Cellular mechanisms and efficacy of hM4Di-dependent chemogenetic silencing in the developing hippocampus

Chemogenetic techniques have broadened our understanding of how early activity shapes developing brain circuits. A widely used chemogenetic actuator is hM4Di, which employs Gi/o-protein signaling for neuronal inhibition. However, its single-cellular mechanisms and efficacy in neurons with immature Gi/o signaling are elusive. Here, we investigate the applicability of transgenically expressed hM4Di for silencing pyramidal cells in the hippocampus of neonatal mice. Electrophysiological recordings from CA1 pyramidal cells demonstrate that hM4Di does not mediate hyperpolarization or shunting and, in addition, leaves action-potential firing unaffected. Instead, hM4Di-induced Gi/o signaling potently restrains synaptic glutamate release by the first postnatal week. Using acousto-optic two-photon Ca2+ imaging, we demonstrate that hM4Di-mediated presynaptic inhibition of excitation suffices to arrest spontaneous synchronized network activity in CA1. Collectively, our data identify presynaptic Gi/o signaling as a potent source of network-level inhibition in the developing hippocampus. Our findings provide insights into the generative mechanisms of spontaneous synchronized activity and are relevant to the application of chemogenetic approaches at early developmental stages.