Developmental Brain Research 143 (2003) 129–136www.elsevier.com/ locate /devbrainres

Research report

C entral expression of c-Fos in neonatal male and female prairie volesin response to treatment with oxytocin

a , a,b b a ,1*Bruce S. Cushing , Yukiyo Yamamoto , Gloria E. Hoffman , C. Sue CarteraDepartment of Biology, University of Maryland, College Park, MD 20742, USA

bDepartment of Anatomy and Neurobiology, University of Maryland Baltimore, Baltimore, MD 21201, USA

Accepted 26 March 2003

Abstract

Early postnatal exposure to both exogenous and endogenous oxytocin (OT) can have long-term effects on behavior and physiology,although the mechanisms of these effects are not known. c-Fos expression was used to investigate the immediate neural effects ofneonatal manipulations of OT in male and female prairie voles. On the day of birth prairie vole pups received an intraperitoneal injectionof OT, a selective OT antagonist (OTA), or saline (vehicle control), while an additional group was handled but not injected. One hourafter treatment brains were collected and fixed via spinning immersion and immunocytochemistry was then used to label for c-Fosimmunoreactivity (IR). There were significant differences between males and females. Handled only females displayed significantlyhigher levels of c-Fos IR in the mediodorsal thalamic nucleus (MD) than males while handled males had higher c-Fos IR in theparaventricular nucleus of the hypothalamus than females. Exogenous OT stimulated neuronal activity in the supraoptic nucleus (SON) inmales, while treatment with OTA increased Fos IR in the SON and was associated with reduced Fos IR in the MD in females. The resultsindicate that neuronal activity and responses to OT are sexually dimorphic in newborn prairie voles. In females changes in Fos expressionwere stimulated by treatment with OTA, suggesting that endogenous OT affects cellular activity while males responded to exogenous OT. 2003 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Hormones and development

Keywords: c-Fos; Oxytocin; Oxytocin antagonist; Mediodorsal thalamic nucleus; Paraventricular nucleus; Supraoptic nucleus; Neonatal; Prairie vole

1 . Introduction growth [34]. Social behaviors also appear to be sensitive tothe effects of OT [9]. In male prairie voles (Microtus

It has been suggested that oxytocin (OT) may have ochrogaster) a single neonatal treatment with OT facili-organizational effects on the central nervous system tated the onset of partner preferences [3], while treatment[32,44]. This hypothesis is supported by findings that early with an OT antagonist (OTA), which blocks the affects ofpostnatal manipulations of OT can have long-term conse- endogenous OT, decreased paternal behavior and increasedquences for adult behavior and physiology. Adult female pup attacks [31] and decreased reproductive competencyrats treated postnatally with OT had significant increases in [6]. In female prairie voles neonatal treatment with OTweight, reduced responses to pain [37] and subsequently facilitated parental care [4], produced an increase induring pregnancy displayed increased placental and fetal aggression following exposure to a male [5] and affected

the frequency of ultrasound vocalizations [25]. Long-last-ing modifications in behavior or physiology in response to

*Corresponding author. Current address: Brain-Body Center, Depart- neonatal OTA support the hypothesis that endogenous OTment of Psychiatry, College of Medicine, University of Illinois at can influence the development of the CNS during theChicago, Chicago, IL 60612, USA. Tel.: 11-312-413-0472; fax: 11-312- postnatal period. Indirect evidence for a developmental996-9358. role for OT also comes from studies in rats. High levels ofE-mail address: bcushing@psych.uic.edu (B.S. Cushing).1 maternal care resulted in an increased expression of OTCurrent address: Brain-Body Center, Department of Psychiatry, Collegeof Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. receptors (OTR) in the adult females [18,19]. In turn, adult

0165-3806/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0165-3806(03)00105-6

130 B.S. Cushing et al. / Developmental Brain Research 143 (2003) 129–136

females that had experienced high levels of maternal Pups were assigned to treatment groups randomly with thestimulation displayed increased social behavior in com- restriction that within each litter and for each sex, thereparison to pups that received low levels of maternal was at least one control (SAL or HAN) and one ex-grooming [17]. perimental (OT or OTA) pup. No treatment was repre-

Analyses of the developmental consequences of neonatal sented more than once per litter. Pups were injected,OT manipulations suggest that the functions of this peptide returned to their parents, and then 1 h later they wereare sexually dimorphic. In prairie voles, neonatal OT deeply anesthetized using a combination of ketamine andtreatment resulted in increased parental behavior [4], post xylazine and decapitated. Brains were removed and fixedcohabitation aggression [5] and affected ultrasound pro- using a spinning immersion fixation technique [11]. Eachduction and corticosterone levels in 8-day-old pups only in brain was placed in a scintillation vial containing 20 ml offemales [25]. In contrast, neonatal OTA inhibited the 4% paraformaldehyde and 5% acrolein in 0.1 M KPBSexpression of alloparental behavior and pup directed (pH 7.6), and then gently spun (50 rpm) for 10 min on aaggression in males but not females [6]. In rats, females magnetic stirring plate using a 1.2-cm stir bar. After 10that received high levels of maternal care displayed an min the brain was removed and blocked, exposing theincrease in OTR while males that received the same care lateral ventricles and then placed back into the fixative anddisplayed an increase in vasopressin (V ) receptors [19]. spun for a total of 4 h. After the first 2 h the fixative1a

Prairie voles were selected as the model system to solution was replaced and fixation continued for an addi-examine the effects of neonatal exposure to OT for several tional 2 h. Brains were post-fixed in 4% paraformaldehydereasons. There is extensive evidence in prairie voles that for 24 h before being placed in 25% sucrose for 24 h, thenOT affects social and reproductive behaviors [9,21,42,43]. coated with 10% gelatin, post-fixed again in 4% parafor-Neonatal manipulation of OT has long-lasting effects on maldehyde for 24 h and then stored in sucrose at 4 8C untilthe expression of behavior and physiology, as stated above sectioning. Brains were sectioned on a freezing sliding[3,5,6,31]. The response to neuropeptides in this species is microtome (30 mm), placed in wells containing cryoprotec-sexually dimorphic, with OT playing a greater role in tant [41], and stored at 220 8C until processing using ABCfemale behavior [42], while the closely related, androgen- immunocytochemistry (ICC) staining for the c-Fos proteindependent neuropeptide arginine vasopressin (AVP) is (conventional method [8]).more important in males [22]. Thus, although there isstrong evidence that manipulations of OT have effects on 2 .1. c-Fos immunocytochemistrythe developing nervous system, and that the effects aresexually dimorphic and species dependent, the neural Floating sections were processed for c-Fos IR. Tissuemechanisms of these effects remain to be identified. sections were rinsed six times (10 min each) in 0.05 MTherefore, the purpose of this study was to examine KPBS. Sections were incubated at room temperature (20immediate neural responses of males and females to early min) in 10% sodium borohydride in 0.05 M KPBS andpostnatal exposure to OT. then rinsed repeatedly in KPBS. To remove residual blood

In the present study, the effects of exposure to OT and sections were incubated for 15 min in 0.014%OTA were measured by examining the expression of c-Fos phenylhydrazine and then rinsed in KPBS. Next, sectionsimmunoreactivity (IR). c-Fos is a marker of non-specific were incubated with rabbit c-Fos polyclonal antibodycellular activity and is a valuable tool for identifying initial (Oncogene Sci anti-cFos A1-2 [4191) at a 1:10,000neural responses. Therefore, by studying the expression of dilution in 0.05 M KPBS–0.4% TritonX (1 h at roomc-Fos IR in brains of newborn prairie voles it was possible temperature and then for 48 h at 4 8C). Sections wereto identify specific regional responses to exogenous OT rinsed six times (10 min each) in KPBS before beingand OTA in males and females. incubated for 1 h at room temperature in biotinylated goat,

anti-rabbit IgG (H1L, BA-1000, Lot LO913, 1:600 dilu-tion in KPBS–0.4% TritonX). Sections were rinsed five

2 . Methods times (10 min each rinse) in KPBS and incubated in anavidin–biotin peroxidase complex (Vectastain ABC kit-

Animals used in this study were laboratory reared F or elite pk-6100 standard 4.5 ml A and 4.5 ml B per 1-ml3F generation prairie voles that originated from wild stock solution) diluted in KPBS–0.4% TritonX (1 h room4trapped near Urbana, Illinois. On the day of birth pups temperature). Sections were rinsed three times in KPBSreceived one of four treatments. Three of the groups followed by three rinses in 0.175 M sodium acetate (5 minreceived one intraperitoneal injection of isotonic saline, 3 each). Finally, c-Fos IR was visualized by incubating in amg OT, or 0.3 mg OT antagonist (OTA) ([d(CH ) , nickel sulfate-diaminobenzine chromogen solution (2502 5

2 8Tyr(Me) , Orn ]-Vasotocin, Peninsula Laboratories, Bel- mg nickel II sulfate, 2 mg DAB, 8.3 ml 3% H O in 10 ml2 2mont, CA), the fourth group was handled but not injected, sodium acetate) for 12 min, and then rinsed three times inwith an n of 5–7 per treatment. OT and OTA were sodium acetate followed by three KPBS rinses. Followingsuspended in isotonic saline and all injections were 50 ml. labeling for c-Fos sections were placed into normal saline,

B.S. Cushing et al. / Developmental Brain Research 143 (2003) 129–136 131

then mounted on subbed glass slides and air-dried over- 4 . Discussionnight. Sections were then dehydrated in ascending ethanolsolutions, cleared in Histoclear, and the slides were Early postnatal exposure to endogenous or exogenouscoverslipped using Histomount. OT can affect the subsequent expression of adult physi-

ology and behavior [3,5,6,19,31,37]. Changes in behaviorare typically associated with changes within the CNS,

2 .2. Statistics supporting the contention that neonatal OT has organiza-tional effects [32,44]. In adults only a small amount of OT

Slides were coded and images captured and analyzed in the general circulation can cross the blood–brain barrierusing IPLab version 3.55 (Scanalytics Inc., Fairfax, VA) by [15,16,23], reducing the capacity of peripheral OT to affectan experimentally blind scorer. IPLab generates a segment the CNS. However, because the blood–brain barrier is notlayer based upon intensity of the staining. The number of fully developed during the first 2 weeks of postnatal lifecells is then determined by counting the distribution of the [38], it is possible that peripheral OT is directly influenc-segmented overlay. The number of cells expressing c-Fos ing the neonatal brain. Alternatively, changes in theIR from a single matched unilateral location per nuclei or expression of c-Fos could be due to an afferent feedbackregion were analyzed by sex and treatment using a one- response associated with the exogenous OT stimulation ofway ANOVA. Although the experiment was designed to peripheral organs. Regardless of the mechanism of thespread possible litter effects across treatments, variance effects, results from the present study support the hypoth-associated with litter was first examined by performing a esis that early exposure to OT can alter neuronal activity inmixed-model ANOVA with litter included as a random newborn prairie voles. The response to OT was sexuallyeffect. In no case was litter a significant effect, so one-way dimorphic with OT increasing neuronal activity in theANOVA results are reported. Whether or not the overall SON of males while in females OTA increased c-Fos IR inANOVA was significant, pre-planned pair-wise compari- the SON and decreased it in the MD.sons were conducted between handled males and handledfemales and for within sex effects between injected 4 .1. Sexually dimorphic neuronal activityanimals versus the controls using an LSD. Differenceswere considered significant at P,0.05. There were significant differences in c-Fos IR between

handled males and females. Females expressed higherlevels of neuronal activity, as measured by c-Fos, in theMD, while males had higher levels than females in the

3 . Results PVN. The PVN coordinates many hormonal and au-tonomic functions, and produces a variety of compounds

Six regions of the brain displayed significant levels of including AVP, OT and corticosterone releasing hormonec-Fos IR and therefore were analyzed for c-Fos IR (CRH) from one of two types of neurons, magnocellularincluding the arcuate nucleus (ARC), anteroventral and paravocellular. While we do not know which cell typesthalamic nucleus (AV), mediodorsal thalamic nucleus are involved, the high levels of c-Fos in the PVN of(MD), supraoptic nucleus (SON), paraventricular nucleus handled controls suggest one of two possibilities. First,of the hypothalamus (PVN) and zona incerta (ZI) (Fig. 1 males may be more sensitive to the effects of stressand Table 1). Significant between sex differences in the associated with handling and treatment than females.expression of Fos IR existed between handled females and Alternatively, neuronal activity in the PVN may be occur-handled males. Females displayed significantly higher ring earlier or at higher levels in males than females.levels of c-Fos IR in the MD (LSD540.6, mean diff.5 Increased neural activity in males could be due to pro-66.5, P,0.01), while expressing significantly lower levels duction of AVP, as AVP production is steroid dependent,in the PVN (LSD510.4, mean diff.5211, P,0.05) (Fig. and males produce higher levels of steroids during the1). postnatal period than females [14]. Additionally, AVP

Within sex effects varied depending upon the sex of the receptors have been detected in many areas of the CNS,pup and the area of the brain. There was an overall including the SON and PVN [29], that are implicated intreatment effect in Fos IR in the SON (F 52.94, P, reproductive and social behaviors [14,12].7,410.05). Treatment with OT increased Fos IR in males in the The MD has been implicated as part of the rewardSON compared to HAN males (LSD5110, mean diff.5 system [20,28] and may play an important role in the161.3, P,0.01). In females treatment with OTA resulted development of social attachment. Specifically, in prairiein an increase in Fos IR in the SON (LSD510.2, mean voles the MD is believed to be part of the neural circuitrydiff.510.8, P,0.05) and, while there was not an overall involved in neuropeptide-dependent pair bond formationtreatment effect, a decrease in the MD (LSD538.7, mean [22]. Since there is evidence for sex differences in prairiediff.543.5, P,0.01) (Figs. 1 and 2) compared with vole social behaviors including pair bonding and parentalhandled females. behavior [9,13], it is possible that early differences seen in

132 B.S. Cushing et al. / Developmental Brain Research 143 (2003) 129–136

B.S. Cushing et al. / Developmental Brain Research 143 (2003) 129–136 133

Fig. 2. Shows images of labelled c-Fos in the SON of handled and OT treated males and in MD of handled and OTA treated females.

T able 1 the MD are associated with the development of sexuallyMean number of cells (6S.E.) expressing c-Fos IR in the arcuate nucleus dimorphic social behaviors.(ARC), anteroventral thalamic nucleus (AV), and the zona incerta (ZI)

Sex Treatment Region 4 .2. c-Fos IR is sexually dimorphic in response totreatment with OTARC AV ZI

Male Han 65614 5469 31614The results from this study suggest that males areSal 3768 3065 2065

OT 103627 2265 1063 responsive to exogenous OT while endogenous OT mayOTA 71628 2166 2868 play a greater role in the expression of neuronal activity in

females. This apparent sex difference may be becauseFemale Han 135632 46613 2667Sal 119650 3269 3363 endogenous OT is either released earlier or plays a largerOT 135626 56624 2366 role in the expression of neuronal activity in females, thusOTA 11865 42617 1867 obscuring the effects of exogenous OT. Treatment with OT

There were no significant differences between sexes or treatment by resulted in increased c-Fos IR in males in the SON, whileregion (n55–6 per group).

Fig. 1. Shows the mean number of cells expressing c-Fos IR by sex and treatment in newborn male and female prairie voles in the SON (a,b), PVN (c,d)and MD (e,f). There were significant differences in the expression of c-Fos between newborn male and female prairie voles, with handled males displayinghigher levels in the PVN and lower levels in the MD than handled females. There were also significant within sex treatment effects in the SON, where OTsignificantly increased c-Fos IR in males, while OTA increased c-Fos expression in females, and the MD where in females OTA decreased c-Fos IR and inmales both SAL and OT increased c-Fos IR compared to handled controls. Differences were considered significant at P,0.05. The number above thebar5n. *Significant difference between male and female handled controls. a5significant within difference between treatment and handled control;b5significant within sex difference between treatment and saline.

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treatment with OTA resulted in changes in c-Fos IR in OTA binding to OTR. However, the possibility that thefemales. The SON is one of the major sites of OT effects of OT and/or OTA on c-Fos and subsequentproduction within the CNS and it is possible that females behavioral patterns are the result of interactions with themay be producing higher endogenous levels of OT and that vasopressinergic or other related system cannot be ruledfollowing blockage of OTR by OTA the system is attempt- out. In addition to binding to OTR, OT and OTA alsoing to compensate by producing OT. shows affinity for V R [7]. The vasopressinergic system1a

Decreased c-Fos expression in the MD also suggests that also begins developing prenatally [2,26,30,36], and inendogenous OT is stimulating activity in the MD in prairie voles V R are apparent on day 1 in a number of1aneonatal females. While in males there was an affect of regions of the brain [40]. However, while V R are present1atreatment with both OT and SAL increasing c-Fos IR at the time of birth there are several pieces of evidencecompared to handled controls. Although the role of the supporting the hypothesis that the observed Fos expressionMD in neonates is not clear, during this period the MD is is more likely associated with interactions with OTR thanbeing actively innervated by the amygdala, a region that is V R. Although both V and OT receptors are potential1a 1avery important in the development of social behavior and sites of action for OTA, the affinity of OTA for OTR isaggression in a number of species [1,27], including prairie 350–450 times higher than for V R [7]. There is increas-1avoles [24]. ing evidence that AVP plays a particularly important role

The effects of OTA in females may reflect the capacity in the expression of male prairie vole behavior andof OTA to inhibit the actions of endogenous OT (see physiology, while OT is more important in femalesbelow). However, OTA and OT also bind to the AVP [10,22]. Therefore, if the observed Fos expression werereceptor (subtype V , albeit at a lower affinity [7]. While due to interactions with V R, then at the very least the1a) 1athe present study does not allow us to differentiate between effects of OTA should have been equal in males andinteractions of OTA with OTR or V R, the results may females if not greater in males; however, this was not the1asupport the hypothesis that endogenous peptides are case.stimulating neuronal activity differentially in neonatal In conclusion neuronal activity is sexually dimorphic inmales and females. newborn prairie voles, especially in nuclei that produce OT

and AVP. The response to OT is also sexually dimorphicwith exogenous OT stimulating neuronal activation in

4 .3. Mechanism of the action of OT males while OTA, possibly by inhibiting the actions ofendogenous OT, influenced c-Fos expression in females.

The blood–brain barrier is not fully developed in These results support the hypothesis that endogenous OTneonates, allowing peptides and related compounds to plays a role in the activity and perhaps organization of thecross from the general circulation into the CNS [38]. Thus, neonatal female brain, with effects that differ from those inc-Fos IR in response to exogenous OT or OTA is probably males.associated with OT and OTA binding directly to receptorsin the brain. Although it is possible the changes seen inc-Fos IR were due to afferent feedback associated with the

A cknowledgementseffects of OT or OTA on peripheral systems. Availabledata indicate that, while perhaps not present in large

We wish to thank Nancy Cushing for her critical reviewnumbers, OTR is present on the day of birth in rodents andof this manuscript and Karyn Levine, Julia Musiker,may occur in the areas of the brain in which changes inUzoma Okorie and the other members of the lab for theirc-Fos were observed. Binding studies in rats have indi-encouragement and support. This research was support bycated that OTR is present in the nucleus accumbens,NIH HD 38490 and MH 01992.anterior olfactory nucleus, and caudate putamen by em-

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