Sex differences in vasopressin 1a receptor regulation of social communication within the lateral habenula and dorsal raphe of mice

Abstract

The neuropeptide arginine-vasopressin (AVP) has long been implicated in the regulation of social behavior and communication in diverse taxa, often through its actions on the V1a receptor (V1aR) and in a sex-different and steroid-dependent way. One source of sex-different brain AVP is the steroid-sensitive and sexually-dimorphic AVP neurons in the bed nucleus of the stria terminalis (BNST), a cell population that regulates social behavior in a sex-dependent manner. Potential targets of these BNST-AVP cells include the lateral habenula (LHb) and dorsal raphe (DR), areas known to be important for social behavior, yet few studies have investigated AVP action within these regions. Consequently, to test if V1aR action in the LHb or DR controls social behavior in a sexually dimorphic manner, we administered a highly-specific V1aR antagonist (or saline vehicle) in the LHb or DR of C57BL/6 male and female mice and tested its effects on social investigation, social communication (urine marking, ultrasonic vocalizations), and territorial aggression. V1aR antagonism of the LHb or DR decreased male urine marking toward unfamiliar males, but not toward unfamiliar females. Additionally, V1aR blockade of the LHb decreased ultrasonic vocalizations generated in the presence of females. Social investigation, locomotion and aggressive behavior were not altered by V1aR antagonism in either area. Blocking V1aR in the LHb or DR of females had no effect, indicating V1aR action in the DR and LHb drives sex differences in social communication.

Introduction

Animals often display profound sex differences in social behavior and communication and especially in reproductively-oriented behaviors such as courtship displays, territorial marking, parenting, and copulation (Darwin 1871; Bradbury and Vehrencamp 1998; Scott et al. 2015; Song et al. 2018). In humans, dysfunction in social communication is a common feature of several psychopathologies, such as autism, that show substantial sex differences in prevalence and impact (Halladay et al., 2015). It is therefore plausible that sex differences in neural circuitry may contribute to sexually differentiated function and dysfunction in social behavior and communication. One of the largest and most evolutionarily conserved sex-different systems in the vertebrate brain is the male-biased and steroid-dependent expression of the neuropeptide vasopressin/vasotocin (AVP) (de Vries, 2008; de Vries and Panzica, 2006; Goodson and Bass, 2001). AVP, working through the vasopressin V1a receptor (V1aR), has been repeatedly implicated in modulation of social behaviors across vertebrate species, including humans, and often in a sex-different way (Choleris et al. 2009; Donaldson and Young 2008; Insel 2010; Duque-Wilckens et al. 2016; Guastella et al., 2010; Rilling et al., 2014). For example, AVP acts in various brain regions to alter vertebrate social communication (Goodson and Bass, 2001; Kelly and Goodson, 2013), maternal care (Bosch and Neumann, 2008), pair bonding (Carter et al., 1995; Jarcho et al., 2011; Young and Wang, 2004), and social recognition (Dantzer et al. 1988; Everts and Koolhaas 1999; Bielsky and Young 2004; Bielsky et al. 2004; Veenema et al. 2012; Johnson and Young 2017).

A number of brain regions involved in social and emotional behavior contain sex-different and steroid-sensitive AVP innervation and V1aR expression, such as the lateral septum (LS), ventral pallidum (VP), lateral habenula (LHb) and several hypothalamic and midbrain areas, including the dorsal raphe nuclei (DR) (Rood et al. 2013; Dumais and Veenema 2016). However, most examinations of the social role of AVP have focused on LS and, to a lesser degree, the VP and hypothalamus (Lim and Young 2004; Bielsky et al. 2005; Ophir et al. 2008; Gobrogge et al. 2009; Bredewold et al. 2014; Otero-Garcia et al. 2014; DiBenedictis et al. 2020). Consequently, we know little about the behavioral role of AVP in other areas previously implicated in social and emotional behavior, such as LHb and DR nuclei (Nagayasu 2017; Balázsfi et al. 2018; Yang et al. 2018; Congiu et al. 2019; Russo et al. 2019) even though they also contain dense, steroid-sensitive and sexually dimorphic AVP innervation (Rood et al. 2013). Moreover, AVP, acting through V1aR, indirectly excites DR serotonin neurons (Rood and Beck 2014) and recent work suggests that increased AVP is associated with decreased LHb output and decreased fear response in stressful situations (Zhang et al. 2016).

Most sex-different brain AVP innervation, including that of LHb and DR, likely originates from sex-different AVP-expressing neurons within the bed nucleus of the stria terminalis (BNST) (de Vries and Panzica, 2006), a key structure in the social behavior neural network. Therefore, we hypothesized that BNST-AVP inputs to DR and LHb regulate sexually differentiated effects of AVP on social communication. Indeed, selective lesions of BNST-AVP neurons reduces social investigation and alters social communication in males, but not females (Rigney et al. 2019). Consequently, we predict that blocking V1aR action in DR or LHb will recapitulate the sex-specific (male, but not female) deficits in social investigation and communication caused by BNST-AVP lesions. To test this, we assessed the effects of injecting a highly-selective V1aR antagonist (Manning et al. 2012) in the LHb or DR on male and female mouse communicative (ultrasonic vocalizations, urine marking, social investigation) and aggressive behaviors that are sexually differentiated (Kimura and Hagiwara 1985; Yang et al. 2013; Palanza and Parmigiani 2017; Zala et al. 2017).

Methods

Animals and husbandry

All mice were maintained at 22°C on a 12/12 h reverse light/dark cycle with food and water available ad libitum, housed in individually ventilated cages (Animal Care Systems), and provided with corncob bedding, a nestlet square, and a housing tube. All animal procedures were performed in accordance with the Georgia State University animal care and use committee regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Subjects

Fifty-six male and female C57BL/6J mice between 4–8 weeks of age were obtained from Jackson Laboratories (stock # 000664) and were singly-housed for a minimum of one week prior to testing.

Stimulus animals

Adult CD1(ICR) mice obtained from Charles River Laboratories were used as stimuli for behavioral testing and to provide male and female subjects with social experience as strain differences between subjects and stimulus mice increase social investigation (Gheusi et al., 1994). Mice were used at 9 –16 weeks of age and were novel and unrelated to the subject to which they were exposed.

Female stimulus mice were group-housed, ovariectomized, implanted with an estradiol capsule (GDX+E), and given two sexual experiences before testing. Two groups of stimulus males were used for behavioral testing differed in their social experience, to either render them more competitive (“dominant”) or less competitive (“subordinate”). Male subordinate mice that were used in the home cage aggression tests and for providing aggressive experience to subjects were group-housed, gonadectomized (GDX), and subjected to two aggressive encounters as an intruder in a resident male’s home cage. Mice in the second group (dominant) were singly-housed, GDX, implanted with testosterone (GDX+T), and given two sexual experiences before testing; these males provided sexual experience to female subjects and served as stimulus animals in the three-chamber social tests.

Surgery

All surgeries were conducted using 1.5–3% isoflurane gas anesthesia in 100% oxygen with 3 mg/kg of carprofen given prior to surgery to reduce pain.

Gonadectomy and hormone treatment

Testes were cauterized and removed at the ductus deferens via a midline abdominal incision. SILASTIC capsules (1.5-cm active length; 1.02-mm inner diameter, 2.16-mm outer diameter; Dow Corning Corporation) were filled with crystalline T (Sigma) and inserted subcutaneously between the scapulae after gonadectomy; this procedure leads to physiologic levels of T (Barkley and Goldman, 1977; Matochik et al., 1994). To reduce aggression and promote submissive behavior in stimulus males (Beeman 1947), these mice were GDX but did not receive a T implant (GDX).

The ovaries of stimulus female mice were removed by cauterization at the uterine horn and attendant blood vessels. SILASTIC capsules (0.7-cm active length; 1.02-mm inner diameter, 2.16-mm outer diameter; Dow Corning Corporation) containing estradiol benzoate (E; diluted 1:1 with cholesterol) were implanted subcutaneously in the scapular region immediately following ovariectomy (GDX+E; Bakker et al. 2002; Ström et al. 2012). To induce sexual receptivity, stimulus females were injected subcutaneously with 0.1 mL of progesterone (500 μg dissolved in sesame oil, Sigma) 4 hours preceding sexual experience and behavioral testing (Veyrac et al. 2011).

Stereotaxic surgery and cannula implantation

Mice were positioned in a stereotaxic frame (David Kopf Instruments) with ear and incisor bars holding the skull level relative to bregma and lambda. After a midline scalp incision, a hand operated drill was used to make holes in the skull, exposing the dura. Subjects were then implanted (LHb animals bilaterally, DR animals unilaterally) with 26-gauge guide cannulas (Plastics One, Roanoke, VA) with a 1mm projection below the pedestal, fixed at a 15° angle 0.99mm anterior to bregma and 1.19mm lateral to the midline suture (LHb) or 4.3mm anterior to bregma and 0.78mm lateral to the midline suture (DR). DR injections were placed at a 15° angle to reduce the potential for V1aR antagonist crossing into the cerebral ventricle. Since crossing the ventricles was unavoidable in LHb injections, we analyzed a separate ‘miss’ group with cannula tracks ending in the lateral ventricle. Guide cannula were secured to the skull with dental cement and a skull screw, and dummy cannula of length equal to the guide cannula were inserted and screwed into place. All subjects were allowed to recover for at least 10 days prior to behavioral testing.

Drug injections

The highly-specific V1aR antagonist (d(CH₂)₅[Tyr(Me)²,Dab⁵] AVP; (a generous gift from M. Manning, University of Toledo)) was diluted in sterile saline to a final injected dose of 450ng/300nL and stored at −20°C until use. This antagonist, modified from the original Manning compound with the addition of diaminobutyric acid (Dab), is exceptionally selective for V1aR, eliciting no detectable anti-OT activity in vitro or in vivo (Chan et al. 1996; Manning et al. 2012); the dose selected here is effective in blocking AVP action in rodents (Lozić et al. 2016).

Forty five to sixty minutes prior to behavioral testing, subjects were briefly anesthetized (1.5–3% isoflurane gas) and a 33 gauge needle was inserted through the guide cannula, extending a total distance of 2.4 mm and 2.3 mm ventral from dura for LHb and DR animals, respectively. Subjects were then injected with 300 nL sterile saline (vehicle) or V1aR antagonist at 100nL/min (10 μL Hamilton syringe; Harvard Apparatus PHD 22/2000 syringe pump) via the guide cannula. The injection needle was left in place for one minute to allow the drug to diffuse away from the tip of the injection needle.

Social experience

As opposite-sex sexual experience and attaining competitive status (“social dominance”) promotes male and female communicative behaviors (Lumley et al. 1999; Roullet et al. 2011), male and female (adjusted for stage of estrous cycle; determined by vaginal lavage) mice received social experience (two sexual and two aggressive encounters on separate days) prior to testing.

Sexual experience

Subjects were given two opportunities to copulate with either a novel stimulus female (for male subjects) or a novel stimulus male (for female subjects). In the first interaction, a sexually experienced stimulus mouse was placed in the subject’s home cage overnight. In the second interaction, a novel stimulus mouse was placed in the subject’s home cage and removed 5 minutes after one ejaculation or 90 minutes in the absence of ejaculation. Subjects that did not engage in any sexual behavior (mounting, intromission, or ejaculation) during the second trial were removed from further testing.

Same-sex (aggressive) experience

Male and female subjects were exposed to two daily interactions with a stimulus animal of the same sex. Male subjects received two interactions with subordinate stimulus males that had 40 μL of freshly-collected GDX+T male urine (pooled from five males) applied to their backs to provide aggression-promoting cues to otherwise non-aggressive stimulus males (Beeman 1947; Connor and Winston 1972; Van Loo et al. 2001).

Stimulus males were placed in the subject’s home cage and removed after the subject’s first offensive attack (lunge with bite) within a 10 minute period. All subject males attacked the intruder male stimulus by the second encounter, and all subordinate stimulus males displayed submissive behavior, defined by defensive postures (e.g., on-back), fleeing, and non-social exploration (Koolhaas et al. 2013). Female subjects received two interactions with a group housed GDX stimulus female in their home cage over a 10 minute period. Female encounters rarely resulted in aggressive responses from either animal; however, if any aggressive behavior, though limited, occurred, the female was removed after the first offensive attack, much like following male-male interactions.

Experimental design

All testing occurred within the first 6 hours of the dark cycle under red light illumination. Subjects were habituated to the testing room and apparatus by handling and placing mice for 5 minutes in the three-chamber apparatus (see below) once a day for 3 days. On experimental days, subjects were adapted to the experimental room for 15 minutes before testing. All tests were scored by an experimenter blind to the drug treatment of the subject.

Subjects received a total of five injections of V1aR antagonist/vehicle across four tests for social communication in the three-chamber apparatus (two antagonist and two vehicle injections) and during one final test (antagonist or vehicle injection) within the subject’s home cage to measure any aggression during same-sex interactions. Each test was separated by at least three days. The order of treatment (saline, V1aR antagonist) and stimulus condition (male, female) was counterbalanced across subjects, except that subjects exposed to a stimulus type on the first test were then given that same stimulus type on the second test (Fig 1). Female subjects were tested irrespective of estrous cycle day; prior research indicates minimal effects of estrous cycle on female mouse communicative behavior (Maggio and Whitney 1985; Coquelin 1992; Moncho-Bogani et al. 2004). Following the final test, subjects were euthanized and injected through guide cannula with dilute India ink.

Figure 1.

Experimental timeline.

Social communication and behavior

Ultrasonic vocalizations (USV), urine marking, and social investigation by subjects was recorded in an acrylic three-chamber apparatus (Crawley 2007; Arakawa et al. 2008; Moy et al. 2009; Harvard Apparatus; dimensions: 20.3 × 42 × 22 cm). Instead of a solid floor, the apparatus was placed on absorbent paper (Nalgene Versi-dry paper, Thermo Fisher Scientific) so as to retain and accurately measure urine marking. Animals were tracked using motion detection software (ANY-maze, San Diego Instruments, RRID:SCR_014289). During testing with stimulus animals, subjects had access to a stimulus animal in a cage (8 cm (D), 18 cm (H); 3-mm diameter steel bars, 7.4 mm spacing) and an empty “clean” cage placed at opposite corners of the outermost chambers of the apparatus. The location of stimulus and clean cages was counterbalanced across animals. After placing the subject in the center of the middle chamber, we measured investigation of clean and stimulus cages, distance traveled throughout the apparatus, time spent in the chambers containing stimulus and clean cages as well as USVs and urine marking across a 5 minute trial. After testing, the apparatus and cages were thoroughly cleaned with 70% ethanol and allowed to dry before further testing.

Social investigation and USVs

Social investigation was defined as time spent sniffing within 2 cm of the stimulus or clean cage; climbing on the cage was not scored as investigation. USVs of subject and stimulus interactions were detected using a condenser microphone connected to an amplifier (UltraSoundGate CM16/CMPA, 10 –200 kHz, frequency range) placed 4 cm inside the apparatus and directly above the center compartment. USVs were sampled at 200 kHz (16-bit) with target frequency set to 70 kHz (UltraSoundGate 116Hb, Avisoft Bioacoustics). Recordings were then analyzed using a MATLAB (MathWorks, RRID:SCR_001622) plug-in that automates USV analysis (Van Segbroeck et al. 2017). Using this program, sonograms were generated by calculating the power spectrum on Hamming windowed data and then transformed into compact acoustic feature representations (Gammatone Filterbank). Each 200-ms window containing the maximum USV syllable duration was then clustered, via machine learning algorithms, into USV syllable types (repertoire units) based on time-frequency USV shape. Repertoire units that appeared as background noise were discarded. Both the number of all USV produced in each condition as well as the number of USV syllable types (using criteria previously described: short, composite, downward, upward, 1 frequency jump, modulated, multiple frequency jumps, u-shape, flat, chevron (Hanson and Hurley 2012)) were counted.

Urine marking

Following testing, the substrate sheet was allowed to dry for 1 hour and then sprayed with ninhydrin fixative (LC-NIN-16; Tritech Forensics Inc.) to visualize urine marks (Meyer 1957; Lehmann et al. 2013). After 24 hours, sheets were imaged (Sony DSC-S700 camera), binarized and analyzed using a computer-aided imaging software (ImageJ, RRID:SCR_003070). Urine marking was measured as both as the total area (cm²) covered and the total count of individual marks throughout the entire arena. Urine spots that were larger than 6 cm² and directed toward corners were counted as eliminative “pools” and were excluded from analysis (Bishop and Chevins 1987).

Same-sex interactions and aggressive behavior

To measure territorial aggression, a subordinate male stimulus animal with applied urine (see above) was placed in a male subject’s home cage and then removed after the subject’s first offensive attack (lunge with bite) within a 10 minute period; the latency to first bite was recorded. Each female subject was presented with a GDX, group housed, female stimulus animal within the subject’s home cage for a 10 minute period. Though rare, if an offensive attack occurred, the stimulus animal was removed, much like following male-male interactions.

Tissue collection and histological analysis

Animals were euthanized by intraperitoneal injection of Beuthanasia-D (150 mg/kg) and subsequently injected through guide cannula with 300nL of dilute India ink. Brains were extracted and flash frozen on dry ice and stored at -80 °C until further processing. Coronal sections were cut at a thickness of 30 μm with a cryostat (Leica CM3050 S, Leica Biosystems) and analyzed for injection placement with reference to anatomical landmarks (ventricles, fiber tracts) and plotted on anatomical plates (Paxinos and Franklin 2012).

Statistical analysis

All data were analyzed and graphed in R (3.4.4; R Core Team, 2017). Social investigation, movement, USV, and urine marking data met the assumptions of parametric statistical tests. Therefore, we analyzed data on social investigation, movement (distance traveled, time in chambers containing stimulus and clean cages), number of USVs, and urine marking with mixed-model ANOVAs [between-subject factor: sex; within-subject factors: treatment (V1aR antagonist, vehicle), sex of stimulus (male, female)] followed by paired t tests assessing treatment effects. The number of specific USV syllables and aggressive behavior (latency to bite) were not normally distributed and could not be transformed, therefore, we analyzed treatment effects using pairwise Mann-Whitney U tests. All post hoc pairwise comparisons report Bonferroni-corrected p values and Cohen’s d for effect size when statistically significant. Results were considered statistically significant if p < 0.05.

Results

Histology

Subjects were included in the main LHb/DR analysis if the tip of the injection cannula was located within LHb bilaterally (10 males, 9 females; Fig. 2a) or the DR (10 males, 8 females; Fig. 2b). Eleven LHb targeted subjects (6 males, 5 females) were analyzed in a separate ‘miss’ group due to placement of injection needle tip outside the LHb, including subjects with only unilateral LHb cannulation (Fig. 2a, Table 5). Similarly, eight DR targeted subjects (males = 4, females = 4) were analyzed in a separate ‘miss’ group due to injection needle placement within the periaqueductal gray (PAG) (Fig. 2b, Table 5).

Figure 2.

Histological identification of injection sites. Coronal sections through the rostral-caudal extent of the mouse brain (relative to bregma) referenced to Paxinos and Franklin (2012). a. Lateral habenula (LHb) targeted subjects. Blue dots represent male subjects with bilateral injections into LHb (‘hits’, n=10), half-filled blue dots represent a subject with a unilateral LHb injection (‘unilateral hit’, n=1), and open blue dots represent bilateral injections outside of LHb (‘bilateral misses’, n=5). Orange dots represent female subjects with bilateral injections into LHb (‘hits’, n=9), half-filled orange dots represent a subject with a unilateral LHb injection (‘unilateral hit’, n=3), and open orange dots represent bilateral injections outside of LHb (‘bilateral misses’, n=5). b. Dorsal raphe (DR) targeted subjects. Blue dots represent male subjects with injections into DR (‘hits’, n=10) and open blue dots represent injections outside of DR (‘misses’, n=4). Orange dots represent female subjects with injections into DR (‘hits’, n=8) and open orange dots represent injections outside of DR (‘misses’, n=4).

Table 5.

Table of median (interquartile range) of urine mark (UM) counts, UM area (in pixels), USV counts, and social investigation (SI, seconds) produced by LHb ‘miss’ and DR ‘miss’ subjects. Male LHb ‘miss’ subjects (n=6) reduced the number of UM (counts, p=0.007) to female stimuli following infusion of V1aR antagonist compared to vehicle injections. Male DR ‘miss’ subjects (n=4) reduced the number of UM (counts, p=0.03) to male stimuli following infusion of V1aR antagonist compared to vehicle injections. Bold numbers represent significant differences between treatment groups.

	male subjects				female subjects
treatment	vehicle		V1aR antagonist		vehicle		V1aR antagonist
stimulus	female	male	female	male	female	male	female	male
LHb ‘miss’ subjects
UM (count)	70 (30–118)	49 (31–134)	(p=0.007) 14 (4–85)	53 (0–112)	0 (0–0)	0 (0–16)	0 (0–112)	0(0–0)
UM (pixels)	7640 (356019277)	19871 (53628798)	12042 (85135031)	8934 (060452)	0 (0–0)	0 (0–3888)	0 (0–323)	0(0–0)
USV (count)	463 (187–848)	24 (9–132)	400 (7–1256)	23 (16–58)	16 (7–70)	24 (17–25)	257 (1–398)	16 (14–21)
SI (s)	37 (25–64)	23 (13–39)	56 (26–91)	16 (11–40)	20 (17–26)	8 (5–113)	18 (12–33)	13 (5–118)
DR ‘miss’ subjects
UM (count)	83 (34–118)	101 (31–136)	30 (0–179)	(p=0.03) 28 (0–57)	0 (0–0)	0(0–0)	0(0–0)	0(0–7)
UM (pixels)	24044 (1238862580)	18865 (556832421)	9509 (023348)	4481 (08962)	0 (0–0)	0(0–0)	0(0–0)	0 (0–1975)
USV (count)	25 (11–155)	1 (0–2)	68 (4–461)	4 (1–14)	5 (2–12)	30 (1–49)	7 (3–17)	8 (4–17)
SI (s)	45 (17–89)	24 (6–36)	39 (23–66)	27 (3–30)	26 (20–34)	18 (11–25)	24 (23–26)	8 (7–19)

V1aR antagonism within lateral habenula

LHb V1aR antagonism decreased male urine marking to males

Male subjects produced more urine marks than female subjects (F(1,17) = 21.25, p = 0.0003) with an interaction between sex of subject and treatment (F(1,17) = 5.37, p = 0.03) and a trend toward an interaction between sex of subject and sex of stimulus (F(1,17) = 4.0, p = 0.06). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marks to male stimuli compared to when given vehicle (saline) injections (t(9) = 3.84, p = 0.004, d = 1.22; Fig. 3a); this effect was not observed in response to female stimuli (t(9) = 0.70, p = 0.50; Fig. 3b). Injections of V1aR antagonist in female subjects did not alter the number of urine marks to male stimuli (t(8) = 1.84, p = 0.10; Fig. 3c) or female stimuli (no marking; Fig. 3d) compared to vehicle injections.

Figure 3.

Effects of V1aR blockade in the lateral habenula (LHb) on urine marking in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. Urine marking was evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) reduced the number of urine marks (counts, p=0.004) as well as area covered by urine marking (pixels, inset; p = 0.02) to (a) male, but not (b) female, stimuli following infusion of V1aR antagonist compared to vehicle injections. c-d. Female subjects (n=9) did not alter the number of urine marks or area covered by urine marking (inset) to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. e. Example images of reduced urine marking by a male subject (toward a male stimulus) following V1aR antagonist or vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

Male subject’s urine marking covered more area than female subjects (F(1,17) = 14.63, p = 0.001) with interactions between sex of subject and sex of stimulus (F(1,17) = 7.41, p = 0.01) and sex of subject and treatment (F(1,17) = 6.37, p = 0.02). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marking area to male stimuli compared to when given vehicle injections (t(9) = 2.79, p = 0.02, d = 0.9; Fig. 3a inset); this effect was not observed in response to female stimuli (t(9) = 2.13, p = 0.06; Fig. 3b inset). Injections of V1aR antagonist in the LHb of female subjects did not alter the urine marking area in the presence of male stimuli (t(8) = 0.83, p = 0.43; Fig. 3c inset) or female stimuli (no marking; Fig. 3d inset).

LHb V1aR antagonism decreased male ultrasonic vocalizations to females

There was no overall difference in USV production by sex of subject (F(1,17) = 0.13, p = 0.73; but there was an interaction between sex of subject and treatment (F(1,17) = 6.57, p = 0.02) and sex of stimulus and treatment (F(1,17) = 5.42, p = 0.03). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased USVs to female stimuli compared to when given vehicle injection (t(9) = 3.18, p = 0.01, d = 0.99; Fig. 4b); this effect was not observed in response to male stimuli (t(9) = 0.67), p = 0.52, Fig. 4a). As expected, males that received vehicle injections produced more USVs to female stimuli than to male stimuli (t(9) = 2.59, p = 0.029; however, when males received the V1aR antagonist, this difference disappeared (t(9) = 1.26, p = 0.24). V1aR injections into LHb of females did not change USV production in the presence of male stimuli (t(8) = 0.68, p = 0.52, Fig. 4c) or female stimuli (t(8) = 1.1, p = 0.30, Fig. 4d) compared to vehicle treatment. Additionally, V1aR antagonism did not change the distribution of USV syllable types in either males or females (Table 1).

Figure 4.

Effects of V1aR blockade in the lateral habenula (LHb) on USVs in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. USVs were evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) did not alter USVs to (a) male stimuli, but did decrease USVs to (b) female stimuli (p=0.01), following infusion of V1aR antagonist into LHb compared to vehicle injections. c-d. Female subjects (n=9) did not alter USVs to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

Table 1.

Table of median (interquartile range) counts of different USV syllable types in male and female subjects. V1aR antagonism in the LHb did not change the composition of USV syllable types produced compared to vehicle treatment.

	male subjects				female subjects
treatment	vehicle antagonist		V1aR antagonist		vehicle		V1aR
stimulus	female	male	female	male	female	male	female	male
short	17 (0–44)	22 (0–56)	24 (0–49)	32 (0–72)	13 (0–38)	13 (0–38)	11 (0–37)	10 (0–32)
down	6 (0–25)	5 (0–23)	6 (0–27)	2 (0–13)	0 (0–13)	0 (0–10)	1 (0–13)	0 (0–9)
upward	22 (0–54)	5 (0–40)	23 (0–50)	3 (0–35)	0 (0–29)	0 (0–38)	8 (0–46)	0 (0–40)
flat	7 (0–33)	20 (0–45)	7 (0–27)	9 (0–50)	25 (7–100)	28 (0–100)	8 (0–100)	27 (0–100)
modulated	7 (0–15)	0(0–8)	4 (0–18)	3 (0–14)	0 (0–25)	2(0–8)	4 (0–13)	4 (0–15)
u shape	7 (0–15)	9 (0–19)	5 (0–13)	3 (0–23)	4 (0–25)	0(0–8)	5 (0–42)	0 (0–8)
chevron	5 (0–10)	0(0–8)	3 (0–15)	0 (0–5)	0 (0–25)	0 (0–29)	4 (0–26)	4 (0–29)
one frequency jump	12 (0–23)	12 (0–28)	9 (0–18)	3 (0–18)	0 (0–20)	3 (0–20)	3 (0–20)	0 (0–16)
composite	3 (0–10)	2 (0–14)	2 (0–6)	2 (0–9)	0 (0–14)	0(0–2)	0(0–7)	0 (0–4)

LHb V1aR antagonism did not alter social investigation in males or females

Subjects spent more time investigating female stimuli than male stimuli (F(1,17) = 25.56, p = 0.001), regardless of treatment (F(1,17) = 0.01, p = 0.92) with no interaction between sex of subject and treatment (F(1,17) = 0.05, p = 0.82), or sex of stimulus and treatment (F(1,17) < 0.01, p = 0.99; Fig. 5a–d).

Figure 5.

Effects of V1aR blockade in the lateral habenula (LHb) on social investigation in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. Social investigation was evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) did not alter social investigation to male (a) or female (b) stimuli following infusion of V1aR antagonist compared to vehicle injections. c-d. Female subjects (n=9) did not alter social investigation to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

V1aR antagonism in LHb did not alter the amount or spatial distribution of activity

Overall, subjects in both treatment conditions traveled similar distances throughout the three-chamber apparatus (F(1,17) = 3.56 p = 0.08) with no evident differences between treatment conditions in time spent in the stimulus chambers (F(1,17) = 0.96, p = 0.34) but with an interaction between treatment and sex in time spent in the clean chamber (F(1,17) = 6.43, p = 0.02). Post hoc comparisons revealed that males receiving V1aR antagonist spent more time in the clean chamber zone in presence of female stimuli compared to when given vehicle injections (t(9) = 2.85, p = 0.01, d = 0.9; Table 2); this effect was not observed in response to male stimuli (t(9) = 0.65, p = 0.53; Table 2) or in female subjects (to male stimuli: (t(8) = 1.65, p = 0.14; to female stimuli: (t(8) = 1.65, p = 0.14; Table 2)).

Table 2.

Table of median (interquartile range) distance traveled (meters) and time spent (seconds) in stimulus or clean cage chamber. Subjects with V1aR antagonist infused into the LHb did not differ in distance traveled or time spent in stimulus chambers compared to when injected with vehicle (saline). However, males receiving V1aR antagonist spent more time in clean chambers during tests with female stimuli compared to when injected with vehicle. Bold numbers represent significant differences between treatment groups.

	male subjects				female subjects
treatment	vehicle		V1aR antagonist		vehicle		V1aR antagonist
stimulus	female	male	female	male	female	male	female	male
distance traveled (m)	0.56 (0.30.74)	0.5 (0.4–0.66)	0.57 (0.340.73)	0.5 (0.390.83)	0.52 (0.340.83)	0.52 (0.340.83)	0.68 (0.540.86)	0.68 (0.54–0.86)
time in stimulus chamber (s)	112 (52–199)	101 (15–138)	174 (62–216)	96 (13–143)	151 (54–188)	151 (54–188)	140 (112–160)	140 (112–160)
time in clean chamber (s)	111 (70–186)	115 (49–193)	p=0.02 82 (21–189)	146 (99–222)	72 (27–117)	73 (27–116)	92 (70–111)	93 (70–111)

V1aR antagonism within dorsal raphe

DR V1aR antagonism decreased male urine marking to males

Male subjects produced more urine marks than female subjects (F(1,16) = 15.02, p = 0.001) with a trend toward an interaction between sex of stimulus and treatment (F(1,16) = 4.4, p = 0.052). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marks to male stimuli compared to when given vehicle injections (t(9) = 3.0, p = 0.015, d = 0.95; Fig. 6a); this effect was not observed in response to female stimuli (t(9) = 0.5, p = 0.63; Fig. 6b). Injections of V1aR antagonist in the DR of female subjects did not alter the number of urine marks in the presence of male stimuli (t(7) = 1.02, p = 0.92; Fig. 6c) or female stimuli (t(7) = 1.54, p = 0.17; Fig. 6d) compared to vehicle injections.

Figure 6.

Effects of V1aR blockade in the dorsal raphe (DR) on urine marking in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. Urine marking was evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) reduced the number of urine marks (counts, p=0.015) as well as area covered by urine marking (pixels, inset; p = 0.03) to (a) male, but not (b) female, stimuli following infusion of V1aR antagonist compared to vehicle injections. c-d. Female subjects (n=8) did not alter the number of urine marks (counts) or area covered by urine marking (pixels, inset) to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. e. Example images of reduced urine marking by a male subject (toward a male stimulus) following V1aR antagonist or vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

Male subject’s urine marking covered more area than female subjects (F(1,16) = 13.61, p = 0.002) with an interaction between sex of stimulus and treatment (F(1,16) = 9.99, p = 0.006) and sex of subject and sex of stimulus and treatment (F(1,16) = 6.69, p = 0.02). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marking area to male stimuli compared to when given vehicle injections (t(9) = 2.57, p = 0.03, d = 0.81; Fig. 6a inset); this effect was not observed in response to female stimuli (t(9) = 1.15, p = 0.28; Fig. 6b inset). Injections of V1aR antagonist in the DR of female subjects did not alter the urine marking area in the presence of male stimuli (t(7) = 0.98, p = 0.36; Fig. 5c inset) or female stimuli (t(7) = 1.11, p = 0.3; Fig. 6d inset).

DR V1aR antagonism did not alter USVs in males or females

V1aR antagonism within DR did not change the number of USVs emitted in either female or male stimulus conditions (treatment: F(1,16) = 0.1, p=0.73; sex: F(1,16) = 4.07, p = 0.06; Fig. 7a–d). Additionally, injections of V1aR antagonist did not change the distribution of USV syllable types produced in these conditions (Table 3).

Figure 7.

Effects of V1aR blockade in the dorsal raphe (DR) on USVs in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. USVs were evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) did not alter USVs to male (a) or female (b) stimuli following infusion of V1aR antagonist compared to vehicle injections. c-d. Female subjects (n=8) did not alter USVs to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

Table 3.

Table of median (interquartile range) number of different USV syllable types in male and female subjects. V1aR antagonism in the DR did not change the distribution of USV syllable types produced compared to vehicle treatment.

	male subjects				female subjects
treatment	vehicle antagonist		V1aR antagonist		vehicle		V1aR
stimulus	female	male	female	male	female	male	female	male
short	19 (0–56)	33 (0–73)	17 (0–67)	35 (0–78)	13 (0–29)	13 (0–22)	15 (0–100)	25 (3–89)
down	10 (0–33)	6 (0–14)	9 (0–21)	0 (0–9)	0 (0–25)	0 (0–11)	0 (0–11)	0(0–6)
upward	24 (0–49)	9 (0–25)	25 (0–63)	4 (0–36)	10 (0–42)	0 (0–40)	0(0–32)	6 (0–38)
flat	6 (0–45)	25 (3–51)	14(7–33)	18 (0–75)	47 (0–63)	56 (0–91)	39 (0–100)	25 (0–68)
modulated	1 (0–11)	0 (0–11)	0 (0–12)	0 (0–0)	0 (0–17)	0 (0–11)	0(0–8)	0 (0–17)
u shape	0 (0–14)	0 (0–17)	0 (0–8)	0 (0–5)	0 (0–0)	0 (0–0)	0(0–0)	0(0–0)
chevron	0(0–5)	0(0–4)	0 (0–4)	0 (0–0)	0 (0–0)	0 (0–18)	0(0–0)	0 (0–18)
one frequency jump	1 (0–40)	0 (0–29)	0 (0–29)	0 (0–14)	24 (0–67)	2 (0–20)	11 (0–31)	8 (0–21)
composite	0 (0–0)	0(0–0)	0 (0–0)	0 (0–0)	0 (0–0)	0 (0–0)	0(0–0)	0(0–0)

DR V1aR antagonism did not alter social investigation in males or females

Subjects spent more time investigating female stimuli than male stimuli (F(1,16) = 15.82, p = 0.001), regardless of treatment (F(1,16) = 3.01, p = 0.10); there was no interaction between sex of subject and treatment (F(1,16) = 0.07, p = 0.79), or sex of stimulus and treatment (F(1,16) = 0.04, p = 0.85; Fig. 8a–d).

Figure 8.

Effects of V1aR blockade in the dorsal raphe (DR) on social investigation in the presence of a confined male (a, c) or female (b, d) within a three-chamber apparatus. Social investigation was evaluated following infusion of V1aR antagonist (450ng/300nL) or vehicle (saline), counterbalanced. a-b. Male subjects (n=10) did not alter social investigation to male (a) or female (b) stimuli following infusion of V1aR antagonist compared to vehicle injections. c-d. Female subjects (n=8) did not alter social investigation to male (c) or female (d) stimuli following infusion of V1aR antagonist compared to vehicle injections. Each point and horizonal line represents individual within-subject data. Overlapping data are represented as one point/line.

V1aR antagonism in DR did not alter the amount or spatial distribution of activity

Overall, subjects in both treatment groups traveled similar distances throughout the three-chamber apparatus (F(1,16) = 0.01 p = 0.12); there were no differences between treatment groups in the amount of time subjects spent in the stimulus chamber zones (F(1,16) = 0.36, p = 0.56) or clean chamber zones (F(1,16) = 0.74, p = 0.40; Table 4).

Table 4.

Table of median (interquartile range) distance traveled (meters) and time spent (seconds) in stimulus or clean cage chamber. Subjects with V1aR antagonist infused into the DR did not differ in distance traveled, time spent in stimulus or clean chambers compared to when injected with vehicle (saline).

	male subjects				female subjects
treatment	vehicle		V1aR antagonist		vehicle		V1aR antagonist
stimulus	female	male	female	male	female	male	female	male
distance traveled (m)	0.35 (0.220.5)	0.34 (0.30.46)	0.36 (0.30.56)	0.3 (0.280.53)	0.47 (0.340.67)	0.43 (0.380.66)	0.55(0.43–0.6)	0.49 (0.260.69)
time in stimulus chamber (s)	194 (151–237)	145 (88–208)	189 (135–229)	97 (29–181)	167 (105–171)	158 (98–184)	162 (142186)	130 (105–202)
time in clean chamber (s)	78 (45–114)	97 (29–181)	78 (47–120)	134 (59–164)	94 (50–104)	70 (55–147)	78 (57–116)	94 (74–119)

V1aR antagonism in LHb or DR did not alter territorial aggression

The attack latency did not differ between treatment groups for LHb or DR animals (LHb: U = 14, p = 0.84; DR: U = 11, p = 0.84; Fig. 9). Female subjects did not attack female intruders.

Figure 9.

V1aR antagonism in LHb (n=5) (A) or DR (n=5) (B) did not alter onset of male-male aggression compared to vehicle (saline) injected subjects (LHb: n=5; DR: n=5). Bar graph and individual data points represent median and range of male subject’s latency to attack a subordinate intruder male.

Effects of V1aR antagonism on areas outside of LHb or DR

LHb or DR targeted subjects with placement of injection needle tip outside of LHb (unilateral hits included) or DR were analysed in a separate ‘miss’ group for urine marking, ultrasonic vocalization (USV), and social investigation behavior. In male subjects with LHb ‘misses’, a treatment and stimulus interaction was found in number of urine marks produced (F(1,9) = 5.76, p = 0.04) but not in urine marking area covered (F(1,9) = .001, p = 0.97). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marks to female stimuli compared to when given vehicle injections (t(5) = 4.45, p = 0.007, d = 1.82; Table 5; this effect was not observed in response to male stimuli (t(5) = 1.57, p = 0.18; Table 5). Injections of V1aR antagonist in female subjects with LHb ‘misses’ did not alter urine mark number in the presence of male stimuli (t(4) = 1, p = 0.37) or female stimuli (t(4) = 1.07, p = 0.35; Table 5). No treament effect was found in USV production (F(1,9) = 0.58, p = 0.47) or social investigation (F(1,9) = 0.03, p = 0.87; Table 5). Therefore, data from LHb ‘misses’ did not match the pattern of behavior following direct LHb injections (reduced urine marking to males and reduced USVs to females).

Injections in subjects that missed the DR were all located above the DR and within the ventrolateral PAG and resulted in a treatment and sex interaction in number of urine marks produced (F(1,6) = 9.11, p = 0.02) as well as urine marking area (F(1,6) = 6.97, p = 0.04). Post hoc comparisons revealed that males receiving V1aR antagonist significantly decreased urine marks to male stimuli compared to when given vehicle injections (t(3) = 3.89, p = 0.03, d = 1.94; Table 5); this effect was not observed in response to female stimuli (t(3) = 0.65, p = 0.50) or urine marking area (to male stimuli: t(3) = 2.53, p = 0.09; to female stimuli: t(3) = 1.38, p = 0.26; Table 5). These results are partially similar to effects of direct DR injections: both reduced male urine marks to other males. V1aR injections in female subjects that missed the DR did not alter urine mark number in the presence of male stimuli (t(3) = 1, p = 0.39) or female stimuli (no marking; Table 5). No treatment effect was observed in USVs (F(1,6) = 0.82, p = 0.40) or SI responses (F(1,6) = 3.56, p = 0.11; Table 5).

Discussion

The importance of central AVP acting on V1aR in the modulation of rodent social behavior has been well-established (Lukas and Neumann 2013) and is often sexually-differentiated (Veenema et al. 2013; Dumais and Veenema 2016; Duque-Wilckens et al. 2016; Rigney et al. 2019). Here, we pharmacologically blocked V1aR in LHb or DR, areas known to have sex-different (male-biased) AVP expression (Rood et al. 2013) and broad involvement in social behavior (Luo et al. 2017; Soutschek 2018), and assessed changes in social behavior and communication. Our results indicate that sex differences in AVP innervation of LHb and DR may contribute to sexually dimorphic expression of social communication, as V1aR blockade in the LHb of males, but not females (who have less LHb and DR AVP innervation), reduced urine marking to unfamiliar males and production of ultrasonic vocalizations (USV) to unfamiliar, sexually receptive females, while V1aR antagonism in the DR of males, but not females, reduced urine marking to unfamiliar males. These changes occurred even though social investigation, locomotion, and territorial aggression were unaffected by V1aR blockade in either area, indicating specific effects on communicative behavior.

V1aR action in lateral habenula

The LHb has long been known to receive sex-different, hormone-sensitive AVP innervation (de Vries and Panzica 2006; de Vries 2008), likely from the BNST cells that show sexually dimorphic AVP expression (de Vries and Panzica 2006). Our previous findings demonstrate that BNST-AVP cells are important for male urine marking and social investigation of unfamiliar male (Rigney et al. 2019) as well as for detection of social novelty (Whylings et al. 2020).

Consequently, our present results suggest that BNST-AVP action on social communication is potentially mediated by the LHb, although the LHb does receive AVP axonal projections from other sources as well (Rood and De Vries, 2011, Zhang et al. 2018). We found that V1aR blockade in the LHb reduced communicative behaviors typical of dominant, territorial male mice (urine marking to unfamiliar males and USVs to unfamiliar females) without concomitant changes in social investigation, locomotion or territorial aggression toward subordinate males. This suggests that AVP may normally act in the LHb to promote specifically male-typical communication, rather than changing all aspects of social behavior. Our experiments do not allow us to fully exclude the possibility that changes in USVs following V1aR blockade in LHb in male subjects are due to changes in USV production by female stimuli. However, given the substantial male bias in USVs in opposite-sex interactions, it is likely that male subjects produced most of the USVs (Warren et al., 2018). Importantly, we did not observe V1aR antagonist effects on social investigation suggesting that AVP/V1aR action does not appear to alter the broader aversive function of LHb (Sparta et al. 2013; Golden et al. 2016; Benekareddy et al. 2018, but see Lecourtier et al. 2004). Moreover, the behavioral effects of V1aR antagonism are likely due to action on LHb directly as injections outside of LHb did not recapitulate the effects of LHb injections in that they did not reduce urine marking to unfamiliar males or production of USVs to unfamiliar, sexually receptive females. It should be noted that our misses were all very close to LHb and so it is possible that observed urine marking reductions in these offsite injections may still have allowed spread of antagonist to LHb.

Recent work on AVP action in LHb suggests that AVP may decrease defensive behavior in response to water deprivation (Zhang et al. 2016), potentially through hypothalamic AVP inputs (Zhang et al. 2018). This reduction in fear-responses, although correlative, combined with the present results, suggests an important role of AVP in LHb in promoting male-typical active responses to social competition and other biological threats. The exact source and nature of AVP action within LHb (BNST, medial amygdala, or hypothalamic) requires further examination.

V1aR action in dorsal raphe

The DR is interconnected with the BNST (Weissbourd et al. 2014; Garcia-Garcia et al. 2018) and contains more steroid-sensitive AVP expression in males than in females, suggesting that BNST AVP and/or medial amygdala AVP cells are the main source of AVP here (Rood et al. 2013). Similarly, sex differences in V1aR expression in DR have been noted in other species (Ross et al., 2019) and AVP has been found to indirectly excite DR serotonin neurons via V1aR action (Rood and Beck 2014), possibly through intrinsic connections of glutamatergic DR cells (Soiza-Reilly and Commons 2011). Our study is the first to examine the role of DR V1aR on social behavior and here we demonstrate that V1aR blockade within DR in males reduces urine marking toward unfamiliar males, but not unfamiliar females, without altering social investigation and other behaviors. The lack of social avoidance following V1aR antagonism is in contrast with previous studies showing that AVP injections within DR facilitate fear-motivated passive avoidance in rats (Kovács et al. 1979; Kovács et al. 1986). This discrepancy may reflect differences in task parameters (social vs. non-social; memory processes), species (rat vs. mouse) as well as the known differences in behavioral effect between exogenous stimulation of the AVP system verses blockade of endogenous action of V1aR action (Engelmann 2008).

V1aR blockade in the ventrolateral periaqueductal gray (PAG) overlying DR also reduced urine marks toward unfamiliar males. As this area is the only nearby region to also contain AVP fibers and V1aR expression, is it possible that V1aR activation in PAG can generate appropriate levels of urine marking in male mice. Although AVP innervation of this relatively caudal region of the PAG is likely of hypothalamic origin and not sex-different or steroid sensitive (Rood et al. 2013), it may play a role in social communication as AVP injected into the PAG stimulates scent marking in hamsters, a species lacking extrahypothalamic AVP (Hennessey et al. 1992). Indeed, the PAG is broadly involved in other aspects of mouse social communication, such as USVs (Tschida et al. 2019). It is also possible that V1aR antagonism could be acting in DR and PAG together to alter behavior since these structures are close in proximity. Additionally, our cannulation approach in LHb or DR does not avoid the possibility that the V1aR antagonist spread into the lateral ventricle (near LHb) or cerebral aqueduct (near DR). Yet, we feel that this is unlikely to fully account for our effects since LHb ‘misses’ did not replicate effects seen in LHb ‘hit’ subjects and a scenario of ventricular leak would require further downstream sex-differences in V1aR responsiveness. Nevertheless, additional experiments targeting different AVP cell populations (hypothalamic vs. limbic) will be required to disambiguate AVP/V1aR action in PAG from DR.

As sex differences in LHb and DR AVP fiber density likely originate from BNST cell bodies (de Vries 2008; Rood et al. 2013; Otero-Garcia et al. 2014), it is surprising that we did not observe alterations in male-male investigation following LHb/DR V1aR antagonism because deletion of BNST-AVP cells reduces male investigation of other males (Rigney et al. 2019). Similarly, BNST-AVP lesions did not alter USVs whereas V1aR blockade within LHb did. These apparent discrepancies may be due to several factors. First, AVP action at other BNST target structures, such as ventral pallidum, may drive social investigation, as V1aR activation in this area regulates male investigatory behavior and partner preference (Lim and Young 2004; DiBenedictis et al. 2020). Second, the source of AVP that regulates USVs in LHb may originate from medial amygdala AVP or hypothalamic AVP cells (Browne et al. 2018). Lastly, it is possible, given the cross-talk between OT and V1aR, that OT projections may be responsible for behavioral action (Song and Albers, 2017).

Our results indicate that the V1aR system plays a sexually dimorphic role in control of social communicative behaviors via LHb and, to a lesser extent, DR. This is largely in keeping with other findings that the AVP/V1aR system is sexually-differentiated not just in anatomy, but also across various domains of behavioral function and across species (Albers, 2015; Dumais and Veenema, 2016; Hammock, 2015), including humans (Meyer-Lindenberg et al. 2011; Rilling et al. 2014). Given that the LHb and DR are strongly interconnected (Pasquier et al. 1976; Aghajanian and Wang 1977; Kalén et al. 1986; Ferraro et al. 1996; Yang et al. 2008) and appear to work as a system to regulate emotional responding (Zhao et al. 2015; Dolzani et al. 2016), concurrent AVP action on V1aR on LHb and DR may coordinate strongly-competitive aspects of social communication, such as territorial scent marking. In contrast, less-competitive aspects of communication, such as sexually-motivated vocalizations, may require direct AVP/V1aR action more specifically at LHb (and perhaps other AVP targets). The present work highlights the need to explore less-investigated targets of sex-different AVP inputs as they may play significant roles in sex-different social and emotional behaviors.

Highlights.

• Blocking vasopressin 1a receptor in the lateral habenula or dorsal raphe reduced male-typical communicative behavior toward males (urine marking)

• Antagonism of vasopressin 1a receptor in lateral habenula decreased male-typical ultrasonic vocalizations to female stimuli in male but not female mice

• Vasopressin 1a receptor antagonism did not alter other aspects of social behavior, including social investigation and territorial aggression) or locomotion