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 non-cholinergic 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 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 interstimulus intervals 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.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.