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

Abstract

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.

Figure 1. ILSCm glutamatergic neurons respond to upper field LS and mediate the LS-triggered innate defensive responses.

(a) Schematic of the looming animation and testing environment. (b) Level of freezing during the 30-s period after stimulus onset. (n=7–8 subjects per group; ***P<0.001 by one-way ANOVA with Holm–Sidak post-hoc test) (c) Confocal images of the SC stained for c-fos 30 min after exposure to upper (left) or lower (right) field LS. The superficial layers (SL) included the superficial grey (SGS) and optic layer (SO); the intermediate layers (IL) included the intermediate grey (SGI) and intermediate white layer (SAI); and the deep layers (DL) included the deep grey (SGP) and deep white layer (SAP). The lateral or medial subdivision of the SC is approximately divided by the horizontal meridian in the collicular map. (green=c-fos; blue=DAPI). (d,e) Comparison of group difference of the c-fos levels in different layers of the SC (d) or in the medial and lateral ILSC (e) (n=24 slices per group; *P<0.05, NS P>0.05 by two-way ANOVA with Holm–Sidak post-hoc test) (f) Cell-type specificity of c-fos-expressing cells. Vglut2+, vesicular glutamate transporter 2 positive; PV+, parvalbumin positive. (g) eNpHR3.0–EYFP expression in CamKIIa neurons in the bilateral ILSCm. The inset shows the schematic of the implanted dual fibres. (h) In vivo electrophysiological identification of optogenetic inhibition of ILSCm neuron activities. (i) Experimental timeline of the optogenetic inhibition of the ILSCm during upper field LS. (j) Levels of freezing elicited by LS combined with (yellow) or without (white) optogenetic inhibition of the ILSCm. Black bars represent the EYFP control group. (n=7–8 subjects per group; **P<0.01, *P<0.05 by two-way ANOVA with Holm–Sidak post-hoc test). Values are represented as mean±s.d.; Scale bars: (c) 250 μm, (g) 500 μm.

Figure 2. Optogenetic activation of ILSCm elicits unconditioned freezing behaviour.

(a) ChR2-mCherry expression in CamKIIa neurons in the ILSCm or ILSCl. (red=mCherry; for number of ChR2+ cells in different layers, see Supplementary Fig. 1c). (b) Cannula tips for ChR2:ILSCm and ILSCl mice are indicated by the red and green dots, respectively. (c) Time courses of the averaged FS revealed that the ChR2:ILSCm (red) US (blue rectangle) elicited UF behaviour when compared against ChR2:ILSCl (green). Grey shaded area, animals are still in freezing states; the bottom red bars, the periods with significant group FS differences (n=15, 11 subjects for each group; P<0.05 by t-test). (d) Box plots of UF durations elicited by the US. (n=15, 11, 8 subjects for each group; ***P<0.001 by Kruskal–Wallis one-way ANOVA with Dunn's post-hoc test) (e) UF elicited by the US adapts significantly after repeated trials (red dots) (n=15 subjects, main effect P<0.001 by repeated one-way ANOVA). Inset, the time courses of the averaged FS before and after the fifth US. (f) Time courses of the normalized distance to the centre of the arena reveal that ChR2:ILSCm mice prefer the periphery of the arena after the UF has stopped. (g) Spatial FS map in the pre- or post-US period (3 min) from a sample ChR2:ILSCm mouse. The ‘light on' symbol indicates the onset of the US. Time spent in the centre of the arena (h) and the moving rate of the tested animals (i) during the post-UF period (subtracted the UF period from the post-US period). (n=15, 11 subjects for each group; ***P<0.001, **P<0.01 by two-way ANOVA with Holm–Sidak post-hoc test). Values are represented as mean±s.d.; Scale bars: (a) 250 μm.

Figure 3. Activation of the amygdala is necessary for the UF elicited by the US in the ILSCm.

(a) Left, schematic of the drug injections into the bilateral BLA via cannulas and fibre optic implantations in the ILSCm. Right, examination timeline of the impact of BLA on the UF, elicited by application of the US on the ILSCm. (b) The duration of UF elicited by application of the US in the ILSCm after BLA infusion (muscimol or PBS) at 20 min and 24 h (repeated test) (n=6–7 subjects per group; ***P<0.001, *P<0.05 by two-way ANOVA with Holm–Sidak post-hoc test). (c) A low-magnification image of the amygdala, stained for c-fos, 30 min after applying the US to the ILSCm. The c-fos-positive cells are mainly located in the LA (green=c-fos; blue=DAPI). Pir, piriform cortex; BA, basal nuclei of BLA; CeA, central amygdala; BMA, basomedial amygdala. (d) High-magnification images of c-fos-positive cells in the bilateral LA. (e) The c-fos levels in the bilateral LA (n=18 slices per group; ***P<0.001, *P<0.05 by two-way ANOVA with Holm–Sidak post-hoc test). (f) Schematic of the stimulation experiments in the ILSCm and simultaneous recording in the LA. (g) Activation of an LA neuron during optogenetic stimulation of the ILSCm (top) and a magnified plot showing 10 light pulses (bottom left). (h) This LA neuron is orthodromically activated by ILSCm pulsed-laser stimuli (blue). (i) Peristimulus time histogram (PSTH) of the example LA neuron reveals the distribution of response times to the upstream ILSCm activation (blue=light, peak response latency≈9 ms). (j) Histogram of the peak response latencies to the onset of light pulses (blue) for 13 identified LA neurons. Values are represented as mean±s.d.; Scale bars: (c) 250 μm; (d) 50 μm.

Figure 4. Activation of the LA is temporally correlated with the UF elicited by applying the US to the ILSCm.

Schematic (a) and photograph (b) of the two-site multi-channel optrode system. The inset in b shows the tip of the optrode (white box). (c) PSTHs of two example neurons from the ILSCm and LA show different firing patterns in response to the US in the ILSCm (1-s bins). The red line represents the estimation of the expected baseline rate and the grey shaded area represents the confidence limit of 99%. (d) Z-scored population PSTH of responsive neurons from the ILSCm (red) and LA (green) (100-ms bins), shaded areas represent the s.e.m. The blue bar indicates the optical stimulation, red bar indicates the period of excitation of ILSCm neurons (n=7 cells, compared with the pre-event reference period, P<0.01 by right-tailed t-test), and green bar indicates the period of excitation of LA neurons (n=11 cells). (e) The normalized post-stimulation mean firing rates of responsive LA neurons (green) show a significant adaptation after each trial (n=11 cells, main effect P<0.001 by repeated one-way ANOVA). Contrarily, the firing rates of responsive ILSCm neurons (red) did not adapt across trials (n=7 cells, main effect P=0.96 by repeated one-way ANOVA). (f) The ILSCm and LA example neuron activity (top; scaled by the hot colour map) were aligned with the FS with time (the bottom red bars represent the freezing state). Values are represented as mean±s.d.

Figure 5. The lateral posterior nucleus of the thalamus is the critical monosynaptic relay underlying the ILSCm–LA circuit.

(a) Schematic of AAV–EYFP-mediated anterograde tracing in the SC and EnvA–RV–mCherry in combination with helper AAV-mediated trans-monosynaptic retrograde tracing in the LA. (b) Coronal brain section shows the anterograde projection pattern from the SC (green) and the distribution of trans-monosynaptically labelled neurons from the LA (red). (c) A magnified view of the LP shows the co-localization of axon terminals from the SC projection neurons and the soma of the LP neurons that project to the LA. Inset white box depicts the area. (d) Schematics of the PRV152–EGFP injection in the LA and of the ibotenic acid injection in the LP. (e) Coronal sections show PRV(+) cells (green) in the SC from an intact (top) and LP-lesioned mouse (bottom). (f) PRV(+) cells are mainly located in the ILSCm (n=12 slices; ***P<0.001 by Kruskal–Wallis one-way ANOVA with Dunn's post-hoc test). (g) Comparison of the PRV(+) cells in the medial and lateral ILSC between intact and LP-lesioned mice (n=12 slices per group; ***P<0.001 by two-way ANOVA with Holm–Sidak post-hoc test). Values are represented as mean±s.d.; Scale bars: (b) 1 mm; (c) 250 μm; (e) 500 μm.

Figure 6. Activation of terminals in the LP that projected from the ILSCm-elicited fast responses in LA neurons and unconditioned freezing behaviour.

(a) Schematic of analysing the function of ILSCm–LP pathway. A optic fibre was implanted in the ipsilateral LP of ChR2:ILSCm mice. In addition, a recording electrode was placed in the LA. (b) Confocal image of ChR2-positive terminals in the LP(LR) that from the ILSCm and the tip of optic fibre (the top dotted rectangle). red=mCherry; blue=DAPI. (c) Time courses of the averaged FS reveals that the US (blue rectangle) of ChR2:ILSCm–LP(LR) terminals elicits UF and a prolonged lower FS (blue) compared with the control (black) (n=8 subjects for per group; P<0.05 by t-test). (d) Spatial FS map of the pre- or post-US period (3 min) from a sample ChR2:ILSCm–LP(LR) mouse. The ‘light on' symbol indicates the onset of the US. (e) The UF elicited by the US of ILSCm–LP(LR) terminals adapts significantly after repeated trials (blue dots, n=8 subjects, main effect P<0.001 by repeated one-way ANOVA). (f) Time courses of the normalized distance to the centre of the arena reveal that animals prefer the periphery of the arena after they are relieved of the UF elicited by the US of ChR2:ILSCm–LP(LR) terminals. (g,h) Time spent in the centre of the arena (g) and the animal speed (h) show a significant reduction in the post-UF period for the US of ChR2:ILSCm–LP(LR) terminals (n=8 subjects for per group; **P<0.01 by two-way ANOVA with Holm–Sidak post-hoc test). (i) Box plots of the duration of UF elicited by the US. The glutamate receptor antagonists NBQX plus AP5 (D(-)-2-amino-5-phosphonovaleric acid) infusion in LP(LR) blocks the UF response (n=7–8 subjects per group; ***P<0.001 by Kruskal–Wallis one-way ANOVA with Dunn's post-hoc test). (j,k) A sample LA neuron is activated by the pulsed laser in the upstream ILSCm–LP(LR) terminals (peak response latency ≈5 ms, (k). Values are represented as mean±s.d.; (b) 250 μm.