Chemogenetic silencing reveals presynaptic Gi/o protein-mediated inhibition of synchronized activity in the developing hippocampusin vivo

The development of neuronal circuits is an activity-dependent process. Research aimed at deciphering the learning rules governing these developmental refinements is increasingly utilizing a class of chemogenetic tools that employ G i/o protein signaling (G i -DREADDs) for neuronal silencing. However, their mechanisms of action and inhibitory efficacy in immature neurons with incompletely developed G i/o signaling are poorly understood. Here, we analyze the impact of G i/o signaling on cellular and network excitability in the neonatal hippocampus by expressing the G i -DREADD hM4Di in telencephalic glutamatergic neurons of neonatal mice of both sexes. Using acousto-optic two-photon Ca 2+ imaging, we report that activation of hM4Di leads to a complete arrest of spontaneous synchronized activity in CA1 in vitro . Electrophysiological analyses demonstrate that hM4Di-mediated silencing is not accounted for by changes in intrinsic excitability of CA1 pyramidal cells (PCs). Instead, activation of hM4Di robustly restrains synaptic glutamate release by the first postnatal week, effectively reducing recurrent excitation in CA1. In vivo , inhibition through hM4Di potently suppresses early sharp waves (eSPWs) and discontinuous oscillatory network activity in CA1 of head-fixed mice before eye opening. In summary, hM4Di dampens glutamatergic neurotransmission through a presynaptic mechanism sufficient to terminate spontaneous synchronized activity in the neonatal CA1. Our findings have implications for designing and interpreting experiments utilizing G i -DREADDs in immature neurons and further point to a potential role of G i/o -dependent endogenous neuromodulators in activity-dependent hippocampal development. Significance Statement Recent advances in understanding how early activity shapes developing brain circuits increasingly rely on G i/o -dependent inhibitory chemogenetic tools (G i -DREADDs). However, their single-cellular mechanisms and efficacy in neurons with immature G i/o signaling are elusive. To close this gap, we analyzed the impact of the G i -DREADD hM4Di in the neonatal murine hippocampus. We report that hM4Di does not mediate hyperpolarization or shunting but rather acts as a potent presynaptic silencer of glutamatergic neurotransmission in CA1 pyramidal cells. Presynaptic inhibition of excitation suffices to arrest spontaneous synchronized network activity in vitro and in vivo . Our findings provide novel insights into the generative mechanisms of spontaneous synchronized activity and bear relevance for applying chemogenetic silencing at early stages of brain development.