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29 30 31 32 33 34 2 35 SUMMARY 36Innate defensive responses are essential for animal survival and are conserved 37 across species. The ventral tegmental area (VTA) plays important roles in learned ap-38 petitive and aversive behaviors, but whether it plays a role in mediating or modulating 39 innate defensive responses is currently unknown. We report that GABAergic neurons 40 in the mouse VTA (VTA GABA+ ) are preferentially activated compared to VTA dopa-41 minergic (VTA DA+ ) neurons when a threatening visual stimulus evokes innate defen-42 sive behavior. Functional manipulation of these neurons showed that activation of 43 VTA GABA+ neurons is indispensable for looming-evoked defensive flight behavior and 44 photoactivation of these neurons is sufficient for looming-evoked defensive-like flight 45 behavior, whereas no such role can be attributed for VTA DA+ neurons. Viral tracing 46 and in vivo and in vitro electrophysiological recordings showed that VTA GABA+ neu-47 rons receive direct excitatory inputs from the superior colliculus (SC). Furthermore, 48 we showed that glutamatergic SC-VTA projections synapse onto VTA GABA+ neurons 49 that project to the central nucleus of the amygdala (CeA) and that the CeA is involved 50 in mediating the defensive behavior. Our findings demonstrate that visual information 51 about aerial threats access to the VTA GABA+ neurons mediating innate behavioral re-52 sponses, suggesting a more general role for the VTA. 53 54 Keywords: Ventral tegmental area; GABAergic neurons; innate fear; superior collic-55 ulus; looming; defensive responses.56 57 109found that VTA GABA+ neurons were significantly activated by the aversive visual stim-110 ulus. Selective optogenetic inhibition and activation of VTA GABA+ neurons showed 111 that they were indispensable and inducing for looming-evoked defensive behavior. 112Tracing and electrophysiological data show that VTA GABA+ neurons received glutama-113 tergic inputs from SC and sent long projections to the CeA, which were also likely in-114 5 volved in the defensive behavior. To the best of our knowledge, this is the first evi-115 dence showing the involvement of VTA GABA+ neurons in an innate, evolutionally con-116 served, visually-evoked defensive responses. 117 118 RESULTS 119 1. VTA GABA+ neurons respond to looming stimulus, which evokes defensive be-120 havior 121According to previous study (Yilmaz and Meister, 2013), mice were placed in an open 122 field with a nest as a hiding place, and the presentation of an upper field expanding 123 dark disc stimulus (looming stimulus) mimicking the approach of an aerial predator 124 triggered transient intermittent periods of immobility (intersperse immobility) follow-125 ing by flight-to-nest and hiding in nest behavior ( Figure 1A). A comparison of vari-126 ous looming stimuli, including front field expanding dark disc stimulus, upper field 127 expanding white disc stimulus, lower field expanding dark disc stimulus and upper 128 field expanding dark disc stimulus, only upper field expand...

Dopamine (DA) system is intriguing in the aspect that distinct, typically opposing physiological functions are mediated by D1 dopamine receptors (Drd1) and D2 dopamine receptors (Drd2). Both Drd1+ and Drd2+ neurons were identified in superior colliculus (SC), a visuomotor integration center known for its role in defensive behaviors to visual threats. We hypothesized that Drd1+ and Drd2+ neurons in the SC may play a role in promoting instinctive defensive responses. Optogenetic activation of Drd2+ neurons, but not Drd1+ neurons, in the SC triggered strong defensive behaviors. Chemogenetic inhibition of SC Drd2+ neurons decreased looming-induced defensive behavior, suggesting involvement of SC Drd2+ neurons in defensive responses. To further confirm this functional role of Drd2 receptors, pretreatment with the Drd2+ agonist quinpirole in the SC impaired looming-evoked defensive responses, suggesting an essential role of Drd2 receptors in the regulation of innate defensive behavior. Inputs and outputs of SC Drd2+ neurons were investigated using viral tracing: SC Drd2+ neurons mainly receive moderate inputs from the Locus Coeruleus (LC), whilst we did not find any incoming projections from other dopaminergic structures. Our results suggest a sophisticated regulatory role of DA and its receptor system in innate defensive behavior.

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