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. Author manuscript; available in PMC: 2024 Aug 1.
Published in final edited form as: Behav Neurosci. 2023 Mar 6;137(4):223–235. doi: 10.1037/bne0000555

The medial preoptic area and its projections to the ventral tegmental area and the periaqueductal gray are activated in response to social play behavior in juvenile rats

Changjiu Zhao 1,*, Lauren V Riters 1
PMCID: PMC10363185  NIHMSID: NIHMS1875551  PMID: 36877484

Abstract

The medial preoptic area (MPOA) is well-known for its role in sexual and maternal behaviors. This region also plays an important role in affiliative social behaviors outside reproductive contexts. We recently demonstrated that the MPOA is a central nucleus in which opioids govern highly rewarding social play behavior in adolescent rats. However, the neural circuit mechanisms underlying MPOA-mediated social play remain largely unresolved. We hypothesized that the MPOA unites a complementary neural system through which social play induces reward via a projection to the ventral tegmental area (VTA) and reduces a negative affective state through a projection to the periaqueductal gray (PAG). To test whether the two projection pathways are activated in response to social play behavior, we combined retrograde tract tracing with immediate early gene (IEG) expression and immunofluorescent labeling to identify opioid-sensitive projection pathways from the MPOA to VTA and PAG that are activated after performance of social play. Retrograde tracer, Fluoro-Gold (FG), was microinjected into the VTA or PAG. IEG expression (i.e., Egr1) was assessed and triple immunofluorescent labeling for mu opioid receptor (MOR), Egr1 and FG in the MPOA was performed after social play. We revealed that play-animals displayed an increase in neurons double-labeled for Egr1+FG and triple-labeled for MOR+Egr1+FG in the MPOA projecting to both the VTA and PAG when compared to no-play rats. The increased activation of projection neurons that express MORs from MPOA to VTA or PAG after social play suggests that opioids may act through these projection pathways to govern social play.

Keywords: opioids, mu opioid receptor, Egr1, Fluoro-Gold

1. Introduction

Affiliative, non-sexual social interactions in groups are critical for establishing bonds and essential for individuals to develop and practice important social skills. For instance, social play in adolescent animals is critical for the development of adult social skills (Vanderschuren et al., 2016), and vocal-social interactions in flocking songbirds are needed for vocal learning and flock cohesion, which offers protection from predators and enhances foraging efficiency (Jullien & Thiollay, 1998; Lazarus, 1979; Riters, Kelm-Nelson, et al., 2019). Although important, thus far, there remain gaps in the knowledge of mechanisms that underly affiliative behaviors outside of reproductive contexts.

Most studies on affiliative behavior focus on the positive affective state induced by social contact that rewards interactions with conspecifics. However, in social animals, affiliative contact is also reinforced because it reduces a negative affective state caused by social exclusion or isolation, thus creating a complementary system (i.e., positive reinforcement from affiliative interactions and negative reinforcement from termination of isolation). This dual-reinforcement network model for social behavior is supported by studies of gregarious birdsong and rat social play. Specifically, conditioned place preference (CPP) studies suggest both gregarious song and play to be highly rewarding (Achterberg et al., 2019; Hahn et al., 2017; Riters & Stevenson, 2012; Riters et al., 2014; Stevenson et al., 2020; Trezza et al., 2009), and both song and play are associated with physical pain reduction (i.e., analgesia) (Kelm-Nelson et al., 2012; Panksepp, 1980).

Songbird studies reveal a crucial role for the medial preoptic area (MPOA) which accesses both the canonical mesolimbic reward pathway via a projection to the ventral tegmental area (VTA) and the periaqueductal gray (PAG), which is well-known for its role in pain, including social pain (Riters, Kelm-Nelson, et al., 2019). We aimed to extend what has been revealed in birds to mammals, to elucidate an intriguing and conserved circuitry across vertebrates. To this end, we proposed to study social play behavior in rats, which, like affiliative birdsong, is a rewarding, non-sexual, affiliative behavior that is important for the development of cognitive and social skills (Riters, Spool, et al., 2019). Recent research from our laboratory for the first time demonstrates a crucial role for the MPOA in social play behavior in rats and that mu opioid receptors (MORs) in this region are critical for play behavior (Zhao et al., 2020). These findings match what we have observed in songbirds, offering initial support for the hypothesis that our studies in birds are revealing an ancient, core, conserved circuitry in which opioids act to facilitate and reward important social behaviors in non-sexual, affiliative contexts. The MPOA to VTA pathway is involved in motivation and reward associated with sexual and maternal behaviors (Fang et al., 2018; McHenry et al., 2017). Additionally, the MPOA projects to PAG, which is well-known for its role in pain and aversive emotional states such as fear and anxiety (Zhang et al., 2021). Given that affiliative social interactions are not only rewarding but also reduce negative affective states, we proposed that social play behavior may be promoted through key output pathways from the MPOA to VTA via a positive and rewarding mechanism and MPOA to PAG via reducing a negative affective state.

The present study was undertaken to test the hypothesis that projection pathways from MPOA to VTA and PAG are active during social play behavior in juvenile rats. We combined retrograde tract tracing with immediate early gene (IEG) expression and immunofluorescent labeling in rats either permitted to play or not. The retrograde tracer, Fluoro-Gold (FG), a widely used fluorescent tracer for retrogradely labeling projection neurons was microinjected into the VTA or PAG. After 10 days, IEG expression (i.e., Egr1) was assessed and triple immunofluorescent labeling for MOR, Egr1 and FG in the MPOA was performed after social play.

2. Materials and methods

2.1. Animals

Forty juvenile Sprague-Dawley (SD) rats purchased from Charles River (Wilmington, MA) were maintained at a controlled temperature (~22°C) and relative humidity (50-55%) on a 12:12 hr reverse light/dark cycle (lights off at 09:00 AM). The subjects had ad libitum access to standard rodent chow and tap water throughout the experiment except during surgery. All experimental procedures were approved by the Animal Care and Use Committee of the University of Wisconsin in strict compliance with NIH guidelines. All efforts were made to minimize any potential pain and to reduce the number of animals used.

2.2. Retrograde tracing

Retrograde tracing with FG (Fluorochrome Inc., Denver, CO) was used to label cells that project from the MPOA to VTA or PAG. A total of 32 male and 8 female rats (16 males/4 females for each brain region, VTA and PAG) at ages of postnatal days 21-22 (PN21-22) were housed in mixed-sex groups of five containing 4 males/1 female. The inclusion of females has been found to stimulate social play in group housed rodents (Argue & McCarthy, 2015a, 2015b), but females were not the focus of this study. Rats received microinfusions of retrograde tracer, following methods similar to those used in our past studies (Zhao et al., 2020; Zhao & Gammie, 2018). In brief, after a 3-day acclimation in our colony (PN24-25, Fig. 1), juvenile male rats were anesthetized with isoflurane at vaporizer setting of 2% and securely placed into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The coordinates for Bregma were determined and infusion cannula (33 gauge, Plastic One, Roanoke, VA) were lowered bilaterally to target either VTA or PAG according to The Rat Brain in Stereotaxic Coordinates (Paxinos & Watson, 2007). VTA: AP=−5.0 mm, ML=±0.8 mm, and DV=8.3 mm; PAG: AP=−6.70 mm, ML=±1.5 mm, and DV=5.5 mm, with an angle of 10°. Choices of coordinates for VTA and PAG injection were based on previous studies showing reliable retrograde labeling in MPOA (Rizvi et al., 1992; Shahrokh et al., 2010). The retrograde tracer, FG (2% in 0.9% saline) was pressure-injected bilaterally into the VTA or PAG in a volume of 0.3 ul per side. After injection, the infusion cannula remained in place for an additional 5 min to minimize backflow of tracer along the cannula track. Cannula were removed and after recovery, rats were returned to their home cages and group-housed as above before testing began.

Figure 1:

Figure 1:

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Experimental timeline for FG injection, behavioral testing and brain tissue collection. FG, Fluoro-Gold; PN, postnatal day.

2.3. Social play behavior test following retrograde tracer injection

The behavioral testing occurred during the active dark phase between 09:00 AM and 10:00 AM under dim red light. On PN33-34 (i.e., 9 days after tracer injection, Fig. 1), rats were housed individually for 24 hrs. This social separation increases affiliative behavior (i.e., social play) once animals are reunited with social groups (van Kerkhof et al., 2014; Vanderschuren, Niesink, et al., 1995a). Following this separation period, half of the animals were reunited with their affiliative group (“play group”, animals were placed in the test cage in groups consisting of 4 males and 1 female) and half remained in isolation (“no-play group”, animals were placed in the test cage alone). In the reunited play group, we video-recorded play behavior for 20 min following identical methods to those described in our recent work (Zhao et al., 2020). We measured indices of social play (i.e., pouncing, pinning, wrestling, chasing and total bouts) and non-play behaviors (i.e., grooming, feeding, motor behaviors). In the no-play group, grooming, feeding, and motor behaviors were recorded for 20 min. To achieve an unbiased observation of social play behavior, behaviors were scored by an experimenter unaware of the experimental manipulations. After the test, animals were placed back into their separate cages for 90 min to ensure the peak expression of play-induced immediate early gene Egr1.

2.4. Preparation of brain sections for single and triple fluorescence immunohistochemistry

Ninety minutes after completion of the social play test, play or no-play male rats were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) (Fig. 1). Brains were dissected and post-fixed overnight in the same fixative and then transferred to 30% sucrose at 4°C for three days. Brains were fast-frozen on dry ice and cut into 40-μm coronal sections and stored in cryoprotectant antifreeze solution at −20°C until processing. Sections encompassing the rostral (Bregma −0.24 to −0.48 mm, Fig. 2A) and caudal (Bregma −0.48 to −0.72 mm, Fig. 2B) MPOA were collected according to The Rat Brain in Stereotaxic Coordinates (Paxinos & Watson, 2007). Sections from the injection sites (i.e., VTA or PAG) were also collected for verification of cannula tip placements.

Figure 2:

Figure 2:

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Illustrations of rat brain showing the approximate location of imaged regions (black boxed areas) for cell counting at the level of the rostral (A) and caudal (B) MPOA. Distance from bregma in the rostrocaudal planes is indicated. Illustrations were modified from Paxinos and Watson (2007).

2.5. Single fluorescence immunohistochemistry of FG with tyramide signal amplification (TSA)

To locate the cannula tip placements of each injection, FG was stained using fluorescence immunohistochemistry. Briefly, brain sections from VTA or PAG were incubated with rabbit anti-FG antiserum (Fluorochrome, LLC, Denver, CO) at a concentration of 1:1000 overnight at 4°C. Following primary body incubation, sections were incubated in HRP-conjugated goat anti-rabbit antiserum (#7074, Cell Signaling Technology, diluted 1:100) for 1 hr, at room temperature and labeled by incubation for 10 min in Cy5-conjugated tyramide (TSA™ Plus Cyanine 5 kit, PerkinElmer; diluted 1:50). Sections were mounted on subbed slides, allowed to dry in a dark room for 24 hrs, and coverslipped using Serva DePeX (Crescent Chemical Company, Islandia, NY).

2.6. Triple fluorescence immunohistochemistry of Egr1, MOR and FG with TSA

Triple-label immunohistochemistry of three target proteins of interest was performed in the MPOA sections according to our recently published research (Merullo et al., 2018; Spool et al., 2019; Zhao et al., 2020; Zhao & Gammie, 2015). For each section, one label was Egr1, which can be used to identify neurons active during behavioral interactions. In the same section the other labels were for mu opioid receptors (MOR), which are considered critical regulators of social play behavior (Trezza et al., 2011; Vanderschuren, Niesink, et al., 1995b), and FG-positive cells (i.e., cells projecting from the MPOA to VTA or PAG). Cells triple-labeled for Egr1, MOR and FG identify MOR projection cells that are activated during performance of affiliative behaviors (i.e., social play behavior). Triple fluorescence immunolabeling was carried out at room temperature unless otherwise indicated. Brain sections were incubated overnight at 4°C with rabbit anti-MOR antiserum (#24216, ImmunoStar; diluted 1:10000). After first primary antibody incubation, sections were incubated for 1 hr with HRP-conjugated goat anti-rabbit antiserum (#7074, Cell Signaling Technology, diluted 1:100), then incubated for 10 min in Cy3-conjugated tyramide (TSA™ Plus Cyanine 3 kit, PerkinElmer; diluted 1:50; red for MOR labeling). Because the MOR, Egr1 and FG antibodies are all raised in rabbit, we block cross-reactivity by heating sections in a citric acid buffer (10 mM, pH 6.0) at 98°C for 5 min. Sections were incubated with rabbit anti-Egr1 (15F7, #4153, Cell Signaling Technology; diluted 1:1000) antiserum overnight at 4°C. Following the second primary incubation, sections were incubated in HRP-conjugated goat anti-rabbit antiserum for 1 hr as above, and labeled by incubation in Alexa Fluor 488-conjugated tyramide (Alexa Fluor 488 TSA kit; Molecular Probes; diluted 1:100; green for Egr1 labeling). Again, we blocked cross-reactivity by heating sections in a citric acid buffer as above. Sections were incubated with rabbit anti-FG (Fluorochrome, LLC; diluted 1:1000) antiserum overnight at 4°C. Following the third primary incubation, sections were incubated in HRP-conjugated goat anti-rabbit antiserum for 1 hr, and labeled by incubation for 10 min in Cy5-conjugated tyramide (TSA™Plus Cyanine 5 kit, PerkinElmer; diluted 1:50; blue for FG labeling). Sections were mounted on subbed slides, allowed to dry in a dark room for 24 hrs, and coverslipped using Serva DePeX (Crescent Chemical Company, Islandia, NY). As controls, quenching of HRP activity prior to incubation with Cy3-, Alexa Fluor 488- or Cy5-conjugated tyramide, or omission of the primary antibodies or HRP-linked secondary antibodies, completely abolished corresponding specific labeling.

2.7. Quantification of Triple Fluorescence Labeling

All images in each sample area were acquired sequentially with a screen resolution of 1024×1024 pixels using an inverted Zeiss LSM 710 Meta laser scanning confocal microscope (Zeiss; Oberkochen, Germany). For quantitative analysis of single-, double- or triple-labeled cells, cell counting was performed using 40× magnification photomicrographs in a counting box of 412 μm × 512 μm within the rostral and caudal MPOA. In each animal, the number of single-labeled cells characterized by clearly stained somata for MOR-expressing cells, nuclei for Egr1-immunoreactive cells and cell bodies and dendrites for FG-labeled cells in both rostral and caudal MPOA were counted bilaterally in a typical section that was anatomically well-matched across all animals (Fig. 2). Simultaneously, double- and triple-labeled cells were also enumerated based on the coincidence of red-labeled somata for MOR, green-labeled nuclei for Egr1 and blue-labeled for FG in the same cells. The counting was performed manually by an experienced investigator unaware of the rats’ behavioral conditions with the aid of MetaMorph software (Molecular Devices, LLC, San Jose, CA). All confocal images were transferred to Adobe Photoshop (Version 22.2), with adjustments of brightness and contrast. The number of single-, double- or triple-labeled cells from bilateral MPOA per rat was averaged.

2.8. Statistical analysis

Statistical analyses were performed using GraphPad Prism Software (version 9.3.1; GraphPad Software, San Diego, CA). All data on social play behavior and number of immunolabeled cells were presented as mean±SEM and analyzed using two-way analysis of variance (ANOVA) with manipulation (2 levels: play and no-play) × region (2 levels: rostral and caudal MPOA) as between-subject factors. When overall significant effects were found, pairwise comparisons of means were assessed using Bonferroni’s post hoc test. Pearson correlations were run to assess relationships between the expression of active MPOA cells, including cells expressing Egr1, Egr1+MOR, Egr1+FG and Egr1+MOR+FG and social play behaviors in play animals. P<0.05 was taken as the significant level of difference.

3. Results

3.1. Verification of FG injections into the VTA and PAG

Cells labeled with FG were found to be predominantly constrained within the VTA (Fig. 3A) or PAG (Fig. 5A), with little or no spread to the immediately adjacent areas in 12 rats for each injection, which were considered to be a good hit for the target (i.e., hit). Three VTA and four PAG injections were off the target as no or few FG-labeled cells were detected in the VTA or PAG, while the majority of FG-labeled cells were identified in remote areas outside the targets (i.e., miss). One no-play rat with VTA injection for unknown reasons had a large lesion in the injection site (i.e., lesion). These miss and lesion animals were eliminated from the data analysis. The number of rats included in data analysis was N=12 (6/6 for play/no-play) for both the VTA and PAG injection groups.

Figure 3:

Figure 3:

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Representative photomicrographs of cells labeled for MOR (B), Egr1 (C) and/or FG (D) in the rostral MPOA of VTA-injected (i.e., FG was injected into the VTA) play rats. FG-positive cells were verified to be restricted to the VTA (A). Arrows indicate cells triple labeled for MOR, Egr1 and FG (B-E). Note that a small portion of cells were triple labeled after social play (E). Scale bars: 100 μm in A, 50 μm in B-E. Egr1, early growth response 1; FG, Fluoro-Gold; MOR, opioid receptor, mu 1; MPOA, medial preoptic area; VTA, ventral tegmental area.

Figure 5:

Figure 5:

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Representative photomicrographs of cells labeled for MOR (B), Egr1 (C) and/or FG (D) in the rostral MPOA of PAG-injected (i.e., FG was injected into the PAG) play rats. FG-positive cells were verified to be restricted to the PAG (A). Arrows indicate cells triple labeled for Egr1, MOR and FG (B-E). Note that a small portion of cells were triple labeled after social play. Scale bars: 100 μm in A, 50 μm in B-E. Egr1, early growth response 1; FG, Fluoro-Gold; MOR, opioid receptor, mu 1; MPOA, medial preoptic area; PAG, periaqueductal gray.

3.2. MPOA neurons project to VTA and PAG

Retrograde tracer FG injected into the VTA retrogradely labeled MPOA cells that project to VTA. FG cells were readily identified based on their labeled neuronal cell bodies and dendrites. FG-labeled cells were present within both the rostral and caudal MPOA (Fig. 3D). No differences in morphology or numbers of FG cells were observed between no-play and play rats (Fig. 4A,4C). Notably, in both no-play and play animals, a greater number of FG-positive cells were found in the rostral MPOA, while few cells were restricted to the caudal portion of MPOA (Fig.4E, both p values <0.0001).

Figure 4:

Figure 4:

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Quantification of cells labeled for MOR, Egr1 and/or FG within the rostral and caudal MPOA of VTA-injected (i.e., FG was injected into the VTA) rats following social play compared to no-play. Note that the number of cells single labeled for Egr1, double labeled for Egr1 and MOR/FG was greatly increased in both the rostral (A,B) and caudal (C,D) MPOA of play animals, while no changes were observed in cells single labeled for MOR or FG, and double labeled for MOR and FG. The number of cells triple labeled for Egr1, MOR and FG was increased in only rostral MPOA (B) of play animals. Moreover, significantly more FG, Egr1+FG, Egr1+MOR+FG cells were observed in rostral MPOA than in caudal MPOA (E,F). Data used for rostral and caudal comparison (E and F) are replotted from A-D. ** P < 0.01, *** P < 0.001, play group versus no-play group (A-D) or rostral versus caudal (E,F).

In a similar vein, FG injected into the PAG retrogradely labeled MPOA cells that project to PAG. FG-labeled cells were observed in both the rostral and caudal MPOA (Fig. 5D). No differences in morphology and number of FG cells between no-play and play rats were found (Fig. 6A,6C). Notably, regardless of play or no-play, animals displayed a larger number of FG-positive cells in the rostral MPOA, with few cells in the caudal part (Fig.6E, both p values <0.0001).

Figure 6:

Figure 6:

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Quantification of cells labeled for Egr1, MOR and/or FG within the rostral and caudal MPOA of PAG-injected (i.e., FG was injected into the PAG) rats following social play compared to no-play. Note that the number of cells single labeled for Egr1, double labeled for Egr1 and MOR, double labeled for Egr1 and FG was greatly increased in both the rostral (A,B) and caudal (C,D) MPOA of play animals, while no changes were observed in cells single labeled for MOR or FG, and double labeled for MOR+FG. The number of cells triple labeled for Egr1, MOR and FG was increased in only rostral MPOA (B) of play animals. Moreover, significantly more FG, Egr1+FG and Egr1+MOR+FG cells were observed in rostral MPOA than in caudal MPOA (E,F). Data used for rostra and caudal comparison (E and F) are replotted from A-D. ** P < 0.01, *** P < 0.001, play group versus no-play group (A-D) or rostral versus caudal (E,F).

3.3. Social play was associated with an increase in Egr1 expression in the MPOA

Consistent with our recent study (Zhao et al., 2020), all the rats with FG injected into the VTA in the play group were actively engaged in play during the testing session (Fig. 7A), and social play upregulated Egr1 expression in the MPOA. Two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=375.3, p<0.0001], a significant effect of region (rostral or caudal) [F(1,18)=150.4, p<0.0001], and a significant interaction between manipulation and region [F(1,18)=27.6, p<0.0001]. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of Egr1-expressing cells in both rostral and caudal MPOA (Fig. 4A,4C, both p values<0.0001). Furthermore, more Egr1 cells were induced in caudal MPOA than in rostral MPOA (Fig. 4E, p=0.0003 for no-play, p<0.0001 for play).

Figure 7:

Figure 7:

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Social play behavior displayed in juvenile male rats who engaged in social play with play partners after social isolation for 24 hrs. Note that the VTA- (A) and PAG-injected (B) play rats exhibited high frequencies of different variables of social play including pouncing, pinning, wrestling, chasing and total play bouts except for biting during the 20-min testing session.

Similarly, all the rats with FG injection into the PAG in the play group were actively engaged in play during the testing session (Fig. 7B), and social play enhanced Egr1 expression in the MPOA. Two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=299.0, p<0.0001], a significant effect of region (rostral or caudal) [F(1,18)=200.8, p<0.0001], and a significant interaction between manipulation and region [F(1,18)=84.4, p<0.0001]. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of Egr1-expressing cells in both rostral and caudal MPOA (Fig. 6A,6C, both p values<0.0001). Moreover, more Egr1 cells were induced in caudal MPOA than in rostral MPOA (Fig. 6E, p=0.0068 for no-play, p<0.0001 for play).

3.4. Social play was associated with an increase in the activation of MOR-expressing cells in the MPOA

Cells double labeled for Egr1 and MOR represent social play-activated MOR-expressing cells. In VTA-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=142.1, p<0.0001], a significant effect of region (rostral or caudal) [F(1,18)=10.62, p=0.0044], but no significant interaction between manipulation and region. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of cells double labeled for Egr1 and MOR in both rostral (Fig. 4B, p<0.0001) and caudal MPOA (Fig. 4D, p<0.0001). Moreover, animals engaged in play displayed a greater increase in Egr1 and MOR double labeling in caudal than rostral MPOA (Fig. 4F, p=0.0051).

In PAG-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=200.7, p<0.0001], no significant effect of region (rostral or caudal) and interaction between manipulation and region. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of cells double labeled with Egr1 and MOR in both rostral (Fig. 6B, p<0.0001) and caudal MPOA (Fig. 6D, p<0.0001). Moreover, no difference was found in double labeled cells between rostral and caudal MPOA in no-play and play animals (Fig. 6F).

3.5. Social play was associated with an increase in the activation of cells in the MPOA projecting to VTA and PAG

In the MPOA, cells double labeled for Egr1 and FG represent play-activated cells projecting from MPOA to VTA or PAG. In VTA-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=442.7, p<0.0001], a significant effect of region (rostral or caudal) [F(1,18)=333.8, p<0.0001], and a significant interaction between manipulation and region [F(1,18)=186.5, p<0.0001]. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of cells double labeled for Egr1 and FG in both rostral (Fig. 4B, p<0.0001) and caudal MPOA (Fig. 4D, p=0.0001). Moreover, play animals displayed a higher increase in rostral than caudal MPOA (Fig. 4F, p<0.0001).

As was observed for the VTA, in PAG-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) [F(1,18)=125.0, p<0.0001], and a significant effect of region (rostral or caudal) [F(1,18)=103.1, p<0.0001], a significant interaction between manipulation and region [F(1,18)=13.64, p=0.0017]. Bonferroni’s multiple comparisons tests showed that social play enhanced the number of cells double labeled for Egr1 and FG in both rostral (Fig. 6B, p<0.0001) and caudal MPOA (Fig. 6D, p<0.0001). Moreover, animals engaged in play exhibited a higher increase in Egr1 and FG double labeling in rostral than caudal MPOA (Fig. 6F, p=0.0007 for no-play, p<0.0001 for play).

3.6. Social play was associated with an increase in the activation of MOR VTA- and PAG-projecting cells in the MPOA

To identify the cell-type specificity of MPOA to VTA or PAG projection participating in social play behavior, we examined the degree to which MOR-expressing cells related to social play with triple fluorescence labeling for MOR, Egr1 and FG. Triple fluorescence labeling showed that a portion of activated MPOA projections cells (cells double labeled for Egr1 and FG) co-expressed MOR after social play (Figs. 3E,4B,5E,6B). In contrast, very few MOR-expressing cells were colocalized with Egr1 and FG in no-play animals (Figs. 4B,6B) due to the low baseline level of Egr1.

In VTA-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) on the number of triple labeled cells [F(1,18)=17.98, p=0.0005], and a significant effect of region (rostral or caudal) [F(1,18)=35.25, p<0.0001], a significant interaction between manipulation and region [F(1,18)=13.24, p=0.0019]. Bonferroni’s multiple comparisons test revealed that the number of cells triple labeled for Egr1, MOR and FG was greatly increased within the rostral (Fig. 4B, p<0.0001), but not caudal MPOA (Fig. 4D) following social play. Moreover, play animals displayed a higher increase in triple labeling in rostral than caudal MPOA (Fig. 4F, p<0.0001).

In PAG-injected rats, two-way ANOVAs revealed a significant effect of manipulation (play or no-play) on the number of triple labeled cells [F(1,18)=29.81, p<0.0001], and a significant effect of region (rostral or caudal) [F(1,18)=69.35, p<0.0001], a significant interaction between manipulation and region [F(1,18)=18.98, p=0.0004]. Bonferroni’s multiple comparisons test revealed that the number of cells triple labeled for Egr1, MOR and FG was greatly increased within the rostral (Fig. 6B, p<0.0001), but not caudal MPOA (Fig. 6D) following social play. Moreover, play animals displayed a higher increase in triple labeling in rostral than caudal MPOA (Fig. 6F, p<0.0001).

3.7. Correlational analysis

To explore the potential relationship between expression for the Egr1 (activated neurons), Egr1+MOR (activated MOR-expressing cells), Egr1+MOR+FG (activated MOR-expressing projection cells) in the MPOA and play behavior in play animals, we performed correlational analysis. Pearson correlation analysis revealed no significant correlations between any of the components of social play behavior, including pouncing, pinning, wrestling, biting, chasing and total play bouts and expression of Egr1, Egr1+MOR or Egr1+MOR+FG in the rostral or caudal MPOA (all p values>0.05)

Discussion

In this study, we identified two neural pathways by which the MPOA may unite a complementary system through one output pathway to VTA and a second projection pathway to PAG in the control of social play behavior in rats. Social play was associated with a robust increase in the number of cells double-labeled for Egr1 and FG (i.e., activated projection neurons) in the MPOA of VTA- and PAG-injected rats, supporting the hypothesis that projection pathways from MPOA to VTA and PAG are involved in social play. Furthermore, social play resulted in an increased number of cells triple-labeled for Egr1, MOR and FG (i.e., activated MOR projection neurons) in the rostral MPOA of VTA- and PAG-injected rats, demonstrating that social play is associated with an increase in the activation of MOR projection cells through the rostral MPOA.

Social play behavior markedly upregulated Egr1 expression and cells double labeled for Egr1 and MOR in both the rostral and caudal MPOA, which was consistent with our recent study demonstrating that opioids in the MPOA are involved in social play (Zhao et al., 2020). At present, how opioids in the MPOA act in the mediation of social play is not clear. Endogenous opioid neuropeptides including β-endorphin, enkephalin and endomorphin, as well as their receptors such as MORs are enriched in the MPOA (Cheung et al., 1995; Eckersell et al., 1998; Gulledge et al., 2000; Martin-Schild et al., 1999; Simerly et al., 1988). Since endogenous opioids are naturally released in the brain when animals are engaged in social play (Panksepp & Bishop, 1981; Vanderschuren, Stein, et al., 1995), a direct mechanism can be readily reasoned that during social play, opioids that bind to MORs are released into the MPOA, where they positively modulate this behavior. Notably, the MPOA is a heterogeneous hypothalamic structure and contains a large cluster of neuronal populations (Simerly et al., 1986; Tsuneoka et al., 2013; Tsuneoka et al., 2017). MPOA neurons send direct projections to approximately 20 brain areas (Fang et al., 2018; Kohl et al., 2018), with many of these regions being known to be involved in social play, including nucleus accumbens (Manduca et al., 2016; Trezza et al., 2011), lateral septum (Bredewold et al., 2018; Bredewold et al., 2015), dorsomedial hypothalamus (Siviy & Panksepp, 1985), amygdala (Meaney et al., 1981; Vanderschuren et al., 2016), VTA (Northcutt & Nguyen, 2014), and paraventricular nucleus of the hypothalamus (Paul et al., 2014; Vanderschuren, Stein, et al., 1995). This suggests that MPOA may regulate social play through its efferent projections to numerous downstream regions. The present data support this hypothesis.

A key finding of the present study is that a greater number of FG-labeled cells were observed in the rostral MPOA of both VTA- and PAG-injected rats, while fewer FG-positive cells were found in the caudal MPOA, suggesting that efferent projections of MPOA to VTA or PAG primarily originate from the rostral MPOA. These findings are in excellent agreement with a past report demonstrating a larger number of FG-positive cells projecting to the VTA are in the rostral MPOA (Tobiansky et al., 2013), and a prior study showing retrogradely labeled neurons shifted medially to laterally along the rostral to caudal axis of the MPOA when a neuronal tracer, wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into the PAG (Rizvi et al., 1992). Previous studies have identified neuroanatomical, neurochemical and functional differences between the rostral and caudal MPOA (Tobiansky et al., 2013), and the rostral MPOA has been linked to the appetitive facets of motivated behavior, whereas the caudal MPOA has been related to consummatory sexual behaviors in birds and mammals (Balthazart & Ball, 2007; Gray & Brooks, 1984; Meddle et al., 1997; Riters & Ball, 1999; Riters et al., 2004; Will et al., 2015). Taken together, our data support this growing body of research demonstrating distinct roles for the rostral and caudal MPOA in regulating social behaviors.

Positive social interactions are well-known to be rewarding, but gregarious species, including humans also engage in social behaviors because social contact reduces the pain and distress of isolation or social rejection (Eisenberger, 2012; Macdonald & Leary, 2005). This suggests that social behaviors may be reinforced positively through rewarding social interactions and negatively through the reduction of the negative state of social disconnection (Riters, Kelm-Nelson, et al., 2019). MORs in the MPOA induce reward and reduce pain (Tseng & Wang, 1992; Tseng et al., 1980) and are necessary for song, song-associated reward, and rat play (Stevenson et al., 2020; Zhao et al., 2020). The MPOA accesses directly both the canonical mesolimbic reward pathway via a projection to the VTA and the well-studied PAG pain pathway (Balthazart et al., 1994; Behbehani, 1995; Kallo et al., 2015; Lieberman & Eisenberger, 2009; Macdonald & Leary, 2005; Riters & Alger, 2004; Tobiansky et al., 2013; Wright & Panksepp, 2011). Our findings reveal that social play behavior increased MOR sensitive projections to both VTA and to PAG, as revealed by neurons triple-labeled for MOR, Egr1 and FG in the MPOA after FG infusions into either VTA or PAG. Collectively, these findings provide neuroanatomical evidence that projections from MPOA to classic reward (VTA) and pain pathways (PAG) are involved in the modulation of social play by MORs in the MPOA. Future research is needed to measure functional roles of these specific pathways for social play, play-related reward and pain.

It has been reported that ontogenetic changes in morphology and structure such as synaptogenesis occur in the MPOA (Gerocs et al., 1986; Lawrence & Raisman, 1980; Reier et al., 1977). However, there is a paucity of literature addressing whether the projection pathways of MPOA to VTA and PAG are fully elaborated by weaning, or still undergo continued development into adolescence. Social play in rats undergoes dramatic changes during development. It occurs around postnatal day 18, peaks during the juvenile and early adolescent period, and declines steadily as animals approach sexual maturity (Panksepp, 1981; Pellis & Pellis, 1997). Given the developmental changes in MPOA and social play, we couldn’t rule out the possibility that the developmental changes may occur in MPOA projections and as such contribute to the developmental changes in social play.

One important caveat to consider when interpreting our data is that a low absolute number of cells (i.e., cells double labeled for Egr1 and MOR/FG, or triple labeled for Egr1, MOR and FG) was activated after social play. Several possible reasons may explain the lower incidence of double or triple labeling. Despite the well-established facts that social behaviors induce IEG expression in distinct brain regions, social behaviors do not always elicit robust IEG expression in a specific type of neurons. For instance, only a small number of tyrosine hydroxylase-immunoreactive cells co-expressed Fos after animals engaged in sexual or non-sexual affiliative social play behaviors (Balfour et al., 2004; Goodson et al., 2009; Northcutt & Nguyen, 2014). Therefore, it is likely that induction of IEGs, including c-Fos and Egr1 following performance of social behaviors in a certain type of cells may not be able to accurately reflect the true activation of cells. More importantly, FG has traditionally been used in conjunction with IEG expression to identify neural circuits activated by pharmacological manipulation and behavioral performance (Flanagan-Cato et al., 2006; Geisler et al., 2008; Hsieh et al., 2011; Miller & Marshall, 2005). However, FG was reported to reduce IEG expression (i.e., c-Fos) at the injection site as well as in FG retrogradely transported areas (Franklin & Druhan, 2000). Thus, the number of functionally activated cells as measured by immediate early gene Egr1 after social play in FG-injected animals might have been underestimated in the present study. Future research is expected to understand whether or how the reduced Egr1 expression, if occurs, affects the cellular activity in the MPOA and social play behavior itself.

Considering the effect of social isolation on IEG expression, caution is warranted when interpreting our results. Social isolation paradigms have been widely employed to enhance the motivation to play in rat social play studies (Niesink & Van Ree, 1989; Panksepp, 1981; Panksepp & Beatty, 1980; van Kerkhof et al., 2014; Vanderschuren et al., 2008); however, social isolation is a robust natural stressor and is associated with neuronal IEG expression. For example, acute social isolation increases c-fos mRNA expression in the hypothalamus and amygdala (Kanitz et al., 2009); by contrast, chronic exposure to isolation downregulates mRNA level of c-fos and Egr1 in the hippocampus and frontal cortex (Ieraci et al., 2016; Matsumoto et al., 2012). The present study was not able to answer the questions of whether or how social isolation for 24 hrs altered IEGs in the MPOA. If effects of social isolation occurred, the Egr1 expression in the MPOA in play rats might reflect the combined results of both social isolation and play.

One limitation of the present study is that it is unclear whether the increase in neuronal activation is due to social play behavior per se, general social contact or motor activity. Social play is a complex behavior, which contains constitutive components of pouncing, pinning, wrestling and chasing (Argue & McCarthy, 2015b; Pellis et al., 2022). As rats exhibit intense locomotor activity and process substantial amount of sensory and emotional information when playing, it is difficult to draw a hard line between social play and non-play social contact or motor activity to disentangle the specific effect of play from that of general social contact or motor activity. Therefore, all sensory, emotional and behavioral components of social play contributed to the observed neuronal activation after exposure to social play. It would be interesting to identify the roles for each sensory and emotional component of social play and non-play behaviors in play-associated neuronal activation in future studies.

There are sex differences in social play behavior, with males playing at high rates relative to females (Argue & McCarthy, 2015a, 2015b; Auger & Olesen, 2009; Pellis et al., 1997). Studying males is a fundamental first step to identify whether the MPOA and its projections to the VTA and PAG are activated in response to social play behavior. Future investigation in females would be an exciting avenue to understand whether the two projection pathways are activated after play, and if activated, how they govern this social behavior.

Conclusions

Collectively, this study provides neuroanatomical evidence supporting the hypothesis that projection pathways from the MPOA to VTA and PAG are involved in the modulation of social play by MORs in the MPOA.

Acknowledgements

This research was supported by the National Institutes of Health Grant (R01 MH119041) and a Kellett Award from the University of Wisconsin-Madison to LV Riters. We thank Alyse Maksimoski, Brandon Polzin, Chinweike Norman Asogwa and Stephen Gammie for comments on an earlier draft of this manuscript. We gratefully acknowledge the expert technical support of Sarah Swanson in confocal fluorescence microscopic imaging and thank Kate Skogen and Jeffrey Alexander for animal care.

Footnotes

Declaration of competing interests

The authors declare no conflict of interests.

Data availability

Data will be available upon request from the corresponding author.

References

  1. Achterberg EJM, van Swieten MMH, Houwing DJ, Trezza V, & Vanderschuren L (2019). Opioid modulation of social play reward in juvenile rats. Neuropharmacology, 159, 107332. 10.1016/j.neuropharm.2018.09.007 [DOI] [PubMed] [Google Scholar]
  2. Argue KJ, & McCarthy MM (2015a). Characterization of juvenile play in rats: importance of sex of self and sex of partner. Biology of Sex Differences, 6, 16. 10.1186/s13293-015-0034-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Argue KJ, & McCarthy MM (2015b). Utilization of same- vs. mixed-sex dyads impacts the observation of sex differences in juvenile social play behavior. Curr Neurobiol, 6(1), 17–23. 10.4172/0975-9042.000117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Auger AP, & Olesen KM (2009). Brain sex differences and the organisation of juvenile social play behaviour. Journal of Neuroendocrinology, 21(6), 519–525. 10.1111/j.1365-2826.2009.01871.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Balfour ME, Yu L, & Coolen LM (2004). Sexual behavior and sex-associated environmental cues activate the mesolimbic system in male rats. Neuropsychopharmacology, 29(4), 718–730. 10.1038/sj.npp.1300350 [DOI] [PubMed] [Google Scholar]
  6. Balthazart J, & Ball GF (2007). Topography in the preoptic region: differential regulation of appetitive and consummatory male sexual behaviors. Frontiers in Neuroendocrinology, 28(4), 161–178. 10.1016/j.yfrne.2007.05.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Balthazart J, Dupiereux V, Aste N, Viglietti-Panzica C, Barrese M, & Panzica GC (1994). Afferent and efferent connections of the sexually dimorphic medial preoptic nucleus of the male quail revealed by in vitro transport of DiI. Cell Tissue Res, 276(3), 455–475. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8062340 [DOI] [PubMed] [Google Scholar]
  8. Behbehani MM (1995). Functional characteristics of the midbrain periaqueductal gray. Progress in Neurobiology, 46(6), 575–605. 10.1016/0301-0082(95)00009-k [DOI] [PubMed] [Google Scholar]
  9. Bredewold R, Nascimento NF, Ro GS, Cieslewski SE, Reppucci CJ, & Veenema AH (2018). Involvement of dopamine, but not norepinephrine, in the sex-specific regulation of juvenile socially rewarding behavior by vasopressin. Neuropsychopharmacology, 43(10), 2109–2117. 10.1038/s41386-018-0100-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bredewold R, Schiavo JK, van der Hart M, Verreij M, & Veenema AH (2015). Dynamic changes in extracellular release of GABA and glutamate in the lateral septum during social play behavior in juvenile rats: Implications for sex-specific regulation of social play behavior. Neuroscience, 307, 117–127. 10.1016/j.neuroscience.2015.08.052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheung S, Salinas J, & Hammer RP (1995). Gonadal-Steroid Hormone-Dependence of Beta-Endorphin-Like Immunoreactivity in the Medial Preoptic Area of the Rat. Brain Research, 675(1-2), 83–88. https://doi.org/Doi 10.1016/0006-8993(95)00042-O [DOI] [PubMed] [Google Scholar]
  12. Eckersell CB, Popper P, & Micevych PE (1998). Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala. Journal of Neuroscience, 18(10), 3967–3976. <Go to ISI>://WOS:000073484300044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eisenberger NI (2012). The pain of social disconnection: examining the shared neural underpinnings of physical and social pain. Nat Rev Neurosci, 13(6), 421–434. 10.1038/nrn3231 [DOI] [PubMed] [Google Scholar]
  14. Fang YY, Yamaguchi T, Song SC, Tritsch NX, & Lin D (2018). A Hypothalamic Midbrain Pathway Essential for Driving Maternal Behaviors. Neuron, 98(1), 192–207 e110. 10.1016/j.neuron.2018.02.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flanagan-Cato LM, Lee BJ, & Calizo LH (2006). Co-localization of midbrain projections, progestin receptors, and mating-induced fos in the hypothalamic ventromedial nucleus of the female rat. Horm Behav, 50(1), 52–60. 10.1016/j.yhbeh.2006.01.012 [DOI] [PubMed] [Google Scholar]
  16. Franklin TR, & Druhan JP (2000). The retrograde tracer fluoro-gold interferes with the expression of fos-related antigens. Journal of Neuroscience Methods, 98(1), 1–8. 10.1016/s0165-0270(00)00168-0 [DOI] [PubMed] [Google Scholar]
  17. Geisler S, Marinelli M, Degarmo B, Becker ML, Freiman AJ, Beales M, Meredith GE, & Zahm DS (2008). Prominent activation of brainstem and pallidal afferents of the ventral tegmental area by cocaine. Neuropsychopharmacology, 33(11), 2688–2700. 10.1038/sj.npp.1301650 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gerocs K, Rethelyi M, & Halasz B (1986). Quantitative analysis of dendritic protrusions in the medial preoptic area during postnatal development. Brain Research, 391(1), 49–57. 10.1016/0165-3806(86)90006-4 [DOI] [PubMed] [Google Scholar]
  19. Goodson JL, Kabelik D, Kelly AM, Rinaldi J, & Klatt JD (2009). Midbrain dopamine neurons reflect affiliation phenotypes in finches and are tightly coupled to courtship. Proceedings of the National Academy of Sciences of the United States of America, 106(21), 8737–8742. 10.1073/pnas.0811821106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gray P, & Brooks PJ (1984). Effect of lesion location within the medial preoptic-anterior hypothalamic continuum on maternal and male sexual behaviors in female rats. Behavioral Neuroscience, 98(4), 703–711. 10.1037//0735-7044.98.4.703 [DOI] [PubMed] [Google Scholar]
  21. Gulledge CC, Mann PE, Bridges RS, Bialos M, & Hammer RP (2000). Expression of mu-opioid receptor mRNA in the medial preoptic area of juvenile rats. Developmental Brain Research, 119(2), 269–276. https://doi.org/Doi 10.1016/S0165-3806(99)00184-4 [DOI] [PubMed] [Google Scholar]
  22. Hahn AH, Merullo DP, Spool JA, Angyal CS, Stevenson SA, & Riters LV (2017). Song-associated reward correlates with endocannabinoid-related gene expression in male European starlings (Sturnus vulgaris). Neuroscience, 346, 255–266. 10.1016/j.neuroscience.2017.01.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hsieh KC, Gvilia I, Kumar S, Uschakov A, McGinty D, Alam MN, & Szymusiak R (2011). c-Fos expression in neurons projecting from the preoptic and lateral hypothalamic areas to the ventrolateral periaqueductal gray in relation to sleep states. Neuroscience, 188, 55–67. 10.1016/j.neuroscience.2011.05.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ieraci A, Mallei A, & Popoli M (2016). Social Isolation Stress Induces Anxious-Depressive-Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice. Neural Plasticity, 2016, 6212983. 10.1155/2016/6212983 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jullien M, & Thiollay JM (1998). Multi-species territoriality and dynamic of neotropical forest understorey bird flocks. Journal of Animal Ecology, 67(2), 227–252. https://doi.org/DOI 10.1046/j.1365-2656.1998.00171.x [DOI] [Google Scholar]
  26. Kallo I, Molnar CS, Szoke S, Fekete C, Hrabovszky E, & Liposits Z (2015). Area-specific analysis of the distribution of hypothalamic neurons projecting to the rat ventral tegmental area, with special reference to the GABAergic and glutamatergic efferents. Front Neuroanat, 9, 112. 10.3389/fnana.2015.00112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kanitz E, Puppe B, Tuchscherer M, Heberer M, Viergutz T, & Tuchscherer A (2009). A single exposure to social isolation in domestic piglets activates behavioural arousal, neuroendocrine stress hormones, and stress-related gene expression in the brain. Physiol Behav, 98(1-2), 176–185. 10.1016/j.physbeh.2009.05.007 [DOI] [PubMed] [Google Scholar]
  28. Kelm-Nelson CA, Stevenson SA, & Riters LV (2012). Context-dependent links between song production and opioid-mediated analgesia in male European starlings (Sturnus vulgaris). PLoS One, 7(10), e46721. 10.1371/journal.pone.0046721 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kohl J, Babayan BM, Rubinstein ND, Autry AE, Marin-Rodriguez B, Kapoor V, Miyamishi K, Zweifel LS, Luo L, Uchida N, & Dulac C (2018). Functional circuit architecture underlying parental behaviour. Nature, 556(7701), 326–331. 10.1038/s41586-018-0027-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lawrence JM, & Raisman G (1980). Ontogeny of synapses in a sexually dimorphic part of the preoptic area in the rat. Brain Research, 183(2), 466–471. 10.1016/0006-8993(80)90482-5 [DOI] [PubMed] [Google Scholar]
  31. Lazarus J (1979). Early Warning Function of Flocking in Birds - Experimental-Study with Captive Quelea. Animal Behaviour, 27(Aug), 855–865. https://doi.org/Doi 10.1016/0003-3472(79)90023-X [DOI] [Google Scholar]
  32. Lieberman MD, & Eisenberger NI (2009). Neuroscience. Pains and pleasures of social life. Science, 323(5916), 890–891. 10.1126/science.1170008 [DOI] [PubMed] [Google Scholar]
  33. Macdonald G, & Leary MR (2005). Why does social exclusion hurt? The relationship between social and physical pain. Psychol Bull, 131(2), 202–223. 10.1037/0033-2909.131.2.202 [DOI] [PubMed] [Google Scholar]
  34. Manduca A, Servadio M, Damsteegt R, Campolongo P, Vanderschuren LJ, & Trezza V (2016). Dopaminergic Neurotransmission in the Nucleus Accumbens Modulates Social Play Behavior in Rats. Neuropsychopharmacology, 41(9), 2215–2223. 10.1038/npp.2016.22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Martin-Schild S, Gerall AA, Kastin AJ, & Zadina JE (1999). Differential distribution of endomorphin 1- and endomorphin 2-like immunoreactivities in the CNS of the rodent. Journal of Comparative Neurology, 405(4), 450–471. https://doi.org/Doi [DOI] [PubMed] [Google Scholar]
  36. Matsumoto K, Ono K, Ouchi H, Tsushima R, & Murakami Y (2012). Social isolation stress down-regulates cortical early growth response 1 (Egr-1) expression in mice. Neuroscience Research, 73(3), 257–262. 10.1016/j.neures.2012.04.004 [DOI] [PubMed] [Google Scholar]
  37. McHenry JA, Otis JM, Rossi MA, Robinson JE, Kosyk O, Miller NW, McElligott ZA, Budygin EA, Rubinow DR, & Stuber GD (2017). Hormonal gain control of a medial preoptic area social reward circuit. Nature Neuroscience, 20(3), 449–458. 10.1038/nn.4487 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Meaney MJ, Dodge AM, & Beatty WW (1981). Sex-dependent effects of amygdaloid lesions on the social play of prepubertal rats. Physiol Behav, 26(3), 467–472. 10.1016/0031-9384(81)90175-x [DOI] [PubMed] [Google Scholar]
  39. Meddle SL, King VM, Follett BK, Wingfield JC, Ramenofsky M, Foidart A, & Balthazart J (1997). Copulation activates Fos-like immunoreactivity in the male quail forebrain. Behavioural Brain Research, 85(2), 143–159. 10.1016/s0166-4328(97)87581-x [DOI] [PubMed] [Google Scholar]
  40. Merullo DP, Spool JA, Zhao C, & Riters LV (2018). Co-localization patterns of neurotensin receptor 1 and tyrosine hydroxylase in brain regions involved in motivation and social behavior in male European starlings. Journal of Chemical Neuroanatomy, 89, 1–10. 10.1016/j.jchemneu.2018.01.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Miller CA, & Marshall JF (2005). Altered Fos expression in neural pathways underlying cue-elicited drug seeking in the rat. European Journal of Neuroscience, 21(5), 1385–1393. 10.1111/j.1460-9568.2005.03974.x [DOI] [PubMed] [Google Scholar]
  42. Niesink RJ, & Van Ree JM (1989). Involvement of opioid and dopaminergic systems in isolation-induced pinning and social grooming of young rats. Neuropharmacology, 28(4), 411–418. 10.1016/0028-3908(89)90038-5 [DOI] [PubMed] [Google Scholar]
  43. Northcutt KV, & Nguyen JM (2014). Female juvenile play elicits Fos expression in dopaminergic neurons of the VTA. Behavioral Neuroscience, 128(2), 178–186. 10.1037/a0035964 [DOI] [PubMed] [Google Scholar]
  44. Panksepp J (1980). Brief social isolation, pain responsiveness, and morphine analgesia in young rats. Psychopharmacology (Berl), 72(1), 111–112. 10.1007/BF00433816 [DOI] [PubMed] [Google Scholar]
  45. Panksepp J (1981). The ontogeny of play in rats. Developmental Psychobiology, 14(4), 327–332. 10.1002/dev.420140405 [DOI] [PubMed] [Google Scholar]
  46. Panksepp J, & Beatty WW (1980). Social deprivation and play in rats. Behavioral and Neural Biology, 30(2), 197–206. 10.1016/s0163-1047(80)91077-8 [DOI] [PubMed] [Google Scholar]
  47. Panksepp J, & Bishop P (1981). An autoradiographic map of (3H)diprenorphine binding in rat brain: effects of social interaction. Brain Research Bulletin, 7(4), 405–410. 10.1016/0361-9230(81)90038-1 [DOI] [PubMed] [Google Scholar]
  48. Paul MJ, Terranova JI, Probst CK, Murray EK, Ismail NI, & de Vries GJ (2014). Sexually dimorphic role for vasopressin in the development of social play. Frontiers in Behavioral Neuroscience, 8, 58. 10.3389/fnbeh.2014.00058 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Paxinos G, & Watson C (2007). The Rat Brain in Stereotaxic Coordinates (6th ed.). Academic Press. [DOI] [PubMed] [Google Scholar]
  50. Pellis SM, Field EF, Smith LK, & Pellis VC (1997). Multiple differences in the play fighting of male and female rats. Implications for the causes and functions of play. Neurosci Biobehav Rev, 21(1), 105–120. 10.1016/0149-7634(95)00060-7 [DOI] [PubMed] [Google Scholar]
  51. Pellis SM, & Pellis VC (1997). The prejuvenile onset of play fighting in laboratory rats (Rattus norvegicus). Developmental Psychobiology, 31(3), 193–205. https://www.ncbi.nlm.nih.gov/pubmed/9386921 [PubMed] [Google Scholar]
  52. Pellis SM, Pellis VC, Burke CJ, Stark RA, Ham JR, Euston DR, & Achterberg EJM (2022). Measuring Play Fighting in Rats: A Multilayered Approach. Curr Protoc, 2(1), e337. 10.1002/cpz1.337 [DOI] [PubMed] [Google Scholar]
  53. Reier PJ, Cullen MJ, Froelich JS, & Rothchild I (1977). The ultrastructure of the developing medial preoptic nucleus in the postnatal rat. Brain Research, 122(3), 415–436. 10.1016/0006-8993(77)90454-1 [DOI] [PubMed] [Google Scholar]
  54. Riters LV, & Alger SJ (2004). Neuroanatomical evidence for indirect connections between the medial preoptic nucleus and the song control system: possible neural substrates for sexually motivated song. Cell Tissue Res, 316(1), 35–44. 10.1007/s00441-003-0838-6 [DOI] [PubMed] [Google Scholar]
  55. Riters LV, & Ball GF (1999). Lesions to the medial preoptic area affect singing in the male European starling (Sturnus vulgaris). Horm Behav, 36(3), 276–286. 10.1006/hbeh.1999.1549 [DOI] [PubMed] [Google Scholar]
  56. Riters LV, Kelm-Nelson CA, & Spool JA (2019). Why Do Birds Flock? A Role for Opioids in the Reinforcement of Gregarious Social Interactions. Frontiers in Physiology, 10, 421. 10.3389/fphys.2019.00421 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Riters LV, Spool JA, Merullo DP, & Hahn AH (2019). Song practice as a rewarding form of play in songbirds. Behavioural Processes, 163, 91–98. 10.1016/j.beproc.2017.10.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Riters LV, & Stevenson SA (2012). Reward and vocal production: song-associated place preference in songbirds. Physiol Behav, 106(2), 87–94. 10.1016/j.physbeh.2012.01.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Riters LV, Stevenson SA, DeVries MS, & Cordes MA (2014). Reward associated with singing behavior correlates with opioid-related gene expression in the medial preoptic nucleus in male European starlings. PloS One, 9(12), e115285. 10.1371/journal.pone.0115285 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Riters LV, Teague DP, Schroeder MB, & Cummings SE (2004). Vocal production in different social contexts relates to variation in immediate early gene immunoreactivity within and outside of the song control system. Behavioural Brain Research, 155(2), 307–318. 10.1016/j.bbr.2004.05.002 [DOI] [PubMed] [Google Scholar]
  61. Rizvi TA, Ennis M, & Shipley MT (1992). Reciprocal connections between the medial preoptic area and the midbrain periaqueductal gray in rat: a WGA-HRP and PHA-L study. Journal of Comparative Neurology, 315(1), 1–15. 10.1002/cne.903150102 [DOI] [PubMed] [Google Scholar]
  62. Shahrokh DK, Zhang TY, Diorio J, Gratton A, & Meaney MJ (2010). Oxytocin-dopamine interactions mediate variations in maternal behavior in the rat. Endocrinology, 151(5), 2276–2286. 10.1210/en.2009-1271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Simerly RB, Gorski RA, & Swanson LW (1986). Neurotransmitter specificity of cells and fibers in the medial preoptic nucleus: an immunohistochemical study in the rat. Journal of Comparative Neurology, 246(3), 343–363. 10.1002/cne.902460305 [DOI] [PubMed] [Google Scholar]
  64. Simerly RB, Mccall LD, & Watson SJ (1988). Distribution of Opioid-Peptides in the Preoptic Region - Immunohistochemical Evidence for a Steroid-Sensitive Enkephalin Sexual Dimorphism. Journal of Comparative Neurology, 276(3), 442–459. https://doi.org/DOI 10.1002/cne.902760309 [DOI] [PubMed] [Google Scholar]
  65. Siviy SM, & Panksepp J (1985). Dorsomedial diencephalic involvement in the juvenile play of rats. Behavioral Neuroscience, 99(6), 1103–1113. 10.1037//0735-7044.99.6.1103 [DOI] [PubMed] [Google Scholar]
  66. Spool JA, Merullo DP, Zhao C, & Riters LV (2019). Co-localization of mu-opioid and dopamine D1 receptors in the medial preoptic area and bed nucleus of the stria terminalis across seasonal states in male European starlings. Horm Behav, 107, 1–10. 10.1016/j.yhbeh.2018.11.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Stevenson SA, Piepenburg A, Spool JA, Angyal CS, Hahn AH, Zhao C, & Riters LV (2020). Endogenous opioids facilitate intrinsically-rewarded birdsong. Sci Rep, 10(1), 11083. 10.1038/s41598-020-67684-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Tobiansky DJ, Roma PG, Hattori T, Will RG, Nutsch VL, & Dominguez JM (2013). The medial preoptic area modulates cocaine-induced activity in female rats. Behavioral Neuroscience, 127(2), 293–302. 10.1037/a0031949 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Trezza V, Damsteegt R, Achterberg EJ, & Vanderschuren LJ (2011). Nucleus accumbens mu-opioid receptors mediate social reward. Journal of Neuroscience, 31(17), 6362–6370. 10.1523/JNEUROSCI.5492-10.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Trezza V, Damsteegt R, & Vanderschuren LJ (2009). Conditioned place preference induced by social play behavior: parametrics, extinction, reinstatement and disruption by methylphenidate. Eur Neuropsychopharmacol, 19(9), 659–669. 10.1016/j.euroneuro.2009.03.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Tseng LF, & Wang Q (1992). Forebrain sites differentially sensitive to beta-endorphin and morphine for analgesia and release of Met-enkephalin in the pentobarbital-anesthesized rat. J Pharmacol Exp Ther, 261(3), 1028–1036. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1318368 [PubMed] [Google Scholar]
  72. Tseng LF, Wei ET, Loh HH, & Li CH (1980). beta-Endorphin: central sites of analgesia, catalepsy and body temperature changes in rats. J Pharmacol Exp Ther, 214(2), 328–332. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=6248633 [PubMed] [Google Scholar]
  73. Tsuneoka Y, Maruyama T, Yoshida S, Nishimori K, Kato T, Numan M, & Kuroda KO (2013). Functional, anatomical, and neurochemical differentiation of medial preoptic area subregions in relation to maternal behavior in the mouse. Journal of Comparative Neurology, 521(7), 1633–1663. 10.1002/cne.23251 [DOI] [PubMed] [Google Scholar]
  74. Tsuneoka Y, Yoshida S, Takase K, Oda S, Kuroda M, & Funato H (2017). Neurotransmitters and neuropeptides in gonadal steroid receptor-expressing cells in medial preoptic area subregions of the male mouse. Scientific Reports, 7(1), 9809. 10.1038/s41598-017-10213-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. van Kerkhof LW, Trezza V, Mulder T, Gao P, Voorn P, & Vanderschuren LJ (2014). Cellular activation in limbic brain systems during social play behaviour in rats. Brain Struct Funct, 219(4), 1181–1211. 10.1007/s00429-013-0558-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Vanderschuren LJ, Achterberg EJ, & Trezza V (2016). The neurobiology of social play and its rewarding value in rats. Neurosci Biobehav Rev, 70, 86–105. 10.1016/j.neubiorev.2016.07.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Vanderschuren LJ, Niesink RJ, Spruijt BM, & Van Ree JM (1995a). Effects of morphine on different aspects of social play in juvenile rats. Psychopharmacology, 117(2), 225–231. 10.1007/bf02245191 [DOI] [PubMed] [Google Scholar]
  78. Vanderschuren LJ, Niesink RJ, Spruijt BM, & Van Ree JM (1995b). Mu- and kappa-opioid receptor-mediated opioid effects on social play in juvenile rats. European Journal of Pharmacology, 276(3), 257–266. 10.1016/0014-2999(95)00040-r [DOI] [PubMed] [Google Scholar]
  79. Vanderschuren LJ, Stein EA, Wiegant VM, & Van Ree JM (1995). Social play alters regional brain opioid receptor binding in juvenile rats. Brain Research, 680(1-2), 148–156. 10.1016/0006-8993(95)00256-p [DOI] [PubMed] [Google Scholar]
  80. Vanderschuren LJ, Trezza V, Griffioen-Roose S, Schiepers OJ, Van Leeuwen N, De Vries TJ, & Schoffelmeer AN (2008). Methylphenidate disrupts social play behavior in adolescent rats. Neuropsychopharmacology, 33(12), 2946–2956. 10.1038/npp.2008.10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Will RG, Nutsch VL, Turner JM, Hattori T, Tobiansky DJ, & Dominguez JM (2015). Astrocytes in the medial preoptic area modulate ejaculation latency in an experience-dependent fashion. Behavioral Neuroscience, 129(1), 68–73. 10.1037/bne0000026 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wright JS, & Panksepp J (2011). Toward affective circuit-based preclinical models of depression: sensitizing dorsal PAG arousal leads to sustained suppression of positive affect in rats. Neurosci Biobehav Rev, 35(9), 1902–1915. 10.1016/j.neubiorev.2011.08.004 [DOI] [PubMed] [Google Scholar]
  83. Zhang GW, Shen L, Tao C, Jung AH, Peng B, Li Z, Zhang LI, & Tao HW (2021). Medial preoptic area antagonistically mediates stress-induced anxiety and parental behavior. Nature Neuroscience, 24(4), 516–528. 10.1038/s41593-020-00784-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Zhao C, Chang L, Auger AP, Gammie SC, & Riters LV (2020). Mu opioid receptors in the medial preoptic area govern social play behavior in adolescent male rats. Genes Brain Behav, 19(7), e12662. 10.1111/gbb.12662 [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Zhao C, & Gammie SC (2015). Metabotropic glutamate receptor 3 is downregulated and its expression is shifted from neurons to astrocytes in the mouse lateral septum during the postpartum period. Journal of Histochemistry and Cytochemistry, 63(6), 417–426. 10.1369/0022155415578283 [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Zhao C, & Gammie SC (2018). The circadian gene Nr1d1 in the mouse nucleus accumbens modulates sociability and anxiety-related behaviour. European Journal of Neuroscience, 48(3), 1924–1943. 10.1111/ejn.14066 [DOI] [PMC free article] [PubMed] [Google Scholar]

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Data Availability Statement

Data will be available upon request from the corresponding author.

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