A Direct Neurokinin B Projection from the Arcuate Nucleus Regulates Magnocellular Vasopressin Cells of the Supraoptic Nucleus

Edinburgh Research Explorer A direct neurokinin B projection from the arcuate nucleus regulates magnocellular vasopressin cells of the supraoptic nucleus

Citation for published version: Pineda Reyes, R, Sabatier, N, Ludwig, M, Millar, RP & Leng, G 2015, 'A direct neurokinin B projection from the arcuate nucleus regulates magnocellular vasopressin cells of the supraoptic nucleus', Journal of Neuroendocrinology. https://doi.org/10.1111/jne.12342

Digital Object Identifier (DOI): 10.1111/jne.12342

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Download date: 02. Oct. 2021 Accepted Article

Abbreviated Title: Abbreviated Africa Cape Town7925, ofCape South Town, Medicine, University Molecular and Diseases Infectious for Institute Unit, Biology Receptor MRC 0001; Pretoria Edinburgh UK EH8 9XD, 2 1 Article type : Original Article 22-Nov-2015 : Date Accepted Revised: 02-Nov-2015 Date : 01-Jul-2015 Date Received This article This protectedis by Allcopyright. rights reserved. 10.1111/jne.12342doi: lead to differences betweenthis version and the Versionof Record. Please cite this article as through thebeen typesetting,copyediting, pa This article hasbeen accepted for publicationand undergone fullpeer review but has not Email: [email protected] [email protected] Email: 3275 650 131 0044 Phone: UK 2XD, EH9 Edinburgh Building, Hugh Robson University of Edinburgh CentreIntegrative for Physiology Mike Ludwig whom to person and author Corresponding Terms: Key A direct neurokinin B projection from magnocellular vasopressinregulates arcuatethe nucleus Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Pretoria, of Entomology, Zoology and of Department University Institute, Mammal Research Square, George Building, Robson Hugh Edinburgh, of University forIntegrative Physiology, Centre Rafael Pineda Reyes Pineda Rafael

electrophysiology, neuroanatomy, magnocellular neurones, hypothalamus, oxytocin oxytocin hypothalamus, neurones, magnocellular neuroanatomy, electrophysiology,

Neurokinin B and vasopressin and B Neurokinin 1 , Nancy Sabatier cells of the cellssupraoptic of nucleus reprint requests should be addressed: be addressed: should requests reprint 1 , Mike Ludwig Mike gination and process, which proofreading may

1 , Robert P. Millar 2 , and Gareth Leng Gareth and ,

Accepted Article agonist senktide potently increases increases electric the senktide potently agonist NK3R of the we injection i.c.v. showthat Finally, SON andPVN. the to project NKB, co-express neur kisspeptin-expressing that found we approach, neurones project to the in the arcuate nucleus trac Retrograde sites. ofthese both at neurones vasopressin with infibresclose in isexpressed juxtaposition and thatNKB immunoreactivity nucleus(SON)and neuronespa supraoptic the in e is forNK3R immunoreactivity rat, in the that, neuroki theactivation of thatdependson mechanism va of secretion and theincreases hypothalamus agonis (NKB) B neurokinin of administration Central Abstract Disclosure statement: This article This protectedis by Allcopyright. rights reserved. re acritical is family, neuropeptide tachykinin express neurokinin B(NKB) and express neurokinin known. not is production steroid and synthesis vasopressin in increase age-related the whether but women, men and both in production steroid sex in decline amarked with associated and thishas been linked secretion, vasopressin vasopressi in an also is increase the SON and in neurons vasopressin pr secretion vasopressin areof chronic attributedto chronically hyponatremia regulator halfof Inhormonal ofall and body content. theelderly, about plasmacases water osmolality (PVN) and paraventricular (SON) the supraoptic Introduction this. of significance possible the andwe discuss rodents, from magnocellular of neuronesneurones regulatethe vasopressin in the arcuate in nucleus secretion no significant effect detected onoxytocin neurons with In this study, we explored neuroanatomical an study, neuroanatomical weexplored In this in is synthesised hormonevasopressin The peptide Nothing to disclose disclose to Nothing ogressively increases with age (2, 3), and the size of magnocellular 3), age (2, size and the magnocellular of with ogressively increases n production (2, 4). In aged rodents, aged In 4). (2, production n magnocellular vasopressin neurons. magnocellular PVN is increased after the age of 60 years, suggesting that there thatthere theage suggesting 60years, of after PVN is increased SON, and in an anterogradeSON, mice, using and in tracing gulator for the central control of reproduction. The ofreproduction. central control the for gulator al activity theSON ofvasopressin neurones in ing in the rat showed that some NKB-expressing showedthatsome NKB-expressing inthe rat ing sopressin from the posterior pituitarysopressin through a to impaired osmoregulation (5).Aging osmoregulation isalso impaired to xpressed in magnocellular vasopressin and oxytocin and oxytocin inmagnocellular vasopressin xpressed raventricular nucleus (PVN) of the hypothalamus, thehypothalamus, of nucleus (PVN) raventricular nuclei ofthe hypothala secretion is linked to age-related decline in sex to islinked age-related in decline secretion ones of the arcuate nucleus, which are known to to are known which nucleus, arcuate the of ones nin nin Herereceptor 3receptors we (NK3R). report elevated secretion of vasopressin (1). In humans, humans, In (1). vasopressin of secretion elevated ts stimulates immediate early gene expression in early gene expression tsimmediate stimulates d functional interactions between neurons that betweenthat neurons interactions d functional . The suggest thatNKB-containing results

magnocellular neurosecretory neurones in neurosecretoryin neurones magnocellular there is also an increase in in increase alsoan there is

NKB, a member of the the of member a NKB, mus, mus, and isthe main in vivo in

Accepted Article is described in detail is describedelsewhere indetail Rats (17). were from thesite, and donot we The detection (16). injection need anfor antibody method injection used studies Tracing/Connectivity Committee. Ethics Edinburgh of University the by reviewed been has that a licence UK Home project Office with accordance in conducted light:dark offatcycle (lights 19.00 h) with fre maleadult transgenic Materials andMethods SON. magnoc of activity electrical the on senktide, agonist, NK3R the the of 2) PVN,and and effects SON the in neurones vasopressin and nucleus arcuate determine 1)whetherthere is an anatomical c reducesosmotically-stimulated antagonist (14). of thesystem hypothalamo-hypophysial andasthe both in can SON and senktide PVN (13), of are senktide The actions effect. have such no whereas (12), neur secretion vasopressin peripheral NK3R agon aselective injection of Central secretion. dendrites and somata the on effects marked increase of and an hypertrophy inneuronal results inmonkeys, ovariectomy where has beendescribed pattern (9). A similar a sex elevated by gonadectomy reversiblein by manner steroids with replacement greatly is expression NKB example, for Thus, steroids. sex circulating of levels to correlated inversely is NKBexpression that the recognition This to led man(8). the aging of nucleus infundibular the in found been since have results Similar rodents. in nucleus arcuate the to homologous nucleus NKBinthe infundibular nucleus – the ofcells and containing hyperplasia hypertrophy demonstrated which came womenand Young reproduction by from (7) postmenopausal Rance a study of NK3R(6). to binds andNKBpreferentially NK3R, are mediated oftachykinins effects by biological This article This protectedis by Allcopyright. rights reserved. For retrograde tracing experiments, we used retro-beads as they show very limited very weForspread limited they retro-beads show retrogradeas used experiments, tracing Experimentsmale onadult were performed Sprague–Dawley rats(body300g) ~ and weight and study, immunocytological using In this that evidence considerable already is There NKB NKB mRNA expression (10). In addition, gonada addition, In (10). expression mRNA Kiss1/ Cre-eGFP mice (body weight ~ 30 Cre-eGFP ~ had g) 30 weight (body mice been housedona h that 12:12 vasopressin secreti vasopressin of arcuate nucleus NKB neurons (11). (11). neurons NKB nucleus arcuate of onnection between the NKB-producing neurones in the neurones in between the NKB-producing onnection e access to food and water. All experiments were experiments All and water. food to e access Conversely, central administration of a NK3R of a administration central Conversely, anesthetized with4% first isoflurane and then likely to be direct, as mRNA for NK3R is expressed NK3Risexpressed as mRNA for direct, be to likely three different neurokinin receptors: NK1R, NK2R, NK2R, NK1R, receptors: neurokinin different three okinin A, substance P and selective NK-1 agonists P and A,substance agonists NK-1 selective okinin The ofa evidence role forNKB human earliest in the NKB/NK3R system may regulate vasopressin vasopressin regulate may system the NKB/NK3R electrophysiological approaches, we aimed to to weaimed approaches, electrophysiological also stimulate vasopressin secretion from explants explants from secretion vasopressin stimulate also ellular vasopressin and oxytocin neurones in the the in neurones oxytocin and vasopressin ellular ist, senktide, induces antidiuresis by increasing increasing by antidiuresis induces senktide, ist, l steroids have been reported to have have reported to l steroids been on inrats(15). Accepted Article microtome. microtome. 30% sucrose in PBS with 0.01% sodium azide. sucrose 0.01% 30% sections (40 sodium brain Coronal inPBSwith overnightat4ºC 2%extracted and post-fixed in phosphate M 0.1 in paraformaldehyde by 4% followed andtran a dose ofsodium lethal with pentobarbitone protocols Immunofluorescence immunofluorescence for removed double below. described and processed as transcardi were perfused mice after injection, weeks Three theAAV-DIO-YFP. come from found any YFP thus, projections nucleus, the to is restricted expression this promoter, kisspeptin the under eGFP expresses line transgenic this mm. Although 5.8 anterioposte the coordinates (28): nucleus following study above, serotype viral titer10 5; Carolina; North of (University Core Vector Center Gene Therapy from was purchased a DIO-AAV, factor1-al elongation expressed the incre only constitutively- cells under specifically to and transduced the correct sense orientated Thus oriented. oppositely sequences arcuat kisspeptin to trace mouse (25) knock-in Using propertythis dynorphin. ofarcuate kiss of the arcuatenucleus in neurons 20). Kisspeptin-expressing (19, neurons kisspeptin nucleus arcuate to specifically YFP deliver system nervous central the into interest of genes Anterograde tracing below. asdescribed for removed immunofluorescence processed and were the brains and perfused, wererats transcardially the after injection, week One subcutaneously. sutured, was wound Once the pressure injection. 9.4 mm; dorsoventral mm. (Lumafl Red retro-beads inSONusing ( implanted the following coordinates was capillary andaglass a stereotaxic frame in placed were They –2% isoflurane. 1.5 on maintained This article This protectedis by Allcopyright. rights reserved. Adult male g)Adult ~300 rats(body weight and male adult We used an AAV where an inverted YFP reporter sequence was floxed by two LoxP by sequence two was floxed YFPan reporter an inverted We AAVwhere used deliver to used commonly have been (rAAVs) vectors virus adeno-associated Recombinant Kiss1/ Cre-eGFP mice were injected with 300 nl of the DIO-AAV into the arcuate the into DIO-AAV the nl of 300 with injected were mice Cre-eGFP 12 Usingsimilar approach inthevg/ml). thatdescribed a retrogradeto tracing pha promoter (Ef1a) pha promoter (26, This27). type known commonly as AAV, of rodents (21, (21, 22), sheep (23), rodents , after the cre-recombinant event in kisspeptin cells, was YFP cells, kisspeptin event in cre-recombinant the after , peptin neurons, we used the transgenic we the neurons, used transgenic peptin e nucleus projections to the PVN. to SON and projections e nucleus PFA + 15% sucrose solution then cryoprotected in then cryoprotected in PFA solution sucrose 15% + rats were given ratswere (0.03mg/kg) buprenorphine in viv 18): anterioposterior -1.3 anterioposterior mm;-1.3 2.0 mediolateral 18): uor, Inc.)uor, were theSON nl) by injected in (100 ally with paraformaldehyde, and the brains were brains the and paraformaldehyde, with ally rior -1.8 mm; 0.3 mediolateral mm; dorsoventral scardially perfused with heparinised 0.9% saline, saline, 0.9% heparinised with perfused scardially o. Here we used a cre-dependent approach to to approach cre-dependent a used we Here o. -buffered saline (pH 7.3-7.4). Brains were Brains (pH7.3-7.4). -buffered saline Kiss1/ and co-expressmonkey and NKB (24) Cre-eGFP mice were injected injected were mice Cre-eGFP μ m) were cut on a freezing afreezing on cut were m) Kiss1/ Cre-eGFP Cre-eGFP Accepted Article channel. colour ina white results green channels overlaymagenta; to the redchannel ofandmagenta was recolored Imag the to exported wereimages Resulting software. maximum inte to stacksviewed and condensed Z stated. unless temperature room were at after(Sigma Fluoromount incubations washing. All Plusslides using SuperFrost in -F4680) mounted were sections Finally, 1). (Table Alexa488 antibody anti-rabbit secondary with incubation were times M four in 0.1 min 10 for washed sections the primarywith PB, followedby antibody, a h 2 NKBantibody primary buffer blocking in 1)diluted rabbit for (Table 2 daysincubation at After 4ºC. were incubated with triton sections X-100 in free-floating serum Mdonkey 3% PB. 0.1 + Next, 0.4% of blocking solution in min 45 by followed M PB 0.1 in min 10 for times four washed and Glycine min 10 Mtimes for 0.1 PB,0.1 andwashedfour in M PB 0.1 M 30minin They were incubated then in borohydride sodium (w/v) 1% freshly made in 30 min for incubated then MPB, 0.1 in 10 min for YFP. of detection the compromises which step, HIER afterthe fibres only label to able were andwe is low, sensitivity its high, is NKB antibody ofthe theWhile specificity by immunohistochemistry. NKB visualize ustosimultaneously use allow not did study The theHIER step. tracing without anterograde but antibodies. primary of theabsence in antibodies secondary applying after detected was TA-030-FM). signal No Scientific, (Thermo Medium Mounting Aqueous PermaFluor using slides 1) in for buffer 1 blocking h at37ºC, then washed three times for10minin PBS, and mounted on (Table antibodies secondary were 10min with incubated in PBS. times Finally, sections three the for washed then 4ºC, at two days for blocking buffer in 1) (Table primary antibodies with wereincubated Triton serum, orgoatdonkey PBS) in at X-100 45min the for sections 0.4% temperature. Next, room (3% blockingbuffer in wereincubated Thesections temperature. PBS atroom min in 5 for washed room wereand then temperature pH6forto cooled 10minat citrate sections Afterwards, 90ºC. retrieva epitope Heat-induced temperature. room for 1 hat were Sections dried then slides 37ºC. This article This protectedis by Allcopyright. rights reserved. For the retrograde tracing study, we sections. Sections were used free-floating twice washed as the study For AAV,we the used above using described same tracing anterograde protocol For studies, NK3R and NKBimmunoreactivity Immunoreactivity was visualized on the Nikon ontheNikon visualized was Immunoreactivity washed inPBS-T (0.1% Tween-20) for 10min at l (HIER) was then performed in 10 mM sodium nsity projections projections using nsity eJ software. To facilitate To readers,color-blind facilitate software. eJ

A1R FLIM confocal system. Images were ImagesA1R FLIM were system. confocal sections were mounted on SuperFrost Plus Plus SuperFrost on mounted were sections NIS-Elements Viewer Viewer NIS-Elements Accepted Article Secondary Abs Code Supplier Dilution Raised in: Raised Dilution Anti-Mouse Alexa Fluor®555 Supplier Anti-Rabbit Code Alexa Fluor®488 Secondary Abs Ab13970 GFP/YFP 1/10,000 Abcam NKB NB300-201 in: Raised Dilution chicken NK3R IS-7/7 Dr neurophysin Novus Supplier Oxytocin- neurophysin Philippe Code Biologicals Vasopressin- Primary Abs 1/800 assays. immunofluorescence the Ciofiin used antibodies secondary and Primary 1: Table rabbit 1/3,000 rabbit This article This protectedis by Allcopyright. rights reserved. techniques. recording extracellular conventional using neurones single from record theSON 0.9% with to NaCl wasin filled placed 1 ~ diameter µm) (tip microelectrode avol in senktide of pmol 600 inject to ventricle thethird to thenlowered stalkandonthemidline into the neural rostral mm placed0.5-1 cannula was fully (29). A as elsewhere described on the midlinemicroinjection weretranspharyngeally, exposed and tothe pituitary the SONand anarea The andtrachea. arostral catheterin cannulated was inserted In vivoelectrophysiology Anti-Mouse Alexa Fluor®555 Anti-Chicken Alexa Fluor®488 Adult male urethane a (1.25 Adult g/kg,anesthetized ratswere with femoral i.p.), vein was A-21422 Life Life A-21422 Technologies Life A-11039 goat 1/500 Technologies Life A-31570 goat 1/500 Technologies Life A-21206 donkey 1/500 Technologies mouse donkey 1/500 1/500 mouse Dr H Gainer 1/500 PS38 Dr H Gainer PS41 ume of 2 µl over 100 s. Under visual control, a glass overcontrol, 100s.Under visual ume of2µl Accepted Article 5]) =5]) (numberof intervals inbin [ bin [ in (hazard tothe formula according functions hazard constructed then and analysed, periods inte weconstructed For frequency. spike neurones, all (AQ= quotient activity the calculated we silences, maximum per burst; spikes 20 s; minimum at st interval 3 length periodminimum 3s;maximums; interburst criteria: interval interspike 1 minimum intraburst For were phasic algorithm neurones, firing bursts (30) recognized conventional by a according the to and significance the min beforeinjection, tested 10 the in activity with the injection after min and 40 20 between theperiod in activity by comparing senktid to arerats. Responses those individual from senktide of oneOnly wa injection injection. senktide after senktide with injection the (basal) firing ra (30). elsewhere described as analysed was rate) firing intraburst and quotient Bachem, Essex, cholecystokinin-(26-33)-sulfated; exclusively) localised to the neuronal cell membrane cell localisedthe to neuronal exclusively) from absent wasmainly PVN, immunoreactivity the a ofNK3 indicating strong and expression specific were zones perinuclear surrounding the whereas PVN. and SON in the NKB immunoreactivity and NK3R Results by andby pattern firing their cells vasopressin asprevishown neurones andoxytocin vasopressin SON patterns of firing the characteristic asthese reveal of cells, recorded the functions hazard and histograms interspike-interval of shape the checked we 3) neurones; vasopressin magnocellular 2) neurons); only magnocellular contains PVN, the onthesurf placetherecordingelectrode visually to wemagnocelluways toensure other recorded from that used theSON.Wetherefore in and electrode a stalk recording neural the electrode on stimulating a place also to space leaveenough not did ventricle 3rd the into ventrally cannula microinjection identify these antidromically neuronesas tothe pituitary posterior projecting gland because inserting a This article This protectedis by Allcopyright. rights reserved. Responses to senktide were analysed by comparing the mean firing rate in 10-min intervals intervals rate10-min in themean comparing firing analysed by were senktide to Responses In this study, unlike our previous electrophysiological studies on weSON neurones, on could not studies our previous study, unlike electrophysiological In this In the SON and PVN (Fig 1,2), we found dense expression of NK3R immunoreactivity NK3R immunoreactivity of wedenseexpression found SONand 1,2), In the PVN (Fig t, t + 5])/(number of intervals of length > > length 5])/(numberintervals of + of their opposite response to intravenous CCK [20 CCK [20 tointravenous opposite their response te measured in a 10-20 min control period before period control min 10-20 a in te measured of differences using a 2-tailed Mann-Whitney test. test. Mann-Whitney a 2-tailed using differences of apparently devoid ofNK3Rimmunoreactivity, devoid apparently ously (30). Oxytocin (30). cells ously UK]. In phasic neurones, burst patterning (activity (activity patterning burst In phasicneurones, UK]. R in magnocellular neurones. Within the SON and the SON Within neurones. magnocellular in R time active /time recorded) and the intraburst ace of the brain just below the SON (which, unlike unlike (which, SON the below just brain the ace of e were very prolonged, effectse wereand wequantified very prolonged, phasically firing can inthisarea be cells only phasically firing s (Fig 1D, 2D). In both nuclei, NK3R expression NK3Rexpression nuclei, In s (Fig 1D, both 2D). s made in each experiment, so the results shown shown results the so experiment, each in s made cell nuclei, and apparently mainly (but not (but mainly apparently and nuclei, cell rspike interval histograms (in 5-ms bins) 5-ms bins) for histograms (in interval the rspike lar 1)theneurones: ventral approach us allows art of burst 0.8 s. Having identified bursts and and bursts identified Having s. 0.8 burst of art t ). were distinguished from were distinguished μ g/kg; g/kg; t, t + Accepted Article (89%), whereas of 128 oxytocin cells, NK3R. co-expressed cells, 36(28%) only oxytocin (89%), whereas 128 of toboththese mice. inthe arcuate nucleus in cells project nuclei kisspeptin-expressing We fibers PVNandthe in 5A). YFP-expressing found thatsome indicating SON (Fig. 5B,C), here the results mice, and present two from Cre/eGFP was target on which theinjection mice (Fig. in Anterograde study of in (27out 238 cells) cells expressing the SON retro-beads into injected of in were found NKB- after injection, 11% cytoplasm the of A beads. week uptake the to likely fibres were a only SON and therefore area few the aonly of small inject to we expected so specificity, achieve higher to retro-beads of volume small a Weused 4A). (Fig. the SON outside spread minimal with target was on injection thewhich rats in two from results nucleus. To test aimed this, we toinject retro-beads found withinthe projections fr SON originated study Retrograde nucleus arcuate fromthe cells NKB-expressing of Projections bodieseither ofthese cell in expressing nuclei. ce tovasopressin closein juxtaposition boutons abunda SONand the both weWithin PVN, found thethird ventricle 3A). (Fig of thewalls thePVN alongside towards dorsally coursed other fibres 3A); be could these of some and arcuate nucleus, rostral the through passed cells these from fibers NKB-positive shown). (not nucleus arcuate caudal the defined as NK3R-positive or NK3R-negative. NK3R-negative. or asNK3R-positive defined expressed vasopressin-neurophysin cells that of sampling threethe rats, PVN from sections weanalysed this, To quantify Fig. 2C). was thePVN in but pref it neurones, all in expressed and intheretrochiasmatic of circularis, part nucleus the in including nuclei magnocellular accessory the all in found also was expression NK3R both vasopressin-neurophy with was co-localized This article This protectedis by Allcopyright. rights reserved. cells in of NKB-expressing population a large we observed previous studies, with Consistent These immunohistochemical studies suggested that at least some oftheNKB at least that suggested studies immunohistochemical These We injected a cre-dependent AAV-DIO-YFP in the arcuate nucleus of three three of arcuate nucleus the in AAV-DIO-YFP Wea cre-dependent injected or oxytocin-neurophysin, and which could be unambiguously unambiguously or oxytocin-neurophysin, andwhichcould be the caudal arcuate nucleus (Fig. 4B-F). the (Fig. 4B-F). caudal arcuate nucleus the SON (images not shown). In the SON, SON, Inthe NK3R was shown). the not SON (images Of 111 vasopressin cell Of 111vasopressin om inthe caudal arcuateNKB-expressing cells lls (Fig. 3B,C). However, we did not anyfind NKB- not we However, did 3B,C). (Fig. lls sin (Fig. 1)and (Fig. sin 2). oxytocin-neurophysin nt expression of NKB-expressingnt expression of fibres with seen to enter the caudal region of the SON (Fig. SON the region of enter thecaudal to seen into here sixone SONside and present rats, thein into erentially expressed in vasopressin cells (Fig cells 1C, expressed erentially vasopressin in s, 99 co-expressed NK3R NK3R co-expressed s, 99 kiss1 - Accepted Article from 6.2 ± 6.2 from ± 7.3 0.5 spikes/s spikes/s(Fig.to 0.7 6F). min aftersenktide injection (Fig. and 6E), intrabur ± The 0.19 frequency. ± AQ was from raised by 0.49 intraburst 0.11 the in to 0.07 ± 0.68 at 20-40 0.1 and time = (AQ active time / recorded) quotient activity intensity the in changes the we analysed burst in a change or/and length burst the in change a to due was senktide to response the whether assess to ac this spikes/s); - 8 0.5 (range spikes/s ± 0.7 3.3 test). Whitney was 2.1 after injection, ± thanthebasal spikes/s rate 0.7 higher (Fig. 6A and C; min over20-40 averaged these, rateof the mean firing and injection, after min 60 and 30 between rate from increasingfiring to 9.7 that its spikes/s 8.0 time. in The other16neurones all were keptfor present vasopressin uniformly in interval interspike from constructed functions hazard firing pattern: their of analysingthe nature response)by orno and (inhibition Those firingcontinuously were identified as vasopressin neurones by theirresponse toCCK firing continuously. were seven and phasically werefiring 10 which of spikes/s), ±0.76 5.1 rate basal neurones andoxytocin SONvasopressin of activity on ofNKB Effect electrical the This article This protectedis by Allcopyright. rights reserved. 6C). Fig. ± -0.2 spikes/s; 0.7 thes activity of ontheelectrical effect significant resulting histograms and interspike-interval their either by toi.v. their excitatory neurones, orbyCCK response and which were identified the shape of range - spikes/s), all ofwhich2.6 were 6.9 record spikes/s. 1.5 at least by was it neurones increased infive the of but spikes/s), one basal; rate ± 1.7 higher in spikes/swas than 0.7 the mean injection, firing senktide after 40min 20and Between - spikes/s). 1.9 10.7 spikes/s (range We also recorded from seven putative oxytocin neurones (mean basal rate 5.25 rate ± neurones(meanbasal from putative 5.25 spikes/s; recorded We also oxytocin seven 0.8 neurones firing vasopressin continuously The seven The nine phasically firing neurones The nine phasically held for firing thatwere more than30minhada meanbasalrate after injection, at10minafter senktide was lost neurone one phasic Of the17neurones, ratswevasopressin 17 (mean recorded neurones from In the SON urethane-anaesthetised of neurones (30) but 6B). mainly neurones(Fig. neurones (30)but oxytocin absent from data revealed the presence of a post-spike hyperexcitability that is that hyperexcitability apost-spike of presence the data revealed tivity comprised bursts and intervening silences, and silences, and intervening comprisedbursts tivity e neurones (mean change at 20-40 min after senktide, after senktide, min at 20-40 change (mean e neurones hazard functions (30). Overall, senktide had no had (30).Overall, no senktide functions hazard ed in close proximity to identified vasopressin vasopressin identified to proximity close ed in st frequency was increased by 1.1 1.1 by st frequencywas increased ± spikes/s 0.6 of these neurones the firing rate was lower (by 0.9 0.9 (by rate lower was the firing neurones of these had a mean firing had rate of7.5 ±basal 1.7 P = 0.008, Mann- = 0.008, Accepted Article for inducing Fos expression in the SON in and PVN(38). Fos expression for inducing thePVN,in with SON or bodies incell either in the accord find NKB expression we to failed va with juxtaposition close fibres expressed in in NKB was NK3R. express cells, oxytocin all including SONitappearedthe neurones, all that butin (32); cells oxytocin some only by but cells vasopressin most by expressed is NK3R PVN, the cells andoxytocin invasopressin expressed densely Discussion This article This protectedis by Allcopyright. rights reserved. component, mediated by GABA, and a slower excitato andaslower mediated GABA, by component, in response resultsa complex in arcuate nucleus fibresACTH-containing originating thefrom arcuate electrophysiologically inprevious studies; inparticular, the SONis innervated by cells. oxytocin in also and perhaps tran a in NK3Rhas role circumstances physiological the P cells SON in and/or nucleus ofvasopressin tr in result senktide as aswell (40) hypotension and 41) (40, stimulation hyperosmotic both Interestingly, (40). nucleus the to translocated are in NK3R receptors cells, In vasopressin senktide. inresponse of oxytocin the PVNwas noreleaseto within the samestudy, microdialysis by found neurones thus itappearsremains uncertain; notto be magnocellular oxytocin The (39). significancein ofNK3R phentolamine expression antagonist effect an appears by as plasma,the into be could be to indirect this theadrenergic but blocked it Hereweus 38). (10, senktide of andan expressi inFos increase (37), NK3R agonists selective of administration i.c.v. after secretion vasopressin in increase an showing studies previous the SON in neurones oxytocin not but neurones vasopressin of activity PVN. and theSON both to project NKB, co-express to known are which cells, kisspeptin-expressing thatarcuate the SON,and in to tracing anterograde mice,by project we found nucleus arcuate rat the in cells NKB-expressing some that showed Wealso (36). PVN neurones and to acontradiction study previous whichreported studies detect which failed to hybridisation It It has previously thati.c.v been reported inje previo as rat, the in that, confirmed we Here, Connections between the arcuate nucleus and the SON have been shown anatomically and anatomically shown have been SON the and nucleus arcuate the between Connections electrical the increases senktide of NK3R the agonist injection thati.c.v. weshowed Finally, ed a ( ofsenktide dose NKB NKB anslocation the cytoplasmof NK3Rfrom the to anslocation co-immunostaining ofNKB co-immunostaining SON neurones characterised by a fast inhibitory a fast inhibitory characterised by SON neurones VN. This raises that the possibility in diverse sopressin cells in both the PVN.However, both cells SON and in sopressin mRNA expression in (33-35), these in nuclei in but mRNA expression in the SON and PVN. We also confirmed that, that, in Weconfirmed also SONand in the PVN. on in on in the SONand i.c.v. PVN after administration scriptional regulation in in regulation scriptional ctions of senktide secretion oxytocin stimulate of ctions ternalised after ligand binding and subsequently subsequently and binding ligand after ternalised usly reported (31), neurokinin receptor NK3R is NK3R receptor neurokinin (31), uslyreported able to activate dendritic oxytocin releaseactivateas, in oxytocin able to dendritic nucleus (43, 44). Electrical stimulation of 44). theElectrical (43, stimulation nucleus ∼ ry component (45, 46). 46). componentThery (45, mediator of this 0.5 0.5 μ g) that has been reported to to g) thathas be maximal been reported . This is consistent with vasopressin cells (42), (42), cells vasopressin and vasopressin inSON and vasopressin β -endorphin- and -endorphin- in situ

Accepted Article reflects upregulation of NK3R expression in response to ligand withdrawal. withdrawal. ligand inresponse to expression NK3R of reflects upregulation lactation in neuronesoxytocin tokisspeptin of may responsiveness it heightened be(50), also the that ki of with a downregulation apparently associated activity supporting bursting in response tosuckli kissp that suggested, authors the as may it be, clear: the SONis apparent late in pregnancy(49). andlactation ofthis The is not significance physiological thatanexcitatory ac by suggested same the authors po were mediated indirectly, kisspeptin intravenous or oxytocin either on kisspeptin ofvasopressina neuronesminority neurones most oxytocin inthe hasexcitatory on only effects on SONbut Intravenous clear. kisspeptin are less kisspeptin effects of the but onbothof (47), thesetypes neuronal are expressed that receptors kappa-opioid at actions its via SON the in neurones and vasopressin bothoxytocin to inhibitory vesicl secretory to peptides of targeting differential on also and depending possibly postsynaptically, are present receptors onwhich depending actions different have may neurones and vasopressin oxytocin to pathway this of projections neurones. vasopressin in activation the of component but presen the excitatoryknown, component isnot This article This protectedis by Allcopyright. rights reserved. together and osteoporosis, confusion, an common include resulting offalls increased from mentalhealth burden: complications incidence serious a can be disorder, electrolyte common most the hyponatremia, elderly, Inthe hyponatremia. in are and vasopressin oxytocin yet oestrogen receptor, the express classical the vasopressin neurones the magnocellular neurones nor oxytocin magnocellular the Neither oxytocin. and/or vasopressin of andsecretion onthe expression steroids gonadal mediates influence of the modulated conspicuously is projection this because vasopressin secretion, and thishas been secretion, vasopressin vasopres in is alsoan increase SONan inthe neurones vasopressin pr secretion vasopressin el chronically to attributed are hyponatremia chronic Vasopressin regulates water uptake in the distal renal tubules and elevated levels can result levels can renal result elevated and the tubules distal in uptake water regulates Vasopressin but to theNKBprojection unclear, role the atpresent, of SON is, thephysiological Thus so and dynorphin, kisspeptin with isco-localised NKB nucleus, arcuate the of In neurones ogressively increases with age (3, 5), and the size of magnocellular 5), age (3, size and the magnocellular of with ogressively increases modulated by oest by modulated sin production (2). In aged rodents, there is also an increase in in increase an also is there rodents, aged In (2). production sin vasopressin neurones inthe SON, vasopressin neurones (48), but same but (48), that study reported d PVN is increased after the age of 60 years, suggesting that there there that suggesting years, 60 age of the after increased is PVN d giving anincreased riskoffrac linked to impaired osmoregulation (54). osmoregulation impaired to linked rogen (51-53). synthesis of oxytocin and the secretion of both and the ofoxytocin secretion synthesis ng and during parturition. However, as lactation is as However, lactation duringng parturition. and sspeptin and NKB expression in the arcuate nucleus arcuate inthe NKBexpression and nucleus sspeptin t data suggest that NKB may contribute to this to contribute thatNKBmayt data suggest es at different sites. Dynorphin is likely to be to likely is sites. Dynorphin different at es tion of i.c.v. kisspeptin onoxytocin neurones of kisspeptin i.c.v. of tion by gonadal steroids, it seems possible that it thatit seemspossible it steroids, bygonadal ssibly via the vagus. However a subsequent study study subsequent a However vagus. viathe ssibly eptin has a specific role in lactation, perhaps in perhaps in in role has lactation, a specific eptin evated secretion of vasopressin (1, 2): in humans, 2): in (1, vasopressin of secretion evated suggesting that the effects of theeffects of that suggesting tures. About halfofall About casestures. of no significant effect of no significant i.c.v. Accepted Article ventricle. third 3V, ME,eminence; median chiasm; optic OC, area. square ofthe enlarged views marked S the both in cells vasopressin to juxtaposition SON(B)a the sections incoronal of apparent andnucleus dorsally SON, towards laterally the to arcuate rostral the from project fibres NKB that Note hypothalamus. anterior rat the of section coronal Figure 3. membranes in the D. arrowed highlighted cells chiasm;in OC, optic thirdventricle.3V, co-localize. immunoreactivity (OT-NP) neurophysin whichNK3Randoxytocin- ofcells in examples arrowsindicate panels ofA The white and C. the right in indicated areas square thehighlighted of BandD andPVN views are (A) C). enlarged Figure 2. ventricle; chiasm. OC, the cell in optic third highlighted D. in arrowed 3V, membranes cell and fibres (VP-NP) co immunoreactivity vasopressin-neurophysin inwhich NK3R and cells examples of indicate arrows A white The panelsof C. and right the in indicated squareareas highlighted the of enlarged views are D and B (C,D). PVN and (A,B) SON Figure 1. Figure legends osteoporosis. new targetsforpreventing may vasopressin secretion lead ininappropriatelyto chain byaging increased the causal whichresults hyponatremia are dueinappropriat to hyponatr with it associated has women (55), been peakin an incidence has Osteoporosis secretion. vasopressin secretion has been causally linked has linked beencausally secretion vasopressin hypona chronic (2). aging In aging, accompanies and to contribute alike, and rodents may the pr leadsan increase to women. This decline in This article This protectedis by Allcopyright. rights reserved. Aging is also associated with a marked decline in sex steroid production menin both production and insex a marked with steroid decline associated also Aging is Expression ofNK3R Expression (green) of (magenta) coronal the in oxytocin sections rat cells SON in rat the coronal of in (magenta) ofNK3R cells sections Expression (green)vasopressin in (A) NKB (green) (A)NKB(green) and vasopr ely elevated secretion ofvasopr secretion ely elevated essin-neurophysin (VP-NP; (VP-NP; magenta) immunoreactivitya in essin-neurophysin the expression ofNKBin ofhumans the expression hypothalamus the nd PVN (C). nd PVN These(C). close fibres found in were ON (B) and PVN (C). The pa and ON PVN (C). (B) ogressive rise in vasopressin secretion that that secretion vasopressin in rise ogressive to osteoporosis, as has to osteoporosis, advanced age, particularly in postmenopausal postmenopausal in age, particularly advanced tremia resulting from from tremia resulting in emia (56), and almost 50% 50% andalmost ofchronicemiaof (56), reports wards the PVN. NKB-expressing fibres (green) are (green) fibres NKB-expressing thePVN. wards Note the expression of NK3R in axons and cell ofNK3R andcell axons in theexpression Note -localize. Note the expression of NK3R in ofNK3Rin Notetheexpression -localize. essin (1, 57). Thus, recognizing recognizing 57). Thus, (1, essin reduced gonadal steroid steroid gonadal reduced appropriately increased appropriately increased nels to the right show nels to Accepted Article showing the increase in electrical inaactivity inelectrical phas the increase showing ofsenktide pmol i.c.v.SON inresponse injection in (600 to 2µl) Figure 6. to cell vasopressin closein juxtaposition fibres YFP-containing show and left, the to panels the in squaresindicated white the of enlargements ofthe sections coronal vasopressin- and (yellow) projections kisspeptin shows B (blue). markerHoechst nuclear with staining Background (yellow). YFP with labeled cells Figure 5. chiasm. OC, channel; optic light TD, cells. transmitted within the presence confirmed beads of wehow illustrating (E), in shown ofthecell reconstruction 3D a F gives and beads, containing cell NKB-expressing aan of example another shows E cell. the within clearly beads these showing D shows arrow) beads(yellow and containsa few NKB expresses that NKB no and a but ofbeads contains a cell arrow) number (white that cell large of injection following arcuateretro-beads nucleus in example an localization showing section of Coronal Figure 4. This article This protectedis by Allcopyright. rights reserved. ( 245009 no. agreement grant under demonstration and development technological Programme research, Framework for Seventh Union’s theEuropean from byfunding part in and supported Grant), Support (Project Neuroendocrinology for by co-funded (Ref. the and Royal Society NF130516), Acknowledgments neurones. phasicvasopressin frequency in intraburst lines.dotted (E) Mean change inactivity in phasic quotient vasopressin neurones; (F) Mean change in recording showing individual spikes; the extract is fromtheportion of the recording indicated by the ne oxytocin and circles) (white vasopressin in circles)and oxytocinneurones (black diamonds). (C) Mean(SE) changefiring in rate per10-min bins senktide: the firing rate is in1-splotted bins. (B) Mean hazard(SE) functions for vasopressin (white from the IMPACT facility at the Univers imaging This work was supported by the Newton International Fellowship program awarded to RPR RPR to awarded program Fellowship International Newton bythe wassupported work This Changes in electrical activity in magnocellu a in nucleus arcuate the of section A coronal retro-beadssite of showinginjection SONtheCoronal the sectionrat (A) (magenta). of (B) kiss1- Cre/eGFP Neurofast

mice in the SON (B) and PVN mice SON(B) inthe (C s. 3V, third ventricle; OC, optic chiasm. ventricle; chiasm. OC, third optic s. 3V, ). We thank Drs Trudi Gillespie and Ulrich Wiegand and Ulrich Gillespie We). Trudi Drs thank urones (D)Extract(blackurones diamonds); ofvoltage neurophysin magenta) immunoreactivity (VP-NP; in neurophysin ity of Edinburgh for technical assistance with with assistance technical for Edinburgh ity of the cell inC plane taken at the cell focal a different ically vasopressin neuroneinresponse firing ically to of NKB (green) and retro-beads (magenta) the in the rat SON. (C) Enlarged view of(B) showing a lar vasopressin and oxytocin neuronesofthe and laroxytocin vasopressin kiss1- the British Academy, and the British Society Academy, and the British the Cre/eGFP mouse showing kisspeptin kisspeptin showing Cre/eGFP mouse in vivo . (A) Representative recording recording Representative (A) . ). The panels onthe rightare Accepted Article macaques. macaques. SJ Krajewski ST, Stalcup T, Sandoval-Guzman cynomolgus young in balance energy and reproduction regulating axes neuroendocrine on the 10. Metab Endocrinol Interactions al. et L, Pinilla F, Ruiz-Pino R, Pineda SM, McConkey JM, VM, Castellano Navarro rat. female the in secretion GnRH of control the in B neurokinin and kisspeptin between 9. Endocrinology Molnar CS, Sipos MT,Vida B, Ciofi P, Borsay BA,Racz etal. K, Morphological evidence for man. aging the of nucleus theinfundibular in Bsignaling neurokinin and kisspeptin enhanced 8. Neuroendocrinol Neuroendocrinol Tac2-enhanced of ovariectomised nucleus the reduces oestradiol Chronic NE. Rance NT, McMullen SJ, Krajewski-Hall M, Cholanian arcuate the in neurones B/dynorphin) (kisspeptin/neurokinin KNDy of density spine dendritic 11. women. ribonucl messenger substance-P and neurokinin-B 7. Rance NE, Young WS, 3rd. Hypertrophy and incr and Hypertrophy 3rd. WS, Young NE, Rance 7. 6. Almeida TA, Rojo J, Nieto PM, Pinto FM, Hernandez M, Martin JD, et al. Tachykinins and and Tachykinins etal. JD, Martin M, Hernandez FM, Pinto PM, Nieto J, Rojo TA, Almeida relationships. and activity structure receptors: tachykinin 6. regulation. fluid on theimpact and menopause during changes Hormonal NS. Stachenfeld Reprod Sci hypothalamus. human the of Ageing DF. 5. Swaab 4. DeSant J, Reichmuth N, Goswami G, Tamma perspectives. current aging: 3. 1985; the in neurons oxytocin and Thevasopressin RW. Verwer CW, Pool DF, E,Swaab Fliers dementia. in senile and with aging changes nucleus; paraventricular and human supraoptic 2. RW Schrier R, Kluge HM, Chung RJ, Anderson vasopressin. thepathogenetic of epidemiologyrole and 1. References antibodies. oxytocin-neurophysin and vasopressin- the us Gainer forkindly providing with kindly for Ciofi Philippe Dr microscopy, confocal This article This protectedis by Allcopyright. rights reserved. 342:

Endocrinology 45-53. 45-53. 2014; J Neuroendocrinol J Neuroendocrinol 2012; 2015; 21: 2011; 2011; 555-561. 153: 1991; 27: 300: 5428-5439. 253-263. Endocrinology E202-210. 128: 2004; 2004; 2239-2247. 16: 146-153. 2015; green fluorescent protein transgenic mice. mice. transgenic protein fluorescent green 156: providing us with the NK3R antibody, and Prof Hal Prof and antibody, NK3R the with us providing eic acids in the hypothalami of postmenopausal postmenopausal of hypothalami the in acids eic , Voytko ML, Rance NE. Effects of ovariectomy ovariectomy of Effects NE. Rance ML, , Voytko o NG, Valenti G. Aquaporins, vasopressin, and and vasopressin, Aquaporins, G. Valenti o NG, . Hyponatremia: a prospective analysis of its its of analysis prospective a Hyponatremia: . 777-788. eased gene expression expression gene eased Horm Res Res Horm Ann Intern Med Curr Med Chem Med Curr 1995; 1995; 1985; 43: 2004; 8-11. 102: of neurons containing containing neurons of 11: 164-168. 2045-2081. J Am JPhysiol Brain Res Brain

Accepted Article 13. Eguchi T, Takano Y, Hatae T, Saito R, Nakayama Y, Shigeyoshi Y, et al. Antidiuretic action of of action Antidiuretic etal. Y, Y, Shigeyoshi Nakayama R, T, Saito Hatae Y, Takano T, Eguchi nucleus. paraventricular therat receptorin NK-3 tachykinin 13. 2010; Durand AR, Adamantidis V, Gradinaru F, Zhang structures. brain mammalian probing for technology circuits: neural of interrogation 20. specific of manipulation functional and labeling High-resolution ZJ. Huang SJ, Kuhlman expression. gene viral Cre-activated by brain mouse in types neuron 19. Academic Press, 2006. 25. Gottsch ML, Popa SM, Lawhorn JK, Qiu J, Tonsfeldt KJ, Bosch MA, et al. Molecular properties properties al. Molecular et MA, Bosch KJ, Tonsfeldt J, Qiu JK, Lawhorn SM, Popa ML, Gottsch themouse. of nucleus thearcuate in neurons of Kiss1 25. nucleus. Ramaswamy S, CiofiSeminaraAli SB, B, P,Amin NA, Plant TM.Neurokinin B stimulates GnRH and mulatta) (Macaca male monkey in the release 24. Endocrinology al. MR, et CV,Jafarzadehshirazi Oliveira de LM, JT,Coolen Smith MN, Lehman RL, Goodman B. neurokinin and A dynorphin both express ewe of the nucleus thearcuate in neurons Kisspeptin 23. mouse. ofthe arcuate nucleus Navarro VM, Gottsch ML, Chavkin the in C,Okamura Bneurons H,Cliftonkisspeptin/dynorphin/neurokinin by DK, Steiner secretion hormone RA. Regulationgonadotropin-releasing of 22. nucleus. arcuate B neurokinin and dynorphin of Coexpression NE. Rance SJ, Krajewski Letts PA, BurkeMC, Morpholo hypothalamus: rat the in immunoreactivity 21. 14. Howe HE, Somponpun SJ, Sladek CD. Role Role CD. Sladek SJ, Somponpun HE, Howe 14. rats. loaded administration Central etal. R, Saito Y, Nakayama M, Tran T, Matsumoto Y, A,Takano Saigo of senktide, tachykinin a NK-3 agonist, has an antidiuretic action by stimulating AVP water- inrelease 12. This article This protectedis by Allcopyright. rights reserved. 8 PaxinosG, Watson C. 18. Cetin A, Komai S, EliavaM,Seeburg PH, Osten P. Stereotaxic gene rodent delivery in the brain. 17. the in pathways tracing for techniques Recent A. Grimaldi D, Garbossa M, Repici A, Vercelli mammals. adult and developing of system nervous central 16. Physiol vasopressin of release systemic to receptorcontribution NK3 Tachykinin FW. Flynn GE, Haley challenge. hypotensive and osmotic to response in and oxytocin 15. neurons. oxytocin and vasopressin 5: Nat ProtocNat 2007; 2007; 439-456. Endocrinology Neurosci Lett 293: 2007; J Comp Neurol J Comp 2006; R931-937. 148: 2010; 2010; 1: 5752-5760. 1993; 3166-3173. 151: The Rat Brain Stereotaxic Coordinates Stereotaxic Brain The Rat J Neurosci J Neurosci 2006; 159: 4494-4503. 4494-4503. J Neurosci 187-190. 498: 2009; 712-726. 2004; 29: of neurokinin 3 rece 3 neurokinin of is colocalized with kisspeptin in the arcuate 11859-11866. 24: gic evidence of interrelated function within the within the function interrelated of gic evidence Endocrinology 10103-10110. R, Airan RD, de Lecea L, et al. Optogenetic Optogenetic al. L, et Lecea de RD, Airan R, Brain Res Bull Res Brain Brain Res Brain Am J Physiol Regul Integr Comp Comp Integr Regul Physiol J Am , 6 2011; PLoS One PLoS 1996; th ptors in supraoptic supraoptic in ptors Edn. San Diego, CA: CA: Diego, San Edn. 2000; 152: 743: 2008; 2008; 51: 4298-4309. 49-55. 11-28. 3: Nat Protoc Nat e2005.

Accepted Article 29. Leng G, Sabatier N. Electrophysiology of Magnocellular Neurons Neurons Magnocellular of Electrophysiology N. Sabatier G, Leng 29. PaxinosG, Franklin KBJ. Press, 2004. 28. 493: paraventricular nucleus of the hypothalamus. hypothalamus. the of nucleus paraventricular nuclear and internalization dendritic Stress-induced A. Lessard FW, Flynn Z, Miklos translocation of the neurokinin-3 (NK3) recept 32. 290-310. the of Localization N. Mizuno S, Nakanishi H, Ohishi M, Takada R, Shigemoto YQ, Ding rat. the of system nervous central the in (NK3) receptor K neuromedin 31. neurones ratsupraoptic in patterning spike Phasic G. Leng M, Ludwig CH, Brown N, Sabatier vitro. in and vivo in 30. 28. Neuroendocrine of Neurophysiology eds. JG, Tasker 27. Tye KM, Mirzabekov JJ, Warden MR, Ferenczi EA, Tsai HC, Finkelstein J, et al. Dopamine Dopamine etal. J, Finkelstein HC, EA, Tsai Ferenczi MR, Warden JJ, Mirzabekov TyeKM, expre and encoding neural modulate neurons 27. 33. Warden MK, Young WS, 3rd. Distribution of cells containing mRNAs encoding substance P P substance encoding mRNAs containing cells of Distribution 3rd. WS, Young MK, Warden system. nervous central rat the in B and neurokinin 33. 34. Lucas LR, Hurley DL, Krause JE, Harlan RE. Localization of the tachykinin neurokinin B neurokinin tachykinin of the Localization RE. Harlan JE, Krause DL, Hurley LR, Lucas 34. Bo GD, Stuber A, Adamantidis F, Zhang HC, Tsai conditioning. behavioral for sufficient is neurons dopaminergic 26. This article This protectedis by Allcopyright. rights reserved. Bealer SL, FW.Central Flynn neurokinin 3receptors systemic increase oxytocin release: interaction with norepinephrine. 39. 882: of senktide- injection al. Intracerebroventricular et XE, Guo CJ, Su JQ, LY, Xu Su J, Shi YD, Ding rat. in the neurons hypothalamic vasopressin-containing in expression Fos induced 38. Lett by induced release Vasopressin M. Massi G, deCaro G, M, Costa Perfumi A, Saija C, Polidori receptors. neurokinin-3 central of toactivation due is tachykinins of injection intracranial 37. receptor. 1 type II angiotensin the in neurons Arginine-vasopressin S. Masuko JE, Krause V, Karpitskiy H, T, Kawano Hatae and receptor NK-3 tachykinin the co-express and B neurokinin produce hypothalamus rat 36. therat B in neurokinin expressing neurons of Distribution JE. G,Krause J, Sperk Marksteiner situ hybridization. in and immunohistochemistry brain: 35. 51: hybridization. situ in and byimmunocytochemistry brain rat in peptide precursor 317-345. 1989; 1989; 537-541. 95-102. 103: 320-325. J Physiol 2004; 2004; Arch Histol Cytol The Mouse Brain in Stereotaxic Coordinates Stereotaxic in Brain Mouse The Exp Neurol Neurol Exp 558: 161-180. 2003; 2003; ssion of depression-relatedssion of behaviour. Brain Res or in vasopressinergic profiles of the rat rat the of profiles or in vasopressinergic 2001; 184: J Comp Neurol Neurons. John Wiley &Sons, Ltd, UK; 2015: 3- 64: nci A, de Lecea L, et al. Phasic firing in in firing Phasic al. et L, Lecea de A, nci 1027-1033. 2014; J CompNeurol 37-44. 1590: Science 1988; 1988; 31-44. 1992; 2009; J Comp Neurol Neurol J Comp in vivo in 272: . Elsevier Academic . 317: 90-113. 324: . ArmstrongIn WE, 341-356. Neuroscience 1080-1084. Brain Res Nature 1996; Neurosci 2013; 364: 2000; 2000; 1992; 1992;

Accepted Article Neuroscience of nuclei to receptor 3 neurokinin tachykinin of Trafficking FW. Flynn Z, D,Zhang Jensen th of nucleus paraventricular the in neurons 41. IntegrPhysiol Comp HaleyGE, Flynn FW. Agonist and hypertonic saline-induced hypothalamus. of the nucleus trafficking theparaventricular within neurons of thevasopressin NK3-receptorson 40. This article This protectedis by Allcopyright. rights reserved. 9 Scott GnRHkisspV, Brown CH. the axis: Beyond 49. Scott V, Brown CH. Kisspeptin activation of supraoptic nucleusEndocrinology neurons invivo. 48. dependence morphine and inhibition opioid Local G. Leng JA, Russell CH, Brown M, Ludwig vivo. in therat in neurones oxytocin nucleus of supraoptic 47. nucleus supraoptic tothe nucleus arcuate the from projection GABAergic G. Leng M, Ludwig in the rat. 46. projection a for evidence Electrophysiological R. Bunting RE, Dyball H, Yamashita G, Leng nucleus. to thesupraoptic from nucleus thearcuate 45. ACTH(1-39)- of origin of cells and distribution The SA. Joseph LW, Swanson PE, Sawchenko nuclei. supraoptic and paraventricular the in varicosities stained 44. parturition. thearcuate in cells Beta-endorphin SL. Meddle JA, G, Russell Leng RJ, Bicknell AJ, Douglas and pregnancy during in expression changes and nucleus supraoptic tothe projections nucleus: 43. challenge. Flynn FW, Jensen DD, Thakar A, Xu X, Flynn SW, Zhang Z. Neurokinin in H4 3receptor and H3 histone forms acetylated a with complex 42. 53. Skowsky WR, Swan L, Smith P. Effects of sex steroid hormones on arginine vasopressin in in vasopressin on arginine hormones steroid sex of Effects P. Smith L, Swan WR, Skowsky rats. female and male castrated and intact 53. from therat. explants neurohypophysial Sladekrole CD, R, steroid of KL, in hormones the Swenson Sidorowicz HE.The Kapoor re oxytocin and vasopressin of regulation 52. Neuroendocrinol magnocellular of regulation Physiological JE. Stern M, Ludwig JS, Bains CH, Brown neurosecretory cell activity: integration of intrinsic, local and afferent mechanisms. 51. Neuroendocrinol nucleus ofarcuate Characterisation MS. Smith KL, Grove P, Ciofi M, C,Kirigiti True rat. the in lactation during regulation and projections Bneuronal kisspeptin/neurokinin 50. lactation. and pregnancy Neurosci Lett Neurosci Am J Physiol Regul Integr Comp Physiol Physiol Comp Integr Regul Physiol J Am J Neuroendocrinol Neuroendocrinol J 2008; 2011; 2013; 2011; 2006; 2006; 155: 152: 23: 25: 2000; 2000; 308-316. Adv Exp Med Biol 3862-3870. 52-64. 678-710. 290: 281: 2002; R1242-1250. 195-197. 14: 768-777. Exp Physiol lease and mRNA expression in hypothalamo- in expression mRNA and lease Endocrinology 2013; e hypothalamus following osmotic challenge. challenge. osmotic following hypothalamus e hypothalamic neurons 784: 2011; Neurosci Lett 2000; 201-218. eptin regulation of th of eptin regulation 301: 1979; J Physiol 85: R822-831. 171S-177S. 104: Brain Res 1988; 1997; 105-108. following hyperosmotic hyperosmotic following 89: 505: 1982; 146-151. e oxytocin system in 145-152. 232: Am JPhysiol Regul J 365-374. J Accepted Article Invest Clin J Eur clinical of Pathogenesis E. E, Ritz Hackenthal A, Schomig W, Rascher H, Pehrisch PA, Gross va of observations hyponatremia: 57. Y, Tian Y, J, Sugimura Barsony JG, Verbalis osteoporosis. 56. future. the and now Osteoporosis: LC. Hofbauer S, Khosla TD, Rachner 1276-1287. 55. in involved Mechanisms etal. A, Aubert J, Dudit N, Mota De K, Palin JC, Delpech J, Sauvant aging. during dysfunction neuron vasopressin/apelin dual 54. This article This protectedis by Allcopyright. rights reserved. J Bone Miner Res Miner J Bone 1987; 1987; 17: 123-129. 2010; sopressin and fluid intake in 100 hyponatremic medical patients. patients. medical hyponatremic 100 in intake andfluid sopressin 25: 554-563. Adams DJ, Carter EA, Carter AdamsDJ, et PLoS One One PLoS 2014; al. Hyponatremia-induced Hyponatremia-induced al. 9: e87421. e87421. Lancet 2011; 377:

Accepted Article