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

Prepulse inhibition (PPI) of the acoustic startle reflex is a widely used operational measure of sensorimotor gating that is disrupted in several neuropsychiatric disorders. PPI deficits are a hallmark feature of schizophrenia, often associated with attentional and cognitive impairments. Although PPI is extensively used in translational and preclinical research, the cellular and circuit mechanisms underlying this phenomenon remain incompletely understood, even under physiological conditions. Recent evidence suggests that noncholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the caudal pontine reticular nucleus (PnC) contribute to PPI. Here, we investigated the role of PPTg glutamatergic neurons in acoustic startle and PPI. Tract tracing, immunohistochemical analyses, and in vitro whole-cell recordings in wild-type male and female mice demonstrated that PPTg glutamatergic neurons innervate the PnC and establish excitatory synapses onto PnC neurons. Optogenetic inhibition of PPTg→PnC glutamatergic synapses in vivo increased PPI across interstimulus intervals (ISIs) without altering baseline startle responses. In contrast, optogenetic activation of this pathway did not modify PPI. However, activation of PPTg-PnC glutamatergic projections instead of an acoustic prepulse bidirectionally modulated startle responses in a time-dependent manner, suppressing startle at short ISIs while enhancing startle at longer intervals. Additionally, while 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 also activate PnC glutamatergic giant neurons. Together, our findings identify a novel temporally dependent excitatory mechanism within the brainstem startle circuit, through which PPTg glutamatergic neurons modulate sensorimotor gating.