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Vasopressin regulates complex behaviors such as anxiety, parenting, social engagement and attachment and aggression in a species-specific manner. The capacity of vasopressin to modulate these behaviors is thought to depend on the species-specific distribution patterns of vasopressin 1a receptors (V1aRs) in the brain. There is considerable individual variation in the pattern of V1aR binding in the brains of the prairie vole species, Microtus ochrogaster. We hypothesize that this individual variability in V1aR expression levels is associated with individual variation in a polymorphic microsatellite in the 5' regulatory region of the prairie vole v1ar gene. Additionally, we hypothesize that individual variation in V1aR expression contributes to individual variation in vasopressin-dependent behaviors. To test these hypotheses, we first screened 20 adult male prairie voles for behavioral variation using tests that measure anxiety-related and social behaviors. We then assessed the brains of those animals for V1aR variability with receptor autoradiography and used polymerase chain reaction to genotype the same animals for the length of their 5' microsatellite polymorphism in the v1ar gene. In this report, we describe the results of this discovery-based experimental approach to identify potential gene, brain and behavior interrelationships. The analysis reveals that V1aR levels, in some but not all brain regions, are associated with microsatellite length and that V1aR levels in those and other brain regions correlate with anxiety-related and social behaviors. These results generate novel hypotheses regarding neural control of anxiety-related and social behaviors and yield insight into potential mechanisms by which non-coding gene polymorphisms may influence behavioral traits.

Receptor autoradiography using selective radiolabeled ligands allows visualization of brain receptor distribution and density on film. The resolution of specific brain regions on the film often can be difficult to discern owing to the general spread of the radioactive label and the lack of neuroanatomical landmarks on film. Receptor binding is a chemically harsh protocol that can render the tissue virtually unstainable by Nissl and other conventional stains used to delineate neuroanatomical boundaries of brain regions. We describe a method for acetylcholinesterase (AChE) staining of slides previously processed for receptor binding. AChE staining is a useful tool for delineating major brain nuclei and tracts. AChE staining on sections that have been processed for receptor autoradiography provides a direct comparison of brain regions for more precise neuroanatomical description. We report a detailed thiocholine protocol that is a modification of the Koelle-Friedenwald method to amplify the AChE signal in brain sections previously processed for autoradiography. We also describe several temporal and experimental factors that can affect the density and clarity of the AChE signal when using this protocol.

Arginine vasopressin and its V1a receptor subtype (V1aR) are critical for pair bond formation between adult prairie voles. However, it is unclear which brain circuits are involved in this vasopressin-mediated facilitation of pair bond formation. Here, we examined mating-induced Fos expression in several brain regions involved in sociosexual and reward circuitry in male prairie voles. Consistent with studies in other species, Fos expression was induced in several regions known to be involved in sociosexual behavior, namely, the medial amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Fos induction also occurred in limbic and reward regions, including the ventral pallidum, nucleus accumbens, and mediodorsal thalamus (MDthal). Next, we infused a selective V1aR antagonist into three candidate brain regions that seemed most likely involved in vasopressin-mediated pair bond formation: the ventral pallidum, medial amygdala, and MDthal. Blockade of V1aR in the ventral pallidum, but not in the medial amygdala or MDthal, prevented partner preference formation. Lastly, we demonstrated that the mating-induced Fos activation in the ventral pallidum was vasopressin-dependent, since over-expression of V1aR using viral vector gene transfer resulted in a proportionate increase in mating-induced Fos in the same region. This is the first study to show that vasopressin neurotransmission occurs in the ventral pallidum during mating, and that V1aR activation in this region is necessary for pair bond formation in male prairie voles. The results from this study have profound implications for the neural circuitry underlying social attachment and generate novel hypotheses regarding the neural control of social behavior.