti-rabbit Single in situ hybridization (ISH) of Amylin mRNA was performed as previously described37 with modifications the nucleotides 70–577 of Amylin (NM_010491) were amplified by PCR from mouse brain cDNA (GenoStaff) and an SP6 RNA polymerase recognition site was added to the 5′ end of the PCR product using specific primers (sense: 5′-CCTCGGACCACTGAAAG-3′; antisense: 5′-ATTTAGGTGACACTATAGAAACATTGACTTCACT CTGAACTTGATCA-3′) The antisense probes were transcribed by SP6 RNA polymerase (P2083 Promega) in the presence of DIG-RNA labeling mix (Roche Diagnostics Switzerland) and isolated by lithium chloride precipitation Brain sections were washed twice with PBST for 5 min treated with PBST containing 20 μg/ml Proteinase K (Invitrogen) for 10 min treated with 4% PFA in PBS for 10 min for post-fixation incubated with methanol containing 0.3% H2O2 for 5 min they were treated with 0.25% acetic anhydride in 0.1 M triethanolamine and washed twice with PBST for 5 min Brain sections were treated with prehybridization solution at 58°C for 30 min and hybridized at 58°C overnight in the probe containing hybridization solution the sections were incubated with 2x SSC containing 50 % formamide twice at 58°C for 10 min with RNase A solution (20 μg/ml) at 37°C for 45 min and finally with SSC 0.2x SSC twice at 37°C for 10 min They were washed with TBST for 5 min and incubated with an alkaline phosphatase-conjugated anti-DIG-AP (1:10000 sections were washed twice with TBST for 5 min and incubated in 100 mM tris-HCl (pH 9.5) for 5 min Signal-positive cells were detected with the BCIP/NBT solution kit (Nacalai Tesque These ISH probes were used in the Allen Mouse Brain atlas Calcr+ cells were detected by DAB reaction without nickel Sections were imaged by NanoZooomer Digital Pathology (Hamamatsu Photonics Japan) with a 20x or 40x objective for bright-field observation and BZ-9000 All-in-one Fluorescence Microscope (Keyence Japan) with a 20x or 40x objective for fluorescence observation Confocal images were acquired using a confocal microscope with a 40x objective (FV1000 To quantify the number of c-Fos or Calcr-ir cells acquired images were binarized and labeled neurons within each conservative contour were automatically or manually counted using ImageJ (NIH) For data shown as densities (N of cells/mm2) the number of labeled neurons was divided by the area of each conservative contour calculated using ImageJ Stereotactic coordinates for AAV injections were taken from the mouse brain atlas103 The coordinates for the cMPOA were AP + 0.1 Virgin female mice were anesthetized with xylazine hydrochloride (0.24 mg Japan) and mounted in a stereotaxic apparatus (Narishige A volume of 100 nl AAVs was injected into the targeted brain region through a pulled fine glass capillary (20–50 μm diameter at the tip) by oil pressure at a rate of 25–50 nl/min Capillaries remained in place for 5 min allowing the solution diffusion and the mice were warmed-up until recovery from anesthesia Subject virgin female Amylin-Cre mice were raised in groups of five or four until they were more than 3 months of age A total of 100 nl of AAV5-hSyn-DIO-hM3D(Gq)-IRES-mCherry (5.6E12 vg/ml USA) was unilaterally injected into the cMPOA at an approximate speed of 50 nl/min each virus-injected Amylin-Cre mouse was transferred into a test cage and cohoused with three wild-type virgin female mice After 1 week of habituation (2 weeks after AAV injection) the mice were subjected to behavioral testing Stocked CNO was dissolved in saline and injected subcutaneously at 10 mg/kg for Gq-DREADD activation prior to somatic isolation or reunion test Control injections were made with 2% DMSO in saline solution The virus-injected mice (one in four mice) were socially isolated in the nestless part of the test cage and biting responses were observed for more than 2 h The expression of mCherry was retrospectively examined using an anti-mCherry antibody (1:5000 USA) were placed stereotaxically and bilaterally into the MPOA (AP Plastics one) protruded 1.0 mm from the tips of the guide cannula Canula-implanted mice were housed individually for 6 days the injector cannula was attached to Hamilton syringes (26201 The bilateral infusion of amylin (300 nl ACSF with 1 μg amylin at a speed of 50 nl/min) was controlled by a microinfusion pump (ESP-36 The brains were fixed and subjected to immunochemical studies 2 h after infusion The correct placement of the injector cannula tip was confirmed in brain sections Virgin female mice were raised in groups of five or four until they were more than 3 months of age A total of 40 or 100 nl of NMDA (Sigma-Aldrich) and NMLA Santa Cruz Biotechnology) dissolved in PBS at 20 mg/ml was injected bilaterally into the cMPOA or the whole posterior MPOA including the medial part DV: -5.1 to -5.4 for the MPOA and AP: + 0.1 behavioral tests followed by immunochemical studies were performed The lesion location was identified by immunostaining with a mouse anti-NeuN antibody (1:5000 ovariectomized mice were treated with 17beta-estradiol (E2 MO) (2 μg/day) via subcutaneously implanted Alzet osmotic pumps (Alzet CA; model 1002; 14-day capacity at 0.25 μl/h infusion rate) E2 was dissolved in USP-propylene glycol (Sigma-Aldrich) Pump implantation was performed soon after the ovariectomy Data were analyzed by unpaired t-test with Excel or nonparametric test with the Prism8 software for single comparisons analysis of variance (ANOVA) with post hoc tests were performed with the software R (R Development Core Team The biological replicate number corresponds to the number of mice Technical replicates were not applicable or performed in this study All fluorescent image analyses were repeated independently at least two times Further information on research design is available in the Nature Research Reporting Summary linked to this article NA. Source data are provided with this paper The materials generated in this study are available from the corresponding author upon reasonable request with a completed Materials Transfer Agreement This study did not generate any unique code Social relationships and mortality risk: a meta-analytic review Social behaviour in mammals (Blackie; Distributed in the USA by Chapman and Hall 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Hazuki Inoue and Hiroka Matsubara for technical assistance Mineko Kengaku for the pAAV-hH1-MCS-CAG-EGFP plasmid Jude Samulski and the Vector Core at the University of North Carolina at Chapel Hill for the AAV2-hSyn-DIO-EGFP Samuel Gebre-Medhin and the European Mouse Mutant Archive for the Amylin KO mouse strain and the RIKEN Research Resource Division for BAC microinjection to create Amylin-Cre mice and maintenance of the animals This research was supported by RIKEN Center for Brain Science (2012-2021 RIKEN Special Post-Doctoral Research fellowship (202001061045 JSPS KAKENHI Grant Number JP16K19011 (K.F.) and partly by 18H02710 and AMED under grant JP20dm0107144 (K.K.) Laboratory for Affiliative Social Behavior Nippon Veterinary and Life Science University Laboratory for Circuit and Behavioral Physiology and with contributions from all the authors The authors declare no competing interests Nature Communications thanks Stefanos Stagkourakis and the other anonymous reviewer(s) for their contribution to the peer review 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/s41467-022-28131-z 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. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. grocery stores are prepping for swarms stocking up on snacks and you better believe avocados will be in high demand as people get ready to make enough guacamole to feed party-sized crowds of hungry football fans it's unlikely anyone will be able to get their hands on the sort of beastly avocados that are currently growing in Hawaii where one farmer has been turning out impressive specimens as large as six pounds each.  has been harvesting the fruits from one tree in particular which his family has tending to for the last 80 years that Fukumitsu ends up giving a lot of them away as he regularly does to the staff at the nearby Kona Urgent Care center.  all falling down," said Fukumitsu in an interview with BIVN so I share them with some of our friends." The sheer size of these things is most astonishing when you set them next to a normal sized specimen And when you consider a typical avocado weighs just six ounces both he and his pals at the Urgent Care center agree they're delicious The current record for the world's largest avocado stands at five pounds The largest of the bunch featured in the clip of Fukumitsu's latest haul (shown above) weighs nearly six pounds two ounces “This baby is 6 pounds!” the Urgent Care center's Dr Fukumitsu is quite humble about it all.  So, if you're hell-bent on sampling some baby-sized avocados, your best bet would be to book a flight to Hawaii and hit up Mr. Fukumitsu. That may seem like a lot of work, but hey, at least getting there has never been cheaper h/t Travel + Leisure Sign up here for our daily Thrillist email and subscribe here for our YouTube channel to get your fix of the best in food/drink/fun TOYAMA — Baseball bat manufacturers in the Fukumitsu district in Nanto are in their busiest time of year ahead of the opening of the new professional baseball season please disable the ad blocking feature and reload the page This website uses cookies to collect information about your visit for purposes such as showing you personalized ads and content By clicking “Accept all,” you will allow the use of these cookies Users accessing this site from EEA countries and UK are unable to view this site without your consent has a thriving woodworking industry as humidity is at a suitable level for woodcraft throughout the year The district is one of the most prominent production areas for baseball bats in the country a major manufacturer that has made bats for such popular players as the Hanshin Tigers’ Takumu Nakano and former star Koji Yamamoto of the Hiroshima Toyo Carp manufactures about 300 bats every day at this time of year The company’s nine employees divide manufacturing procedures among themselves such as carving maple wood imported from North America or coating the bats “I hope that the players do a great job using the bats we’ve made with such care,” said the head of the factory Our weekly ePaper presents the most noteworthy recent topics in an exciting © 2025 The Japan News - by The Yomiuri Shimbun Today's print edition Home Delivery Why don't Japanese audiences turn up in big numbers for Hollywood superhero movies The rare success in Japan of the Spider-Man series suggests one answer: Japanese like superheroes just fine as long as they're flawed humans as well as heroic fighters for justice Another case in point is "Hero Mania," Keisuke Toyoshima's black comedy about everyday heroes who are not just flawed And though they may have superior fighting skills they are not super-powered.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); Based on Shigeyuki Fukumitsu's alternative manga "Seikatsu" ("Life") the film is funny in smart off-kilter ways while making astute observations about Japanese society in particular and human nature in general though produced by the major Japanese film companies — Nikkatsu and Toei — "Hero Mania" has an indie It may look like a typical genre entertainment the characters arrive at various destinations not all of which are predictable — or perhaps even legal In a time of both misinformation and too much information quality journalism is more crucial than ever.By subscribing Your subscription plan doesn't allow commenting. To learn more see our FAQ Sponsored contents planned and edited by JT Media Enterprise Division Electronic Arts has just announced the four new Swap Deals of the month of March for the FIFA 19 Ultimate Team mode The object is obtained for free simply by entering FUT The object is achieved by a SBC. Here you can find various possible solutions Requirements: Score using Colombian players in 3 separate Rivals wins during the week of 5.4-12.4 Requirements: Play 13 online matches during the week of 5.4-12.4 Requirements: Finish Silver 3 or higher in Squad Battles for the week of 7.4-11.4 Requirements: Win 8 online matches this week to earn a Swap Deals Player Requirements: Score using a UEFA Champions League Rare player in 4 separate wins across any FUT game modes to earn a Swap Deals Player The object is achieved by a SBC. 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