Monosynaptic inputs to ventral tegmental area glutamate and GABA co-transmitting neurons

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of their functional capabilities. By coupling monosynaptic rabies tracing with intersectional genetic targeting in male and female mice, we found that VTA VGluT2+VGaT+ neurons received diverse brain-wide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus, lateral hypothalamus, midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from dorsal raphe nucleus, lateral habenula, and ventral tegmental area. Based on these and prior data, we hypothesized that lateral hypothalamus and superior colliculus inputs were glutamatergic neurons. Optical activation of glutamatergic lateral hypothalamus neurons activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic superior colliculus neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high stimulation frequency and resulted in head rotation and arrested ambulatory behavior (freezing). Stimulation of glutamatergic lateral hypothalamus neurons, but not glutamatergic superior colliculus neurons, was associated with VTA VGluT2+VGaT+ footshock-induced activity. In addition, inhibition of lateral hypothalamus glutamatergic neurons disrupted VTA VGluT2+VGaT+ tailshock-induced activity. We interpret these results such that inputs to VTA VGluT2+VGaT+ neurons may integrate diverse signals related to the detection and processing of motivationally-salient outcomes. Significance Statement VTA glutamate neurons have roles in motivated behavior and unique neurotransmission capabilities. A specific VTA glutamate neuron subtype, those that co-transmit glutamate and GABA, have unique outcome signaling properties compared to other VTA cell-types. However, the circuits that regulate these neurons are unclear. We identified the whole-brain inputs to VTA glutamate and GABA co-transmitting neurons. We also identify two distinct glutamatergic inputs that activate VTA glutamate and GABA co-transmitting neurons and result in different behavioral repertoires suggestive of threat processing. Together, these results provide novel insights into the circuit and cell-type specific influences on VTA glutamate and GABA co-transmitting neuronal activity as integrators of motivationally salient outcomes.