Divergent outputs of the ventral lateral geniculate nucleus mediate visually evoked defensive behaviors

Abstract

Rapid alternations between exploration and defensive reactions require ongoing risk assessment. How visual cues and internal states flexibly modulate the selection of behaviors remains incompletely understood. Here, we show that the ventral lateral geniculate nucleus (vLGN)-a major retinorecipient structure-is a critical node in the network controlling defensive behaviors to visual threats. We find that vLGN^GABA neuron activity scales with the intensity of environmental illumination and is modulated by behavioral state. Chemogenetic activation of vLGN^GABA neurons reduces freezing, whereas inactivation dramatically extends the duration of freezing to visual threats. Perturbations of vLGN activity disrupt exploration in brightly illuminated environments. We describe both a vLGN→nucleus reuniens (Re) circuit and a vLGN→superior colliculus (SC) circuit, which exert opposite influences on defensive responses. These findings reveal roles for genetic- and projection-defined vLGN subpopulations in modulating the expression of behavioral threat responses according to internal state.

Keywords: anxiety; fear; retina; thalamus; vLGN; visual.

Figure 1.. vLGN neurons modulate the duration of visual-threat-evoked freezing

(A) Experimental paradigm for assessing responses to an overhead looming stimulus. (B) Ethogram of behavioral responses during (left of blue line) and after (right of blue line) the looming threat. Black circles above the graph represent the looming stimulus (50 expansions in 82 s). Pink lines represent freezing. Black symbols represent tail rattling. (C and D) Quantification of freezing (C, duration) and tail-rattling (D, incidence) behaviors in response to the looming stimulus (n = 14 mice; Wilcoxon test). (E) Cumulative frequency distribution plot of the time to habituate to the looming stimulus (n = 14 mice). Blue line indicates the total duration of the looming stimulus (82 s). (F) Example image of vLGN^GABA neurons (pink) from a Gad2-Cre;Ai9 mouse. DAPI in blue. Coronal, bregma −2.5 mm. Scale bar, 100 μm. (G) Viral strategy for inactivation and activation of vLGN^GABA neurons. (H) Representative image of vLGN^GABA neurons labeled with hM4Di-mCherry (pink) from an injected Gad2-Cre mouse. DAPI in blue. Coronal, bregma −2.6 mm. Scale bar, 1 mm. (I) Time spent freezing in response to the looming stimulus in all three treatment groups (n = 9 controls, n = 7 inactivate, n = 7 activate; one-way ANOVA). (J) Incidence of tail rattling events in response to the looming stimulus in all three treatment groups (n = 9 controls, n = 7 inactivate, n = 7 activate; Kruskal-Wallis test). (Kand L) Ethograms of responses during (left of blue line) and after (right of blue line) the looming stimulus in mice with their vLGN^GABA neurons inactivated (K) or activated (L). (M) Cumulative frequency distribution plots of the time to habituate to the looming stimulus for all three treatment groups (n = 9 controls, n = 7 inactivate, n = 7 activate; Kolmogrov-Smirnov test). For all figure panels, data are mean ± SEM. Dots on bar plots in (I) and (J) represent data from individual animals. Blue line in (E), (I), and (M) indicates the total duration of the looming stimulus (82 s). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. If data met normality and homogeneity of variance assumptions, parametric tests were used (ex. ANOVA). If not, nonparametric tests were used (ex. Kruskal-Wallis). See Table S1 for further details of the statistical analyses. vLGN, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus. See also Figure S1, Table S1, and Video S1.

Figure 2.. vLGN neural activity scales with environmental illumination

(A) Experimental configuration for fiber photometry recordings from vLGN^GABA neurons. (B) Representative image of vLGN^GABA neurons labeled with GCaMP (green). The location of the fiber optic tract is indicated in yellow. DAPI in blue. Coronal, bregma −2.7 mm. Scale bar, 100 μm. (C) Mean vLGN^GABA recording trace during looming stimuli in freely behaving animals. (D and E) Mean vLGN^GABA recording traces (D) and population activity (E) during looming-evoked freezing behaviors (n = 5 mice GCaMP, n = 5 mice GFP control; repeated-measures ANOVA). (F) Experimental paradigm for assessing vLGN^GABA responses to luminance changes. (G) Representative vLGN^GABA recording trace from a mouse presented with increments and decrements of full-field illumination. (H and I) Mean vLGN^GABA recording trace (H) and vLGN^GABA population activity (I) during increments and decrements of full-field illumination (n = 5 mice GCaMP, n = 5 mice GFP control, 3 trials per mouse; repeated-measures ANOVA). (J) Representative vLGN^GABA recording trace from a mouse presented with blue light flashes of varying intensities from 10 lux (dark blue lines) to 10,000 lux (light blue lines above trace). (K and L) Mean vLGN^GABA recording traces (K) and population activity (L) during light flashes of varying intensities (n = 5 mice GCaMP, n = 5 mice GFP control, 3 trials per mouse; repeated-measures ANOVA). (M) Schematic depicting rapid luminance changes during real-world threats. For all figure panels, data are mean ± SEM. Thin lines in (E), (I), and (L) represent data from individual animals. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. vLGN, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus. See also Figure S2 and Table S1.

Figure 3.. Inactivation of vLGN neurons reduces exploration in brightly illuminated environments

(A) Experimental paradigm of the burrow emergence assay (BEA) performed under brightly illuminated (top; condition A, ~1,000 lux) or dark conditions (bottom; condition B, ~0 lux). (B) Representative trace of a mouse in the top chamber during the BEA under the two lighting conditions. (C) Cumulative percentage of time spent in the top chamber that is either brightly illuminated (in yellow, n = 20 mice) or dark (in black, n = 7 mice; one-way ANOVA). (D) Mean latency to enter the top chamber that is either brightly illuminated (yellow bar, n = 20 mice) or dark (gray bar, n = 7 mice; Mann-Whitney U test). (E) Viral strategy for inactivation and activation of vLGN^GABA neurons. (F) Percentage of time spent in the top chamber under bright (left) and dark (right) conditions in all three treatment groups (n = 12 control, n = 5 inactivate, n = 5 activate; one-way ANOVA). (G) Mean latency to enter the top chamber under bright (left) and dark (right) conditions in all three treatment groups (n = 12 control, n = 5 inactivate, n = 5 activate one-way ANOVA). (H) Experimental paradigm for the light-dark test. (I) Percentage of time spent in the light chamber during the light-dark test in all three treatment groups (n = 12 control, n = 5 inactivate, n = 5 activate; one-way ANOVA). (J) Mean latency to enter the light chamber during the light-dark test in all three treatment groups (n = 12 control, n = 5 inactivate, n = 5 activate; Kruskal-Wallis test). (K) Experimental paradigm for the open field test under brightly illuminated conditions. (L) Percentage of time spent in the center of the arena in the open field test in all three treatment groups (n = 12 control, n = 5 inactivate, n = 5 activate; one-way ANOVA). For all figure panels, data are mean ± SEM. Dots on bar plots represent data from individual animals. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Table S1 and Video S2.

Figure 4.. vLGN neurons modulate duration of threat responses independently of their influence on levels of autonomic arousal

(A) Viral strategy for inactivation and activation of vLGN^GABA neurons. (B-D) Heart rate responses of mice following CNO injections in control mice (B), mice with their vLGN^GABA neurons inactivated (C), and mice with their vLGN^GABA neurons activated relative to saline injections (D; n = 11 control, n = 7 inactivate, n = 7 activate). (E) Mean heart rate of mice with and without CNO across all three treatment groups (n = 11 control, n = 7 inactivate, n = 7 activate; paired t test). (F and G) Viral strategy (F) and experimental paradigm (G) for optogenetic activation of vLGN^GABA neurons via stabilized-step function opsin (SSFO). (H) Heart rate responses of mice prior, during, and after induction of vLGN^GABA neuron activation (n = 8 mice). (I and J) Experimental protocol for prior activation (I) and concurrent activation (J) of vLGN^GABA neuron relative to the presentation of the looming stimulus. (K and L) Ethograms of responses in mice with their vLGN^GABA neurons activated before (K, prior activation) or during (L, concurrent activation) the looming stimulus presentation. Gray line represents the onset (left line) and blue line represents the offset (right line) of the looming stimulus. Pink lines represent freezing. Black symbols represent tail rattling. (M) Time spent freezing under conditions in which vLGN^GABA activation occurred before or during the looming stimulus. Thin lines represent paired data from individual animals (n = 8 mice; Wilcoxon test). For all figure panels, data are mean ± SEM. *p < 0.05; **p < 0.01; ns, not significant. See also Table S1.

Figure 5.. Divergent vLGN outputs impart opposing influences on freezing behaviors

(A) Viral strategy for circuit tracing experiments of vLGN^GABA neuron output projections. (B and C) Example images of vLGN^GABA neuron axons in the SC (B) and in the Re portion of the vMT (C). DAPI in blue. Coronal, bregma −4.2 mm (B) and −0.9 mm (C). Scale bars, 100 μm. (D and E) Experimental schematic (D) and quantification (E) of retrograde-labeled vLGN neurons that project to the SC (green, vLGN→SC), to the Re (pink, vLGN→Re), or both (yellow). (F) Viral strategy for inactivation and activation of vLGN→Re neurons (left) and vLGN^GABA→Re neurons (right). (G and H) Quantification of freezing (G, duration) and tail-rattling (H, incidence) behaviors in response to the looming stimulus for all vLGN→Re treatment groups (left vLGN→Re: n = 9 control, n = 6 inactivate, n = 9 activate; freeze and rattle: Kruskal-Wallis test; right vLGN^GABA→Re: n = 7 control, n = 5 inactivate, n = 5 activate; freeze and rattle: one-way ANOVA). (I) Cumulative frequency distribution plots of the time to habituate to the looming stimulus for vLGN^GABA→Re neuron treatment groups (n = 7 control, n = 5 inactivate, n = 5 activate; Kolmogrov-Smirnov test). (J) Viral strategy for inactivation and activation of vLGN→SC neurons (left) and vLGN^GABA→SC neurons (right). (K and L) Quantification of freezing (K, duration) and tail rattling (L, incidence) behaviors in response to the looming stimulus for all vLGN→SC treatment groups (vLGN→SC; n = 5 control, n = 6 inactivate, n = 6 activate; freeze: one-way ANOVA; rattle: Kruskal-Wallis test; vLGN^GABA→SC; n = 8 control, n = 6 inactivate, n = 6 activate; freeze and rattle: Kruskal-Wallis test). (M) Cumulative frequency distribution plots of the time to habituate to the looming stimulus for vLGN^GABA→SC neuron treatment groups (n = 8 control, n = 6 inactivate, n = 6 activate; Kolmogrov-Smirnov test). (N) Schematic depicting the influence of activating vLGN→Re and vLGN→SC projection neurons during the looming stimulus. (O) Viral strategy for simultaneous activation of vLGN→Re and vLGN→SC neurons. (P and Q) Quantification of freezing (P, duration) and tail rattling (Q, incidence) behaviors in mice with simultaneous activation of vLGN→Re and vLGN→SC neurons during the looming stimulus (n = 6 control, n = 5 activate; freeze: unpaired Student’s t test; rattle: Man-Whitney test). (R) Cumulative frequency distribution plots ofthetimeto habituation in mice with simultaneous activation of vLGN→Re and vLGN→SC neurons (n = 6 control, n = 5 activate; Kolmogrov-Smirnov test). For all figure panels, data are mean ± SEM. Dots on bar plots represent paired data from individual animals. Blue line in (G), (I), (K), (M), (P), and (Q) indicates the total duration of the looming stimulus (82 s). *p < 0.05; **p < 0.01; ***p < 0.00; ns, not significant. vLGN, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus; Xi, xiphoid; Re, nucleus reuniens; ventral midline thalamus, vMT; SC, superior colliculus; sSC, superficial SC; dSC, deep SC. See also Figures S3, S4, S5, S6, and S8; Table S1 ; and Video S3.

Figure 6.. Activation of vLGN^Glut outputs trigger defensive behaviors in the absence of threat stimuli

(A) Schematic depicting the influence of inactivating vLGN→SC and vLGN→SC GABA-specific projection neurons during the looming stimulus. (B) Viral strategy for mapping the cell types of vLGN→SC neurons in Gad2-Cre mice. (C) Example image of vLGN Gad2+ (green) and Gad2− (pink) neurons that project to the SC. DAPI in blue. Coronal, bregma −2.5 mm. Scale bars, 100 μm. (D) Fraction of vLGN Cre+ (green) and Cre− (pink) neurons that project to the SC from Gad2-Cre, Vgat-Cre, and Vglut2-Cre mice. (E) Viral strategy for inactivation and activation of vLGN→SC glutamatergic neurons. (F and G) Quantification of freezing (F, duration) and tail-rattling (G, incidence) behaviors in response to the looming stimulus for all glutamatergic vLGN→SC treatment groups (n = 7 control, n = 5 inactivate, n = 6 activate; vLGN→SC; freeze: one-way ANOVA; rattle: Kruskal-Wallis test). (H) Cumulative frequency distribution plots of the time to habituate to the looming stimulus for all glutamatergic vLGN→SC treatment groups (n = 7 control, n = 5 inactivate, n = 6 activate; Kolmogrov-Smirnov test). (I) Time spent freezing before, during, and after the looming stimulus for all treatment groups (n = 7 control, n = 5 inactivate, n = 6 activate; Friedman test with Dunn’s correction). (J) Ethograms of behavior responses in the home cage without a threat present in mice with their vLGN→SC glutamatergic neurons activated. Pink lines represent freezing. Black symbols represent tail rattling. Green circles represent running. (K and M) Quantification of freezing (K, percentage of time), tail-rattling (L, incidence), and running (M, incidence) behaviors in the home cage without a threat present for all glutamatergic vLGN→SC treatment groups (n = 7 control, n = 5 inactivate, n = 6 activate; Kruskal-Wallis test). (N) Experimental paradigm of the BEA performed under brightly illuminated conditions (~1,000 lux; condition A). (O) Percentage of time spent in the top chamber under brightly illuminated conditions for all glutamatergic vLGN→SC treatment groups (n = 7 control, n = 5 inactivate, n = 6 activate; one-way ANOVA).

Figure 7.. Summary: The vLGN bidirectionally regulates responses to visual threats

For all figure panels, data are mean ± SEM. Dots on bar plots represent paired data from individual animals. Blue line in (F) and (H) indicates the total duration of the looming stimulus (82 s). *p < 0.05; **p < 0.01; ***p < 0.00; ns, not significant. vLGN, ventral lateral geniculate nucleus; vLGNe, vLGN external subdivision, vLGNi, vLGN internal subdivision; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus; SC, superior colliculus. See also Figures S7 and S8 and Table S1.