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.

Keywords: bed nucleus of the stria terminalis; mice; sex differences; social behaviour; vasopressin.

FIGURE 1

Histology. (A) Adenoassociated virus (AAV) expressing a short hairpin RNA (shRNA) to target Avp mRNA (AAV8‐GFP‐U6‐mAVP‐shRNA) or a control AAV expressing a scramble shRNA sequence (AAV8‐GFP‐U6‐scramble‐shRNA) and location of bilateral injection site in the bed nucleus of the stria terminalis (BNST). (B) Example image showing AAV‐GFP‐labelled cells in the BNST. (C) Example images of AVP‐immunoreactive (ir) cells within the BNST following a scramble shRNA AAV (left) or Avp‐shRNA AAV injections (right). Rectangles indicate position of AVP‐ir cells (D) Boxplots showing that AVP‐ir cell number is significantly lower in AVP shRNA‐injected male (n = 14, p < 0.00001) and female (n = 10, p = 0.0002) subjects compared to scramble shRNA‐injected controls (males: n = 13; females n = 11). (E) Example images of AVP‐immunoreactive (ir) fibres within the LS following scramble shRNA AAV (left) or Avp‐shRNA AAV (right) injections into the BNST. Images were taken at 10x magnification from sections from the same scramble shRNA or Avp‐shRNA AAV‐injected male subjects. Rectangles indicate position of AVP‐ir fibres. Scale bar = 50 µm (F) Boxplots showing that AVP‐ir fibre density is significantly lower in Avp‐shRNA‐injected male (p = 0.00002) and female (p = .05) subjects compared to scramble shRNA‐injected controls. Boxplots indicate individual data points, median, first, and third quartiles

FIGURE 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

FIGURE 3

Ultrasonic vocalizations (USV) and urine marking within the three‐chamber apparatus. (A) BNST AVP knockdown reduced USVs produced during male‐male conditions (p = 0.05). (B) Avp‐shRNA and scramble shRNA‐ injected females did not differ in USVs produced during three‐chamber testing. (C, D) Avp‐shRNA and scramble shRNA‐injected males (C) and females (D) did not differ in urine marking (area covered) toward male or female stimuli during three‐chamber testing. Boxplots indicate individual data points, median, first and third quartiles

FIGURE 4

Aggressive behaviour. In males, BNST AVP knockdown did not alter the latency to bite (A) or latency to rolling attack (B) but did reduce the number of tail rattles during encounters with male intruders (C), p = 0.048. Boxplots indicate individual data points, median, first and third quartiles

FIGURE 5

Copulatory behaviour. Avp‐shRNA and scramble shRNA‐ injected males (A) and females (B) did not differ in number of mounts performed (males) and number of times mounted (females). (C) Avp‐shRNA reduced the number of intromissions performed by males compared to controls (p = 0.002). (D) The number of intromissions received by females was unaltered. Pie chart summarizing the proportion of male subjects that ejaculated (E) and the proportion of male stimulus animals that ejaculated with female subjects (F). BNST AVP knockdown resulted in fewer males ejaculating (p < 0.00001). Boxplots indicate individual data points, median, first and third quartiles

FIGURE 6

Anxiety‐like behaviour in the elevated plus maze (EPM). BNST AVP knockdown did not alter anxiety‐like behaviour. (A) Avp‐shRNA and scramble AAV‐injected males and females did not differ in time spent in the open arm (A), the number of stretch attend postures (B), or head dips (C). Boxplots indicate individual data points, median, first and third quartiles