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Peptides 57 (2014) 122–128

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

ffects of sex and reproductive experience on the number of orexin-immunoreactive cells in the prairie vole brain

ichael Donlin, Breyanna L. Cavanaugh, Olivia S. Spagnuolo, Lily Yan, Joseph S. Lonstein ∗

euroscience Program, Michigan State University, 108 Giltner Hall, East Lansing, MI 48824, United States

r t i c l e i n f o

rticle history:eceived 25 March 2014eceived in revised form 7 May 2014ccepted 7 May 2014vailable online 27 May 2014

eywords:ormonesypothalamusonogamyeuropeptideseproductioneward

a b s t r a c t

Large populations of cells synthesizing the neuropeptide orexin (OX) exist in the caudal hypothalamusof all species examined and are implicated in physiological and behavioral processes including arousal,stress, anxiety and depression, reproduction, and goal-directed behaviors. Hypothalamic OX expression issexually dimorphic in different directions in laboratory rats (F > M) and mice (M > F), suggesting differentroles in male and female physiology and behavior that are species-specific. We here examined if thenumber of hypothalamic cells immunoreactive for orexin A (OXA) differs between male and femaleprairie voles (Microtus ochrogaster), a socially monogamous species that pairbonds after mating and inwhich both sexes care for offspring, and if reproductive experience influences their number of OXA-immunoreactive (OXA-ir) cells. It was found that the total number of OXA-ir cells did not differ betweenthe sexes, but females had more OXA-ir cells than males in anterior levels of the caudal hypothalamus,while males had more OXA-ir cells posteriorly. Sexually experienced females sacrificed 12 days after thebirth of their first litter, or one day after birth of a second litter, had more OXA-ir cells in anterior levelsbut not posterior levels of the caudal hypothalamus compared to females housed with a brother (incest

avoidance prevents sibling mating). Male prairie voles showed no effect of reproductive experience butshowed an unexpected effect of cohabitation duration regardless of mating. The sex difference in thedistribution of OXA-ir cells, and their increased number in anterior levels of the caudal hypothalamusof reproductively experienced female prairie voles, may reflect a sex-specific mechanism involved inpairbonding, parenting, or lactation in this species.

© 2014 Elsevier Inc. All rights reserved.

. Introduction

Very large populations of cells synthesizing the neuropeptiderexin (OX), also known as hypocretin, are found in the lateralnd perifornical/dorsomedial regions of the caudal hypothala-us of all mammals examined. These include laboratory rats

46,52,63], mice [69], grass rats (Arvicanthis niloticus) [15,49], Syr-an hamsters (Mesocricetus auratus) [44], degus (Octodon degus)47], domesticated cats [69], African green monkeys (Cercopithe-us aethiops) [16] and humans [20]. OX exists in two isoformsA and B) that are produced by the prepro-OX polypeptide andhese isoforms have differential affinity for the OX receptors,ith OXA preferentially binding the OX1 receptor but OXA and

XB having similar affinity for the OX2 receptor [66]. Through

heir widespread inputs and outputs [46,52,66], hypothalamicX cells integrate a broad range of internal and external signals

∗ Corresponding author. Tel.: +1 517 353 8675; fax: +1 517 432 2744.E-mail address: lonstein@msu.edu (J.S. Lonstein).

ttp://dx.doi.org/10.1016/j.peptides.2014.05.004196-9781/© 2014 Elsevier Inc. All rights reserved.

underlying arousal, stress, emotions and mood, reward processing,and the performance of goal-directed behaviors [2,58,66]. Studiesof mice with null mutations of the OX receptor genes demonstratethat OX’s role in arousal is mediated by the OX2 receptor whileits other effects (e.g., reward processing and goal-directed behav-iors) are more likely controlled by the OXA-preferring OX1 receptor[43].

The goal-directed behaviors influenced by OX include the socialinteractions necessary for reproduction. For example, infusion ofOXA into the mPOA (a site well-studied for its role in copulatorybehaviors [26,30,54]) facilitates sexual motivation and perfor-mance in male rats [27]. Conversely, antagonism of OX1 receptorsby systemic injection of SB-334867 increases the latency for malerats to intromit and decreases their frequency of ejaculation [45;but see 4]. Furthermore, expression of the immediate-early genec-fos increases in hypothalamic OXergic cells when male rats

are exposed to conspecific female sensory cues, sexually inter-act with a female, or are placed in a chamber associated withsexual reward [17,18,45]. Given these findings it is surprisingthat destruction of hypothalamic OXergic neurons does not affect

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exual performance or motivation in experienced male rats, but itoes facilitate the onset of a sexual interaction in inexperiencedales, which has been attributed to the disrupted OX signal-

ng reducing their novelty-induced anxiety [17]. In postpartumaboratory mice intracerebroventricular infusion of OXA dose-ependently decreases maternal attacks on an unfamiliar maleouse and disrupts nursing behavior [14]. Consistent with the lat-

er result, inhibiting endogenous OX signaling with a peripheralnjection of SB-334867 tends to increase some aspects of nursing14].

Sex and endocrine state are two factors affecting OX synthesisnd, therefore, its influence on reproductive and other behav-ors. Hypothalamic prepro-orexin mRNA has been seen to be >50%igher and OXA content almost twice as high in virgin female ratsompared to males [34,64], but the direction of this sex differ-nce may be species specific because female mice have 15–20%ewer OXA-ir cells in the hypothalamus than do male mice [8].onadal and other hormones circulating during adulthood con-

ribute to the magnitude of this sex difference. In male rats theumber of hypothalamic prepro-OX-ir cells decreases after cas-ration in conjunction with the decrease in copulation, but bothan be maintained by exogenous estradiol, presumably naturally

result of the aromatization of endogenous testosterone [45]. Theore numerous studies on how gonadal hormones affect OXA-ir

ells in females are equivocal. Hypothalamic OXA content has beeneen to be particularly low during late proestrus or in response toxogenous estradiol, perhaps reflecting increased OXA release andepletion of intracellular stores [57], but others have found prepro-X mRNA or OXA levels to be at their peak during late proestrus

53,60]. Still others have found that OX synthesis does not dif-er across the estrus cycle [70]. Pregnancy and lactation also havenclear effects on OX in female laboratory rats, being associatedith higher, lower, and no change in expression [7,9,23,36,61,70].

All of the studies described above examined polygamous labora-ory rodents, but it would be interesting to study the relationshipsmong the hypothalamic OX system and social interactions, sex,nd reproductive experience in a monogamous rodent such as therairie vole (Microtus ochrogaster). Male and female prairie voleshare more aspects of their behavioral repertoires when comparedo most polygamous rodents, with both sexes of prairie vole form-ng lifelong pairbonds after mating and later contributing parentalare to their offspring [11,76]. Furthermore, reproductive experi-nce and pairbonding alter anxiety-related behavior [32,37,39] andnergy balance [10] in male prairie voles in ways that are more typ-cally found in postpartum female mammals [see 40, 74]. Prairieoles also have smaller sex differences compared to polygamousodents in some (but certainly not all) aspects of their hypotha-amic anatomy and neurochemistry [38,59]. Finally, OX interacts

ith the oxytocin and vasopressin neuropeptide systems [1,3,67]hat have been so well studied for their contributions to prairie voleehavior and physiology [11,75].

Considering these points above, it is reasonable to suggest thatX could be a component of the neural systems necessary for main-

aining the rewarding pair bond, parental caregiving, emotionaltate, or energy balance in prairie voles. If so, one might expectpregulated OXA in the hypothalamus of both sexes of pair bondednd parental prairie voles. If OXA is instead more involved in sex-pecific processes in this species, any influence of reproductivexperience and parental state might be expected to differ betweenale and female prairie voles. To begin evaluating which of these

ossibilities is more likely, we first determined if the number ofXA-ir cells in the prairie vole hypothalamus is sexually dimor-

hic. We then determined if reproductive experience and its suitef behavioral and physiological changes produce a similar or dif-erent influence on the number of OXA-ir cells in male and femalerairie voles.

57 (2014) 122–128 123

2. Materials and methods

2.1. Subjects

Subjects were male and female prairie voles (M. ochrogaster)from our breeding colony that descended from wild voles cap-tured in Urbana, Illinois and described previously [13]. Animalswere maintained on a 14:10-h light:dark cycle, an ambienttemperature of 21 ± 1 ◦C, and housed in clear plastic cages(48 cm × 28 cm × 16 cm) containing wood chips, wood shavings,and a substantial covering of hay. They were provided water anda food mixture containing cracked corn, whole oats, sunflowerseeds, and rabbit chow (Tekland rodent diet No. 2031) in a ratioof 1:1:2:2 ad libitum. Subjects were weaned from their parents at20 days of age and housed in mixed-sex sibling groups, where theyremained until being used in the experiment when they reached70–95 days of age. Prairie voles are induced ovulators and incestavoidance prevents them from mating with siblings, even if the sib-lings are separated for at least up to a week before a reunion [24];this mixed-sex housing after weaning permitted us to use famil-iar opposite-sex sibling pairs as the reproductively inexperiencedcontrol groups (see below). All procedures were in accordance withthe Institutional Animal Care and Use Committees at Michigan StateUniversity.

2.2. Housing and reproductive experience conditions

Subjects (N = 25) were taken from their home cages and sociallyisolated in a clean cage for four days. They were then placed ina soiled cage containing a similarly separated unfamiliar prairievole of the opposite sex to elicit female estrus and mating [11]or they were placed in a soiled cage containing a similarly sepa-rated familiar opposite-sex sibling. For the sexually experiencedsubjects, cages were inspected daily starting 2–3 days before theexpected day of parturition for the presence of pups. The sexuallyexperienced females and males were sacrificed 12 days after thebirth of their first litters (n = 9 females, 5 males) or one day after afemale give birth to a second litter (n = 6 females, 5 males). Thus,the first group of dams were both lactating and pregnant and thesecond group of dams were lactating, recently parturient, and ina postpartum estrus. In the group sacrificed one day after birth ofthe second litter, the older litter of pups remained in the homecage until the time parents were sacrificed. The reproductivelyinexperienced females and males were yoked to mated animalsand sacrificed between 35 and 40 days after rehousing with a sib-ling (shorter cohabitation: n = 5 females, 8 males) or 42–50 daysafter rehousing with a sibling (longer cohabitation: n = 6 females, 6males). At sacrifice, it was confirmed that the uterine horns of thesexually experienced females killed 12 days after the birth of theirfirst litter contained fetuses, and that the uterine horns of sexu-ally inexperienced females housed with their brothers contained nofetuses and that no pups were living in those home cages. Althoughwe confirmed that these sibling pairs did not have fetuses or pupsat the time of sacrifice, so were reproductively inexperienced, wecannot be absolutely certain they were also sexually inexperiencedbecause it is conceivable that mating between siblings could occurwithout producing fetuses or live offspring.

2.3. Perfusion, tissue collection and immunocytochemistry

Subjects were overdosed with an anesthetic containingketamine (62.5 mg/kg), xylazine (7.5 mg/kg), and acepromazine

(0.8 mg/kg) and perfused through the heart with 100 mL of 0.9%saline followed by 100 mL of 4% paraformaldehyde in sodium phos-phate buffer (NaPB; pH = 7.6). Brains were removed, post-fixedovernight in 4% paraformaldehyde in NaPB, and submerged in a

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0% sucrose/NaPB solution for at least 3 days before sectioning into0 �m sections. Immunocytochemistry for OXA was performedn every other section through the brain. The free-floating sec-ions were rinsed three times with 0.1 M Trisma-buffered salineTBS; pH = 7.4) between each incubation. Sections were incubatedn 0.1% sodium borohydride in TBS for 15 min, 1% hydrogen per-xide in 0.3% Triton X-100 in TBS for 10 min, blocked with 5%ormal goat serum (NGS) in 0.3% Triton X-100 for 60 min, and

ncubated overnight with slow agitation at room temperatureith rabbit anti-Orexin A polyclonal primary antiserum (H-003-

0, 1:20,000; Phoenix Pharmaceuticals, Burlingame, CA) in 3% NGSnd 0.3% Triton X-100. The next morning sections were incubatedn goat anti-rabbit biotinylated secondary antiserum in 3% NGS and.3% Triton X-100 for 60 min at room temperature (1:500; Vec-or Laboratories, Burlingame, CA) and then avidin–biotin complexVectastain Elite, Vector Laboratories) for 60 min. Vector SG wassed as the chromagen (Vector Laboratories) and provided a blueytoplasmic label. Sections were mounted on microscope slides,ehydrated, and coverslipped.

.4. Tissue analysis

Slides were coded and randomized for analysis, which was per-ormed by one person (O.S.) using a Nikon E600 microscope at 200×

agnification with the aid of Nikon NIS-Elements software. Fouroronal sections through the region of the hypothalamus contain-ng OXA-ir cells were analyzed bilaterally and all cells containingetectable OXA immunoreactivity were counted within an areatandardized for all subjects that covered the entire hypothalamicegion. The levels analyzed corresponded to Plates 27, 28, 30, and2 from Swanson’s atlas of the rat brain [62], which is where mostXA-ir cells were found in the caudal hypothalamus of our prairieoles and as is true for other laboratory rodents [44,49,52,69].

.5. Data analyses

Our preliminary data indicated that a potential sex differenceas related to the rostrocaudal level of analysis, so a repeated-easures ANOVA was first used to compare the sexes across the

our levels analyzed and t-tests were then used to compare theexes at each rostrocaudal level. Following that, two-way ANOVAsere conducted within sex to determine the effect of reproduc-

ive experience (no, yes) and duration of cohabitation (shorter,onger) on the total number of OXA-ir cells in the two anterior lev-ls combined and in the two posterior levels combined. Statisticalignificance was indicated by p < 0.05.

. Results

.1. Sex difference in OX-ir cells

The lateral and perifornical/dorsomedial regions of the caudalypothalamus contained very large numbers of OXA-ir cells in bothexes of prairie vole (Fig. 1). There was no main effect of sex on theumber of OXA-ir cells (F(1,144) = 0.44, p > 0.50) but there was aain effect of rostrocaudal level on the number of OXA-ir cells, with

ewer cells at the most rostral and caudal levels compared to thewo levels in between (F(3,144) = 51.48, p < 0.0001). There was alson interaction between sex and rostrocaudal level (F(3,144) = 6.91,

< 0.0002). Females tended to have more OXA-ir cells than males in

he most rostral level analyzed (t48 = 1.84, p = 0.07) and significantly

ore OXA-ir cells than males in the subsequent level analyzedt48 = 2.56, p < 0.02), but significantly fewer OXA-ir cells in the mostaudal level compared to males (t48 = 2.80, p < 0.007) (Fig. 2).

57 (2014) 122–128

3.2. Reproductive experience effects on OXA-ir cells in females

At anterior levels of the female prairie vole caudal hypotha-lamus, there was a main effect of reproductive experienceon the number of OXA-ir cells, with the reproductively expe-rienced females having ∼18% more cells than inexperiencedfemales (F(1,22) = 4.71, p < 0.05; Fig. 3). There was no significanteffect of cohabitation duration (F(1,22) = 2.06, p > 0.16) or interac-tion between reproductive experience and cohabitation duration(F(1,22) = 1.79, p > 0.19).

In contrast, within the posterior levels of the caudal hypothala-mus there was no significant main effects of females’ reproductiveexperience (F(1,22) = 0.22, p > 0.64; Fig. 3) or cohabitation duration(F(1,22) = 0.92, p > 0.34) and no interaction between these factors(F(1,22) = 0.29, p > 0.59).

3.3. Reproductive experience effects on OXA-ir cells in males

In the anterior levels of the caudal hypothalamus, the maineffect of reproductive experience on the number of OXA-ir cellsin male prairie voles was not significant (F(1,20) = 0.08, p > 0.78),but the effect of cohabitation duration was unexpectedly signif-icant, with males (reproductively inexperienced or experienced)sacrificed after the shorter cohabitation having ∼30% fewerOXA-ir cells compared to the males sacrificed after the longercohabitation (F(1,20) = 5.59, p < 0.03). The interaction betweenreproductive experience and cohabitation duration was not signif-icant (F(1,20) = 0.54, p > 0.46; Fig. 4).

Within the posterior levels of the caudal hypothalamus therewas no significant main effects of reproductive experience(F(1,20) = 0.30, p > 0.58) or cohabitation duration (F(1,20) = 0.01,p > 0.91) and no interaction between these factors (F(1,20) = 0.19,p > 0.66; Fig. 4).

4. Discussion

Central OX signaling influences a diverse array of physiologicaland behavioral processes [2,58,66] and many of these are knownto be sexually dimorphic. For example, female mice show higherbehavioral arousal on some measures compared to males [73] andwomen have an earlier onset of the arousal disorder, narcolepsy,than do men [42]. Furthermore, the greater incidence of anxietyand depression in women is well known [19,48] and there are sexdifferences in responses to rewarding stimuli [21,33,71]. Only afew studies have examined sex differences in central OX systems,though. Two studies reported that female laboratory rats had con-siderably higher hypothalamic prepro-orexin mRNA or OXA levelswhen compared to males [34,64], but another study of rats brieflystated they found no difference between the sexes in the number ofOX-ir cells [22]. In laboratory mice, females have fewer OXA-ir cellsin the hypothalamus than males, which is the opposite direction ofthe sex difference sometimes found in laboratory rats [8].

We herein found a different type of sex difference in the numberof OXA-ir cells in prairie voles, with females having more OXA-ircells than males at relatively rostral levels of the caudal hypothala-mus, while males had a greater number of OXA-ir cells posteriorly.Taheri et al. [64] found a sex difference (F > M) in the concentrationof OXA in the rat posterior hypothalamus, but not in the anteriorhypothalamus where OX-synthesizing cells are rather rare, but weare unaware of any other study comparing the sexes at differentrostrocaudal levels within the posterior hypothalamus. This sug-

gests that making a rostrocaudal distinction within the posteriorhypothalamus could be particularly fruitful for revealing sex dif-ferences in the OX system and not doing so may have contributedto the inconsistencies in the literature on other rodents. Factors

M. Donlin et al. / Peptides 57 (2014) 122–128 125

F us of

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ig. 1. Photomicrographs of OXA-ir cells at four levels (A–D) of the hypothalamammillothalamic tract.

riving this rostrocaudal sex difference in prairie voles remain toe determined but it not due to a sex difference in their overallrain size [59] and any functional significance of this sex differenceequires further study.

In addition to the sex difference, we hypothesized that repro-uctive experience could influence the number of OXA-ir cells inoth sexes of prairie vole. We examined this by comparing vir-in voles housed with a familiar opposite-sex sibling with matedoles sacrificed 12 days after the birth of their first litter (shorterohabitation) or one day after a female gave birth to their sec-

nd litter (longer cohabitation). These time points were of interestecause of differences in the voles’ endocrine states and degree ofeproductive experience. The reproductively experienced femalesn the shorter cohabitation condition were concurrently lactating

representative male prairie voles. Schematics modified from [62]. f, fornix; mtt,

and pregnant, which involves low circulating estradiol and mid-to-high levels of progesterone. The reproductively experiencedfemales in the longer-cohabitated group were both lactating and ina postpartum estrus, so likely had low circulating progesterone andhigh circulating estradiol [12,29,56]. The long-cohabitated volesalso had more parental experience. We had no a priori predictionsabout the effect of reproductive experience or its duration giventhe contradictory literature from laboratory rats on the effect ofpregnancy and lactation on OX expression [7,9,23,36,61,70]. Wefound that the two reproductively experienced groups of females

did not differ from each other, and that both had significantlymore OXA-ir cells in anterior levels of the caudal hypothala-mus compared to inexperienced females. This suggests as long asfemale prairie voles have cohabitated with an unfamiliar male,

126 M. Donlin et al. / Peptides

Fig. 2. Number (mean ± SEM) of OXA-ir cells in female and male prairie voles atfpc

moecn

Fo*

Fo*

our rostrocaudal levels through the hypothalamus. * = comparison between sexes < 0.05, += p < 0.08. Not indicated is a significant main effect of rostrocaudal levelollapsed across sex (see text for details).

ated, given birth, and become mothers that the consequences

f multiparity (including endocrine state and degree of maternalxperience) do not further affect their number of OXA-synthesizingells. We previously found a similar lack of multiparity on theumber of steroid-sensitive cells in the principal bed nucleus of

ig. 3. Number (mean ± SEM) of OXA-ir cells in the anterior (left panel; rostrocaudal levef reproductively inexperienced and experienced female prairie voles that were sacrificed

= significant main effect of reproductive experience, p < 0.05.

ig. 4. Number (mean ± SEM) of OXA-ir cells in the anterior (left panel; rostrocaudal levef reproductively inexperienced and experienced male prairie voles that were sacrificed

= significant main effects of cohabitation duration, p < 0.05.

57 (2014) 122–128

the stria terminalis (pBST) that synthesize tyrosine hydroxylase.Both primiparous and multiparous prairie voles had more tyro-sine hydroxylase-immunoreactive cells compared to virgin females[13].

Our finding that reproductive experience affected the number ofOXA-ir cells only in the anterior levels of the female prairie vole cau-dal hypothalamus indicates subregional differences in the largerpopulation of OXA-ir cells not only related to their distributionbased on the animals’ sex (male or female), but also in their sensi-tivity to reproductive experience within females. How anterior andposterior subpopulations of OXA cells differ from each other has notoften been studied in any species, but there is a rostrocaudal dif-ference in the degree of loss of OX cells in patients with narcolepsywithout cataplexy [68]. Various afferents and efferents of thehypothalamic OX field in rats are also known to be topographicallyorganized [75], so perhaps the rostral and caudal populations arethe brain’s sources of the ascending and descending OX fiber projec-tion paths, respectively [52]. It would be interesting if the anteriorand posterior OXA-ir populations differ in their peptide or steroidhormone receptor expression in a way that permits differentialcontribution to female reproductive physiology and behavior. Inlaboratory mice, oxytocin and vasopressin depolarize OX cells [67].Furthermore, OX receptors are found on magnocellular oxytocinand vasopressin cells [3] and can stimulate vasopressin synthesis

[1] in rats. Perhaps oxytocin and vasopressin influence reproduc-tively experienced female prairie voles by acting preferentially onthe anterior subpopulation of OX cells. This does not need to beexclusive to prairie voles, and such interactions with OX may also

ls A + B) and posterior (right panel; rostrocaudal levels C + D) caudal hypothalamus after a shorter or longer cohabitation with their male cage mate (brother or mate).

ls A + B) and posterior (right panel; rostrocaudal levels C + D) caudal hypothalamusafter a shorter or longer cohabitation with their female cage mate (sister or mate).

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nderlie how oxytocin or vasopressin facilitate social behaviorsven in animals that are only highly prosocial seasonally, such aseadow voles [6,50,51].In male prairie voles, an increased number of OXA-ir cells after

eproductive experience might have been expected because cir-ulating testosterone tends to rise after males cohabitate with aemale and mate [5,25,72], and that testosterone maintains highrepro-OX immunoreactivity in the hypothalamus of male rats45]. The effects of exogenous OX on maternal behaviors in rats14], the high metabolic rate and food intake of prairie vole fathers10], and changes in their anxiety- and depressive-like behaviorsfter mating [32,37,39] further contributed to the possibility thathere would be more OXA-ir cells in our mated male prairie volesompared to unmated males. We found no such effect of repro-uctive experience, though. Perhaps this is due to a ceiling effect,uch that an increase above normal physiological titers of testos-erone in gonadally intact males has no effect on their number ofXA-ir cells, but it certainly remains possible that characteristicsf these OX cells other than their number is affected by such a risen testosterone. There may also be a ceiling effect if OX expressions closely related to parenting in prairie voles because many virgin

ales are spontaneously paternal [41], although sometimes not tohe same degree as sires [65], so the OXA system may already bepregulated in virgin males of this species. In fact, the upregula-ion of OXA expression in our reproductively experienced femalerairie voles may be related to their much more dramatic increase

n maternal responsiveness after giving birth compared to before-and. Indeed, most virgin female prairie voles are infanticidal andnly after giving birth show high levels of pup caregiving behaviors28,41].

We unexpectedly found a significant effect of cohabitation dura-ion in males, such that both mated and unmated males in the longohabitation group had more OXA-ir cells compared to males inhe short cohabitation group. There was an inkling of a similar dif-erence between the short- and long-cohabitated reproductivelynexperienced female prairie voles (see Fig. 3). As with the effectsf reproductive experience in females, this increase in the long-ohabitated males was only found anteriorly, again suggestingifferential sensitivity of OXA subpopulation to our manipulations.e can offer no simple explanation for the overall finding. The

nmated males were housed with familiar sisters (i.e., ones theyere also housed with before being involved with the experiment)

nd we cannot think of any obvious change in that relationshipccurring across time. Also, there was only a 7–10 day differenceetween the short and long cohabitation groups in the number ofays between their re-housing and sacrifice, making an age effecteem unlikely. Another probably unlikely possibility is that rehous-ng with either a familiar sister or unfamiliar female is stressful andhis increased OXA expression only in the long cohabitation males.ypothalamic OX expression is upregulated in response to various

tressors in laboratory rats [31] and, in turn, OXA can stimulatehe hypothalamic-pituitary axis and other physiological stress sys-ems [35]. If so, this effect would have had to begin 35–42 daysfter rehousing with the female, which was the range of days afterehousing when the long-cohabitated males were sacrificed, andhis time course seems inconsistent with a stress effect.

In conclusion, there is a sex difference in the rostrocaudal distri-ution of OXA-ir cells in the prairie vole caudal hypothalamus andhe number of OXA-ir cells at relatively anterior levels is signif-cantly upregulated in reproductively experienced female prairieoles compared to inexperienced females. This upregulation is notbserved in reproductively experienced male prairie voles, which

ad more OXA-ir cells in anterior levels of the caudal hypothala-us after a longer duration of cohabitation with either a sister

r a mate. It would be valuable to determine if these results areruly species-specific or if they extend to the OX system of other

[

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57 (2014) 122–128 127

monogamous and biparental rodents such as pine voles (Microtuspinetoum), California mice (Peromyscus californicus), or Djungar-ian hamsters (Phodopus campbelli). The difference between maleand female prairie voles in how reproductive experience alters thenumber of OXA-ir cells may be due to female-specific changes inphysiology and behavior occurring after mating and giving birth.These include the obviously female-specific lactation, which pro-duces mixed effects on OX expression in laboratory rats, and aparticularly dramatic shift in positive responsiveness to neonates infemale prairie voles when compared to that occurring in the alreadyhighly parental males. Future studies manipulating OX receptor sig-naling in reproductively experienced female prairie voles will helpreveal which physiological and behavioral systems depend most onthis upregulated OXA expression.

Acknowledgements

The authors thank Dr. Eman Ahmed and Chelsea Saleeby for theirassistance with this project. This work was supported in part by NSFgrant # 0515070 to JSL.

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