Differential Effects of Relaxin-3 and a Selective Relaxin-3 Receptor Agonist on Food and Water Intake and Hypothalamic Neuronal

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Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats Camila de Ávila, Sandrine Chometton, Christophe Lenglos, Juliane Calvez, Andrew L. Gundlach, Elena Timofeeva

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Camila de Ávila, Sandrine Chometton, Christophe Lenglos, Juliane Calvez, Andrew L. Gundlach, et al.. Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on foodandwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, Elsevier, 2017, 336, 10.1016/j.bbr.2017.08.044. hal-01603277

HAL Id: hal-01603277 https://hal.archives-ouvertes.fr/hal-01603277 Submitted on 27 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., laect hsatcea:de as: article this 28-8-2017 cite 21-8-2017 Please 5-6-2017 date: Accepted date: http://dx.doi.org/10.1016/j.bbr.2017.08.044 Revised S0166-4328(17)30952-X 11063 date: BBR Received in: appear To Reference: DOI: PII: Elena Gundlach, Timofeeva L. Andrew Calvez, Juliane Lenglos, de Camila Authors: hypothalamic rats and in intake activity water neuronal and food relaxin-3 on selective agonist a receptor and relaxin-3 of effects Differential Title: Manuscript Accepted rosmyb icvrdwihcudafc h otn,adallgldsliesthat disclaimers process legal production all the and pertain. during content, journal that the the affect note to could Please apply which manuscript. form. discovered the ﬁnal be its of may in errors version publication. proof published early for resulting is this the accepted of it providing review been before and are typesetting, has copyediting, we that undergo customers will manuscript manuscript our The unedited to an service of a ﬁle As water PDF and a rats. is food This in on activity agonist receptor Elena.Differential neuronal relaxin-3 Timofeeva hypothalamic selective http://dx.doi.org/10.1016/j.bbr.2017.08.044 L, a and Andrew and intake relaxin-3 Gundlach of Juliane, effects Calvez Christophe, Comment citer cedocument: vl,Snrn hmto,Christophe Chometton, Sandrine Avila, ´ eaiua ri Research Brain Behavioural

vl aia hmto adie Lenglos Sandrine, Chometton Camila, Avila ´ eaiua ri Research Brain Behavioural Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., E Phone: (1 Sainte 2725 Chemin Centre de de recherche de l’Institut pneumologie cardiologie et de Y3106, Québec, Elena Timofeeva, Ph.D. 1 Melbourne, Australia. Victoria 3010, and b (QC), G1V 0A6, Canada. Québec Laval, Université Québec, de pneumologie de et cardiologie de universitaire l’Institut a *Thesecontributed authors equally tothis Differential effectsDifferential relaxin of

Corresponding Corresponding Faculté de Médecine, Département de Psychiatrie et de Neurosciences, Centre de recherche de de recherche de Centre Neurosciences, de et Psychiatrie de Département Médecine, de Faculté - The Florey Institute of Neuroscience and Mental Health, Mental and Neuroscience of Institute Florey The Camila deCamila Ávila* mail: mail:

lry eatet of Department Florey [email protected] - 418) 656 author: - a 8711 ext. 3749; Fax: (1 3749; ext. 8711 - , Sandrine Chometton , Sandrine Foy, QuébecG1VFoy, (QC), 4G5; Canada, intake and and intake Comment citer cedocument:

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1 , The University of Melbourne, Melbourne, of University The ,

a , Juliane Calvez , Juliane

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AndrewL.

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[ 3 - , 3; foodintake; 7 ] Comment citer cedocument: Hmn and Humans . - 1 [

N3 3

seven peptides known initially for their major roles in reproduction, in roles major their for initially known peptides seven - (H1), 5 the ] – .

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simultaneous activation of RXFP3 and RXFP1 by RLN3 on food and water and food on RLN3 by RXFP1 and RXFP3 of activation simultaneous

RLN3 [ n RXFP1, and a

administration

role treatment of treatment Comment citer cedocument:

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o hormone (LH) and follic and (LH) hormone

[ od intake food -

- 28 selective A2, - novmn o RXFP3 of involvement opr te fet of effects the compare the the RXFP3 A2 produced inhibitory effects inhibitory produced A2 ] .

mediated by RXFP1 by mediated

the specific activation of RXFP3 by RXFP3 of activation specific -

n diin chronic addition, In tuctd L3 nlge with analogue RLN3 truncated a fos the eety a Recently, - a A2

mRNA pplication of pplication analogue 2( expression

from male rats male from

[ increased 21

- to h i The 23

increases expression

the ] high

and

ntracerebroventricular RXFP3

pharmacological food intake in satiated rat satiated in intake food f c of RLN3 ular -

affinity

ae intake water and/ [ L3 a RLN3,

lsa corticosterone plasma

icv - stimulates - stimulating hormone hormone stimulating Gvn h possible the Given . [ F ] 27 -

. r XP i these in RXFP1 or os

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on RXFP3 RXFP3 protein 32 ,

was non oxyto represent -

36 its selective selective its

the [

24 ] - effects produced selective ,

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( high high ] icv

an an of in 6 s )

by our institutional animalby care ourinstitutional committee. the levels of levels

n ae rats male in

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mRNA . n addition, In

2. in

- the MATERIALS ANDMATERIALS METHOD pituitar

expression otaa days postnatal ),

regulation of food intake a intake food of regulation with testosterone o the new environment. Rats had free had Rats environment. new the o y

h atvto b RN ad RXFP3 and RLN3 by activation the (St

- an ambient temperature of 23 ± 1°C. Rats were left were Rats 1°C. ± 23 of temperature ambient an oaa (P) axes (HPG) gonadal - . Constant, QC, Canada). The rats were rats The Canada). QC, Constant,

and

, 57

oxytocin, vasopressin, epinephrine, and and epinephrine, vasopressin, oxytocin, -

maintained on a 12:12 h 12:12 a on maintained 59 250 , for [ 30 - 275 , and the present protocol was was protocol present the and ,

] nd and handled according to the the to according handled and , activity on S

g, the

food and water intake and water food . = 32) = n 1 kcal/g, Harlan Teklab, Teklab, Harlan kcal/g, 1 hypothalamic

were access to standard standard to access

were dark - investigated A2

- of light - purchased purchased housed pituitary h key the

cycle

and

by by in 7 -

- administration gauge) that projected 1 projected that gauge) Icv

was measured immediately before the before immediately measured was

- was rat Each - were Rats nitni II angiotensin A2 A2 injections peptide

were dissolved in dissolved were (n

of Neuroscience and Mental Health Mental and Neuroscience of

12) to measure chow intake, water intake, and c and intake, water intake, chow measure to

of infused

BW. or RLN3 andRLN3 anl Patc One (Plastics cannula

deeply anesthetized anesthetized deeply

ad libitum ad h cnua oiin a vrfe b a oiie isgnc epne to response dipsogenic positive a by verified was position cannula the injection Comment citer cedocument:

a , Belmont, CA, USA CA, Belmont, , – CSF

E a 7 days. days. 7 mm caudal to the bregma, 1.5 bregma, the to caudal mm icv

ach rat received rat ach mm below the tip of the guide cannula. guide the of tip the below mm between

(n a with CSF

RXFP3

3 n pr rat, per ng (30 - = fed rats in the early light phase (8 phase light early the in rats fed R

10)

. The . ats were randomly divided randomly were ats riiil cerebrospinal artificial - ujc de subject

were administered over 1 min 1 over administered were - A2

dose rats using , Roanoke ,

in rats three icv ) or )

were returned to their home cage home their to returned were used used sign. sign.

injections to assess the food the assess to injections

Sigma 4

1.1

injections isoflurane % [ 30 was

, I nmol nmol , VA, USA VA, , ] Parkville cv . -

Aldrich

chosen chosen

netos f 5 of injections the to lateral mm of RXFP3 of li ( fluid

separated by separated - fos in , Victoria, Australia Victoria, ,

S. oi, MO Louis, St. , based on an earlier an on based and to three groups and all and groups three to ) aimed at the lateral ventricle ventricle lateral the at aimed )

a mRNA CSF)

mlne wt a 26 a with implanted After injection, the injector injector the injection, After

:00 :00 - via stainless steel injectors injectors steel stainless via A2 ( A2

–

μ one week one expression

l Dr 11:00 midline, and 3.5 and midline,

1.1 f RLN3 of and water and

MA Hossain, The Hossain, MA

s ). ml f human of nmol ). . All .

A ). Body weight Body report

of of

. RLN3 and and RLN3 dipsogenic

One week One injections (n

the same the intake in in intake peptides

- = o gauge n

10) mm

the 8 ,

Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., 4% of ml 200 by followed collected was blood intracardial euthanasia. before cages home their to transferred were RXFP3 RLN3, 2. 5. the difference ofthe chowweight inthe post RXFP3 test the after andcontainerbefore to was intake water 2. 4. Only > injection; after min 2 least at continuously drank and delay without water drink to started rat the if validated was response m 5 chow - paraformaldehyde for one week. one for paraformaldehyde B injection Water rats l Cnua lcmns ee liaey ofre hsooial a te n o te study. the of end the at histologically confirmed ultimately were placements Cannula . lood sample lood and b t h ed f xeiet rt wr sciie 3 mn after min 30 sacrificed were rats experiment, of end the At - were Rats . A2 Water intakeWater with correct cannula placementwith correct cannula were included

(n and chow i chow and

in home cages home in = - 2 r CF uig h ery ih phase. light early the during aCSF or A2

12) and and 12) measured at 0, 30, 60 30, 0, at measured

injected R ats were ats was measured bywas Comment citer cedocument: a ntake a 4% rain preparationrain

CSF (n CSF icv

parafo with a volume of volume a with deep

without without

with RLN3 with , .

= ly anesthetized (60 mg/kg ketamine plus 7.5 mg/kg xylazine), mg/kg 7.5 plus ketamine mg/kg (60 anesthetized ly and they were perfused intracardially with 100 ml of saline saline of ml 100 with intracardially perfused were they and One week later, the r the weeklater,One

rmaldehyde solution. rmaldehyde 10) and chow intake was measure was intake chow and 10) ,

where where access to access B

and

rains were then transferred to a solution containing solution a to transferred then were rains remaining remaining calculating the difference in the weight of the cage water cageweight water the calculating difference of inthe the (n

120 min 120

they

water consumed during consumed water water 10

had no acce no had ), and thatprovided immediately thetest before

post RXFP3 . Food intake was measured by measured was intake Food . ats wereats in

- B injection

the data analysis.the data rains were removed removed were rains meitl atr neto, h rats the injection, after Immediately - A2 ss to chow and water and chow to ss icv

in home cages home in

injected with RLN3with (n injected =

d at 0, 30, 60 and and 60 30, 0, at d 1 h final the the 2 ) or 30 min min 30

a CSF ( CSF and post and icv without post in the in

n neto of injection calculating

= - injection -

120 fixed in fixed

10 30 min 30

access access = ) and )

10), min

4% 4% for for 9 .

S; estvt: .4 gm) eiehie n nrpnprn (Abnova, norepinephrine and epinephrine pg/ml), 2.84 sensitivity: USA; n situ n until eemnto o pam co plasma of Determination µ L (3 centrifuged were samples Blood g/m evels the

02% n 0.1 in (0.25% 00 Heidelberg 2000, hybridization for l

n 100 in as described as vity: and36pg/ml,respectively) 10pg/ml determination of determination f c of – , 2

USA; sensitivity: 0.13 ng/ml), vasopressin (Enzo Life Science, Life (Enzo vasopressin ng/ml), 0.13 sensitivity: USA; 0 °C °C

- An ro, I USA MI, Arbor, Ann , plasma levels plasma fos

M Tris mM 20% sucrose, 20% Comment citer cedocument:

n cl seie cryoprotection sterile cold a in RA xrsin n the in expression mRNA

[

32 Gray. Thirty Germany). , 4% titoaie p 8.0 pH trietholamine, M

- , c

C cnann 50 containing HCl

- 37 corticosteron fos paraformaldehyde, digested for 30 min at 37°C with proteinasewith 37°C at min 30 fordigested paraformaldehyde,

] in duplicate in estvt, 5. sensitivity,

mRNA Briefly, the sections were mounted on poly on mounted were sections the Briefly, before being before ncentration , and , 000 g for 15 min at 4 at min 15 for g 000 20% glycerol20%

sniiiy 5 sensitivity, ; e, e,

testosterone using using

- pg/ml), 7 s micron sectioned

M DA p 8.0 pH EDTA, mM of hypothalamus

commercial testosterone, . .

thick , n dhdae truh graded through dehydrated and ), ouin containing solution

, xtcn Ponx Pharmaceuticals, (Phoenix oxytocin 12 h later using a sliding microtom sliding a using later h 12

.0 oxytocin, vasopressin, epinephrine, vasopressin, oxytocin,

pg/m coronal °C) and plasma was stored was plasma and °C)

ELISA kit ELISA were l ) corticosterone ,

, ctltd ih acetic with acetylated ), t sectio sotrn (no Life (Enzo estosterone eetd using detected of s ns were collected collected were ns 50 mM sodium sodium mM 50 - L

c -

Farmingdale, Farmingdale, lysine coated coated lysine orticosterone api City Taipei , oxytocin, oxytocin,

n situ in at 10 e - ,

sitive hybridization signal hybridization sitive bregma) m Image analyses s l (PVNm) ,wse n ecnigcnetain o oim chloride sodium of concentrations descending in washed ),

r track beta track r before S lides were examined with dark with examined were lides

7 prevent saturation of the integrated signal; and the and signal; integrated the of saturation prevent and the OVLT (from 0.15 mm to 0.00 mm rostral to rostral mm 0.00 to mm 0.15 (from OVLT the and hog an through

cpm/m developing

( (DVC Company Inc., Austin, TX, USA) TX, Austin, Inc., Company (DVC from - ethanol ethanol , 2 nuclear emulsion (Eastman Kodak (Eastman emulsion nuclear 2 Richmond l

) 1.72

for Comment citer cedocument: tao gradient. ethanol . , cleared withcoverslipped mounting intoluene andmedium. . Sections were incubated overnight with antisense with overnight incubated were Sections .

mm Tissues were counterst were Tissues c Wlitn V, USA VT, Williston, , - in fos

to 1.80 mm 1.80 to

sections of the parvocellular PVN (PVNp) and the magnocellular the and (PVNp) PVN parvocellular the of sections il O, Canada ON, Hill, gnrtd rm h 2116 the from (generated

- oim irt slto, ietd ih ribonuclease with digested solution, citrate sodium -

field microscopy using an Olympus BX61 microscope BX61 Olympus an using microscopy field hratr s Thereafter,

caudal to bregma),to caudal I

Verma, Salk Institute, La Jolla, CA) at 60°C. 60°C. at CA) Jolla, La Institute, Salk Verma, ained with thionin with ained . mgs ee curd ih a with acquired were Images ). . The ). - fos , Rochester, NY, USA Rochester,NY, , ie wr dfte i tlee dpe in dipped toluene, in defatted were lides

mRNA

system was calibrated for each set of of set each for calibrated was system n nlzdwt See Investigator Stereo with analyzed and - p rget f a c rat of fragment bp the SON the optical density ( density optical

- bregma) the MPA the oim ci sodium e , dehydrated through graded through dehydrated , (from 1.32 to 1.56 caudal 1.56 to 1.32 (from the

[

of OD 38 ), and exposed for exposed and ), [ (from 0.3 (from rt ouin, and solutions, trate 35 ] S OD , ] and subtracting and - labeled cRNA cRNA labeled

) pi

DVC - reading fos es f the of xels

rostral to rostral (opaque) - cDNA; cDNA; - 2000C A (20 (20 A S lides

was was 11 2

fos 0.05 <

Food water intake and Statistical analyses mRNA A

intake water and intake food cumulative For SEM. ± mean as presented are Results statistical software to 0.4 to

threshold depicting positive hybridization signal was set was signal hybridization positive depicting threshold n fr c for and Post a cniee saitcly significant statistically considered was a

- CSF expression was assessed using using assessed was expression a repeated way mm caudal to caudal mm -

hoc for all micrographs captured with the same luminosity same the with captured micrographs all for - injected group (mean value of a rat/mean value of value rat/mean a of value (mean group injected

- comparisons between the groups were performed using using performed were groups the between comparisons fos - Comment citer cedocument: fos

mRNA ,

test when the main and inte and main the when test mRNA. V6 - RXFP3

measures ANOVA was used to detect the main and interactive interactive and main the detect to used was ANOVA measures bregma) .04 (

analyses GraphPad Software

and the LHA the and

and one , 3.

a CSF) and CSF) the the RESULTS - way ANOVA was used to detect the effect of of effect the detect to used was ANOVA way l score of each rat was normaliz was rat each of score l uuaie ra f h pstv hybridization positive the of area cumulative

. Statistical analyses were performed using using performed were analyses Statistical . bilaterally (from 2.9 to 3.4 mm caudal to bregma to caudal mm 3.4 to 2.9 (from

ractive ANOVA effects were significant were effects ANOVA ractive I

nc., La USA) CA, nc., Jolla, time (0, 30, 60 30, (0, time

on two or three or two on for a

CSF group) to obtain the obtain to group) CSF OD with pixels . or 120 min) 120 or

The OD measurements OD The Fisher’s consecutive brain brain consecutive .

ed to the mean the to ed . For plasma plasma For . protected > 5 times times 5 ) [ 38 12 ] . ,

. Compared to RLN3, RLN3, to Compared . = 4.342, p < 0.0006) on water intake. water on 0.0006) < p 4.342, = - c The c The 3, 116 3, A2 (1.1 ± 0.5 mg/g BW, 5.6 ± 1.4 mg/g BW, mg/g 1.4 ± 5.6 BW, mg/g 0.5 ± (1.1 A2 - revealed measures c effects after 30 min 30 after effects c 9.5 ± 1.5 mg/g BW mg/g 1.5 ± 9.5 the between

) 2, 2, = 31.63, p < 0.0001) < p 31.63, =

umulative water intake was significantly increased by RLN3 (12.2 ± 2.7 mg/g BW, BW, mg/g 2.7 ± (12.2 RLN3 by increased significantly was intake water umulative (Fig. 1A). 1A). (Fig. mltv fo itk ws infcnl icesd by increased significantly was intake food umulative c 108 umulated icv 30, 60, and and 60, 30,

) and RXFP3 and ) = 30.88, p < 0.0001) and a significant interaction significant a and 0.0001) < p 30.88, =

that compared to the to compared that

ANOVA revealed significant effects of time (F time of effects significant revealed ANOVA injections.

, ie n tetet (F treatment and time

and Comment citer cedocument:

The t The food intake food the

27.9 ± 4.9 mg/g BW 27.9 ±4.9mg/g administration of RXFP3 of administration 120 min 120 wo

the

- a (p < 0.0023) and 60 min (p < 0.0384), 0.0384), < (p min 60 and 0.0023) < (p A2 (11.3 ± 1.6 mg/g BW, 12.6 ± 1.4 mg/g BW, and 13.3 ± 1.4 ± 13.3 and BW, mg/g 1.4 ± 12.6 BW, mg/g 1.6 ± (11.3 A2 fter 30, 60, and 120 min, respectively) min, 120 and 60, 30, fter - way administration of RXFP3 of administration and

after 30 min min 30 after , respectively , repeated a treatment (F treatment a CSF CSF control group, control CSF

< 6, 116 116 6,

- 0.0001 at each time each at 0.0001 injected group (2.8 ± 0.8 mg/g BW, 5.6 ± 1.2 mg/g1.2 ± 5.6 BW, mg/g 0.8 ± (2.8 group injected

- after Post measures 555 p 000) on 0.0001) < p 5.555, = (p < 0.0054 < (p ) compared to compared ) - 30, 60, and 120 min, respectively) but not by by butnot respectively) 120min, and 30, 60, 2, 116 2, hoc

- and A2 significantly increased significantly A2

analy ANOVA revealed significant effects of of effects significant revealed ANOVA = 43.16, p < 0.0001) and a significant a and 0.0001) < p 43.16, =

10.6 ± 2.3 mg/g BW mg/g 2.3 ± 10.6 - ) A2 produced significantly stronger stronger significantly produced A2 the the , s

60 min 60 es injection of RLN3 significantly significantly RLN3 of injection the - revealed 3, 1 3, point) compared to compared point) between 08

control injection control

but ( the

p < 0.0001 < p

= 32.22, p < 0.0001) < p 32.22, = (Fig. 1B). 1B). (Fig.

not

od intake. food icv 30, 60, and 60, 30, that compared to the the to compared that

time and treatment and time

after 120 min (p > > (p min 120 after diitain of administration

after 30, 60, and 60, 30, after 30, 60, and 60, 30,

the ) , The t The

and 120 min min 120 and

s umulative umulative the

120 min 120 Post of wo

a a - CSF CSF - CSF

way way 120 hoc and 13 ,

or - p The - The Plasma - - A2 (5.3 ± 0.5 ng/ml) compared to the to compared ng/ml) 0.5 ± (5.3 A2 A2 (17.1 ± 3.4 ng/ml) compared to compared ng/ml) 3.4 ± (17.1 A2 way ANOVA revealed a significant effect of the treatme the of effect significant a revealed ANOVA way way ANOVA way

RXFP3 ) levels of levels , but , a lasma corticosterone lasma CSF and RXFP3

p < 0.0001 < p they concentrations - 2 Tsotrn ws nrae b RN (19 13 gm) u nt by not but ng/ml) 1.3 ± (11.9 RLN3 by increased was Testosterone A2. According to According the plasma hormones plasma Post were Comment citer cedocument: revealed injection of RLN3 significantly increased water intake water increased significantly RLN3 of injection - hoc not ), , and (p< compared 120min toRXFP3 0.0001) and 120 min 120 and -

A2 groups (p >A2 0.05). analys different

significant

levels the of

s post

testosterone,

- were measured measured were detected 2 and A2 between were increased by RLN3 (51.9 ± 6.2 ng/ml) but not by not but ng/ml) 6.2 ± (51.9 RLN3 by increased were

- (p effects of treatment on plasma corticosterone (F corticosterone plasma on treatment of effects hoc the

< 0.0001). Conversely, no significant difference (p (p difference significant no Conversely,0.0001). < a

a CSF analys CSF

a the RXFP3 the significant differences between the RLN3 and and RLN3 the between differences significant CSF - - injected g injected

i corticosterone njected group (18.4 ± 5.6 ng/ml; 5.6 ± (18.4 group njected e - s, at netd groups injected

the the 3 0 min min 0 - A2 plasma corticosterone plasma - injected rats after 30 min (p < < (p min 30 after rats injected roup (6.1 ± 0.7 ng/ml) ( ng/ml) 0.7 ± (6.1 roup - A2 groups (p < 0.0001) < (p groups A2 - nt on plasma testosterone (F testosterone plasma on nt

and after - A2 , a

CSF the xtcn vasopressin, oxytocin,

at (p < 0.0001) and 0.0001) < (p

any time point. point. time any icv - - injected groups (p > > (p groups injected A2 administration.

administration of administration after 30 min 30 after

levels Fig. Fig. , but not but

a 2, 29 were 2A CSF 2B (p < (p The The 2, 29 14 ). ). ). =

c mRNA was observed observed was mRNA -

or fos ± At of injection after vasopressin plasma in decrease a for tendency a was there Although O 16.4 a V adfrteSN (F SON the for and PVN 49.3 x CSF. RXFP3

tcn lsa levels plasma ytocin mRNA expression in thePVN in mRNA SON and expression 3

min 0 - Fig. ± way ANOVA; Fig. 2C). 2C). Fig. ANOVA; way

p 14.6 O g/ml

relative to levels relative - ne 3 A2 2, 29 2,

C, E C, -

after ), these these ), p way ANOVA revealed a significant effect of the treatment on c on treatment the of effect significant a revealed ANOVA way . g/ml

) compared to compared ) and and

Comment citer cedocument:

1.832

2, 29 29 2, the ) or RXFR3 or ) post in these regions these in changes did not reach significance (Fig. 2D). O 2D). (Fig. significance reach not did changes

= 26.93, p < 0.0001) and parvocellular (F parvocellular and 0.0001) < p 26.93, = icv , p = 0. = p , -

Fig.

hoc

ee significantly were diitain f RLN3, of administration in aCSF in 2, 29 29 2,

3 analyses did not did analyses 1817 a A, D A, - A2 CSF = 14.32, p < 0.0001). 0.0001). < p 14.32, = Post pnprn (F epinephrine -

; Fig. 2F) Fig. ; (49.0 injected group (10.8 ± 0.9 ng/ml). ng/ml). (10.8 ±0.9 groupinjected ) and parvocellular ( parvocellular and ) - injected rats. However, no difference in the levels of levels the in difference no However, rats. injected between - - hoc A2 (8.4 ± 0.4 ng/ml) but not by RLN3 (p > 0.05; > (p RLN3 by not but ng/ml) 0.4 ± (8.4 A2

analyses 18.7

levels

fetd y treatments by affected

rats reveal

p , 29 2,

g/ml levels that received that were not affected by affected not were the revealed

= significant effects of effects significant )

The The relative to relative Fig. 0.35

xrsin f c of expression in

p h RLN3 the p 0. = p , 3

ost B, D B, ht o that

2, 29 2, - icv hoc

) parts of the PVN and and PVN the of parts ) aCSF control injection injection control aCSF

injection = 3.867, p = 0.0324) 0.0324) = p 3.867, = ne

xytocin 7096 analy However, t However, ( - - F

way ANOVA way , 29 2, - n RXFP3 and icv fos Fg 2E) Fig. ; s treatments on treatments es

s 38, = p 3.89, = injections o injections RA was mRNA levels

- of fos revealed here was here was RXFP3

mRNA

were

A2 and (F 15

2, 2, a - - f

was RXFP3 c c ). . s but not lateral LHA (p > 0.05) > (p LHA lateral not but - - RXFP3 es

fos fos The e The I cv altered the the revealed

mRNA expression in theventral in mRNA MPA expression the in mRNA expression - A2 injection xpression of c of xpression RLN3 and and RLN3 - Conversely A2 group compared to comparedgroup A2

RXFP3 group (p < (p group by

significant a

but Fig. the s

not the lateral the not Comment citer cedocument: f L3 and RLN3 of A2 (p < 0.05) groups compared to compared groups 0.05) < (p A2 - icv 4 fos RXFP3 , no significant variation was observed in observed was variation significant no , , C B,

0.0001 for the PVNm and the SON; the and PVNm the for 0.0001 -

diitain f RLN3 of administration fos mRNA expression in the RLN3 group compared to compared group RLN3 the in expression mRNA

), ), y larger ly mRNA in the ventral MPA ventral the in mRNA - 2 groups A2 but

treatment on treatment

LH . No significant No . the a the part o i te aea LA ( LHA lateral the in not

RXFP3 A nrae in increase

(F CSF

2, 26 2, in the the in - - injected rats (p rats injected

c induced A2 = 1.123, p = 0.3406) = p 1.123, = the ,

and OVLT and and p < 0.001 for the SON) and compared to compared and SON) the for 0.001 < p and perifornical

. c c differences - - O fos fos ne

- RA xrsin n h L3 p < (p RLN3 the in expression mRNA

levels in levels a way ANOVA revealed a significant significant a revealed ANOVA way (Fig. CSF

Fig. Fig. - p < 0.05 for the PVNp) PVNp) the for 0.05 < p (p > 0.05) 0.05) > (p fos

5 0.05 for the for 0.05 injected injected in c in A, C) and C) A,

the perifornical perifornical the 4 the RA xrsin n the in expression mRNA

- , C A, of the LHA the of fos

- mRNA rats in the perifornical the in rats fos ). or the OVLT (Fig. (Fig. OVLT the

PVNm, O mRNA expression mRNA lateral ne the - . were way ANOVA ANOVA way The part a

PVNp, and PVNp, CSF group CSF p > (p

observed for p (F ost

2, 2 2, 0.05) these - hoc 5 3 B, B, 16

(p < 0.01 for the ventral MPA MPA ventral the for 0.01 < (p - study This A2 mRNA expression in these regions in the RLN3 the in regions these in expression mRNA ,

the

on food and water intake water and food on

nrae n od n wtr intake water and food in increase that we and others and we that the min min

central administration of administration central [ , 7 stimulation of stimulation RXFP3 post , 2, 28 2, 24 investigated ifferent concentrations of RLN3 produced differential effects on cumulative on effects differential produced RLN3 of concentrations ifferent - A2 and aCSF A2 - - = 35.95, p < 0.0001) < p 35.95, = 26 injection than to than injection Comment citer cedocument: ] - , this study this , A2, and p < 0.001 for the OVLT)for the 0.001 < p and

their the

and

c RXFP3 reported

, - tissue penetration penetration tissue

h efcs of effects the fos reflect but did not affect water intake water affect not did but the native RLN3 peptide, which activates which peptide, RLN3 native the - control groups groups control

expression expression and has

and p < 0.001 for the OVLT the for 0.001 < p and

RLN3 is

4. equimolar amounts of of amounts equimolar “ a earlier

demonstrated on in agreement with agreement in DISCUSSION . cei The

hypothalamic . ig effect” ling

Differential effects of RLN3 and RXFP3 and RLN3 of effects Differential [

. the for the ventral MPA ventral the for

21 p Conversely, ost in the ventral MPA capacity - 23 central - hoc ,

. No significant differences were observed weresignificant differencesNo . 30 th

at analy - ]

injected . However, the absence of differences differences of absence the However, . . or c

( the However, - icv the orexigenic effect orexigenic the

fos

rats the s RLN3 and RXFP3 and RLN3

) es icv

. mRNA expression mRNA administration

The observed The

reaching reaching detected administration

group compared to the aCSF the to compared group injection of injection ) and RXFP3 and )

(F or a 2, 2 2, direct comparison of the the of comparison direct OVLT 8

= 5.155, p = 0.0124) = p 5.155, = a significant increase significant a

a RXFP1 and RXFP1 maximum in food food in maximum

(p > 0.05). A2 RLN3 increase in food in increase of of

- s - A2 of

A2 revealed revealed A2 of RLN3 of . at h selective the In line with line In

RXFP3

group

30 and 60 and 30 induced a a induced -

A2 may may A2 RXFP3

(p < < (p - and A2 17 a

he blockade of of blockade he

- u not but - G are RXFP3 and RXFP1 22R interfere induced s

[

revealed 3 , the n od intake food in ] the . inhibitory inhibitory h a The RXFP3

ciain of activation s inhibition was dependent on dependent was inhibition parvocellular and magnocellular magnocellular and parvocellular

bath to its cognate receptor RXFP3 receptor cognate its to with - A2; the an G tvto o RF1 nue a ices i itaellr cAMP intracellular in increase an induces RXFP1 of ctivation i/o

- application of RLN3 RLN3 of application Comment citer cedocument:

inhibition of oxytocin and vasopressin PVN neurons in neurons PVN vasopressin and oxytocin of inhibition A2 activation of RXFP3 alone RXFP3 of activation - however proteins in PVN neurons prevented the inhibitory action of RXFP3 of action inhibitory the prevented neurons PVN in proteins r offsets or impact on target cells, respectively cells, target on impact , which might be associated with associated be might which , rdcd by produced

the , when [ PVN -

40 protein via h effects the ] .

The present results support results present The RXFP3 action eas a ices i c in increase an because RLN3 and and RLN3 - coupled receptors receptors coupled caused an increase increase an caused , reduce food intake in male rats inmale food intake reduce

an an

s but in the early dark phase dark early the in

within within was b was

outward calcium outward and of . at of parts not In addition, induction of expression of c of expression of induction addition, In

RXFP3

“pure” to

l at 120 min min 120 at ocked h PVN the RXFP1 via V after PVN

A2 activation of RXFP1 in the OVLT the in RXFP1 of activation

the [ by RXFP3 3 ht induce that , , the native receptor for relaxin, for receptor native the , might 11 n cin oeta firing potential action in activation of both RXFP1 and RXFP1 both of activation the because - dependent potassium c potassium dependent

- or ] the fos .

specific RXFP3 specific

Recent

later later

the [ activation be be potential mRNA expression was was expression mRNA 39

] ifrn intracellular different the rather icv , [

22 [ and there is a high high a is there and neurophysiological 39

administration administration , response ]

24 . involvement of involvement . eed on depend T ] n fact In his .

Significant

antagonist hl the while anorectic

to urrent

, [

both

31 - -

the the A2 fos icv 18 of of ] , .

[ be induced by the binding of this this of binding the by induced be In the current study, current the In mRNA in the PVN, consistent with consistent PVN, the in mRNA 27

[ ] may

51 . a stronger potential of RXFP3 of potential stronger a

ovrey t Conversely, ] the neuron

. yoi at a a aoetc inl n oh nml ad humans and animals both in signal anorectic an as acts xytocin affect food intake food affect is

vasopressin Therefore, CRF ciain f h RF3 niioy pathway inhibitory RXFP3 the of activation also

populations , neurons the osdrd n nrxgnc factor anorexigenic an considered were rxgnc fet o RLN3 of effects orexigenic Comment citer cedocument:

the increase in c in increase

lead to lead

the

and increases the levels of CRF mRNA CRF of levels the increases and not significantly not

rdcin f slcie XP agonist RXFP3 selective a of production n h PVN the in induction

their i

cv and energy expenditure energy and ,

, nldn CRF, including reduction a administration of RXFP3 of administration

leading to leading

nadir the -

peptide fos - f c of A2

RXFP1 [ s soitd with associated is the 52 mRNA expression in the PVN the in expression mRNA - than altered fos a highera

] Future . in food intake food in

inhibition of PVN neurons after neurons PVN of inhibition to

- to response in PVN the in expression mRNA

RLN3

related activation activation related RXFP1 . oxytocin

by ovrey te fet o RXFP3 of effects the Conversely, the increase in food intake. food in increase

due to due estimation either to decrease to

expression ofexpression oxytocin in the OVLT the in [ - 43 ,

A2 did not induce induce not did A2

decrease n vasopressin and [

] RLN3 RLN3 the the 49 , i G via and ,

involvement 50 f the of

of plasma oxytocin levels oxytocin plasma in t in i/o ] or icv

. s the CRF neurons CRF the

Administration of RLN3 of Administration rti coupling protein he parvocellular PVN in PVN parvocellular he in RXFP3 [

, 41

r nrprtna ( intraperitoneal or effects of RLN3 and and RLN3 of effects R3/I5 od intake food in response to response in ] .

P T

arvocellular of the he present results present he neur - when the plasma plasma the when the

, A2. and vasopressin

one or more of more or one by

expres ons activation of of activation [ Th 42 an

- e [ [ A2 44 [

27 46 42 [

lack of of lack sion of sion adeno 11 RLN3 , . ] PVN - -

may 48 45 44 The The ] that

ip 19 in ] ] ] - ) . . .

] administration of relaxin, the cognateadministration of relaxin, RXFP1 ligand of , there is there , - with associated was NI the of activation Although are PVN parvocellular the in neurons CRF the intake, food on effects their to addition In - A2 on the levels of plasma vasopressin should be performed during the late light phase phase light late the during performed be should vasopressin plasma of levels the on A2 A2. byrted

thyroid (ACTH), plasma the

currently RXFP1 because RXFP1 regulation of the HPA axis. This axis includes the hypophysiotropic parvocellular includesthe hypophysiotropic axis This axis. HPA ofthe regulation an earlier earlier an - t ees CF rm xnl emnl a te ee o mda eiec from eminence median of level the at terminals axonal from CRF release at stimulating hormone hormone stimulating

levels of levels the dark phase,the dark

that which stimulates stimulates which synthesis and releaseand synthesis no no Comment citer cedocument: - study that study direct PVN RLN3 RLN3 PVN the -

epinephrine and norepinephrine after norepinephrine and epinephrine plasma the decreased significantly RLN3 of administration PVN ciain f h HA xs n epne o L3 eed on depends RLN3 to response in axis HPA the of activation

the evidence that RLN3 RLN3 that evidence

when plasma vasopressin increases and stabilises stabilises whenvasopressinand plasma increases

activation of RXFP3 by its selective agonist selective its by RXFP3 of activation rep [ [ orted the 55 55 or ] ] adrenal . Similarly,. , oxygen consumption and respiratory exchange ratio ratio exchange respiratory and consumption oxygen , intracranial administration of RLN3 of administration intracranial

[ c

44 - an increase in plasma corticosterone level corticosterone plasma in increase an fos , injections injections 53

mRNA expression in the PVN. inthe mRNA expression ] . Inaddi . lccriod crio i hmn and humans in (cortisol glucocorticoid alters the current the

[ significantly increased the levels of of levels the increased significantly the activity of the autonomic nervous nervous autonomic the of activity the 36 tion to tion egtnd rua ad locomotion and arousal heightened the ] .

reported activation of the HPA the of activation reported

rdcn adrenocorticotropic producing study did not detect signidetect not did study icv the

administration of RLN3 of administration activation of c of activation [

22 This conclusion This conclusion RXFP3 , 27 [ - 52 fos s ,

] 36 after .

mRNA A2

] ficant . The .

did the its its 20 or

administration icv icv

and in the OVLT the in and n PVN and , as

Notably , I administration of RLN3 of administration

as on activation of the of activation on effect dipsogenic led toa strong n agreement with agreement n administration

y RXFP3 by produced by equimolar

directly involved in fluid regulation fluid in involved directly the OVLT OVLT the

the of , relaxin stimulates stimulates relaxin ,

RXFP1 [ possibility that the magnocellular regions were activated by RLN3 RLN3 by activated were regions magnocellular the that possibility 25 of of - , 2 i nt change not did A2 displayed RLN3 41 and

Comment citer cedocument: in response to response in of of

c [ , 60 n ae intake water in - Fos expression Fos 56

relaxin had comparable had relaxin previous report previous not RXFP3, activation. RXFP3, not ] . ,

] organ subfornical and OVLT the as such organs circumventricular . tog ciain f c of activation Strong but not RXFP3 not but

h mgoellr ern poue aorsi ad oxytocin and vasopressin produce neurons magnocellular The

a significant induction of c of induction significant a

was observed was amounts of the the

water a intravenous infusion of infusion intravenous disrupted blood disrupted expression of c of expression

RLN3 but not RXFP3 not but RLN3 . .

Conversely, the intake but not but intake in magnocellular neurons magnocellular in n fact, In s

[ RLN3 25 ae intake water - . However, the presen the However, . A2. , [ 42 26

neto o RN ta activates that RLN3 of injection

The OVLT project OVLT The ,

This conclusion This ] in rats. [ , a significant increase in water intake after intake water in increase significant a , - 57 25 - fos - fos the the ri barrier brain , ]

o food intake food RA n h mgoellr V and PVN magnocellular the in mRNA mRNA in the the in mRNA selective r

]

relative -

. stronger fos

- relaxin,

There is evidence that activation of of activation that evidence is There

mRNA expression in response to response in expression mRNA ,

suggests the dipsogenic effects dipsogenic the suggests

stimulation of RXFP3stimulation

to [

is supported by observation by supported is

t study t [ 59 [ while ablation of the OVLT OVLT the of ablation while [ 41 24 39 s the the n is isgnc effects dipsogenic its and

] magnocellular neurons of of neurons magnocellular ] . In fact, In . directly ] ] . The present results do results present The .

. effect a The a The CSF suggests

iveness - to the SON and and SON the to dministration of dministration netd control injected ablation of the the of ablation

XP and RXFP1 a particular a

on ,

via but not but not

water water

the the 21 s ,

] , , brain 64 but there is evidence is there but s at

] h c The within results particularly results - .

concentrating hormone (MCH) hormone concentrating

LHA The to the lateral the expression of orexin mRNA in the perifornical but not but perifornicalthe in mRNA orexin of expression in activity of orexin neurons in the perifornical but not not but perifornical the in neurons orexin of activity n epne o L3 n RXFP2 and RLN3 to response in structures

orexin orexin RXFP3 in this region.this in RXFP3 the perifornical but not but perifornical the obesity the the entral [

rats 65 the LHA the

was not observed not was HA produces anorectic effects anorectic produces HA ] . dark dark

ht overeat that neurons contains oevr obesity Moreover, administration - LHA, where LHA, prone rats prone , f the of

rdtoal called traditionally Comment citer cedocument: - , the brain region brain the , active A2

of implicate

n h prfria bt not but perifornical the in Both perifornical and lateral LHA lateral and perifornical Both RXFP3 but not RXFP1 not but RXFP3

gustatory a particular role of role particular a

have circadian phase when their their when phase circadian high a . a

In It is also possible that possible also is It

f L3 and RLN3 of

significant

in the perifornical the more the presentthe

the the did not affect not did - n mtblc ewrs nldn the including networks metabolic and , induced induced a diet fat which are which strongly

orexin lateral subdivision of LHA of subdivision lateral the the - A2 effect

hne center,” “hunger [ study the the ipae elevated displayed by - 61

RXFP3

positive a dpn o mliyatc circuitry multisynaptic on depend may involved , neuropeptides with strong orexigenic effects orexigenic strong with neuropeptides

LHA sub LHA a LHA,which

binding sites binding 62 food intake intake food of of ,

high c ] -

. RLN3 or or RLN3 f induction of c of induction

the - os

The A2 neurons

in fat diet significantly increased increased significantly diet fat mRNA aea LHA lateral -

may regions regions LHA appetite eating contain the the

is [ [ nrae appetite increases

21 RX in the perifornical the in 20 tmlt food stimulate

lateral LHA lateral

strongly lateral LHA lateral expression was expression neurons express neurons

] eairl rua and arousal behavioral ]

, FP3

after after in

behavior behavior - orexin and MCH and orexin that but these injections were were injections these but regulation fos

eating -

A2 [ mRNA expression in expression mRNA may limit may icv 65

interconnected ]

administration I addition, In .

administration of of administration behavior infralimbic and and infralimbic was was [ increases . 67

The e The intake significantly ] ,

detected

orexin and orexin than in than the effects the The d The while

lectrical s neurons . Thus, Thus, . u to due

[ with with irect irect 66 The The

the the the the the

on an an an 22 in in ] .

] mRNA also significantly also diitain f 0 po RN i ml rt icesd c increased rats male in RLN3 pmol 800 of administration . a In addition to RXFP3, to addition In increases RLN3 o nme o GnRH of number low

RXFP3 and , -

the expression A2 - significant n n atvto of activation no and neurons, fos

that

the LHA by RLN3 may RLN3 by LHA the likely has P ai b icesn the increasing by axis HPG substantia innominata substantia of perifornical LHA perifornical of

mRNA expression expression mRNA A2. GnRH release from hypothalamic explants hypothalamic from release GnRH

RXFP1 may be be may RXFP1

also y 14% by

depend on depend

may reinforce its orexigenic effects. Conversely effects. orexigenic its reinforce may increased c increased

in the MPA in response to response the MPA in in . In the present study present the In .

a potential a Comment citer cedocument:

the the

[ MPA MPA oprd to compared

28 eta nces f h amygdala, the of nucleus central - icv muoecie neurons immunoreactive the ] . -

fos GnRH is expressed is GnRH role in reproductive in role h PVN the neurons express neurons involved neto o RN ws lce by blocked was RLN3 of injection

stimulation of GnRH neurons because because neurons GnRH of stimulation a neurons, which contain the contain which neurons, n h prfria LHA perifornical the in

CSF mRNA expression in the perifornical LHA. perifornical the in expression mRNA and and counteract - the parabrachial nucleus nucleus parabrachial the injected group. injected

wih contains which , the , LH n P activation. MPA in

a higher ( higher

control

[ the 28

its orexigenic effects orexigenic its

RXFP1 ]

icv

and by neurons by

function. 1.1

administration of RLN3 notRXFP3 administration of but [ aot 3%) (about 23 T FSH

nmol he same he mRNA ] ,

;

and an increase in plasma LH and and LH plasma in increase an and nrxgnc R, o CRF, anorexigenic

by however,

[ orexigenic neuropeptides orexin orexin neuropeptides orexigenic ) The 2

h a The

in the MPA the in - 9 amount the 47 fos

] [ [

19 dose i lsa levels plasma %, which which %, co

, cv ] RA expression mRNA e nces f h stria the of nucleus bed a , tvto o te P by MPA the of ctivation . - ,

20 administration of RLN3 RLN3 of administration xrs RF3 mRNA RXFP3 express In coinciding which were which

this this ( of RLN3 RLN3 of 1.1 ] pre the a .

The i The , previous study previous

- and in this this in and increase nmol ramn wt a with treatment administration of of administration was statistically statistically was

The a The nduction of c of nduction

n ae rats. male in xytocin ) activation of of activation significantly

of RXFP3 of lower lower

ctivation was

r egion n the in ,

, than

and and not the 23 - - - ,

activation of the HPA and HPG activation theHPA of

anorexigenic effects. effects. anorexigenic , with In the summary - - in the MPA

A2 may involve involve may A2 2 a dpn on depend may A2 icv uronal groupsuronal ttsial non statistically

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Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., 6 Saapo F Sprvc , ae D Te ua isln uefml o polypeptide of superfamily insulin 1 2009; 80: hormones. VitamHorm. human The JD. Wade F, Separovic F, Shabanpoor [6] Relaxin MA. Hossain family peptides: structure RAD, Bathgate JD, Wade F, Separovic KJ, Rosengren NA, Patil [5] the relaxin family 389 Pharmacol peptides. Rev.2015;67: of understanding the in b advances and pharmacology Recent XCV. Pharmacology. Clinical and Basic of Union International RJ. Summers TB, Dschietzig SW, Sutton RA, Bathgate ML, Halls [4] f Relaxin RJ. Summers M, Kocan GE, Callander ET, Westhuizen der van ML, Halls RA, Bathgate [3] Neurochem.J 82:1553 2002; gene rat novel the of expression abundant, AL,Gundlach S, Layfield M, Macris RA, Bathgate TC, Burazin [2] peptide family. Biol 277:1148 Chem. 2002; J relaxin the of members Novel gene. (M3) relaxin mouse equivalent the and (H3) 3 gene relaxin TC, Burazin CS, Samuel RA, Bathgate [1] REFERENCES thank Geneviève Guèvremont 1067522). grant (A.L.G.; Australia of Council Research Medical and Health National and amily and their peptides 405 receptors.Rev. 2013;93: Physiol

ooia rls f eai fml ppie eetr 1 receptors peptide family relaxin of roles iological Comment citer cedocument: - activityre -

155 for

valuable 7.

lationship studies. Br J Pharmacol. 2017; 174: 950 Br 174: Pharmacol. studies. lationship J 2017; - 31.

- technical assistance. 3 (R3) relaxin in the dorsal tegmental region of brain. brain. of region tegmental dorsal the in relaxin (R3) 3 Layfield S, Claasz AA, Reytomas IG, et al. Human al. et IG, Reytomas AA, Claasz S, Layfield - 11 57.

- 440.

Tregear GW. Restricted, but Restricted, GW. Tregear - 4 80. -

4, the receptors for for receptors the 4, - 9 61.

We 25

Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., relaxin Blasi CM, Smith S, Ma [15] 572 2015;466: Commun. Res Biophys Biochem neurons. in genes early immediate of expression the on signals cAMP Fukuchi M,Kanesaki K,Takasaki[14] I, A, Tsuda effects M. Convergent Tabuchi ofCa(2+)and Ther. Exp Pharmacol J genes. reporter using RXFP2, and RXFP1 receptors, peptide family relaxin the by activated pathways signaling of Comparison RJ. Summers RA, Bathgate ML, Halls [13] that relaxin (GPCR) receptor G al. et J, Zhu J, Wu D, Nepomuceno C, Kuei P, Bonaventure SW, Sutton [12] 50754 2003; 278: G orphan the for ligand endogenous an as al. et C, Kuei B, Roland J, Chen S, Sutton E, Eriste Liu C, [11] forebrain on influence circuits via G widespread suggests incertus nucleus of neurons projection GABA al. et RA, Bathgate TC, Burazin PJ, Shen T, Ferraro P, Bonaventure S, Ma [10] teleosts. Good S, Yegorov [9] family.BMC evolutionary 14. 2005;5: biology. relaxin the of Evolution RA. Bathgate GW, Tregear TP, Speed TN, Wilkinson [8] years.10 Chem Neuroanat. 42:262 J 2011; Relaxin AL.Gundlach S, IT,Ma Ryan Hosken CM, PJ, Smith [7]

2007; 320: 281 2007; 320: - 3/RXFP3 systemsinbrain. 174:1034 Pharmacol. 2017; Br J Ann N Y Acad Sci. 2009; 1160:42 Sci. 2009; Ann N YAcad - 3 isthep - protein - - 142 does not contribute to relaxin to contribute not does 142 507 - hysiological Neuroendocrinology. GPCR135. ligandfor 139 2005;82: - 2 coupled receptorcoupled 64. - 90. vl S, ar L Wlo B. Rela BC. Wilson L, Parry SV, Avila Comment citer cedocument:

67 ak A, Gundlach AL. Distribution, physiology and pharmacology of of pharmacology and physiology Distribution, AL. Gundlach A, ak 7.

- 135 in the rat. 135 inthe - - 2 - 4 75. protein 4.

- 3 binding in the mouse brain: further support furthersupport mousebrain: the in binding 3 - ope rcpo G receptor coupled Neuroscience. 165 2007;144: i fml gns n uas and humans in genes family xin Identification of relaxin of Identification - 3 systems in the in systems 3 - 10 48. C15 J il Chem. Biol J PCR135.

- protein

brain - - Relaxin 1 like peptide like 90. - - 3/INSL7 -

coupled coupled the first first the - - 1 3 in 3 50. 26

Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., femaleBehav. 550 Brain 14: Genes rats. 2015; effe relaxin of Differential administration central E. Timofeeva G, Guevremont C, Avila de C, Lenglos J, Calvez [23] ofexpression corticotropin Lenglos[22] administration inmale Wistar Physiol. E913 rats.2007; 292: AmJ Fos and action orexigenic of mapping Hypothalamic [21] - Relaxin AL. Gundlach S, Ma DE, Ganella [20] Proc Natl A.1991;88:6413 Acad Sci US bi relaxin of localization Autoradiographic HS. Phillips PL,Osheroff [19] 298 2004; 80: relaxin for role a suggests G of Distribution al. et D, Nepomuceno J, Chen B, Roland C, Kuei P, Bonaventure SW, Sutton [18] 138 ERK1/2 with receptors Long Gutierrez [17] dependent Pharmacol. Mol mechanism. 71:1618 2007; activates 3 receptor peptide family relaxin The RJ. Summers PM, Sexton TD, Werry ET, Westhuizen der van [16]

A perspective hypothalamic onextrinsic control. 11. - McGowan BM, Stanley SA, White NE, Spangeus A, Patterson M, Thompson EL, et al. al. et EL, Thompson M, Patterson A, Spangeus NE, White SA, Stanley BM, McGowan - protein atn bhvoa rsoss o s to responses behavioral lasting

- C, Calvez J,Timofeeva Calvez C, Sex E. coupl - - 307. Mecinas M, Trollope AF, Collins A, Morfett H, Hesketh SA, Kersante F, et al. et F, Kersante SA, Hesketh H, Morfett A, Collins AF, Trollope M, Mecinas

ed receptor (GPCR)135 binding sites and receptor mRNA in the rat brain brain rat the in mRNA receptor and sites binding (GPCR)135 receptor ed xrclua signal extracellular Comment citer cedocument: - MSK1 - - releasing Endocrinology. factor 523 inrats. 2015;156: i nuonorn ad esr poesn. Neuroendocrinology. processing. sensory and neuroendocrine in 3 - Elk - 3 on food intake and hypothalamic neuropeptides in male and male in neuropeptides hypothalamic and intake food on 3 -

1 signaling. Proc Natl Acad Sci U S A. 2011; 108: 13806 108: 2011; A. S U Sci Acad Natl Proc signaling. 1 rs ivle drc itrcin f glucocorticoid of interaction direct a involve tress - - 641 euae kns 12 hog a rti kns C kinase protein a through 1/2 kinase regulated - specific effectsspecific relaxin of - 7. 5 - 3/RXFP3 signaling and neuroendocrine function neuroendocrine and signaling 3/RXFP3 63. Front Endocrinol

- like immunoreactivity following relaxin following immunoreactivity like - 16 29.

- E91 . 2013; 4: 1 . 2013;4: - 3 on food intake and brain 3 onfood intake and brain 9.

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Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., 3 relaxin by nucleus paraventricular hypothalamic rat in activity neuron vasopressin and oxytocin Z, Rajfur T, Blasiak C, Avila de A, Grabowiecka A, Gugula A, Kania [31] al. relaxin et M, Kocan S, Layfield A, Belgi M PJ, Ryan M, Hossain Akhter F, Shabanpoor [30] in rat Sex E. Timofeeva relaxin of administration G, Guevremont C, Avila de J, Calvez [29] E2 Relaxin al. et NM, Semjonous M, Patterson EL, Thompson J, Donovan SA, Stanley BM, McGowan [28] response systemtherathypothalamus. in Relaxin M. Tanaka T, Matsuda Y, Miyamoto Y, Watanabe [27] recep high its of demonstration and strategy synthesis improved Relaxin al. et C, Giannakis A, Guidolin Jr., L, Otvos NF, Hanson F, Lin RA, Bathgate [26] facilitates 31:1124 2010; water intake. Peptides. al. et M, Todoroki T, Ohbuchi relaxin A, administered Katoh H, Suzuki T, Onaka H, Otsubo [25] 146: 3295 relaxin Central al. et EL, Thompson MM, Connolly NE, White KL, Smith SA, Stanley BM, McGowan [24] - RXFP3 Physiol. signalling. J 3425 2017;595: inimization ofinimization humanrelaxin 86. s. J Neuroendocrinol. 28.dio: 2016; s. J tor LGR7 and Biochemistry. invitro both invivo. tor 1043 2006;45:

- family Med 55:1671 receptor Chem. 2012; peptide(RXFP3) J 3 RXFP1. over - 3 stimulates the hypothalamic the stimulates 3 - 3 300. - 3 admi 3

- idcs o epeso i te soestv aes n a brain rat in areas osmosensitive the in expression Fos induces 3 nistration causes hyperphagia in male Wistar rats. Endocrinology. rats. Wistar male in hyperphagia causes nistration Comment citer cedocument: - 3 on food intake, body weight and hypothalamo and weight body intake, food on 3 - 3 leading high to

- pituitary J Mol Neurosci. Mol 43:169 J 2011; 10.1111/jne.12439. - - - 34 - gonadal axis. Am J Physiol. 2008; 295: E278 295: 2008; Physiol. J Am axis. gonadal 11 affinity analogues with increased selectivity for for affinity selectivityanalogues withincreased 47. 30.

- fiiy neato wt te relaxin the with interaction affinity

- 3/INSL7 regulates the stress the regulates 3/INSL7 - pcfc effe specific - 10 - 1 53. 74. - pituitary

et al. Inhibition of Inhibition al. et t o chronic of cts - gonadal axis axis gonadal - 16 Centrally 81. 2005; 2005;

and and - 28 3: 3: - - -

Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., Obes Metab. 1090 2010;12: relaxin human of administration peripheral and Central al. et SA, Stanley D, Roy NE, White KG, Murphy JS, Minnion BM, McGowan [39] 6 Coordinates. Elsevier; 2007. Stereotaxic in Brain Rat The C. Watson G, Paxinos [38] and decreases anorectic Neurosci.41:420 effects2015; EurJ ofstress. A, Mitra [37] 337 2014; 221: thestimulates neuro McGowan Murphyet BM,[36] al.Relaxin JS, Minnion WS, KG, RoyD,Stanley Dhillo SA, disorders. relaxin and network incertus nucleus the targeting for case al.et S, Ma Hong JM, S, Marwari Rajkumar JayakodyT, R, JR, Kumar [35] 145 2010; 58: relaxin of induction rapid and incertus nucleus of excitation stress Swim AL. Gundlach RA, Bathgate S, Ma PJ, Shen A, Banerjee [34] "floxed al. et CM, Smith E, Relaxin Timofeeva RAD, Bathgate C, Zhang G, Guevremont M, Haidar [33] 207 2016; 102: of Role E. r Timofeeva AL, Gundlach G, Guevremont LO, Matte C, Avila de J, Calvez [32] elaxin - - 3/RXFP3 system in stress in system 3/RXFP3 - RXFP3" mice impairs spatial 529 RXFP3" micememory.2017;27: Hippocampus. impairs ipt tre hpoapl nenuos n deletion and interneurons hippocampal target inputs 3

Br J Pharmacol. J 174:1061 Br 2017; - Lenglos C, Timofeeva E. Inhibition in the lateral septum increases sucrose intake intake sucrose increases septum lateral the in Inhibition E. Timofeeva C, Lenglos 1

- - 3 55. 2 46. 15.

endocrine viacorticotrophin stress axis Comment citer cedocument: - 109 6. - induced binge induced

- 3 expression vi expression 3 - 10 - 2 to adult male rats inhibits food intake. Diabetes Diabetes intake. food inhibits rats male adult to 2 76. - like eating in female rats. Neuropharmacology. rats. female in eating like

a CRF1 activation. CRF1 a - - 3/RXFP3 system in neuropsychiatric in system 3/RXFP3 releasing hormone.

f ia relaxin hilar of - 4 33.

Neuropharmacology. - Relaxin' the brain: a brain: the Relaxin' 5 th 46.

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- rcpos in receptors 3 - 3 . 29

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Comment citer cedocument: Front Endocrinol - 3 - 6. protein

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Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., 2006; 136: 72 2006; 136: al. et EL, relaxin chronic and acute of Effects Thompson J, Donovan JS, Minnion KL, Smith SA, Stanley BM, McGowan [55] 222 2017; Funct. Struct Brain arousal. behavioral and desynchronization cortical promotes incertus Ong G, Allocca S, Ma [54] 1 the in roles their receptors: CRF corticotropin The E. Timofeeva Q, Lin D, Richard [53] Liferats. 31:2843 Sci. 1982; G [52] therapy. 703 2013;20: relaxin a of expression rAAV chronic by feeding of Modulation RA. Bathgate AL, Gundlach CR, Bye S, Ma GE, Callander DE, Ganella [51] Vet 951 MedSci.2004;66: endogenous of Involvement osmolality plasma high M. in vasopressin Nishihara K, Yamanouchi T, Matsuwaki R, Ikemura [50] Physiol. 465 1989;74: Langhans AH, Meyer [49] 35. satiety. and feeding, Oxytocin, J. Menzies G, Leng N, Sabatier [48] 97.

: 515

reeley GH, Jr., Morris M, Eldridge JC, Kizer JS. A diurnal plasma vasopressin rhythm in rhythm vasopressin plasma diurnal A JS. Kizer JC, Eldridge M, Morris Jr., GH, reeley - 5 37. -

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- 3 on food intake and energy expenditure in rats. Regul Pept. Regul rats. in expenditure energy and intake food on 3 - induced anorexia via V1 receptor V1 via anorexia induced - 3 peptide agonist in rat hypothalamus. Gene Gene hypothalamus. rat in agonist peptide 3 - releasing factor family of peptides and peptides of family factor releasing Front Endocrinol Front - mediated mechanism. J J mechanism. . 2013; 4: 2013; . 31 -

Version postprint Gundlach, A.L.,Timofeeva, E.(Auteur decorrespondance) (2017). Differentialeffects ofrelaxin-3 and aselective relaxin-3 receptor agonist onfood andwater intake and hypothalamic neuronal activity in rats. Behavioural Brain Research, 336, 135-144. , DOI : 10.1016/j.bbr.2017.08.044 de Ávila, C.(Co-premier auteur),Chometton, S.(Co-premier auteur), Lenglos,Calvez, J., characterization. J Comp Comp characterization.Neurol. J 218 1992;319: al. et JL, Nahon concentrat J, Vaughan C, Peto C, Arias F, Presse JC, Bittencourt [64] 10015. systems. neuronal multiple to project (orexin) hypocretin containing A Pol den van DK, Tighe C, Peyron [63] 375 1962; 135: self and feeding of control Hypothalamic P. Teitelbaum BG, Hoebel [62] drinking afterlaterallesions. hypothalamic synd hypothalamic lateral The AN. Epstein P, Teitelbaum [61] lamina and nerve vagus the of terminalis tobraininduced activation by Neurosci.22:1489 2005; Eur refeeding. J Contribution D. Richard ED, Baraboi E, Timofeeva [60] PharmacolExp Physio Neural al. et D, Sly BJ, pathways laminaterminalis from influencing the fluidhomeostasis.Clin cardiovascularand body Oldfield RM, McAllen CN, May AM, Allen MJ, McKinley [59] the by regulation Neuroendocrinol. 16:340 2004; hormonal and osmotic secretion: Vasopressin al. et GL, Pennington RR, Miselis RC, McClear RM, McAllen ML, Mathai MJ, McKinley [58] insignals.dissimilarities Physiol Behav. 731 2002;77: vasop of Controls AF. Sved EM, Stricker [57] consciouscentrally in rats.Neuroendocrinol. J relaxin administered 9:431 1997; by induced hypothalamus and terminalis lamina the in immunoreactivity Fos of Oldfield Distribution LM, Colvill P, Burns MJ, McKinley [56]

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