Co-localization of NK(1)-receptor mRNA with glutamate immunopositivity in cat hypothalamic neurons by the combination of in situ hybridization and immunohistochemistry.

Previous studies had demonstrated that, in the cat, aggression is mediated by glutamatergic neurons in the anterior medial hypothalamus which project to the periaqueductal gray. Additionally, NK(1) receptor activation in the medial hypothalamus plays a role in the regulation of aggressive behavior by the medial amygdala. In the present study, in situ hybridization and immunohistochemistry were combined in order to provide neurochemical characterization of medial hypothalamic neurons containing NK(1)-receptor mRNA. In order to identify NK(1) receptors in cat brain, a 650-bp fragment of the cat NK(1) cDNA was cloned. This fragment was used to synthesize a riboprobe for in situ hybridization. Partial DNA sequence analysis of the fragment indicated a 90% homology with human cDNA. In situ hybridization revealed the presence of NK(1)-receptor mRNA in cat hypothalamic neurons. Tissue used to localize NK(1) receptors was also processed for glutamate immunopositivity. The results demonstrated that NK(1)-receptor mRNA is present in glutamate-immunopositive neurons in the anterior medial hypothalamus of cat, thus reinforcing the hypothesis that NK(1) receptors play an important role in this neural circuit.

Neurokinin-1 expression and co-localization with glutamate and GABA in the hypothalamus of the cat.

Recent behavioral studies using pharmacological techniques have demonstrated that the high affinity substance P (SP) receptor, neurokinin-1 receptor (NK-1), in the medial hypothalamus could be important in mediating defensive rage behavior in the cat. These observations prompted us to use molecular techniques to determine the distribution of NK-1 in the hypothalamus and in other regions of the forebrain relevant to the control of rage behavior. We cloned a 650 bp fragment of the cat NK-1 cDNA. Partial DNA sequence analyses of this fragment indicate 90% homology with the human cDNA. By in situ hybridization (ISH), we showed that NK-1 mRNA was localized in the cytoplasm but not nuclei of cat forebrain neurons. Furthermore, NK-1 mRNA was co-localized in neurons that displayed positive immunolabeling for glutamate or GABA. Moderate labeling was visualized in the anterior medial hypothalamus which receives significant SP input via the stria terminalis from the medial amygdala. Strong labeling was also observed in the basal amygdaloid complex. The functional significance of this labeling pattern is suggested from the observation that both the medial and basal complex of amygdala serve as powerful modulators of defensive rage behavior. Weaker labeling was seen over the posterior medial and lateral hypothalamus. The distribution of NK-1 in the hypothalamus was matched by that of SP-immunoreactive axons and pre-terminals that were observed in the hypothalamus. The overall findings provide anatomical evidence to show that the high affinity SP receptor, NK-1, is linked to glutamate and GABA neurons in the anterior medial hypothalamus and further suggests its likely role in the regulation of feline aggression.