Knockdown of sexually differentiated vasopressin expression in the bed nucleus of the stria terminalis reduces social and sexual behaviour in male, but not female, mice

Abstract The neuropeptide arginine‐vasopressin (AVP) has long been implicated in the regulation of social behaviour and communication, but the sources of AVP release relevant for behaviour have not been precisely determined. Ablations of the sexually dimorphic AVP cells within the bed nucleus of the stria terminalis (BNST), which are more numerous in males, affect social behaviour differently in males and females. However, it is unknown whether these behavioural effects are caused by a reduction of AVP or of other factors associated with these cells. To test the role of AVP specifically, we used an shRNA viral construct to knock down AVP gene expression within the BNST of wild‐type male and female mice, using scrambled sequence virus as a control, and evaluated subsequent changes in social behaviours (social investigation, ultrasonic vocalization (USV), scent marking, copulation, and aggression), or anxiety‐like behaviours (elevated plus maze). We observed that, in males, knockdown of AVP expression in the BNST strongly reduced investigation of novel males, aggressive signalling towards other males (tail rattling, USV), and copulatory behaviour, but did not alter attack initiation, other measures of social communication, or anxiety‐like behaviours. In females, however, BNST AVP knockdown did not alter any of these behaviours. These results point to differential involvement of AVP derived from the BNST in social behaviour.

septum, lateral habenular nucleus, and ventral pallidum 10 affect social behaviour differently in males and females in rats and mice. [12][13][14][15] In addition, partial knockdown of AVP gene expression in the BNST reduces male, but not female, social interactions, while increasing male, but not female, aggression in finches. 4,16,17 In rats, intermale aggression correlates with AVP release in the septum and is reduced by intraseptal AVP antagonist application. 18 Overall, these studies indicate that BNST AVP is important for male-male interactions and for certain aspects of male prosocial communication, while playing a lesser role in female social behaviour and communication.
Recently, we directly tested whether cells expressing AVP in the BNST are involved in social behaviours in mice. 19,20 We found that ablations of these cells reduced male-male investigation and increased male scent marking toward female stimuli. 19 These studies, however, left the critical question unresolved as to whether AVP or other neuroactive substances are responsible for the effects seen after removal of BNST AVP cells. Consequently, in order to specifically target AVP, we reduced AVP-expression in the BNST using viral expression of a short hairpin RNA (shRNA) targeted against AVP mRNA and tested the effects of this manipulation on social investigation, ultrasonic vocalization (USV), and urine marking, all aspects of mouse communication that show pronounced sex differences. [21][22][23] 2 | ME THODS

| Animals and husbandry
All mice were maintained at 22°C on a 12:12 reverse light cycle with food and water available ad libitum and housed in individually

| Subjects
Forty-eight male and female C57BL/6J mice between 8 and 12 weeks of age were obtained from Jackson Laboratories (stock # 000664) and were singly-housed for a minimum of one week prior to testing. were used as stimuli for behavioural testing and to provide male and female subjects with social experience, because strain differences between subjects and stimulus mice increase social investigation. 24 Mice were used at 9-16 weeks of age and were novel to the subject to which they were exposed.
Female stimulus mice were grouped-housed, ovariectomized, and implanted with an estradiol capsule (GDX+E; see below), and given two sexual experiences before testing. Two groups of stimulus males were used for behavioural testing. Mice that were used as subordinates in the home cage aggression tests and to provide aggressive experience to subjects, were grouped-housed, gonadectomized (GDX), and subjected to two aggressive encounters with a dominant male (see below). Mice in the second group, which provided sexual experience to female subjects and served as sexual partners during copulatory tests as well as stimulus animals in the three-chamber social test, were singly-housed, gonadectomized, implanted with testosterone (GDX+T; see below), and then given two sexual experiences before testing.

| Surgery
All surgeries were carried out using 1.5%-2.5% isoflurane gas anesthesia in 100% oxygen; 3 mg/kg of carprofen was given subcutaneously before surgery to reduce pain.

| Urine collection
Pooled urine samples were collected from stimulus females induced into estrus and from stimulus males (5-10 mice per sample). Estrous state was verified by color, swelling, and expanded size of the vaginal opening. 31 To collect urine, mice were picked up by the tail base and held by dorsal neck skin; this method typically induced urination.
If the mouse did not urinate, stroking its belly from an anterior to posterior direction stimulated bladder voiding. Each mouse provided 40-80 μl of urine that was pooled into a 1.5 ml Eppendorf tube.
Urine samples were used fresh within 1 h of collection to prevent chemosignal degradation. 32

| Social experience
As opposite-sex sexual experience and attaining competitive status ("social dominance") promote communicative behaviours, 32

| Aggressive experience
Male subjects were exposed to two interactions with a subordinate stimulus male treated with 50 μl of GDX+T male urine applied to its back, a manipulation which elicits offensive aggression in subjects. 27 36 ). Female subjects were exposed to a female intruder; however, this did not elicit attacks from either animal.

| Experimental procedure
All testing occurred within the first 5 h of the dark cycle under red light illumination (27 lux), with the exception of the elevated plus maze (EPM), which took place in bright illumination (435 lux). All tests were scored by an experimenter blind to the genotype of the subject and testing occurred across multiple cohorts of subjects.
Three weeks after viral injections, subjects were habituated to the testing room and apparatus by handling and placing mice for 5 min in the three-chamber apparatus (see below) each day for three days.
On experimental days, subjects were adapted to the experimental room for 15 min prior to testing. First, we tested mice on an EPM to test for anxiety-related behaviour. 37 Mice were then tested in the three-chamber apparatus over six days with a one day break on the fourth day. Lastly, copulatory and aggressive behaviour were measured sequentially, with a day in between, in the subject's home cage.
Female subjects were tested irrespective of oestrous cycle day, except during copulation testing, when they were in behavioural estrus. Prior research indicates minimal effects of oestrous cycle on female mouse communicative behaviour, [38][39][40] nevertheless the oestrus cycle was monitored throughout the study. Following testing, subjects were sacrificed and their brain tissue was processed using immunohistochemistry to detect AVP, oxytocin (OT), galanin (GAL), and GFP-immunoreactive (-ir) cells and fibres in the BNST, paraventricular nucleus of the hypothalamus (PVN), and lateral septum (LS). cess to 50 μl of fresh urine from a stimulus animal or 50 μl saline pipetted onto a clean piece of filter paper (3 cm 2 ) that was taped on the outside of cages. The location of stimulus and the "clean" cage were counterbalanced across animals. After placing the subject in the center of the middle chamber, we measured, across a 5-min trial, close investigation of clean and stimulus cages as well as USV and urine marking, as described below. After testing, the apparatus and cages were thoroughly cleaned with 70% ethanol and allowed to dry before further testing. In all cases, male or female urine stimulus was presented first (day one), followed by exposure to a stimulus animal of the same sex the following day (day two); this order was then repeated one day later for the opposite sex. In this fashion, mice first experienced a weak stimulus (urine), then a stronger social stimulus (live animal). The order of male and female stimuli presentation was counterbalanced across subjects.

| Investigation and ultrasonic vocalizations
Close 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 were detected using a condenser microphone con-

| Urine marking
Following testing, the substrate sheet was allowed to dry for 1 h and then sprayed with ninhydrin fixative (LC-NIN-16; Tritech Forensics Inc.) to visualize urine marks. 22,45 After 24 h, sheets were imaged (Sony DSC-S700 camera), binarized and analyzed using computeraided imaging software (ImageJ, RRID:SCR_003070). Urine marking was measured as the total area (pixels) of visualized ninhydrin urine marks in the entire arena.

| Copulatory and aggressive behaviour
To measure copulatory behaviour, a stimulus mouse was placed in the subject's home cage and then removed after 90 min had elapsed.
The number of mounts, intromissions, and ejaculations and their latencies were recorded, along with mount rejections (female kicking male off during mounting attempt) by female subjects. To measure territorial aggression, subordinate stimulus males were placed in the subject's home cage and then removed after the subject's first offensive attack (biting and rolling) within a 10-min period; the latency to first bite and to first rolling-attack was recorded.

| Elevated plus maze
The manually scored from video. 46 Subjects were removed from EPM data analysis if they fell off the EPM during testing.

| Histology and immunohistochemistry
Mice were transcardially perfused with 50 ml of 0.1 M PBS (pH 7.

| Tissue analysis
Bilateral BNST images were taken at 10× magnification using a Zeiss Specific AVP-ir fibre density was calculated by subtracting the average nonspecific background from the AVP-ir density measurement (pixels). and therefore attention towards a distal stimulus, similar to social vigilance measurements that indicate increased anxiety in stressed animals. 50 We measured this orientation behaviour when the subject's nose was oriented toward the centre of the circular region of interest and was a distance of 9-30 cm away from the centre of the circular region of interest.

| Statistical analysis
All data were analysed and graphed in R  Figure S1). ward the stimulus cages at a 9-30 cm distance ( Figure S4).

| BNST AVP knockdown reduced male-male USVs, but did not alter urine marking
Most vocalizations were produced during male-female interactions and least during female-female interactions ( Figure 3A

| BNST AVP knockdown did not alter anxietylike behaviour in the elevated plus maze
We did not find sex

| DISCUSS ION
Previously, we found that removal of AVP-expressing cells in the BNST of male, but not female, mice reduced investigation of F I G U R E 2 Social investigation. Boxplots indicate individual data points, median, first and third quartiles for time spent investigating wire cages containing male or female stimulus animals, or an empty wire cage within the three-chamber apparatus. BNST AVP knockdown in males (A), but not females (B), decreased investigation of male (p < 0.00001) stimuli compared to controls. BNST AVP knockdown in males (C), but not females (D), decreased investigation of male urine (p = 0.012) and female urine (p = 0.0003) compared to controls same-sex conspecifics and altered social communication, while minimally affecting female social behaviour. 19 What was left unresolved was whether these results were due to the loss of AVP signalling from BNST cells or of other factors, such as other neuropeptides and neurotransmitters, associated with these cells. Here, we found that shRNA knockdown of AVP in BNST largely replicated the effects of BNST AVP cell ablations (e.g., on social investigation), although some effects diverged (e.g., on urine marking and copulatory behaviour), which may be related to AVP cell ablation affecting more than just AVP signalling. Additionally, BNST  or mounting behaviours toward receptive females after AVP knockdown, the effects on copulatory behaviour are more likely to be due to deficits in bridging the appetitive and consummatory phases of sexual behaviour, rather than to changes in sexual motivation. 72 These results do not match the effects of ablating BNST AVP cells, which reduced female, but not male, copulatory behaviour. 19 One possible explanation for this discrepancy is that AVP ablation left the expression of galanin, a neuropeptide colocalized with AVP in the BNST 73 intact in the present study. This may have increased overall inhibitory signalling to targets of BNST AVP cells, as galanin promotes neuronal inhibition 74 whereas AVP promotes excitation. 65 Indeed, ICV injections of galanin strongly inhibited male copulatory behaviour in rats, 75 and galanin has been shown to block AVPinduced flank marking in golden hamsters. 76 Future studies may help unravel the physiological and behavioural significance of coexpression of AVP and other signalling molecules.

| CON CLUS ION
In summary, our results indicate that sexually dimorphic AVP expression within the BNST contributes to sex differences in social behaviour. More specifically, BNST AVP knockdown in male, but not female, mice reduced investigation and communicative behaviours directed toward same-sex conspecifics as well as male sexual behaviour, without changing offensive attacks, anxiety-related behaviours, and overall activity. These results match those of other studies that have shown a sex-specific role of AVP in behavior. For example, AVP and its antagonists have different effects on aggressive play behaviour in rats, 77 territorial aggression in hamsters, [78][79][80] and social communication in humans. [81][82][83] Together, these studies point toward a sexually differentiated role of AVP in vertebrate social behaviour.
By ablating specific AVP cell groups in the BNST, PVN, SCN, 19,68,69 or knocking down AVP specifically in the BNST (present results), we have started addressing directly the question as to which AVP cell groups contribute to sex differences in social behaviour and its regulation.

ACK N OWLED G EM ENTS
This work was supported by National Institutes of Health (R01 MH121603) to AP and GJD, (F31 MH125659) to NR, and the Center for Behavioural Neuroscience at Georgia State University. We would like to thank Maria Olifer for her assistance with histology and immunohistochemistry.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/jne.13083.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.