The ability of animals to respond to life-threatening stimuli is essential for survival. Although vision provides one of the major sensory inputs for detecting threats across animal species, the circuitry underlying defensive responses to visual stimuli remains poorly defined. Here, we investigate the circuitry underlying innate defensive behaviours elicited by predator-like visual stimuli in mice. Our results demonstrate that neurons in the superior colliculus (SC) are essential for a variety of acute and persistent defensive responses to overhead looming stimuli. Optogenetic mapping revealed that SC projections to the lateral posterior nucleus (LP) of the thalamus, a non-canonical polymodal sensory relay, are sufficient to mimic visually evoked fear responses. In vivo electrophysiology experiments identified a di-synaptic circuit from SC through LP to the lateral amygdale (Amg), and lesions of the Amg blocked the full range of visually evoked defensive responses. Our results reveal a novel collicular–thalamic–Amg circuit important for innate defensive responses to visual threats.

The dorsal striatum integrates inputs from multiple brain areas to coordinate voluntary movements, associative plasticity, and reinforcement learning. Its projection neurons consist of the GABAergic medium spiny neurons (MSNs) that express dopamine receptor type 1 (D1) or dopamine receptor type 2 (D2). Cholinergic interneurons account for a small portion of striatal neuron populations, but they play important roles in striatal functions by synapsing onto the MSNs and other local interneurons. By combining the modified rabies virus with specific Cre- mouse lines, a recent study mapped the monosynaptic input patterns to MSNs. Because only a small number of extrastriatal neurons were labeled in the prior study, it is important to reexamine the input patterns of MSNs with higher labeling efficiency. Additionally, the whole-brain innervation pattern of cholinergic interneurons remains unknown. Using the rabies virus-based transsynaptic tracing method in this study, we comprehensively charted the brain areas that provide direct inputs to D1-MSNs, D2-MSNs, and cholinergic interneurons in the dorsal striatum. We found that both types of projection neurons and the cholinergic interneurons receive extensive inputs from discrete brain areas in the cortex, thalamus, amygdala, and other subcortical areas, several of which were not reported in the previous study. The MSNs and cholinergic interneurons share largely common inputs from areas outside the striatum. However, innervations within the dorsal striatum represent a significantly larger proportion of total inputs for cholinergic interneurons than for the MSNs. The comprehensive maps of direct inputs to striatal MSNs and cholinergic interneurons shall assist future functional dissection of the striatal circuits.

BackgroundHerpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer.MethodsBased on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity.ResultsH129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results.ConclusionsWith the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0179-7) contains supplementary material, which is available to authorized users.

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The lateral entorhinal cortex (LEC) receives direct input from olfactory bulb mitral cells and piriform cortical pyramidal cells and is the gateway for olfactory input to the hippocampus. However, the LEC also projects back to the piriform cortex and olfactory bulb. Activity in the LEC is shaped by input from the perirhinal cortices, hippocampus, and amygdala, and thus could provide a rich contextual modulation of cortical odor processing. The present study further explored LEC feedback to anterior piriform cortex by examining how LEC top-down input modulates anterior piriform cortex odor evoked activity in rats. Retrograde viral tracing confirmed rich LEC projections to both the olfactory bulb and piriform cortices. In anesthetized rats, reversible lesions of the ipsilateral LEC increased anterior piriform cortical single-unit spontaneous activity. In awake animals performing an odor discrimination task, unilateral LEC reversible lesions enhanced ipsilateral piriform cortical local field potential oscillations during odor sampling, with minimal impact on contralateral activity. Bilateral LEC reversible lesions impaired discrimination performance on a well learned, difficult odor discrimination task, but had no impact on a well learned simple odor discrimination task. The simple discrimination task was impaired by bilateral reversible lesions of the anterior piriform cortex. Given the known function of LEC in working memory and multisensory integration, these results suggest it may serve as a powerful top-down modulator of olfactory cortical function and odor perception. Furthermore, the results provide potential insight into how neuropathology in the entorhinal cortex could contribute to early olfactory deficits seen in Alzheimer's disease.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK −/− mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. Highfrequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after lowfrequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory. cholecystokinin | NMDA receptor | long-term potentiation | memory | entorhinal cortex M emory is stored in neural networks through changes in synaptic strength (1). Long-term potentiation (LTP) and long-term depression (LTD) are two forms of synaptic plasticity that are believed to represent a neural basis of memory in different brain regions (2-5). The major form of LTP in the hippocampus and neocortex is induced through theta burst stimulation or highfrequency stimulation (HFS) (2, 3). Previous studies have shown that NMDA receptors (NMDARs) play a crucial role in HFSinduced LTP in the hippocampus (6-9) and neocortex (2, 10), and in the formation and consolidation of associative memory (11,12).Serving as the gateway from the hippocampus to the neocortex, the entorhinal cortex forms strong reciprocal connections with the neocortex (13, 14) and shows extensive cholecystokinin (CCK) labeling (15-17) with projections to neocortical areas, including the auditory cortex (13,14,18). CCK is the most abundant cortical neuropeptide (19), and mice lacking the CCK gene exhibit poor performance in a passive avoidance task and display impaired spatial memory (20). Although many studies have focused on GABAergic CCK neurons (21-24), many glutamatergic neurons in the neocortex express CCK (25, 26). We previously found that local infusion of CCK into the auditory cortex of anesthetized rats induces plastic changes that enable auditory cortical neurons to start responding to a light stimulus after its pairing with an auditory stimulus (18). Activation of the entorhinal cortex potentiates neuronal responses in the auditory cortex, and this effect is suppressed by infusion of a CCK-B receptor (CCKBR) antagonist (18), suggesting that the entorhinal cortex enables neocortical plasticity via CCK-containing neurons projecting to the neocortex.If CCK enables cortical neuroplasticity and associative memory formation, then we would expect CCK-induced neuroplasticity to affect LTP. The release of neuropeptides occurs slowly in response to repetitive firing (27,28)....

Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue-induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.

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...