Midbrain Glutamatergic Neurons Modulate the Acoustic Startle Reflex and Prepulse Inhibition in Mice

Abstract Prepulse inhibition (PPI) of the auditory startle reflex task is a widely recognized operational measure of sensorimotor gating. PPI deficits are a hallmark feature of schizophrenia, often associated with attentional and cognitive impairments. Despite its extensive use in preclinical research for screening antipsychotic drugs, the precise cellular and circuit mechanisms underlying PPI remain unclear, even under physiological conditions. Recent evidence suggests that non-cholinergic inputs from the pedunculopontine tegmental nucleus (PPTg) to the caudal pontine reticular nucleus (PnC) mediate PPI. In this study, we investigated the contribution of PPTg glutamatergic neurons to acoustic startle and PPI. Tract-tracing, immuno-histochemical analyses, and in vitro whole-cell recordings in wild-type mice confirmed that PPTg glutamatergic neurons innervate the PnC. Optogenetic inhibition of PPTg-PnC glutamatergic synapses in vivo resulted in increased PPI across various interstimulus intervals. Notably, while optogenetic activation of this pathway had no additional effect on startle and PPI, activation of this connection alone before startle stimulation reduced startle at short interstimulus intervals and increased startle at longer intervals. Furthermore, although PPTg glutamatergic inputs target PnC glycinergic neurons, our in vitro whole-cell recordings combined with optogenetic stimulation at PPTg-PnC synapses revealed that PPTg glutamatergic inputs activate PnC glutamatergic giant neurons. Our findings identify a feed-forward excitatory mechanism within the brainstem startle circuit, whereby PPTg glutamatergic inputs modulate PnC neuronal activity. These results provide new insights into the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological disorders. Significance Statement The pedunculopontine tegmental nucleus (PPTg) contributes to prepulse inhibition (PPI) of startle, a translational measure of sensorimotor gating that is impaired in neuropsychiatric disorders, including schizophrenia. While PPTg cholinergic neurons have been shown not to contribute to PPI, the mechanisms by which non-cholinergic PPTg neurons modulate PPI remain incompletely understood. Here, combining tract-tracing, optogenetics, in vitro electrophysiology, and in vivo startle testing in mice, we show that PPTg glutamatergic neurons that innervate startle-mediating neurons of the caudal pontine reticular nucleus (PnC) play a critical role in PPI. Their activation modulates startle responses in a time-dependent manner, either decreasing or increasing startle magnitude. These findings refine our understanding of sensorimotor gating neural mechanisms and may inform therapeutic strategies.