AT1aR dependent GABAa inhibition in the MnPO

Background: The median preoptic nucleus (MnPO) receives input from other circumventricular organs (e.g. SFO and the OVLT) sensitive to circulating Angiotensin II (Ang II) and plasma Na+ concentrations suggesting an involvement in hydromineral balance and blood pressure regulation. Additionally, evidence suggest the SFO synthesizes Ang II and releases it on the MnPO as a neurotransmitter suggesting the role of the MnPO in hydromineral balance and blood pressure regulation is mediated in part by Ang II. The Ang II activation of AT1aR has also been shown to influence the function of GABAaRs though the mechanisms are still unclear. Here we investigate the role of Ang II signaling via the AT1aR in the MnPO and its influence on excitatory/inhibitory balance. Methods: Male Sprague-Dawley rats received infusions of an AAV construct containing GFP reporter and shRNA against AT1aR (shAT1a) or a shRNA scramble (shScr) targeted to the MnPO. Two weeks following AAV infusion, slices containing the MnPO were cut using standard in vitro slice procedures followed by loose patch recordings obtained from GFP labeled neurons. Spontaneous action potential firing was recorded in response to focal application of Ang II or muscimol. Additionally, activity of MnPO neurons in response to muscimol was observed in the presence of a PLC or PKC activator. The GABAa mediated effects in AT1a KD were compared to acute blockade of AT1aRs in rats that did not receive AAV infusions. Western blot and RT-qPCR analyses were used to investigate the effect of AT1a KD on GABAa and KCC2 protein and mRNA expression. Results: Brief focal application of Ang II produced a time dependent increase in spontaneous firing of MnPO neurons. The Ang II dependent enhancement of spontaneous activity was blocked by bath application of the AT1aR antagonist Losartan. Additionally, Ang II failed to alter firing rate of MnPO neurons in shAT1a KD rats. In control animals, the GABAa agonist muscimol decreased action potential activity. In rats that received microinjections of the shAT1a muscimol failed to decrease action potential activity. In AT1a KD rats, RT-qPCR analysis shows a reduction in AT1a and KCC2 mRNA but no reduction in GABAa Beta subunit mRNA. Conclusions: The current findings demonstrate Ang II dependent increases in the excitability of MnPO neurons are mediated by activation of AT1aRs. Moreover, AT1aRs activation also mediates the inhibitory effects of GABAaR activation. The current study suggests the reduction in GABAa dependent inhibition following AT1a KD is mediated by a down regulation of KCC2 and subsequent disruption of intracellular Cl- homeostasis. AT1aR function can modulate the balance of excitatory and inhibitory activity within the MnPO and efferent nuclei involved in the regulation of blood pressure and hydromineral balance. However, mechanisms underlying the dual excitatory/inhibitory functions of AT1aR activation remain unclear.