NPFFR2 Activates the HPA Axis and Induces Anxiogenic Effects in Rodents

International Journal of Molecular Sciences

Article NPFFR2 Activates the HPA Axis and Induces Anxiogenic Effects in Rodents

Ya-Tin Lin 1, Yu-Lian Yu 2, Wei-Chen Hong 2, Ting-Shiuan Yeh 3, Ting-Chun Chen 2 and Jin-Chung Chen 1,4,5,* ID

1 Graduate Institute of Biomedical Sciences, Department of Physiology and Pharmacology, Chang Gung University, No. 259 Wenhwa 1st Road, Guishan, Taoyuan 333, Taiwan; [email protected] 2 Department of Biomedical Sciences, Chang Gung University, Taoyuan 333, Taiwan; [email protected] (Y.-L.Y.); [email protected] (W.-C.H.); [email protected] (T.-C.C.) 3 Department of Medicine, Chang Gung University, Taoyuan 333, Taiwan; [email protected] 4 Neuroscience Research Center, Chang Gung Memorial Hospital, No. 5, Fusing St., Guishan, Taoyuan 333, Taiwan 5 Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan * Correspondence: [email protected]; Tel.: +886-3-211-8800; Fax: +886-3-211-8700

Received: 9 June 2017; Accepted: 18 August 2017; Published: 21 August 2017

Abstract: Neuropeptide FF (NPFF) belongs to the RFamide family and is known as a morphine- modulating peptide. NPFF regulates various hypothalamic functions through two receptors, NPFFR1 and NPFFR2. The hypothalamic-pituitary-adrenal (HPA) axis participates in physiological stress response by increasing circulating glucocorticoid levels and modulating emotional responses. Other RFamide peptides, including neuropeptide AF, neuropeptide SF and RFamide related peptide also target NPFFR1 or NPFFR2, and have been reported to activate the HPA axis and induce anxiety- or depression-like behaviors. However, little is known about the action of NPFF on HPA axis activity and anxiety-like behaviors, and the role of the individual receptors remains unclear. In this study, NPFFR2 agonists were used to examine the role of NPFFR2 in activating the HPA axis in rodents. Administration of NPFFR2 agonists, dNPA (intracerebroventricular, ICV) and AC-263093 (intraperitoneal, IP), time-dependently (in rats) and dose-dependently (in mice) increased serum corticosteroid levels and the effects were counteracted by the NPFF receptor antagonist, RF9 (ICV), as well as corticotropin-releasing factor (CRF) antagonist, α-helical CRF(9-41) (intravenous, IV). Treatment with NPFFR2 agonist (AC-263093, IP) increased c-Fos protein expression in the hypothalamic paraventricular nucleus and induced an anxiogenic effect, which was evaluated in mice using an elevated plus maze. These ﬁndings reveal, for the ﬁrst time, that the direct action of hypothalamic NPFFR2 stimulates the HPA axis and triggers anxiety-like behaviors.

Keywords: neuropeptide FF (NPFF); NPFFR2; HPA axis; anxiety; PVN; CRF

1. Introduction The founding member of the RFamide family, FMRF-amide, was originally isolated from the clam, Macrocallista nimbosa, and was reported to be a cardioexcitatory peptide [1]. Subsequently, two mammalian FMRF-amides were purified from bovine brain, and were named neuropeptide FF (NPFF, FLFQPQRF-NH2) and neuropeptide AF (NPAF, AGEGLSSPFWSLAAPQRF-NH2)[2]. NPFF is sometimes referred to as morphine modulating peptide, but it exhibits no significant binding affinity towards opiate receptors [3]. Two G protein-coupled receptors were identified as NPFF receptors, namely NPFFR1 and NPFFR2 (also called OT7TO22 and HLWAR77, respectively) [4–6]. Both of these receptors are coupled to Gi/o protein upon activation and are highly expressed in the central nervous system, predominantly in the posterior pituitary, thalamus and hypothalamus [3,7,8]. Both NPFFR1

Int. J. Mol. Sci. 2017, 18, 1810; doi:10.3390/ijms18081810 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1810 2 of 13 and NPFFR2 are expressed in various subregions of the hypothalamus, including paraventricular nucleus (PVN), which is upstream of the hypothalamic-pituitary-adrenal (HPA) axis [7,8]. NPFF was then characterized in the context of the RFamide peptide family. There are five subtypes of mammalian RFamide peptides, including NPFF, RF-amide-related peptide (RFRP), pyroglutamylated RF-amide peptide (QRFP), prolactin releasing peptide (PrRP) and kisspeptin (KISS) [9]. Among these subtypes, NPFF-derived peptides include NPFF and NPAF, while Neuropeptide SF (NPSF, also known as RFRP-1) and Neuropeptide VF (NPVF, also known as RFRP-3) are NPVF-derived peptides [9–11]. Both NPFF- and NPVF-derived peptides show binding affinity with NPFF receptors. NPFF-derived peptides display higher affinity with NPFFR2 than NPFFR1, while NPVF-derived peptides display high affinity with NPFFR1 and poor affinity with NPFFR2 [12]. Initial studies of NPFF were mainly focused on its interaction with opioid signaling and the subsequent modulation of antinociceptive effects [3,13,14]. However, emerging evidence has emphasized the modulatory role of NPFF on hypothalamus-related functions, including cardiovascular and neuroendocrine regulation [11]. The HPA axis mediates physiological stress response. Activation of the HPA begins with stimulation of the PVN in the hypothalamus, which releases corticotropin-releasing factor (CRF) to activate pituitary corticotrophs. Upon CRF stimulation, adrenocorticotrophic hormone (ACTH) is released from the pituitary into circulation. ACTH stimulates the secretion of glucocorcorticoid from the adrenal cortex [15], which modulates a wide variety of physiological functions. The HPA axis is known to control metabolism, the immune system, the reproductive system, as well as growth and feeding [15,16]. Importantly, chronic stress and the hyperactivity of HPA axis are highly related to depressive and anxiety-like behaviors [17,18]. NPFF has been reported to regulate hypothalamic PVN neurons [11]. NPFF and NPVF disinhibit γ-aminobutyric acid-ergic (GABAergic) projections to the parvocellular PVN, thereby activating downstream neural circuits [19]. On the other hand, NPFF augments GABAergic neurotransmission to magnocellular PVN neurons, inhibiting the downstream release of vasopressin [20]. These opposing functions may be attributable to individual receptor activities. Other RF-amide peptides have also been reported to regulate the activity of HPA axis and increase the blood corticosteroid (CORT) levels, including NPAF, NPSF, kisspeptin-13 and RFRP-3 [21–24]. The stimulation of NPFFR1 was reported to enhance circulating CORT levels [24], while chronic activation of NPFFR2 leads to HPA axis dysfunction [25]. Continuous stimulation of NPFFR2 possibly disrupts the negative feedback pathway of the HPA axis since the expression of hippocampal glucocorticoid receptors was reduced while serum CORT level increased. Consequently, these mice displayed depressive and anxiety-like behaviors [25]. Nevertheless, direct observations that describe the function of NPFFR2 in regulating the HPA axis are lacking. In an attempt to pinpoint the role of NPFFR2 in HPA axis regulation, NPFFR2 was modulated using pharmacological approaches in rodents, and the effects on HPA axis activity were measured.

2. Results

2.1. Activation of NPFFR2 Stimulates the HPA Axis in Mice and Rats A NPFFR2 non-peptide agonist AC-263093 was used to stimulate the HPA axis. Mice were injected with AC-263093 (5, 7.5, 10, 20 or 30 mg/kg; intraperitoneal, IP) or vehicle and then sacrificed after 1 h by decapitation to collect trunk blood. Serum CORT was measured by a commercially available ELISA kit. The results showed that AC-263093 dose-dependently increased the serum CORT level, an effect that reached statistical significance at 10, 20 and 30 mg/kg compared to vehicle control (Figure1). The data were analyzed by one-way ANOVA (F(5, 24) = 10.81, p < 0.0001) followed by Newman-Keuls post hoc tests. Another NPFFR2 agonist was used to confirm the observations made using AC-263093. Int. J. Mol. Sci. 2017, 18, 1810 3 of 13 Int. J. Mol. Sci. 2017, 18, 1810 3 of 13

Mice(30 mg/kg, wereinjected IP) or vehicle with CFMHC and sacrificed (compound after 31 inh. Ref.The [result26]) (30 showed mg/kg, that IP) CFMHC or vehicle increased and sacriﬁced serum afterCORT 1 h.(Figure The result S3A). showed that CFMHC increased serum CORT (Figure S3A).

400 *** 300 ***

200 *

100 Serum (ng/ml) CORT

0 Vehicle 5 7.5 10 20 30 AC-263093 (mg/kg) FigureFigure 1.1. EffectEffect ofof AC-263093AC-263093on onthe thehypothalamic-pituitary-adrenal hypothalamic-pituitary-adrenal (HPA)(HPA) axisaxis inin mice.mice. MiceMice werewere intraperitonealintraperitoneal (IP)(IP) injectedinjected withwith vehicle or AC-263093 (5, (5, 7.5, 10, 10, 20, 20, 30 30 mg/kg) mg/kg) and and sacrificed sacriﬁced after after 1 1h. h. Levels Levels of of serum serum corticosteroid corticosteroid (CORT) (CORT) were were measured measured by by ELISA. ELISA. Data Data are are expressed expressed as as mean mean ± ±standardstandard error error of ofthe the mean mean (SEM) (SEM) and and were were analyzed analyzed using using a a one-way ANOVA followedfollowed byby Newman-KeulsNewman-Keuls post post hoc hoc tests. tests. *,*,p p< < 0.05;0.05; ***,***, pp << 0.001, compared toto vehiclevehicle controlscontrols( (nn= = 55 perper group).group).

Further,Further, consciousconscious ratsrats werewere utilizedutilized toto monitormonitor thethe changeschanges ofof serumserum CORTCORT atat differentdifferent timetime pointspoints following local local administration administration of of NPFFR2 NPFFR2 agonist. agonist. The The selective selective NPFFR2 NPFFR2 agonist, agonist, dNPA, dNPA, was wasadministered administered to the to lateral the lateral ventricle ventricle (10 nmol, (10 nmol, intracerebroventricular, intracerebroventricular, ICV) ICV)and blood and blood samples samples were werecollected collected from fromthe tail the tailvein vein by byintravenous intravenous (IV) (IV) catheter catheter at at10 10 min min intervals intervals up up to 70 minmin post-administration.post-administration. The results showed that dNPAdNPA time-dependentlytime-dependently elevatedelevated thethe serumserum CORTCORT level,level, reachingreaching aa plateauplateau 4040 minmin afterafter drugdrug treatmenttreatment (Figure(Figure2 2A).A). Analysis Analysis with with two-way two-wayANOVA ANOVA indicatesindicates thatthat therethere werewere statisticallystatistically significantsignificant effectseffects ofof drugdrug treatmenttreatment (F(1,(F(1, 5)5) == 17.28,17.28, pp == 0.0001)0.0001) andand timetime (F(1,(F(1, 5)5) == 2.442,2.442, pp == 0.0443).0.0443). There was no significantsignificant effecteffect ofof thethe interactioninteraction betweenbetween treatmenttreatment andand time.time. Bonferroni Bonferroni post post hoc hoc tests tests show show the the CORT CORT level level was was significantly significantly increased increased at at40 40min min post-drug post-drug administration administration when when comparing comparing dNPA-treated dNPA-treated rat ratto vehicle to vehicle controls controls (p < (0.05).p < 0.05). The Theeffect effect of dNPA of dNPA was was inhibited inhibited by by the the pre-treatmen pre-treatmentt with with NPFF NPFF receptor receptor antagonist, antagonist, RF9 RF9 (10 nmol, ICV),ICV), inin thethe laterallateral ventricleventricle 1515 minmin priorprior toto dNPAdNPA treatmenttreatment (Figure(Figure2 A).2A). Comparing Comparing the the RF-9 RF-9 pretreatedpretreated andand dNPAdNPA alonealone groups,groups, aa two-waytwo-way ANOVAANOVA testtest revealedrevealed aa statisticallystatistically significantsignificant effecteffect ofof drugdrug treatmenttreatment (F(1,(F(1, 5)5) == 17.06,17.06, pp == 0.0001).0.0001). There There was no significantsignificant effect from time,time, oror thethe treatmenttreatment andand timetime interaction.interaction. BonferroniBonferroni postpost hochoc teststests indicateindicate thethe CORTCORT levellevel waswas reducedreduced andand differencesdifferences reached reached statistical statistical significance significance at timeat time points points of 40 of and 40 60and min. 60 Themin. area The under area curveunder (AUC) curve values(AUC) furthervalues showedfurther showed that the serumthat the CORT serum was CORT increased was increased after treatment after treatment with dNPA with and dNPA remained and aremained vehicle-controlled a vehicle-controlled level in rats level pre-treated in rats pr withe-treated RF9 (Figurewith RF92B). (Figur A one-waye 2B). A ANOVA one-way indicated ANOVA thatindicated there that was there a statistically was a statistically significant significant effect of drugeffect treatmentof drug treatment (F(2, 13) (F(2, = 8.122, 13) =p 8.122,= 0.0051). p = 0.0051). Rats treatedRats treated with RF9with alone RF9 exhibitedalone exhibited decreased decreased serum CORTserum levels CORT (Figure levels S2A),(Figure with S2A), a one-way with a ANOVAone-way indicatingANOVA indicating a statistically a statistically significant significant effect of drug effect treatment of drug treatment (F(3, 20) = (F(3, 5.697, 20)p == 0.0055).5.697, p = 0.0055). TheThe activityactivity ofof HPAHPA axisaxis waswas alsoalso evaluatedevaluated afterafter administrationadministration ofof thethe non-peptidenon-peptide NPFFR2NPFFR2 agonist,agonist, AC-263093.AC-263093. Rats were injected withwith AC-263093AC-263093 (30(30 mg/kg,mg/kg, IP) and bloodblood samplessamples werewere collectedcollected fromfrom thethe tailtail veinvein ofof consciousconscious animals,animals, viavia IVIV cathetercatheter atat 1010 minmin intervalsintervals upup toto 9090 minmin post-administration.post-administration. The The results results showed showed that that activation activation of NPFFR2 of NPFFR2 time-dependently time-dependently enhanced enhanced serum CORT,serum withCORT, a peak with level a peak at 60 level min afterat 60AC-263093 min after treatmentAC-263093 (Figure treatment3A). A(Figure two-way 3A). ANOVA A two-way test indicatesANOVA theretest indicates was a statistically there was significant a statistically effect significant of AC-263093 effect treatment of AC-263093 (F(1, 9) treatment = 29.31, p <(F(1, 0.0001), 9) = but29.31, there p < was 0.0001), no significant but there effect was ofno time, significant or treatment effect and of timetime, interaction.or treatment Bonferroni and time post interaction. hoc tests showBonferroni that the post CORT hoc leveltests wasshow increased that the at CORT 60 and level 80 min was post-drug increased treatment at 60 and when 80 comparingmin post-drug the AC-263093-treatedtreatment when comparing group to vehicle the AC-263093-treated controls. The effect group was inhibitedto vehicle by thecontrols. pre-treatment The effect of CRFwas antagonistinhibited byα-helical the pre-treatment CRF (9-41) (200of CRFµg, IV)antagonist (Figure 3αA).-helical A two-way CRF (9-41) ANOVA (200 test μg, indicates IV) (Figure there 3A). was A atwo-way statistically ANOVA significant test effect indicates of CRF there antagonist was treatmenta statistically (F(1, 7)significant = 16.91, p

Int. J. Mol. Sci. 2017, 18, 1810 4 of 13

Int. J. Mol. Sci. 2017, 18, 1810 4 of 13 Int. J. Mol. Sci. 2017, 18, 1810 4 of 13 due to time or the interaction of treatment and time. The AUC calculations indicate that serum CORT group, but was reduced to vehicle-controlled level in animals that were pre-treated with CRF wasgroup, increased but was in the reduced AC-263093-treated to vehicle-controlled group, but leve wasl in reduced animals to that vehicle-controlled were pre-treated level with in animalsCRF antagonist (Figure 3B). The one-way ANOVA indicates that there was a statistically significant effect thatantagonist were pre-treated (Figure 3B). with The CRF one-way antagonist ANOVA (Figure indicates3B). The that one-way there was ANOVA a statistically indicates significant that there effect was from drug treatment (F(2, 18) = 3.762, p = 0.0431). Rats treated with CRF antagonist alone showed a statisticallyfrom drug treatment significant (F(2, effect 18) from = 3.762, drug p treatment = 0.0431). (F(2,Rats 18)treated = 3.762, withp CRF= 0.0431). antagonist Rats treatedalone showed with CRF decreased levels of serum CORT (Figure S2B). A one-way ANOVA indicated that there was a antagonistdecreased alone levels showed of serum decreased CORT levels(Figure of serumS2B). A CORT one-way (Figure ANOVA S2B). Aindicated one-way that ANOVA there indicatedwas a significant difference between drug treatments (F(3, 20) = 5.347, p = 0.0072). thatsignificant there was difference a significant between difference drug treatments between drug (F(3, treatments20) = 5.347, (F(3,p = 0.0072). 20) = 5.347, p = 0.0072). ABVehicle ABVehicledNPA 350 dNPARF9+dNPA 1200 350 RF9+dNPA 1200 300 * * 300 RF-9 (ICV ) * * 250 RF-9 (ICV ) 900 250 900 200 dNPA or Vehicle (ICV) 200 dNPA or Vehicle (ICV) 600 150 600 150 100 100 300

## # CORTSerum (AUC) 300 50 Serum CORT level (%) level CORT Serum

## # CORTSerum (AUC) 50 Serum CORT level (%) level CORT Serum 0 0 0 -15 0 10 20 40 60 70 0 -15 0 10 20 40 60 70 PA Time (min) dNPA PA Vehicle Time (min) dNPA Vehicle RF9+dN RF9+dN Figure 2. Time-dependent effects of treatment with dNPA on serum CORT in rats. Rats were injected FigureFigure 2. 2.Time-dependent Time-dependent effects effectsof of treatmenttreatment with dNPA dNPA on on serum serum CORT CORT in in rats. rats. Rats Rats were were injected injected with vehicle or dNPA (10 nmol, intracerebroventricular, ICV) and serum CORT was monitored up to withwith vehicle vehicle or or dNPA dNPA (10 (10 nmol, nmol, intracerebroventricular,intracerebroventricular, ICV) ICV) and and serum serum CORT CORT was was monitored monitored upup to to 70 min following injection. (A) Levels of serum CORT were measured to indicate the activity of the 7070 min min following following injection. injection. ((AA)) LevelsLevels ofof serum CORT CORT were were measured measured to to indicate indicate the the activity activity of ofthe the HPA axis. Neuropeptide FF (NPFF) receptor antagonist RF9 (10 nmol, ICV) was administered 15 min HPA axis. Neuropeptide FF (NPFF) receptor antagonist RF9 (10 nmol, ICV) was administered 15 min HPAprior axis. to NeuropeptidedNPA treatment. FF Data (NPFF) are receptorexpressed antagonist as mean ± RF9 SEM (10 and nmol, were ICV) analyzed was administered using a two-way 15 min prior to dNPA treatment. Data are expressed as mean ± SEM and were analyzed using a two-way priorANOVA to dNPA following treatment. by Bonferroni Data are post expressed hoc tests. as * mean p < 0.05, SEMcompared and weredNPA analyzed to vehicle using controls. a two-way # p < ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls. # p < ANOVA0.05; ## followingp < 0.01, compared by Bonferroni dNPA postto RF9+dNPA hoc tests. group; * p < ( 0.05,B) The compared results of dNPAarea under to vehicle curve (AUC) controls. 0.05; ## p < 0.01, compared dNPA to RF9+dNPA group; (B) The results of area under curve (AUC) # pcalculation.< 0.05; ## p Data< 0.01, are compared expressed dNPA as mean to RF9+dNPA ± SEM and group; were (B )analyzed The results using of areaa one-way under curve ANOVA (AUC) calculation. Data are expressed as mean ± SEM and were analyzed using a one-way ANOVA calculation.following Databy Bonferroni are expressed post ashoc mean tests.± *SEM p < 0.05, and werecompared analyzed dNPA using to vehicle a one-way controls ANOVA (n = 4–7 following per following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls (n = 4–7 per bygroup). Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls (n = 4–7 per group). group). A Vehicle B A VehicleAC-263093 B AC-263093CRF antagonist + AC-263093 500 CRF antagonist + AC-263093 3000 500 3000 * -helical CRF9-41 (IV) 2500 * 400 -helical CRF (IV)  9-41 * * 2500 400 * * Vehicle or AC-263093 (IP) 2000 300 Vehicle or AC-263093 (IP) 2000 300 1500 200 1500 200 1000 1000 serum CORT (%) serum 100 Serum CORT(AUC) Serum serum CORT (%) serum 100 500 Serum CORT(AUC) Serum 500 0 0 0 -15 0 10 20 30 40 50 60 70 80 90 0 icle -15 0 10 20 30 40 50 60 70 80 90 h Time (min) Ve icle Time (min) h Ve AC-263093 AC-263093t + AC-263093 nis + AC-263093 o t nis tago tag CRF an CRF an Figure 3. Time-dependent effects of treatment with AC-263093 on serum CORT in rats. Rats were FigureFigure 3. 3.Time-dependent Time-dependent effectseffects ofof treatmenttreatment with AC-263093 AC-263093 on on serum serum CORT CORT in in rats. rats. Rats Rats were were injected with vehicle or AC-263093 (30 mg/kg, IP) and serum CORT was monitored for up to 90 min injectedinjected with with vehicle vehicle or or AC-263093 AC-263093 (30 (30 mg/kg,mg/kg, IP) IP) and and serum serum CORT CORT was was monitored monitored for for up up to to90 90min min post-administration. (A) Levels of serum CORT were measured by ELISA. Corticotropin-releasing post-administration.post-administration. (A ()A Levels) Levels of of serum serum CORT CORT were were measured measured by ELISA.by ELISA. Corticotropin-releasing Corticotropin-releasing factor factor (CRF) antagonist α-helical CRF(9-41) (200 μg, intravenous, IV) was applied 15 min prior to (CRF)factor antagonist (CRF) antagonistα-helical α CRF(9-41)-helical CRF(9-41) (200 µg, (200 intravenous, μg, intravenous, IV) was appliedIV) was 15applied min prior 15 min to AC-263093prior to AC-263093 treatment. Data are expressed as mean ± SEM and analyzed using a two-way ANOVA treatment.AC-263093 Data treatment. are expressed Data are as expressed mean ± SEM as mean and analyzed± SEM and using analyzed a two-way using a ANOVA two-way following ANOVA by following by Bonferroni post hoc tests. * p < 0.05, compared AC-263093 to vehicle controls; (B) The Bonferronifollowingpost by Bonferroni hoc tests. *postp < hoc 0.05, tests. compared * p < 0.05, AC-263093 compared to AC-263093 vehicle controls; to vehicle (B) Thecontrols; results (B of) The AUC results of AUC calculation. Data are expressed as mean ± SEM and were analyzed using a one-way calculation.results of DataAUC arecalculation. expressed Data as mean are expressed± SEM and as mean were analyzed± SEM and using were a analyzed one-way using ANOVA a one-way following ANOVA following by Bonferroni post hoc tests. * p < 0.05, compared dNPA to vehicle controls (n = byANOVA Bonferroni following post hoc by tests. Bonfe * rronip < 0.05, post compared hoc tests. dNPA* p < 0.05, to vehicle compared controls dNPA (n =to 4–11vehicle per controls group). (n = 4–11 per group). 4–11 per group).

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2.2. NPFFR2 NPFFR2 Agonist Agonist Induces Activity of Hypothalamic PVN Neurons in Mice Neuronal activity in the hypothalamic PVN was evaluatedevaluated to verify the role of NPFFR2 on the HPA axis. The The levels levels of of c-Fos c-Fos protein protein were were measur measureded 1 h 1 after h after mice mice were were treated treated with with AC-263093 AC-263093 (30 mg/kg,(30 mg/kg, IP) or IP) vehicle. or vehicle. The The results results showed showed that that c-Fos c-Fos protein protein was was upregula upregulatedted in in the the hypothalamic hypothalamic PVN ofof NPFFR2NPFFR2 agonist-treated agonist-treated mice mice (Figure (Figure4A). Quantifying4A). Quantifying the number the number of c-Fos-positive of c-Fos-positive neurons neuronswithin the within PVN the indicated PVN indicated that treatment that treatmen with NPFFR2t with agonistNPFFR2 increased agonist PVNincreased activity PVN (unpaired activity (unpairedStudent’s tStudent’s-test, t(6) =t-test, 3.809, t(6)p = = 0.0089) 3.809, (Figurep = 0.0089)4B). This (Figure ﬁnding 4B). demonstrates This finding thatdemonstrates stimulation that of stimulationNPFFR2 enhances of NPFFR2 the activity enhances of hypothalamicthe activity of PVNhypothalamic neurons. PVN neurons.

Figure 4. EffectsEffects of of AC-263093 AC-263093 treatment treatment on on c-Fos c-Fos ex expressionpression in in paraventricular nucleus nucleus (PVN) (PVN) neurons. Mice Mice were were injected injected with with vehicle vehicle or orAC-263093 AC-263093 (30 (30 mg/kg, mg/kg, IP) and IP) andsacrificed sacrificed after after 1 h. 1(A h.) Immunofluorescence(A) Immunofluorescence staining staining was was used used to todetect detect c-Fos c-Fos expression expression in in the the PVN. PVN. Nuclei Nuclei were counterstained with DAPI; ((B)) QuantificationQuantification ofof c-Fos-positivec-Fos-positive cellcell numbers numbers in in the the PVN. PVN. Histogram Histogram B Bshows shows the the c-Fos c-Fos immunoreactive immunoreactive neurons neurons counts counts expressed expressed asas sumsum ofof bilateralbilateral frontal cross section surface areas of PVN. Data are expressedexpressed asas meanmean ±± SEMSEM and and were analyzed using an unpaired Student’s t-test. **, **, p < 0.01, 0.01, compared compared to to vehicle vehicle control control ( n = 4 4 per per group). group). Scale Scale bar bar = = 100 100 μµm.m.

2.3. NPFFR2 NPFFR2 Stimulation Stimulation Induces Anxiety-Like Behavior Anxiety-like behavior was monitored after 1 h, in mice treated with AC-263093 (30 mg/kg, IP). IP). The elevated plus maze (EPM) was used to evaluate anxiety-like behavior. The duration in the open arm waswas reducedreduced (unpaired (unpaired Student’s Student’st-test, t-test,t(13) t(13) = 3.406, = 3.406,p = 0.0047),p = 0.0047), while while the duration the duration in the closedin the closedarm was arm increased was increased in AC-263093-treated in AC-263093-treated mice, compared mice, to compared vehicle controls to vehicle (unpaired controls Student’s (unpairedt-test, Student’st(13) = 2.532, t-test,p = t(13) 0.0251) = 2.532, (Figure p =5 A).0.0251) In addition, (Figure the5A). number In addition, of open the arm number entries of wasopen decreased arm entries in wasmice decreased treated with in AC-263093mice treated (unpaired with AC-263093 Student’s (unpairedt-test, t(13) Student’s = 3.489, tp-test,= 0.0040) t(13) (Figure= 3.489,5 pB), = but0.0040) the (Figureadministration 5B), but of the AC-263093 administration had no of influence AC-263093 on thehad locomotor no influence activity, on the as evaluatedlocomotor by activity, total time as evaluatedspent with by movement total time in spent the EPM with (Figure movement5C) as in well the as EPM open (Figure field (Figure 5C) as S1). well These as open results field were (Figure then S1).confirmed These usingresults another were then NPFFR2 confirmed agonist, using CFMHC another (Figure NPFFR2 S3). The agonist, findings CFMHC indicate (Figure that activation S3). The of findingsNPFFR2 indicate induces anxiety-likethat activation behavior. of NPFFR2 induces anxiety-like behavior.

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A B C

400 Vehicle 20 400 AC-263093 * 300 15 300

200 10 200 90 ** ** 60 5 100 Open arm entries arm Open Time in arms (sec) arms Time in 30 ** (sec) Total activity 0 0 0 Open arm Close arm Vehicle AC-263093 Vehicle AC-263093 Figure 5.5.Effects Effects of AC-263093of AC-263093 treatment treatment on anxiety-like on anxiety- behavior.like behavior. Mice were Mice injected were with injected AC-263093 with (30AC-263093 mg/kg, (30 IP) 1mg/kg, h prior IP) to 1 behavioral h prior to testing.behavioral Anxiety-like testing. Anxiety-like behavior was beha evaluatedvior was by evaluated the elevated by plusthe elevated maze (EPM). plus (mazeA) Time (EPM). that mice(A) Time stayed that in themice open stayed arms in or th closede open arms; arms (B or) Number closed arms; of entries (B) Numberinto open of arms; entries (C )into Total open time arms; spent (C with) Total movement. time spent Data with are movement. expressed Data as mean are expressed± SEM and as mean were ±analyzed SEM and using were an analyzed unpaired using Student’s an unpairedt-test. *,Student’sp < 0.05; t-test. **, p <*, 0.01,p < 0.05; compared **, p < to0.01, vehicle compared controls to (vehiclen = 7–8 controls per group). (n = 7–8 per group).

3. Discussion In this study, we utilized a pharmacological approach to uncover the direct involvement of NPFFR2 in determining stress-dependent biochemi biochemicalcal and behavioral changes in rodents. NPFFR2 agonists, dNPA or AC-263093, administeredadministered to mice and rats, evoked increased serum CORT levels in a dose- and time-dependent manner. The phenom phenomenonenon was validated by reversal using the NPFF receptor antagonist, RF9. The NPFFR2 agonist-indu agonist-inducedced elevation of serum CORT was also inhibited by a CRF antagonist. Mice treated with NPFFR2 agonistagonist showed anxiety-like behavior (assessed by EPM) andand increasedincreased c-Fosc-Fos expression expression in in hypothalamic hypothalamic PVN PVN neurons. neurons. These These findings findings suggest suggest that that the thestimulation stimulation of NPFFR2 of NPFFR2 within within the PVNthe PVN activates activates the HPAthe HPA axis. axis. This mightThis might act concomitantly act concomitantly with withother other limbic limbic structures structures to trigger to trigger the anxiogenic the anxiogenic behavior. behavior. In thisthis study,study, mice mice were were used used to evaluate to evaluate the dose-response the dose-response of NPFFR2 of NPFFR2 agonist-induced agonist-induced changes changesin serum in CORT, serum c-Fos CORT, expression c-Fos expression in PVN and in PVN anxiety-like and anxiety-like behaviors. behaviors. Blood samples Blood weresamples collected were collectedupon decapitation, upon decapitation, precluding precluding time course time measurements course measurements on these animals. on these Because animals. it wasBecause technically it was technicallynot feasible not to insert feasible tail to vein insert catheters tail vein into mice,catheters time-course into mice, studies time-course on NPFFR2 studies agonist-induced on NPFFR2 agonist-inducedchanges of serum changes CORT were of serum performed CORT in were rats. Interestingly,performed in ourrats. results Interestingly, show that our the results responses show of thatserum the CORT responses to NPFFR2 of serum agonist CORT were to comparable NPFFR2 betweenagonist were both rodentcomparable species. between Furthermore, both rodent dNPA (aspecies. peptide Furthermore, with poor blood-brain dNPA (a barrierpeptide penetrationwith poor thatblood-brain was administered barrier penetration ICV) and AC-263093 that was (aadministered small molecule, ICV) administeredand AC-263093 by (a IP small injection) molecule, were similarlyadministered effective by IP at injection) enhancing were the similarly levels of effectivecirculating at CORT. enhancing Together, the theselevels findings of circulatin confirmedg CORT. that NPFFR2Together, acts these in the findings central nervousconfirmed system that NPFFR2of both rats acts and in the mice central to regulate nervous the system HPA axis. of both rats and mice to regulate the HPA axis. There is a growing body of evidenceevidence describingdescribing the involvement of RFamide peptides in controlling anxiety-related behaviors behaviors and and HPA HPA axis activity, but the roles for NPFFR1 and NPFFR2 remain unclear. RatsRats treatedtreated withwith NPAF,NPAF, NPSF, NPSF, kisspeptin-13 kisspeptin-13 and and RFRP-3 RFRP-3 all al showl show enhanced enhanced levels levels of ofcirculating circulating CORT CORT [21– 24[21–24].]. With theWith exception the exception of NPSF, of these NPSF, neuropeptides these neuropeptides also induce also anxiety-like induce anxiety-likebehaviors. It behaviors. was speculated It was thatspeculated NPSF may that exhibitNPSF may relatively exhibit weak relatively binding weak affinity binding to both affinity NPFF to bothreceptors. NPFF Thus, receptors. the physiological Thus, the effectsphysiological of NPSF mayeffects be mediatedof NPSF throughmay be acidmediated sensing through ion channels acid sensingrather than ion NPFFchannels receptors rather than [23,27 NPFF]. Because receptors NPAF [23,27]. and RFRP-3 Because exert NPAF different and RFRP-3 and complementary exert different andbinding complementary affinities toward binding NPFFR2 affinities and NPFFR1 toward [ 10NPFFR2,28], their and similar NPFFR1 effects [10,28], suggest their that similar both receptors effects suggestmay be involvedthat both in receptors the regulation may ofbe theinvolved HPA axis in andthe relevantregulation behavioral of the HPA changes. axis Theand effectsrelevant of behavioralRFRP-3 on thechanges. HPA axis The were effects inhibited of RFRP-3 by a newlyon the developed HPA axis NPFF were receptorinhibited antagonist by a newly GJ14 developed [24]. This NPFFantagonist receptor displays antagonist higher GJ14 affinity [24]. toward This antagoni NPFFR1st (IC displays50, 21.08 higher nM) than affinity NPFFR2 toward (IC 50NPFFR1, 387.34 (IC nM),50, 21.08suggesting nM) than that activationNPFFR2 (IC of50 brain, 387.34 NPFFR1 nM), stimulatessuggesting the that HPA activation axis [24 of]. Tobrain address NPFFR1 the role stimulates of NPFFR2 the HPAin HPA axis axis [24]. activation, To address we treated the role rats of with NPFFR2 the highly in HPA selective axis NPFFR2activation, agonist we treated dNPA, rats administered with the highly selective NPFFR2 agonist dNPA, administered directly into the lateral ventricle of conscious animals. dNPA time-dependently increased the level of serum CORT and this phenomenon was

Int. J. Mol. Sci. 2017, 18, 1810 7 of 13 directly into the lateral ventricle of conscious animals. dNPA time-dependently increased the level of serum CORT and this phenomenon was inhibited by intra-ventricular pre-treatment of the NPFFR antagonist, RF9. The effect of NPFFR2 activation was confirmed using another NPFFR2 agonist, AC-263093, which dose- and time-dependently enhanced serum CORT. These data support a positive regulatory role for NPFFR2 on the activity of the HPA axis. Another reported NPFFR2 agonist, CFMHC, was also used to confirm the effect of AC-263093. The results indicate the administration of CFMHC not only increases the level of serum CORT but also induces anxiety-like behavior. However, it should be noted that the selectivity of CFMHC between NPFFR1 and NPFFR2 is not high [26]. When we measured NPFFR2 agonist-induced changes in serum CORT, the rats were conscious and gently restrained by hand with cotton gloves. Prior to the day of the experiment, the experimental operator handled the rats daily, lightly restraining the animals by hand and gently touching their faces to reduce anxious behaviors. On the day of the experiment, rats were accustomed to the touch of the operator, and exhibited greatly reduced physical struggles and flinching behavior, compared to the initial acclimation sessions. With this experimental protocol, we were able to continuously monitor the drug effects without the influence of anesthesia. Despite our efforts to acclimate the animals, the potential for restraint-induced stress response was still of concern. According to our CORT ELISA measurements, the concentration of serum CORT was approximately 200 ng/mL at time zero. This relatively elevated level of serum CORT at the initiation of the experiment suggests that the effects of dNPA and AC-263093 were in addition to an underlying elevation of serum CORT from handling stress. However, in our experiments with mice, AC-263093 was clearly demonstrated to activate the HPA axis in the absence of additional stressors (Figure1). On the other hand, the injection of CRF antagonist and RF9 reduced the levels of serum CORT. This result implies that experimental rats were under handling stress, since the inhibition of NPFFR2 could reduce the activity of HPA axis and stress responses. RF9 was first synthesized in 2006 and was recognized as an NPFF receptor antagonist [29]. It had been reported to inhibit opioid-induced hyperalgesia, development of tolerance toward opioids, NPFF-induced hypothermia and lipopolysaccharide (LPS)-triggered fever in mice [29–31]. However, RF9 was recently been reported to act as an agonist of kisspeptin [32–34]. In this study, RF9 was used to inhibit HPA axis activity that was stimulated by dNPA. Although we observed a significant inhibitory effect on the action of NPFFR2 agonist dNPA, the possibility that RF9 produces this effect despite acting on other receptors cannot be excluded. NPFF and other RFamide neuropeptides were reported to modulate various functions of the hypothalamus, including cardiovascular and neuroendocrine regulation, energy consumption, food intake and reproduction [10,11]. Both NPFFR1 and NPFFR2 are highly expressed in hypothalamic nuclei [11] and may modulate these effects. Furthermore, NPFF was reported to regulate GABA neurotransmission within parvocellular and magnocellular PVN neurons, and in turn, to modulate downstream neural pathways [19,20]. The regulation of parvocellular PVN neurons was further shown to be due to a reduction of inhibitory postsynaptic currents, causing neuronal depolarization [19]. NPFF was also shown to activate the oxytocin-containing hypothalamic PVN neurons that project to the brainstem and increase arterial blood pressure [35]. Besides modulating GABA, NPFF may affect CRF in the PVN. CRF is synthesized and expressed in the parvocellular PVN neurons [36,37], where it acts as an upstream regulator of the HPA axis [15]. It was previously reported that NPFF-induced c-Fos expression is not predominantly colocalized with CRF containing neurons in the PVN [35]. However, comparing a recent published article with our IHC figures, we notice that AC compound-induced c-Fos proteins distribute similarly among the PVN subregions, including CRF, vasopressin and oxytocin-containing neurons [38]. Nevertheless, we observed that NPFFR2 agonist increased c-Fos expression in the PVN, and that a CRF inhibitor could attenuate NPFFR2 agonist-induced CORT release. The results suggest that NPFFR2 may inactivate PVN GABA neurons, and as such, it is possible that NPFFR2 evokes hypothalamic CRF release from parvocellular neurons. It was noted that PrRP also increases CRF neural activity in the PVN [39]. Interestingly, the effects of PrRP were suggested to be mediated through NPFFR2, since PrRP exhibits Int. J. Mol. Sci. 2017, 18, 1810 8 of 13 high binding affinity toward NPFFR2 [40,41]. Therefore, our study and others suggest that NPFFR2 plays a positive modulatory role in hypothalamic CRF neurons. It was noted that the c-Fos positive neurons were slightly more numerous in the vehicle control group (approximately 100 neurons per brain slice). This might be due to the 5% dimethyl sulfoxide (DMSO) that was contained in the vehicle, which had been reported to have predominated influence on neural activities [42–45]. The handling stress may also have an influence on the basal c-Fos cell counts; however, that phenomenon does not affect our main conclusion. NPFF receptor antagonists have been reported to confer anxiolytic effects. Treatment with RF9 reverses amphetamine withdrawal-induced anxiety-like behavior [46], and dansyl-PQRamide (a putative NPFF antagonist) reduces anxiety responses triggered by ethanol withdrawal [47]. We have recently reported that chronic stimulation of NPFFR2 triggers hyperactivity of the HPA axis and disrupts hippocampal feed-back regulation resulting in depression-like behaviors in mice [25]. These results suggest an anxiogenic effect from brain NPFFR2, as reported in the present study. However, another study showed that intra-ventral tegmental area (VTA) injection of NPFF prevents mild stress-induced locomotor activity [48]. The inconsistent regulation of NPFF in different aspects of stress response might be due to the involvement of different neural circuitries that impinge on the VTA and hypothalamus.

4. Materials and Methods

4.1. Animals Sprague Dawley (SD) rats (250–300 g) and C57BL/6 (B6) mice (8–10 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and acclimatized to the room with controlled temperature, air humidity and a 12 h day-night cycle (light on at 7:00 AM). Mice were housed 5–6 per cage and rats were housed 2–3 per cage, with food (Western Lab 7001, Orange, CA, USA) and water ad libitum. All the animals used in this study were male. Animal handling and drug treatments were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Animal Care Committee of Chang-Gung University (CGU 13-014, approved on 1 April 2013).

4.2. NPFFR2 Agonists Non-peptide NPFFR2 agonists, AC-263093 (2-[(3,4-dibromophenyl) methylene]-hydrazine- carboximidamide hydrochloride) and CFMHC (2-[[4-chloro-3-(triﬂuoromethyl)phenyl]methylene]- Hydrazinecarboximidamidehydrochloride) were used in current study [26]. The preparation of AC-263093 and CFMHC was described previously [25,49]. The drugs were dissolved in a vehicle solution consisting of DMSO: Tween 20: saline at a 1:2:17 ratio. Selective peptide NPFFR2 agonist dNPA (D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) was commercially synthesized by Genemed Synthesis (San Antonio, TX, USA) and dissolved in 20% methanol with PBS, then diluted to 10% methanol before use.

4.3. Implantation and Cannulation in the Lateral Ventricle Rats were anesthetized with pentobarbital (45 mg/kg, IP) and atropine (0.1 mg/kg, IP), and then placed into a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). The animals were implanted with a 23 G cannula positioned in the lateral ventricle at the coordinates: 0.8 mm posterior to the bregma, 1.4 mm lateral from the midline, and 3.7 mm ventral from the dura [50]. A stainless tube was left inside of cannula (0.5 mm protrusion) to prevent the cannula blocked by blood clog. After experiments, the brain was collected and sectioned by the rat brain matrices to validate the position of cannula using the anatomic microscope. Ampicillin (1 mg/kg, IP) was daily applied to prevent infection after the surgery. Rats were allowed to recover for 5 d before receiving ICV injections of either vehicle or dNPA. Int. J. Mol. Sci. 2017, 18, 1810 9 of 13

4.4. Pharmacological Intervention Studies Rats were anesthetized with 2% isoflurane and an IV catheter (24 G, Terumo, Laguna, Philippines) was inserted into the tail vein. The catheter was flushed with 150 µL Heparin solution (50 U/mL, China Chemical & Pharmaceutical Co., Taipei City, Taiwan) to prevent blood clotting. After removal from the isoflurane anesthesia, rats were gently restrained by hand with cotton gloves to reduce anxious behavior (gentle body movement was allowed). A rest time of 30 min elapsed prior to drug treatment. To reduce the stress responses, the experimental operator needs to remain in daily close contact with rats by holding, slightly restraining by hand and gently touching the faces of rats. The rats became much less anxious during experimental days. The cotton gloves were put into the home cages of experimental rats daily for 20 min to get used by rats and acquired the smell of rat bedding on the same time. A 30 min intervals after rats recovered from isoflurane anesthesia but prior to drug treatment was also important to reduce the stress-induced responses. Two tubes of unused blood (50 µL) were collected within this rest period to ensure the smooth functioning of the IV catheter. After treatment with vehicle or AC-263093 (30 mg/kg, IP), approximately 80 µL blood was collected at 10 min intervals until 90 min had elapsed. In the CRF antagonist-pretreated group, α-helical CRF(9-41) (200 µg in 300 µL saline, Phoenix Pharmaceuticals, Burlingame, CA, USA) was injected into the tail vein 15 min prior to AC-263093 treatment (blood samples at 20 and 40 min post-injection of AC-263093 were not collected). In addition, NPFFR2 selective agonist dNPA was applied to stimulate the HPA axis. After ICV vehicle or dNPA (10 nmol) injection, approximately 80 µL blood was collected at 10 min intervals until 70 min had elapsed. In the NPFFR antagonist-pretreated group, RF9 (10 nmol, Tocris Bristol, UK) was injected (ICV) 15 min prior to dNPA treatment. Time zero indicates the time point immediately before the treatment of NPFFR2 agonists (AC-263093 or dNPA). Due to the individual difference between rats, the levels of serum CORT were normalized to percentage and time zero served as 100% in each rat.

4.5. Immunohistochemistry (IHC) Mice were anesthetized with 45 mg/kg pentobarbital and then perfused with 4% paraformaldehyde after treatment with AC-263093 (30 mg/kg, IP) or vehicle for 1 h. The brain was then removed and cryoprotected with 20% sucrose in potassium phosphate buffer saline (KPBS) overnight at 4 ◦C. Brain tissues were sliced into 30 µm sections using a freezing microtome (LEICA CM3050S, Bannockburn, IL, USA). After rinsing with KPBS, sections were incubated in primary antibody (anti-c-Fos, 1:1000; Santa Cruz, CA, USA) in 2% normal serum/0.3% Triton X-100/KPBS at 4 ◦C for 24 h. Afterward, sections were incubated with 1:200 FITC-conjugated secondary antibody (FITC anti-rabbit antibody; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Sections were then incubated with 5 µg/mL 40,6-diamidino-2-phenylindole (DAPI, Roche, Switzerland) for 5 min before mounting with glycerol. Immunofluorescent staining were observed by fluorescence microscopy (Olympus, Tokyo, Japan). The immunofluorescent signals were analyzed by ImageJ software (NIH, Bethesda, MD, USA). The result was presented as c-Fos+ cell number in one brain slice of bilateral PVN. The specificity of c-Fos antibody (sc-52, Santa Cruz, CA, USA) in mouse brain had been tested in a previous report using synthetic c-Fos control peptide [51].

4.6. CORT ELISA Assay To obtain serum samples, blood was clotted at room temperature for 30 min and centrifuged at 15,000 rcf for 15 min. Serum CORT were analyzed by ELISA kits (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocols.

4.7. Elevated Plus Maze (EPM) EPM was used to test anxiety-like behavior. The EPM chamber contains two open arms and two closed arms, and is raised 100 cm off the ground. Light intensities in open arms and close arms are Int. J. Mol. Sci. 2017, 18, 1810 10 of 13 between 80–85 Lux. A test mouse was placed on the middle of the elevated maze (face to the open arms) and movement was tracked by video recording for a session of 6 min. The time spent in open and closed arms, and the numbers of entries into open arms were calculated from the video recording replay. The total activity was also recorded as the total time spent in movement within 6 min. Mice were considered to have entered the arms once the middle part of their body passed the border of the arms.

4.8. Open Field Activity Mice were transferred to an open ﬁeld chamber (40 cm × 40 cm) 60 min after injection with AC-263093 (30 mg/kg, IP). The activity was then evaluated by tracking the body movement within 30 min (EthoVision, Noldus, Wageningen, The Netherlands). The results are presented as the distance that mice moved in every 5 min for a total session of 30 min.

4.9. Statistical Analysis The effect of NPFFR2 agonist-induced CORT elevation in mice was analyzed by one-way ANOVA followed by Newman-Keuls post hoc tests (independent factors). The effect of NPFFR2 agonist-induced CORT elevation in rats was analyzed by the repeated measures two-way ANOVA followed by Bonferroni post hoc tests (dependent factor was level of serum CORT, independent factor was drug treatment). The following AUC was analyzed by one-way ANOVA with Bonferroni post hoc tests (independent factors). The Immunostaining assay and behavioral test were analyzed by the unpaired Student’s t-test (independent factors). All the statistical analyses were performed using GraphPad Prism 5 software with statistical signiﬁcance set as p < 0.05.

4.10. Experimental Summary The experimental details are summarized below (Table1).

Table 1. The experimental summary.

Experiment in Figure Subject Experimental Groups n Numbers Vehicle 5 Figure1 Mouse AC-263093 5 Vehicle 5 dNPA 7 Figure2 Rat RF9 + dNPA 4 RF9 (Figure S2) 6 Vehicle 7 AC-263093 11 Figure3 Rat α-helical CRF(9-41) + AC-263093 4 α-helical CRF(9-41) (Figure S2) 6 Vehicle 4 Figure4 Mouse AC-263093 4 Vehicle 7 Figure5 Mouse AC-263093 8

5. Conclusions In summary, we demonstrate, for the ﬁrst time, that NPFFR2 positively modulates the HPA axis via hypothalamic CRF. Our ﬁnding that NPFFR2 regulates stress-dependent neural pathways advances our understanding of neuroendocrine regulation and opens new avenues for probing the functions of NPFF. Int. J. Mol. Sci. 2017, 18, 1810 11 of 13

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/8/1810/s1. Acknowledgments: We thank M. Calkins for providing English editing. This work was supported by the Chang Gung University, Healthy Aging Research Center (EMRPD1G0171) and Ministry of Science and Technology (105-2320-B-182-012-MY2). Author Contributions: Ya-Tin Lin performed data acquisition and analysis, drafted the initial manuscript, and reviewed and revised the manuscript. Yu-Lian Yu, Wei-Chen Hong, Ting-Shiuan Yeh and Ting-Chun Chen contributed to the data acquisition and analysis. Jin-Chung Chen conceived, designed, and oversaw the execution of the study and revised the manuscript. All authors discussed the results and commented on the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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