Gq DREADD activation of CaMKIIa MnPO neurons stimulates nitric oxide activity

Abstract

Designer receptors exclusively activated by designer drugs (DREADDs) modify cellular activity following administration of the exogenous ligand clozapine-N-oxide (CNO). However, some reports indicate CNO may have off-target effects. The current studies investigate the use of G_q DREADDs in CaMKIIa-expressing neurons in the median preoptic nucleus (MnPO). Male Sprague-Dawley rats (250 g) anesthetized with isoflurane were stereotaxically microinjected in the MnPO with the G_q DREADD (AAV5-CaMKIIa-HM3D-mCherry) or control virus (AAV5-CaMKIIa-mCherry). Following a 2-wk recovery, rats were used for either immunohistochemical Fos analysis or in vitro patch-clamp electrophysiology. In G_q DREADD-injected rats, CNO induced significant increases in Fos staining in the MnPO and in regions that receive direct or indirect projections from the MnPO. In electrophysiological studies, CNO depolarized and augmented firing frequency in both G_q DREADD-positive neurons (G_q DREADD) as well as unlabeled MnPO neurons in slices from G_q DREADD-injected rats (G_q DREADDx). G_q DREADDx neurons also displayed increases in spontaneous postsynaptic current (sPSC) frequency in response to CNO. Additionally, CaMKIIa-positive MnPO neurons, which also express nitric oxide synthase (NOS), were treated with N^ω-nitro-l-arginine (l-NNA; competitive inhibitor of NOS) and hemoglobin (NO scavenger) to assess the role of NO in G_q DREADDx neuron recruitment. Both l-NNA and hemoglobin blocked CNO-induced effects in G_q DREADDx neurons without affecting G_q DREADD neurons. These findings indicate that G_q DREADD-mediated activation of CaMKIIa/NOS expressing neurons in the MnPO can influence the activity of neighboring neurons. Future studies utilizing the use of Gq DREADDs will need to consider the potential recruitment of additional cell populations.NEW & NOTEWORTHY Rats were injected in the median preoptic nucleus (MnPO) with either an adeno-associated virus (AAV) and excitatory (G_q) designer receptor exclusively activated by designer drugs (DREADD) construct or a control AAV. In the G_q DREADD-injected rats only, clozapine-N-oxide (CNO) increased Fos staining in the MnPO and its targets and increased neuron action potential frequency. In electrophysiology experiments with slices with DREADD cells, unlabeled cells were activated and this was likely due to nitric oxide release by the DREADD cells.

Keywords: DREADDs; MnPO; NOS1; nitric oxide.