The neuropeptide arginine-vasopressin (AVP) has been strongly implicated in the regulation of social behavior and communication across species (Caldwell et al., 2008; Goodson and Bass, 2001; Insel, 2010; Kelly and Goodson, 2014; Rigney et al., 2022a), including humans (Oztan et al., 2020; Parker et al., 2019), and often in sex-specific ways. Indeed, one of the largest and most evolutionarily conserved sex different systems in the vertebrate brain is the male-biased and steroid-dependent AVP innervation of limbic structures (de Vries, 2008; De Vries and Panzica, 2006; Goodson and Bass, 2001), which originates from neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (MA) (De Vries and Buijs, 1983; Rigney et al., 2023a; Rood et al., 2013). Removing BNST AVP cells or knocking down AVP within BNST reduces male social behavior, indicating that this system is a powerful regulator of male social behavior (Kelly and Goodson, 2014; Rigney et al., 2023b). BNST AVP cells send denser projections to several brain regions in males than in females, including the ventral pallidum (vPal) (DiBenedictis et al., 2020; Rigney et al., 2023a), a key node in the mesolimbic reward system (Morais-Silva and Lobo, 2024; Root et al., 2015; Soares-Cunha and Heinsbroek, 2023) implicated in reward seeking (Ottenheimer et al., 2018), hedonic impact (Smith and Berridge, 2005), emotional regulation (Knowland et al., 2017), and social investigation (Wang et al., 2020).

The vPal also expresses high levels of the vasopressin 1a receptor (V1aR) (Dumais and Veenema, 2016; Freeman et al., 2020), the dominant AVP receptor in the nervous system (Koshimizu et al., 2012), and one that mediates AVP effects on social recognition (Bielsky et al., 2005; Landgraf et al., 1995), social interest (Rigney et al., 2024), play behavior (Bredewold et al., 2014; Veenema et al., 2013), aggression and territoriality (Albers, 2012; Gobrogge et al., 2009), parental care (Bayerl and Bosch, 2019; Wang et al., 1994), and anxiety-like behavior (Landgraf et al., 1995; Liebsch et al., 1996). Pharmacological blockade of V1aR or viral-mediated V1aR partial knockdown in the vPal impairs formation of pair-bond mate-preferences in male prairie voles (Barrett et al., 2013; Lim and Young, 2004), whereas upregulation of vPal V1aR facilitates it (Lim et al., 2004; Pitkow et al., 2001). In adult rats, blocking V1aR in the vPal reduces male attraction to conspecific females but, surprisingly, increases female interest in male conspecifics (DiBenedictis et al., 2020). In juvenile rats, V1aR activation in the vPal decreases play fighting in males while increasing it in females (Lee et al., 2024). Taken together, these results suggest that AVP/V1aR interactions in the vPal result in sex differences in the regulation of social behavior.

To test this hypothesis, we examined whether reducing V1aR expression in the vPal, using viral expression of a short hairpin RNA (shRNA) targeted against V1aR mRNA, would impact social investigation, adult ultrasonic vocalizations, and urine marking in male and female mice, all aspects of mouse communication that show greater levels of expression in males than in females (Kas et al., 2014; Lehmann et al., 2013; Wöhr, 2014). In addition, given the role of the vPal in motivation (Reichard et al., 2019; Soares-Cunha and Heinsbroek, 2023), we tested whether vPal V1aR knockdown would more generally alter social (copulatory and agonistic behavior) as well as non-social (anxiety-like behavior, hedonic processing) behavior.