AT1aR dependent GABAa inhibition in the MnPO
Authors: Farmer George E , Little Joel T. , Bachelor Martha E. , Cunningham J. Tom 
Published: November 2023

The median preoptic nucleus (MnPO), located along the anterior wall of the third ventricle, 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 that although the MnPO is not directly sensitive to circulating Ang II, 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 AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance. Male Sprague‐Dawley rats (250–350g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (shAT1a) or an AAV construct containing the GFP reporter and a shRNA scramble (shScr) targeted to the MnPO. Two weeks following AAV injection, coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Western blot and RT‐qPCR analyses of punch samples containing the MnPO were used to investigate the effect of AT1a KD on GABAa receptor subunits and KCC2 protein and mRNA expression. Brief focal application of Ang II produced a time dependent increase in action potential frequency of MnPO neurons (n = 12). The Ang II dependent increase in activity was blocked by bath application of the AT1aR antagonist Losartan (n = 12). Additionally, Ang II failed to alter firing rate of MnPO neurons in slices prepared from shAT1a injected rats (n = 12). In animals injected with the control vector, the GABAa agonist muscimol decreased action potential activity (n = 10). In rats that received microinjections of the shAT1a, muscimol failed to decrease action potential frequency (n = 10). In AT1a KD rats, RT‐qPCR analysis shows a reduction in AT1a and KCC2 mRNA but no reduction in GABAa Beta subunit mRNA in the MnPO. The current findings demonstrate that Ang II dependent increases in the excitability of MnPO neurons are mediated by AT1aRs. Moreover, AT1aRs are also necessary for the inhibitory effects of GABAaR activation by muscimol. The current study suggests the reduction in GABAa dependent inhibition following AT1a KD could be mediated by a down regulation of KCC2 and subsequent disruption of chloride homeostasis. Taken together, AT1aR in the MnPO contribute to a balance of excitatory and inhibitory activity in MnPO neurons. This balance could be important for the MnPO's role in the regulation of blood pressure and hydromineral balance. However, the signaling mechanisms underlying theses contributions of AT1aRs to MnPO activity remain to be determined.

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Supported by P01 HL088052