The ventromedial hypothalamic circuitry and male alloparental behavior in a socially monogamous rodent species

Abstract

As prairie voles (Microtus ochrogaster) display spontaneous bi-parental care, and the ventromedial hypothalamus (VMH) has been implicated in reproductive behavior, we conducted experiments to test the hypothesis that the VMH neurochemical circuitry is involved in alloparental behaviors in male prairie voles. We compared alloparental behaviors of adult, sexually-naïve male and female voles – both displayed licking/grooming, huddling, and retrieving behaviors toward conspecific pups. We also stained for the immediate-early gene encoded early growth protein Egr-1 in the vole brain. The pup-exposed animals showed levels of Egr-1 staining that was higher in the VMH but lower in the amygdala compared to animals exposed to a pup-sized piece of plastic (control). A retrograde tracer, Fluoro-Gold (FG), was injected into the VMH of male voles that were subsequently tested in the pup-exposure or control condition. More FG/Egr-1 cells were detected for glutamatergic (GLU) staining in the ventral bed nucleus of the stria terminalis (BNSTv) and medial amygdala (MeA), whereas less FG/Egr-1 cells were stained for gamma-aminobutyric acid (GABA) in the MeA of the pup-exposed group compared to the control group. Further, the ratio of GLU:GABA expression in FG/Egr-1 projection neurons from both the BNSTv and MeA to the VMH was increased following pup exposure. Finally, pharmacological blockade of either dopamine D1 receptor or oxytocin receptor in the VMH impaired the onset of male alloparental behavior. Together, these data suggest that the VMH may be involved in the onset of alloparental care and play a role in regulating social approach in male prairie voles.

Introduction

A large body of research demonstrates the importance of both the mother and the father’s involvement for successful offspring development (Kentner et al., 2010). Although the critical role of parental involvement in the physical and psychological well-being of children is well documented in humans, parents are often not the sole caregivers. Care provided to offspring by those that are not the biological parents, known as alloparenting, is widespread throughout human society. Given their cooperative and social nature, humans have consistently relied on alloparenting in communities for optimal outcomes (Hrdy, 2007). In fact, there is an increasing presence of non-biological caregivers in the household responsible for taking on the caregiving role (Hynes & Dunifon, 2007; Center, 2015). This additional caregiving, whether from siblings or others in the community, typically leads to more successful outcomes for offspring (Sear & Mace, 2008). Conversely, the presence of non-biological parents may also result in increased maltreatment of children (Daly & Wilson, 1988; Kenkel et al., 2017). Although the specific behavior exhibited by alloparental caregivers may be similar to those of biological parents, the reasoning, motivations, and thus neurochemical circuits underlying alloparental care may be exceedingly different from those of parents. Therefore, an understanding of the neural mechanisms underlying alloparental behavior, along with the mechanisms underlying the decision to either provide or deny care to non-biological offspring, is important for the physical, mental, and overall health status of both individuals and their children. Given that traditional laboratory rodent models do not normally display biparental and alloparental care similar to humans, there is still much left to uncover about the underlying neurobiology differentiating these two types of behaviors.

While progress has been made in uncovering the underlying circuits of parental behavior (Dulac et al., 2014; Bendesky et al., 2017), rodent models have also been used to begin studying the neural mechanisms underlying alloparental behavior. More specifically, changes in the underlying neural circuitry that occur upon the induction of alloparental behavior and in response to pup exposure have been examined in biparental rodent models. Several brain areas and neurochemicals have been implicated in various types of alloparental behaviors (Kenkel et al., 2017). For example, pup exposure increases neuronal activation in both the bed nucleus of the stria terminalis (BNST) and the medial amygdala (MeA) in male prairie voles (Kirkpatrick et al., 1994; Northcutt & Lonstein, 2009). In California mice, pup exposure increases Fos expression in the BNST as well (Horrell et al., 2017). These data, together with those from other previous studies, indicate a role for both the BNST and the MeA along with other key brain regions in the regulation of alloparental care (Kenkel et al., 2017). Data also pinpoint specific neurochemicals involved in alloparental behavior, including dopamine (DA), oxytocin (OXT), vasopressin (AVP), glutamate (GLU), and gamma-aminobutyric acid (GABA) (Northcutt & Lonstein, 2009; Lambert et al., 2011; Kenkel et al., 2017). The published data suggest that alloparental behavior, like other social behaviors, is regulated by multiple neurochemicals that interact with each other in a complex neural circuitry. The ventromedial nucleus of the hypothalamus (VMH), which is well known for its involvement in various social behaviors, including parental behavior, receives inputs from both the BNST and the amygdala (AMYG) (Numan & Numan, 1997; Gu et al., 2003; Pardo-Bellver et al., 2012). Given the involvement of the BNST and the MeA in alloparental behavior, an examination of the role of the BNST-AMYG-VMH circuitry in alloparental behavior and how this may be uncoupled from its involvement in reproductive and parental behaviors is needed.

Since only a small percentage of mammals display cooperative breeding, animals that naturally display this caregiving strategy must be used to study the underlying neurobiology of alloparental behavior (Russell, 2004; Russell & Lummaa, 2009). The socially monogamous prairie vole (Microtus ochrogaster) has emerged as an appropriate, alternate animal model for studying biparental and alloparental behaviors (Young et al., 2011). Prairie voles display bi-parental care toward offspring after a female’s parturition (Ahern et al., 2011; Young et al., 2011). In the wild, prairie voles typically live in small communities, allowing for a cooperative breeding environment in which alloparental care is frequently displayed (Getz & McGuire, 1993). For example, juvenile voles display alloparental behavior toward their younger siblings (Wang & Novak, 1994), adolescent males and females show spontaneous alloparental behavior toward conspecific pups (Lonstein & De Vries, 1999; Lonstein et al., 2002; Bales et al., 2004b; Olazabal & Young, 2005), and such behaviors can be enhanced by sexual and social experience with a partner (Bamshad et al., 1994). The display of alloparental and parental behavior by male prairie voles is associated with changes in several neurochemical systems, such as OXT and DA (Lonstein, 2002; Bales et al., 2004b). The display of alloparental behaviors is also related to the reduced survival of new neurons in the MeA, VMH, and hippocampus of the adult brain and is associated with increased anxiety-like and depressive-like behaviors, further indicating a role for the VMH in the alloparental neural circuitry (Lieberwirth et al., 2013). This naturally occurring display of alloparental behavior by male prairie voles provides an opportunity to keep expanding upon this underlying neural circuitry (Wang et al., 1998; Young et al., 2011).

Since many sexually-naïve prairie voles exhibit spontaneous alloparental behavior, they allow for the study of this behavior in the absence of other social behaviors and physiological processes (e.g., mating, pregnancy, lactation) that often affect parental behavior. Further, this provides the opportunity to differentiate the underlying circuits of biparental and alloparental behavior. In the present study, we compared sexually-naïve male and female prairie voles to characterize their alloparental behavior toward conspecific pups and resulting brain region-specific neuronal activation. Because Egr-1, an immediate-early gene (IEG) encoded early growth protein, is rapidly induced by neuronal activity and plays an important role in cellular responses that contribute to complex forms of cognitive and behavioral functions, Egr-1 has been used as an indirect marker for neuronal activation in the study of neurobiology of behaviors – including parental behaviors in rats and prairie voles (Numan et al., 1998; Northcutt & Lonstein, 2009; Barry et al., 2016; Duclot & Kabbaj, 2017). Egr-1 has also been shown to have rapid expression in the brain and to function as a transcriptional regulator for the expression of many other genes (Beckmann & Wilce, 1997; Yan et al., 2000). Thus, we used Egr-1 staining as an indirect measure of neuronal activation for the present study. As the VMH serves as a node for parental behavior and receives input from brain regions known to be involved in alloparental behavior, we tested the hypothesis that the VMH neurochemical circuitry is involved in male alloparental behavior. We examined projection neurons from the BNST and MeA to the VMH, their activation by pup exposure, and their GLU-ergic and GABA-ergic phenotypes in male voles. We also illustrated the effects of pharmacological receptor blockades on the onset of alloparental behavior.

Materials and methods

Subjects

Subjects were sexually-naïve male and female prairie voles (Microtus ochrogaster) from a laboratory breeding colony descended from populations in southern Illinois. The breeding colony was outbred in 2017 using the F1 generation of field-captured prairie voles. The gestation period for prairie voles is about 21–22 days. Subjects were weaned at 21 days of age prior to the birth of their younger siblings and housed in same-sex sibling pairs in plastic cages (12 W × 28 L × 16 H cm) containing cedar chip bedding without any other nesting materials. Water and food were provided ad libitum. All cages were maintained under a 14:10 light:dark cycle, and the temperature was kept at 20°C. Subjects were tested at 90–120 days of age. All experimental procedures were approved by the Florida State University Institutional Animal Care and Use Committee and were in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23).

Alloparental behavior test

The alloparental behavior of sexually naïve, male and female prairie voles was tested in a Plexiglas cage (20 W × 45 L × 25 H cm) with a thin layer of cedar chip bedding and ad lib food and water as described for the housing cages. Behavioral tests were conducted between 0900–1300 h during the light phase. After a 15 min habituation, a 1–3 day-old stimulus pup (PUP) or an odorless, dark brown, pup-sized piece of plastic (CONTROL for novelty) was introduced into the testing cage at the opposite corner from the subject, and the subject’s behaviors were digitally recorded for 60 min. The plastic piece is a dark brown ABS Rod (ASTM D4673, UL 94HB, https://www.mcmaster.com/#abs-rods/=1e548fx) that was cut into a pup-sized cylinder 25 mm long and 12 mm in diameter. The plastic pieces were cleaned by 70% ethanol after each test. The 60-min pup exposure was chosen to induce Egr-1 expression in the brain, and was kept consistent across all behavioral experiments in the study. Some subjects showed aggression toward the pup. In these cases, the test was immediately terminated, and the pup was removed. These subjects were removed from the experiments (Table S1).

Brain tissue preparation

Subjects in each experimental group (PUP or CONTROL) were deeply anesthetized with an i.p. injection of sodium pentobarbital (60mg/kg body weight) immediately after the 1-h behavioral test and were then perfused through the ascending aorta with pre-cold 0.9% saline followed with 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4). Thereafter, brains were extracted and post-fixed in 4% paraformaldehyde in PB for 2 h at 4°C and were then transferred into 30% sucrose in PB at 4°C. Brains were cut coronally into 40 μm sections via Microm HM 450 and stored in 0.1M PB with 1% sodium azide at 4°C.

Immunocytochemistry

Egr-1 staining was performed on a set of coronal sections with 200 μm intervals. Sections were rinsed in 0.1M PB 3 times for a total 15 min (each rinsing was kept at the rate of 3 times for a total of 15 min below) and then treated with 0.3% H₂O₂ in 0.1M PB for 20 min. After rinsing in 0.1M PB, sections were incubated in 10% normal goat serum (NGS, Sigma-Aldrich, St. Luis, MO) in 0.3% Triton X-100 in 0.1M PB (TPB) for 1 h at room temperature. Subsequently, sections were incubated in rabbit anti-Egr-1 (1:3000, cat# 4153S, lot: 3, Cell Signaling Technology, Inc., Danvers, MA) in 0.3% TPB with 2% NGS at 4°C overnight. Sections were then rinsed in 0.3% TPB and incubated in biotinylated goat anti-rabbit IgG (1:300, Vector Laboratories, Burlingame, CA) for 2 h at room temperature. Thereafter, sections were rinsed in 0.3% TPB followed by 0.1M PB and then incubated in the ABC Elite HRP Kit (Vector Laboratories) in 0.1M PB for 90 min at room temperature, and Egr-1 immunostaining was revealed using 3´-diaminobenzidine (DAB, Sigma-Aldrich). For FG/Egr-1 double labeling, a set of brain sections with 200 μm intervals were processed for Egr-1 labeling as described above, followed by a blocking in 5% NGS in 0.3% TPB for 30 min, and were then incubated in rabbit anti-FG (1:10K, lot: 52–9600, Fluorochrome, LLC) in 0.3% TPB with 2% NGS at 4°C overnight. Thereafter, sections were rinsed in 0.3% TPB and incubated in goat anti-rabbit followed by ABC (as described above). FG immunostaining was visualized using VECTOR SG peroxidase (HRP) Substrate Kit (Vector Laboratories). Sections from both Egr-1 single and FG/Egr-1 double labeling were mounted on slides, dehydrated in an ethanol series, clarified in xylene, and cover slipped in permount. Finally, GLU/FG/Egr-1 and GABA/FG/Egr-1 triple labeling was performed on one brain section from both the BNST and AMYG of each subject. After a 1 h-blocking in 10% normal donkey serum (NDS, JacksonImmuno, West Glove, PA) in 0.3% TPB, the floating sections were incubated in a cocktail of rabbit anti-FG (1:1000) and either chicken anti-GLU (1:100, cat# 62668, lot: GR102316–4, Abcam, Cambridge, MA) or guinea pig anti-GABA (1:100, cat# AB175, lot: 2524679, Millipore, Temcula, CA) in 0.3% TPB with 2% NDS overnight at 4°C. Sections were then rinsed in 0.3% TPB and incubated in a secondary antibody cocktail of 1:200 Cy2 conjugated donkey anti-rabbit IgG (JacksonImmuno) and either Cy3 conjugated donkey anti-chicken IgG (for GLU, JacksonImmuno) or Cy3 conjugated donkey anti-guinea pig IgG (for GABA, JacksonImmuno) in 0.3% TPB with 2% NDS for 2 h at room temperature. Thereafter, the sections were rinsed in 0.3% TPB and then incubated in rabbit anti-Egr-1 (1:300) in 0.3% TPB with 2% NDS overnight at 4°C. The sections were then incubated in Cy5 conjugated donkey anti-rabbit IgG (JacksonImmuno) in 0.3% TPB with 2% NDS for 2 h at room temperature, rinsed in 0.3% TPB and then 0.1M PB, and finally mounted on slides and cover slipped in slow fade. The slides were examined using a Zeiss LSM 880 confocal microscope. The FG and GABA antibodies have been successfully used in our previous studies in prairie voles (Smith et al., 2016; Gobrogge et al., 2017). To validate the glutamate antibody, the antibody was pre-absorbed with 10μg L-Glutamate acid (Sigma G1251) or omitted in ICC, which both led to the absence of positive staining in the vole brain tissue. The Egr-1 immunocytochemistry has also previously been conducted in prairie voles (Northcutt & Lonstein, 2009). Based on manufacturer details (Cell Signaling Technology, Inc.,), the Egr-1 antigen sequence and the corresponding sequence in prairie voles show 100% sequence homology.

Cannulation and microinjections

Subjects were anesthetized with sodium pentobarbital (60mg/kg body weight, i.p.) and then stereotaxically implanted with bilateral guide cannula (26 gauge, Plastics One, Roanoke, VA) aimed at the VMH (nose bar −2.5mm; coordinates from bregma: caudal −1.4mm, lateral ±0.6mm, and ventral 6.0mm). After being lowered into the correct position, the cannula was attached to the skull firmly by applying liquid dental cement (Stoelting Co, Wood Dale, IL) around the base of the cannula. When the dental cement solidified, the animal was removed from the stereotaxical instrument, a dummy cannula and cap were applied to prevent needle track blockage, and ketoprofen (5mg/kg in 0.05 ml saline) was injected for post-operative pain relief. Post-operative care was provided by lab personnel, which included daily monitoring of the animals to ensure normal food and water intake and absence of infection for at least 3 days following surgery. FG injections were conducted on animals in Experiment 2 using an established procedure (Gobrogge et al., 2017). Briefly, under anesthesia, subjects received 50 nl of 4% FG (Fluorochrome, LLC, Denver, CO) in sterile 0.01M phosphate buffer (pH 7.4) into the VMH (coordinates from bregma: caudal −1.4mm, lateral ±0.6mm, and ventral 6.9mm) bilaterally using a pre-calibrated glass capillary micropipette. The injection was achieved by pressuring the micropipette at 5 nl/pulse/minute/side spanning a 10 min period through a PV 830 Pneunatic PicoPump (World Precision Instruments, Inc., Sarasoto, FL) connected to a CO₂ tank. The micropipette was left in the brain for 5 min before removal. Alloparental behavior tests were conducted 10 days after the FG injection. In our previous study, 10 days were a sufficient amount of time to allow FG to be expressed in the cell bodies of projection neurons (Gobrogge et al., 2017). For the pharmacological manipulation of neurochemical receptors in the VMH in Experiment 4, subjects were allowed to recover for a week following the cannulation. Right before habituating in the testing cage, subjects received microinjections of either: a CSF-DMSO mix (containing 50% of artificial CSF and 50% of DMSO, 200nl/side); a CSF-DMSO mix containing a low or high dose of a glutamate blocker cocktail of MK-801 (NMDA blocker, Sigma-Aldrich) and CNQX (AMPA/Kainate blocker, Sigma-Aldrich) (Kirkpatrick & Kakoyannis, 2004; Bredewold et al., 2015); D1R antagonist SCH23390 (Sigma-Aldrich); or OXTR antagonist (des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT; kindly provided by Dr. Maurice Manning, Toledo, OH). Microinjections were achieved by a 33-gauge needle that extended 1 mm below the guide cannulae and were delivered at a rate of 200nl/min as previously described (Lei et al., 2017). Drug doses were determined based on previous vole studies (Aragona et al., 2003; Liu & Wang, 2003; Aragona et al., 2006). After the 1-h behavioral test, all subjects were decapitated, and their brains were extracted to verify injection sites histologically. Subjects with misplaced cannulae were excluded from data analyses (Table S1).

Data quantification and analyses

All behaviors were scored by JWatcher software program (V1.0, Macquarie University and UCLA: http://www.jwatcher.ucla.edu/). The first 10 min of the subject’s interactions with the stimulus pup or the pup-sized plastic piece were quantified, as we were mainly interested in the initial choice to approach and begin displaying alloparental behavior for the current study using an established behavioral paradigm in our previous study (Lei et al., 2017). Prairie voles displayed pup licking/grooming, huddling (crouching over the pup without engaging in any other activities), and retrieving. Specific parental behaviors examined were as described in previous vole studies (Lonstein & De Vries, 1999; Lonstein et al., 2002; Lei et al., 2017). These behaviors were quantified by an experienced lab personnel who was blind to treatment groups. Egr-1, an immediate early gene and indirect measure of neuronal activation, increased expression in the brain following pup exposure in the present and in previous studies (Northcutt & Lonstein, 2009). Egr 1-labeled cells were quantified bilaterally (using Zeiss Axioskop 2, Carl Zeiss Microscopy Ltd, Germany) in the entire NAcc, VMH, paraventricular nucleus of the hypothalamus (PVN), and dentate gyrus of the hippocampus (DG) using three sections per brain area in each subject. Data quantification in the BNST was conducted in both the posterior dorsal (BNSTd) and ventral (BNSTv) nuclei, while the AMYG quantification included the anterior cortical (CoA), medial (MeA), and central (CeA) nuclei. These brain areas were chosen based on their roles in parental, alloparental and other social behaviors as found in previous studies (Numan et al., 1985; Numan & Numan, 1997; Lonstein et al., 1998; Sheehan et al., 2001; McHenry et al., 2015). The numbers of FG and FG/Egr-1-labeled cells were also counted in the BNST and AMYG. In addition, GLU/FG/Egr-1 and GABA/FG/Egr-1-labeled cells were quantified in the BNSTv and MeA, respectively. Brain sections were anatomically matched across subjects. All statistical analyses were performed using SPSS Statistic software (IBM), and p < 0.05 was deemed statistically significant. Data were checked for homogeneity of variance and normalized when necessary by square root transformations. The duration and frequency of each behavior were analyzed by two-way ANOVA (sex × group) followed by the Student Newman-Keuls (SNK) post hoc test. Group differences in the proportion of animals displaying alloparental behavior were compared using Fisher’s Exact test. Some animals showed aggression toward the pup or had misplaced cannula in the VMH and were therefore excluded from the data analyses (Table S1). The mean cell count per area for each subject was calculated and converted into a percent change over the average of the control group. Egr-1 single-labeled cells were analyzed by two-way ANOVA (sex × group). Group differences in FG and FG/Egr-1 labeling in male voles were analyzed by two-tailed t tests. The numbers of GLU/FG/Egr-1 and GABA/FG/Egr-1-labeled cells were converted into percentages of FG/Egr-1-labeled cells, and group differences were analyzed by two-tailed t tests. Finally, Cohen’s d for the t tests and partial eta squared (η_p²) for ANOVAs were used to calculate effect sizes.

Experimental Design

Experiment 1 compared male and female prairie voles in their alloparental behavior toward a conspecific pup. After behavioral testing, brain sections from those subjects (PUP) and sections from subjects that were exposed to a piece of pup-sized plastic (CONTROL) were processed for immunocytochemical labeling of Egr-1. Experiment 2 was designed to map the VMH neural circuitry and to examine the neuronal activation and neurochemical phenotypes associated with pup exposure in male prairie voles. Males were injected with FG into the VMH. Ten days later, they were exposed to a conspecific pup or a piece of pup-sized plastic for 60 min. Subjects’ brain sections were subsequently processed for FG single, FG/Egr-1 double, or either GLU/FG/Egr-1 or GABA/FG/Egr-1 triple labeling, respectively. Experiment 3 examined the effects of pharmacological blockades of neurochemical receptors in the VMH on male alloparental behaviors. Subjects were implanted with bilateral guide cannula aimed at the VMH. One week later, they were randomly assigned into experimental groups that received intra-VMH injections of either vehicle (CSF-DMSO mix), vehicle containing a low or high dose of the glutamate blocker cocktails (MK-801:CNQX; 10ng, 100ng), D1R antagonist SCH23390 (10ng, 50ng), or OXTR antagonist des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT (10ng, 50ng) followed by alloparental behavior testing.

Results

Alloparental behavior

As we were interested in the neuronal and neurochemical regulation of alloparental behaviors, we focused solely on voles that displayed alloparental behavior. Sexually-naïve male and female prairie voles were tested for alloparental behavior toward a conspecific pup (PUP). Both males (n=8) and females (n=6) displayed pup licking/grooming, huddling, and retrieving without any sex differences in the duration and frequency of these behaviors (Figure 1). Pup exposure also altered several other behaviors (Table S2). Subjects spent more time investigating a stimulus pup in comparison to a piece of plastic (novelty CONTROL, males, n=8, females, n=7; F_1,25 = 6.55, p = 0.01, η_p² = 0.208.). The duration (F_1,25 = 45.20, p = 0.01, η_p² = 0.0.644) and frequency (F_1,25 = 12.66, p = 0.01, η_p² = 0.336) of the subject’s locomotor activity were lower in the PUP group compared to the CONTROL group. No group differences were found in self-grooming (duration: F_1,25 = 0.18, p = 0.67, η_p² = 0.007; frequency: F_1,25 = 0.14, p = 0.72, η_p² = 0.006), and no sex or sex-by-group interactions were found in any of those behaviors (Table S2).

Figure 1.

Pup-directed behavior displayed by sexually-naïve prairie voles. Female and male prairie voles did not differ in the duration (A) or frequency (B) of pup licking/grooming, huddling, retrieving, or in the total score of their alloparental behavior. It should be noted that these behavioral data were obtained from 8 of 11 males and 6 of 10 females that displayed alloparental behavior toward conspecific pups.

Pup-induced neuronal activation

We performed immunocytochemical staining of Egr-1 on vole brain sections associated with pup exposure. Pup-exposed voles showed higher numbers of Egr-1-positive cells in the VMH (F_1,25 = 10.06, p = 0.01, η_p² = 0.287), but lower numbers in the AMYG (F_1,25 = 5.41, p = 0.03, η_p² = 0.178) (particularly in the anterior cortical nucleus of the AMYG (CoA; F_1,25 = 7.70, p = 0.01, η_p² = 0.235)) in comparison to control voles (Figure 2, Table S3). This effect was significant for both males and females. No group differences were found in Egr-1 labeling in other brain areas examined (NAcc: F_1,25 = 4.04, p = 0.06, η_p² = 0.139; BNST: F_1,25 = 2.22, p = 0.15, η_p² = 0.082; PVN: F_1,25 = 2.70, p = 0.11, η_p² = 0.097; DG: F_1,25 = 0.01, p = 0.91, η_p² = 0.000; Figure 2; Table S3).

Figure 2.

Egr-1 labeling in the brain of male and female prairie voles. Exposure to a conspecific pup (Pup) for 60 min induced a significant increase in Egr-1-positive cells in the ventromedial hypothalamic nucleus (VMH) and a decrease in Egr-1-positive cells in the amygdala (AMYG) compared to exposure to a pup-sized piece of plastic (Control) (top panel). No differences were found in Egr-1 labeling in other brain areas examined. Additionally, no sex or sex-by-group differences were found in Egr-1 labeling in any of the measured brain areas. The bottom panel consists of composed photomicrographs illustrating Egr-1-labeled cells in the bed nucleus of the stria terminalis (BNST), VMH, and AMYG in prairie voles under the Control and Pup conditions. ac: anterior commissure; BNSTd: BNST dorsal part; BNSTv: BNST ventral part; CoA: anterior cortical nucleus of the AMYG; DG: dentate gyrus of the hippocampus; MeA: medial nucleus of the AMYG; opt: optical tract; VMHc: VMH central part; VMHd: VMH dorsal part; VMHv: VMH ventral part; 3V: 3^rd ventricle. *: p < 0.05; **: p < 0.01. Scale bar = 1 mm.

The VMH-BNST-AMYG circuitry

Our tract-tracing data revealed FG-stained neurons in several brain areas, including the BNST, AMYG, lateral septum (LS), prefrontal cortex (PFC), and ventral tegmental area (VTA). We focused on the BNST and AMYG projection neurons to the VMH; no group differences between subjects exposed to a conspecific pup (PUP; n=9) or a piece of pup-sized plastic (CONTROL; n=7) were found in the number of FG-labeled cells in the BNST (t₁₄ = 0.93, p = 0.37, d = 0.50) and AMYG (t₁₄ = 1.96, p = 0.07, d = 1.048) (Figure 3A). For the neurons in the AMYG that were labeled for Egr-1, the PUP group had a lower percentage that were co-labeled with FG (t₁₄ = 3.11, p = 0.01, d = 1.66) (Figure 3B), particularly in the MeA, but not in the CoA and CeA (Table S4), compared to the CONTROL group. No group differences were found in the numbers of FG/Egr-1 over Egr-1 labeling in the BNST (t₁₄ = 1.01, p = 0.13, d = 0.54; Figure 3B) or in the BNSTd and BNSTv subnuclei (Table S4). For the FG-labeled cells, no group differences were found in the percentages of those cells co-labeled for Egr-1 either in the AMYG (t₁₄ = 0.21, p = 0.84, d = 0.11) or BNST (t₁₄ = 1.62, p = 0.14, d = 0.87; Figure 3C), or in any of the subnuclei measured (Table S5).

Figure 3.

Numbers of FG-labeled (A) and FG/Egr-1 double-labeled (B & C) cells in the bed nucleus of the stria terminalis (BNST) and amygdala (AMYG) in the brain of male prairie voles. (A) No group differences were found in the numbers of FG-labeled cells in both the BNST and AMYG. (B) Exposure to a conspecific pup (Pup) for 60 min induced a significant decrease in the percentage of cells double-labeled for FG/Egr-1 over a total number of Erg-1 cells in the AMYG, but not BNST, compared to exposure to a pup-sized piece of plastic (Control). (C) No group differences were found in the percentage of cells double-labeled for FG/Egr-1 over total FG-labeled cells. **: p < 0.01.

We also examined the activated projection neurons (FG/Egr-1 double-labeled) from the BNSTv and MeA to the VMH for their GLU-ergic and GABA-ergic phenotypes. In both the BNSTv and MeA, a higher percentage of FG/Egr-1 neurons expressed GLU in the PUP group than the CONTROL group (BNSTv: t₁₄ = 3.64, p = 0.01, d = 1.95; MeA: t₁₄ = 5.82, p = 0.01, d = 3.11; Figure 4A). In contrast, less FG/Egr-1-labeled neurons expressed GABA in the MeA of the PUP group compared to the CONTROL group (t₁₄ = 3.17, p = 0.01, d = 1.69), but no group difference was found in the BNSTv (t₁₄ = 1.83, p = 0.09, d = 0.98; Figure 4B). Finally, group differences in the ratio of GLU:GABA expression in the FG/Egr-1 projection neurons from the BNSTv and MeA to the VMH were examined by t tests. Our data showed that the PUP group had a higher GLU:GABA ratio in the FG/Egr-1 projection neurons from the BNSTv (0.78 ± 0.04) to the VMH than that of the CONTROL group (0.52 ± 0.04) (t₁₄ = 4.20, p = 0.01, d = 2.24). Similarly, the PUP group also had a higher GLU:GABA ratio in the FG/Egr-1 labeled neurons in the MeA (2.26 ± 0.45) compared to the CONTROL group (1.03 ± 0.03) (t₁₄ = 2.75, p = 0.02, d = 1.47).

Figure 4.

Numbers of GLU/FG/Egr-1 (A) and GABA/FG/Egr-1 (B) triple-labeled cells in the bed nucleus of the stria terminalis ventral part (BNSTv) and medial nucleus of the amygdala (MeA) of male prairie voles exposed to a conspecific pup (Pup) or a pup-sized piece of plastic (Control) for 60 min. Pup exposure significantly increased the number of GLU/FG/Egr-1 triple-labeled cells in the BNSTv and MeA (A) and decreased the number of GABA/FG/Egr-1 triple-labeled cells in the MeA (B). *: p < 0.05. Photomicrographs illustrate fluorescent-labeled cells in the BNSTv and MeA. Cells (indicated by arrowheads) were labeled for Egr-1 (blue), FG (green), and either Glu (red; top panels) or GABA (red; bottom panels). Arrows indicate triple-labeled cells. Scale bar = 10 μm.

Neurochemical receptor blockades and male alloparental behavior

Pharmacological blockades of neurochemical receptors in the VMH affected the onset of alloparental behavior in male prairie voles (Figure 5). In comparison to vehicle controls (n=13), intra-VMH injections of a glutamate blocker cocktail (MK-801 and CNQX, 50:50) both at a low (10 ng, n=7, p=0.59) and a high (100 ng, n=9, p=0.07) dose did not change the percentage of animals displaying alloparental behavior. However, injections of the D1R antagonist (SCH23390) at a high (50 ng, n=7; p=0.03), but not a low (10 ng, n=6; p=0.54), dose decreased the number of animals displaying alloparental behavior. Similarly, administration of the OXTR antagonist (des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT) at a high (50 ng, n=10; p=0.01), but not a low (10 ng, n=8; p=0.13), dose was effective at impairing the onset of alloparental behavior. However, patterns of specific alloparental behaviors, such as pup licking/grooming, huddling, and retrieving were not significantly affected by any of the treatments.

Figure 5.

Effects of neurochemical receptor blockades in the VMH on the percentage of male prairie voles displaying alloparental behavior. Intra-VMH microinjections of a high dose of D1R antagonist (50ng) or a high dose of OXTR antagonist (50ng) resulted in significant decreases in the number of males approaching the pup compared to vehicle-injected controls. *: p < 0.05.

Discussion

In the present study, we used socially monogamous prairie voles to characterize alloparental behaviors displayed by both males and females and to examine neuronal activation induced by pup exposure in selected brain regions. We also examined pup exposure-induced activation of projection neurons from the BNSTv and MeA to the VMH and revealed their GLU-ergic and GABA-ergic neurochemical phenotypes in male prairie voles. Finally, we pharmacologically blocked GLU-ergic receptors, D1R, and OXTR in the VMH to examine their functional roles in the onset of social approach behavior. Together, our data indicate that the VMH is an important node for regulating social approach behavior towards pup-related stimuli in male prairie voles and may play a role in the onset of alloparental behavior.

Our data showed that sexually-naïve, male prairie voles displayed all aspects of maternal behaviors (except nursing), including pup licking/grooming, huddling, and retrieving. It should be mentioned that although some sexually-naïve prairie voles display alloparental behaviors, others attack the conspecific pups (Bamshad et al., 1994; Lonstein & De Vries, 1999; Lei et al., 2017) – this was also found in the present study (Table S1). The high levels of alloparental behaviors and their equivalency to that of maternal behaviors are consistent with previous findings (Lonstein et al., 2002; Bales et al., 2004b) and further demonstrate the utility of the prairie vole model for studying the differences between parental and alloparental behavior (Wang et al., 1998). Notably, pup exposure and the display of alloparental behavior had similar effects on regional Egr-1 staining in the brain of both males and females, supporting the notion that the neural substrates are preserved to mediate similar social behaviors displayed by both male and female voles (Kirkpatrick et al., 1994). The decreased number of Egr-1-positive neurons in the AMYG associated with pup exposure also supports the findings that activation of the AMYG inhibits parental behavior (Sheehan et al., 2001). Interestingly, mating and cohabitation that leads to male-female pair bonding are associated with increased neuronal activation in the AMYG (Wang et al., 1997; Curtis & Wang, 2003), whereas pup exposure was related to a decrease in neuronal activation in the present study. Therefore, the AMYG may have differential, context-specific effects on social behaviors – it facilitates pair bonding, but inhibits male alloparental care in prairie voles.

The VMH has been implicated in a variety of social behaviors – including parental behavior (Bridges et al., 1999). As in rats and mice (Numan & Numan, 1997; Gu et al., 2003; Pardo-Bellver et al., 2012), neurons from the BNST and AMYG project to the VMH in the prairie vole brain. The BNSTv plays a role in processing chemosensory cues associated with pup exposure and in facilitating alloparental behavior (Northcutt & Lonstein, 2009; Horrell et al., 2017). Further, activation of the ERα system in the AMYG, and more specifically the MeA, inhibits alloparental behavior (Lei et al., 2010). Therefore, the combination of increased excitatory input from the BNSTv and decreased inhibitory input from the MeA may have contributed to the increased neuronal activation in the VMH. The activation of VMH neurons may influence the perception of chemosensory and somatosensory stimuli from pups (Pfaff et al., 1994) and facilitate the onset of male alloparental behavior. One possibility is that pup-associated stimuli elevate the ratio of GLU:GABA inputs from the BNSTv and MeA to the VMH, ultimately resulting in the activation of other neurochemical systems such as DA and OXT that have been implicated in maternal behavior in rats (Numan & Stolzenberg, 2009) and parental/alloparental behavior in both male and female prairie voles (Lonstein, 2002; Bales et al., 2004a). Although not tested in the VMH, previous studies have shown that enhanced glutamatergic signaling upregulates OXT mRNA expression in the brain (Yoshimura et al., 2015) and facilitates OXT release (Busnardo et al., 2012). In addition, glutamatergic neurotransmission and its interactions with DA plays an important role in a variety of cognitive and behavioral functions (Wan et al., 1995; Wiedholz et al., 2008). Our data also add to previous literature demonstrating that the balance of excitatory and inhibitory input to various brain regions involved in sensory processing plays an important role in social behavior, including maternal behavior (Rubenstein & Merzenich, 2003; Tyzio et al., 2014; Marlin et al., 2015).

Our pharmacological manipulations resulted in some intriguing effects on male alloparental behavior. Blockade of D1R or OXTR dose-dependently decreased the number of males that approached the pup to begin displaying alloparental care, but it had no effects on any specifically measured alloparental behaviors in male voles. These data indicate a functional role of those receptors in the VMH in the onset of approach to the social stimulus, but not in the expression of specific alloparental behaviors. In rats, D1R stimulation in the NAcc and medial preoptic area (MPOA) promotes the onset of maternal behavior in pregnancy-terminated females (Stolzenberg et al., 2007), and intracerebroventricular (icv) administration of OXT facilitates the onset of maternal behavior in estrogen-primed females (Fahrbach et al., 1984) (but see (Rubin et al., 1983)). In prairie vole parents, intraperitoneal administration of DA receptor antagonists impairs both maternal and paternal behaviors (Lonstein, 2002). Further, icv administration of OXTR and V1aR antagonists reduce alloparental behavior in reproductively naïve males, although the action sites are unknown (Bales et al., 2004a). Our findings on the BNST-AMYG-VMH circuitry, along with previous alloparental studies that implicate other key brain areas (i.e. NAcc, PVN, LS), support the notion that male alloparental behavior is regulated by a neural circuitry with multiple brain regions and neurochemicals that differentiate from neurochemical circuits underlying parental care (Kenkel et al., 2017). The VMH may serve as a gate to turn on or off approach behavior for social stimuli, whereas other brain areas may be involved in mediating the expression of the specific behaviors. As not examined in the present study, we can only speculate that concurrent activation of the D1R and OXTR may be necessary for the onset of alloparental behavior, and, therefore, blockade of either type of the receptors may impair the approach to pup-associated stimuli. This notion is supported by previous data showing that blockade of either OXTR or V1aR in the LS impairs AVP-facilitated pair bonding (Liu et al., 2001), and concurrent activation of OXTR and D2R (DA D2-type receptors) in the NAcc is essential for partner preference formation (Liu & Wang, 2003) in prairie voles. Alternatively, these neurochemical systems may interact in sequence in the regulation of alloparental behaviors.

Taken together, our data suggest that the VMH may serve as a node in a neural circuitry that involves inputs from both the BNST and AMYG, as well as local activation of OXTR and D1R, to regulate the onset of social approach behavior, leading to alloparental care in sexually-naïve, male prairie voles (see Figure 6). Several issues should be noted. First, as the control for these experiments did not include a social stimulus, we cannot confidently state that the pharmacological manipulation specifically prevented the onset of alloparental care as opposed to other social behaviors. Nevertheless, the VMH may serve as a functional node for the decision to approach and provide alloparental care versus the decision to attack. These experiments may serve as a foundational starting point for this specific line of research. Second, although our focus was on the BNST-AMYG-VMH circuitry, we also observed FG-labeled cells in other brain areas, such as the LS, PFC, and VTA (indicating their local projection neurons to the VMH). The LS, for example, is implicated in maternal behaviors in mice (Curley et al., 2012) and paternal behaviors in prairie voles (Wang et al., 1994). Therefore, the roles of those brain areas in the regulation of male alloparental behavior should not be ignored. Third, previous studies have shown that AVP in the LS facilitates pair bonding and paternal behavior (Wang et al., 1994; Liu et al., 2001), and AVP in the anterior hypothalamus (AH) facilitates aggression associated with mate guarding (Gobrogge et al., 2009; Gobrogge et al., 2017). The role of the brain AVP system in the regulation of social behaviors, including alloparental care, associated with the monogamous life strategy is a focus in one of our ongoing investigations. Fourth, although the spontaneous alloparental behavior displayed by sexually-naïve, male voles provides an opportunity to study alloparental behavior apart from other social behaviors/experience, the neurochemistry of father voles likely differs from that of sexually naïve voles. This is supported by data showing that social interactions with a female not only facilitates alloparental behaviors, but also affects brain neurochemical systems implicated in parental behaviors in prairie voles (Bamshad et al., 1994; Lei et al., 2017). Therefore, the neurochemical underpinnings of parental behaviors between sexually-naïve and father voles may be different and should be examined in future studies. Finally, some variations were noticed in the percentage of male voles that displayed alloparental behaviors. More specifically, 67% of intact males (Expt 1) displayed alloparental behavior, whereas 83% of males that received intra-VMH injections of FG (Expt 2) or vehicle (Expt 4) were alloparental. Interestingly, lowering cannulae or infusions of saline into the VMH facilitate the onset of maternal behavior in steroid-primed, nulliparous female rats (Bridges et al., 1999). It remains to be determined whether stimulation of the VMH by cannulation and vehicle infusion can alter social approach and the onset of alloparental behavior in male prairie voles.

Figure 6.

Schematic illustration of the data from the present study illustrating the role of the BNSTv-MeA-VMH pathway in the regulation of alloparental behavior in male prairie voles. Sexually naïve, adult males were either exposed to a piece of plastic (to control for novelty) or to a conspecific pup for 60 mins. Males that were exposed to a conspecific pup showed an increase in GLU expression in FG/Egr-1 labeled projection neurons from the BNSTv and MeA to the VMH as well as a decrease in the expression of GABA in FG/Egr-1-labeled projection neurons from the MeA to the VMH. Pup exposure also increased the GLU:GABA ratio in both the BNSTv and MeA FG/Egr-1-labeled projection neurons to the VMH compared to controls. The onset of social approach towards a pup was associated with the activation of D1R and OXTR within the VMH, as pharmacological blockade of either D1R or OXTR in the VMH decreased the percentage of male voles that approached the pup to begin displaying alloparental care.

Supplementary Material

Supporting information

Additional supporting data can be found in the online version of this article.

In sexually naïve male prairie voles, pup exposure increased the number of GLU/FG/Egr-1-labeled cells in the BNSTv and MeA (A) and decreased the number of GABA/FG/Egr-1-labeled cells in the MeA (B) that projected to the VMH. Intra-VMH microinjections of high doses of either a D1R or an OXTR antagonist decreased the number of males approaching the pup (C). Schematic illustration summarizes the role of the BNSTv-MeA-VMH pathway in regulating the onset of social approach towards a pup (D).

Abbreviations

ADHD

attention-deficit/hyperactivity disorder

AMYG

amygdala

AVP

vasopressin

BNST

bed nucleus of the stria terminalis

BNSTd

posterior dorsal BNST

BNSTv

posterior ventral BNST

CeA

central nucleus of the amygdala

CoA

anterior nucleus of the amygdala

CSF

cerebrospinal fluid

DA

dopamine

DG

dentate gyrus of the hippocampus

D1R

dopamine D1-type receptor

D2R

dopamine D2-type receptor

Egr-1

early growth response 1

FG

Fluoro-Gold

GABA

gamma-aminobutyric acid

GLU

glutamatergic

icv

intracerebroventricular

IEG

immediate-early gene

LS

lateral septum

MeA

medial nucleus of the amygdala

NAcc

nucleus accumbens

OXT

oxytocin

OXTR

oxytocin receptor

PFC

prefrontal cortex

PUP

pup-exposed animals

PVN

paraventricular nucleus of the hypothalamus

SNK

Student Newman-Keuls

VMH

ventromedial hypothalamus

VTA

ventral tegmental area

Data accessibility

All relevant data within the document will be made available on demand by email zwang@psy.fsu.edu.