Processing of visually evoked innate fear by a non-canonical thalamic pathway

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.

and ILSCm (or LP) respectively. Four screws were fixed on the skull around the craniotomies, and packed with dental cement to make a firm steel frame. The two-site multi-channel optrode system was lowed through the drilled holes slowly until the two adjustable MEA were placed 1mm above ILSCm (or LP) and LA, then sealed the holes with softened paraffin and weld the reference to the wire linked to the screw insert in the skull. Last, the whole system was secured with dental cement linked to the steel frame mentioned above. At the end of the experiment, recording sites were marked through electrolytic lesions for further histological verification using fluorescent Nissl staining.

Multi-channel recording. 72 hours later after the surgery, electrophysiological activities of the animal were acquired by OmniPlex® D Neural Data Acquisition
System. The micro-drive screw was turned 1/4 cycle one time to advance the electrode 70 μm deeper until the electrode tips reached the ILSCm (or LP) and the LA. Then the screw was turned a few times again at a rate of less than 1/16 cycle one time to try to detect the good signal-to-noise (SNR, generally > 3 was kept) spikes, thus this step may carry on a couple of days with patience. When the stable spontaneous spikes appeared in both two sites, we started to record the data and optogenetics US (20-Hz frequency with 2 ms pulse width for 2.5 s) was delivered in the ILSCm (or ILSCm-LP terminals) during the animal freely exploring.
Single-unit spikes sorting and analysis. Continuous wide-band data (anesthetized recording using the stereotrodes) or discrete spike data (freely moving recording using To assess the response latencies of the LA neurons to ILSCm somata or ILSCm-LP terminals light stimulation, the PSTH in 40 ms (1-ms bins) following the light pulses during 20-Hz stimulation was calculated and the latency detection procedure is same as mentioned above.
Behavior assay. Looming. Looming apparatus and the testing paradigm was mentioned in 5 . In brief, the apparatus was a 40 length × 40 cm width × 30 cm height closed box, which consisted of a white frosted organic plastic floor and ceiling and 4 transparent plexiglass side walls. All the side walls were coated to be unidirectional perspective, so that the mouse couldn't see the outside but its movement can be  For lesion studies, ibotenic acid (1% in dH2O, 300 nl) was injected ipsilaterally into the LP using similar method as with PRV/CTb injections. Ibotenic acid was injected 9 days before the injection of PRV/CTb in the LA (n = 3, mice were sacrified at 65h post-infection). As for the control test, an equal volume of 0.9 % saline was injected ipsilaterally into LP (n = 4, mice were sacrificed at 60h (n = 1) or 65h (n = 3) post-infection). The stereotaxic coordinate of LP is: (AP: -2.50mm; ML:1.50 mm; DV:-2.40 mm) .
Tissue section preparation and confocal imaging. All mice were deeply anaesthetized with an overdose of urethane (1g/kg) and were transcardially perfused with 400 ML of 0.9 % saline followed by 300 ML of 4 % paraformaldehyde (PFA) solution. The brain was peeled out, post-fixed in PFA overnight and dehydrated in 30% (W/V) sucrose solution for 3 days. The brain was sectioned coronally with Leica freezing microtome. Sequential brain sections, with a thickness of 40μm, were collected in 24 well plates (each 4 serial sections per well), and were stored at -20 °C. coronal slices from the SC (AP from approximately -3.08 mm to -4.48 mm, spaced 320 µm from each other) per mouse were included (LP intact group: 60h (n = 1) or 65h (n = 2); LP lesioned group 65h (n = 3)). Confocal images (20× objective) were acquired and cell counting was automatic performed using image pro plus (IPP).
Statistical Analyses. One-way or two-way analysis of variance (ANOVA), Kruskal-Wallis one-way ANOVA (an extension of rank test to 3 or more groups), student's t-tests, Mann-Whitney U-tests were used to determine statistical differences using SigmaPlot (version 12.0). Post hoc analysis was applied to test individual differences between subgroups. Detailed statistical analyses are provided in supplementary information. Statistical significance was set at *** P < 0.001; ** P <