Shared behavioural impairments in visual perception and place avoidance across different autism models are driven by periaqueductal grey hypoexcitability in Setd5 haploinsufficient mice

Abstract

Despite the diverse genetic origins of autism spectrum disorders (ASDs), affected individuals share strikingly similar and correlated behavioural traits that include perceptual and sensory processing challenges. Notably, the severity of these sensory symptoms is often predictive of the expression of other autistic traits. However, the origin of these perceptual deficits remains largely elusive. Here, we show a recurrent impairment in visual threat perception that is similarly impaired in 3 independent mouse models of ASD with different molecular aetiologies. Interestingly, this deficit is associated with reduced avoidance of threatening environments-a nonperceptual trait. Focusing on a common cause of ASDs, the Setd5 gene mutation, we define the molecular mechanism. We show that the perceptual impairment is caused by a potassium channel (Kv1)-mediated hypoexcitability in a subcortical node essential for the initiation of escape responses, the dorsal periaqueductal grey (dPAG). Targeted pharmacological Kv1 blockade rescued both perceptual and place avoidance deficits, causally linking seemingly unrelated trait deficits to the dPAG. Furthermore, we show that different molecular mechanisms converge on similar behavioural phenotypes by demonstrating that the autism models Cul3 and Ptchd1, despite having similar behavioural phenotypes, differ in their functional and molecular alteration. Our findings reveal a link between rapid perception controlled by subcortical pathways and appropriate learned interactions with the environment and define a nondevelopmental source of such deficits in ASD.

Fig 1. ASD mice exhibit delayed and less vigorous looming escape responses.

(A) LER paradigm showing the shelter’s location and the threat zone (left) and LER example (right). (B) Paradigm schematic. Day 0 (D0) was used for acclimatisation. D1-D5 consisted of an acclimatisation period (grey) followed by 3 LER tests (red). The looming stimulus consisted of 5 consecutive looms (right). (C) Raster plot of mouse speed during LER (white, dotted vertical lines denote the start of each loom; solid white line denotes the end of the stimulus) for Setd5^+/+ (upper, n = 14, 60 trials) and Setd5^+/− (lower, n = 14, 198 trials), sorted by reaction time. Bottom, distribution of reaction times for all Setd5^+/+ (black) and Setd5^+/− (red) trials (p < 0.001, two-sample Kolmogorov–Smirnov test). (D) Left, example trials based on whether the mouse responds within 1 of the 5-loom stimuli, after the fifth (>5), or not at all (NR, no response) from one Setd5^+/− mouse. Grey shaded areas represent the frames used to calculate the speed at stimulus onset (S_at) and the immediate response speed (S_im). Right, proportion of escapes to loom presentations. (E) Total looms triggered across the 5 test days (Setd5^+/+, 4.36 looms; Setd5^+/−, 16 looms, P = 0.013). (F) Total shelter exits across the 5 test days (Setd5^+/+, 6.0; Setd5^+/−, 24.5, P = 0.019). (G) Average reaction time (left) and maximum escape speed (right) per animal (reaction time, P = 0.001; max. escape speed, P < 0.001). (H) As (G), but only for the very first loom presentation (reaction time, P = 0.046; max. escape speed, P = 0.166). (I) Average immediate speed change following the stimulus presentation for all trials where the mice escape within the first loom presentation (left, Setd5^+/+, n = 14, black, p < 0.001; right, Setd5^+/−, n = 14, red, p < 0.001). S_at is the mean speed of the animal ±50 ms of stimulus onset, and S_im is the mean speed of the animal 300–800 ms after stimulus onset. (J) As (I) but for trials where the mice escape during or after the second loom (left, Setd5^+/+, n = 3, black, p = 0.007; right, Setd5^+/−, n = 11, red, p < 0.001). (K) Proportion of response types per genotype (X² = 103.9, p < 0.001, X² test of independence). P-values: Wilcoxon’s test, p-values: paired t test, unless specified. The data underlying this figure can be found in S1 Data.

Fig 2. Altered adaptation and repetitive behavioural phenotype to the LER.

(A) Left, graphic depicting exits where the mouse enters (dotted line, filled dot) or does not enter (dashed line, open dot) the threat zone. Right, ethogram of exploratory shelter exit behaviour during the prestimulus acclimatisation, the first and last test of the LER paradigm. Each row represents one animal. (B-D) Adaptation in the number of shelter exits, average number of looms triggered and reaction times across days (B, Setd5^+/+: p = 0.2189, Setd5^+/−: p = 0.4974; C, Setd5^+/+, p = 0.0087; Setd5^+/−, p = 0.2192; D, Setd5^+/+, p = 0.890; Setd5^+/−, p < 0.001). (E) Relationship between the number of shelter exits and the average reaction time per animal (Setd5^+/+, p = 0.626; Setd5^+/−, p = 0.002). (F) As (E) but for maximum escape speed per animal (Setd5^+/+, p = 0.547; Setd5^+/−, p = 0.053). (G) Left, reward trial example showing the location of the food reward within the threat zone. Right, example trajectories during the reward trials show the mouse’s position for the 3 s before triggering the loom (light grey) and the 6 s following the stimulus start (black or red). Filled dots represent the position of the mouse when the stimulus was triggered, grey square represents the shelter, and the yellow star shows the position of the food reward. (H) Number of looms triggered during the reward trial (Setd5^+/+, 1 bout; Setd5^+/−, 8 bouts, P = 0.005). (I) Ethograms of exits, as in (A), during the reward trials show an increased probability of Setd5^+/− mice leaving the shelter during a trial (number of exits, 2.12 for Setd5^+/+, 6.88 for Setd5^+/−, p = 0.057). (J) Reaction time (top panel, Setd5^+/−, 44 trials, r = 0.828, p = 0.0001) and escape vigour (bottom panel, Setd5^+/−, 44 trials, r = 0.184, p = 0.5116) during repeated presentations of the loom. Trials when the animal was interacting with the reward were excluded. P-values: Wilcoxon’s test, p-values: Pearson’s correlation test, unless specified. Plotted linear fits depict the statistically significant correlations. The data underlying this figure can be found in S2 Data.

Fig 3. Conserved behavioural changes across etiologically distinct autism models.

(A) Raster plot of mouse speed in response to the looming stimuli for Ptchd1^Y/+ (upper, n = 9, 49 trials) and Ptchd1^Y/− (lower, n = 9, 259 trials), sorted by reaction time. Bottom, distribution of reaction times for all Ptchd1^Y/+ (black) and Ptchd1^Y/− (orange) trials. (B) Left, example trials based on whether the mouse responds within 1 of the 5-loom stimuli, after the fifth (>5), or not at all (NR, no response) from 1 Ptchd1^Y/− mouse. Right, proportion of escape to loom presentations. Trials where mice escaped within the first loom: Ptchd1^Y/+, 0.826, Ptchd1^Y/−, 0.374, p = 0.004. (C) Average reaction time and (D) maximum escape speed per animal, for all trials where the mice escaped (reaction time; Ptchd1^Y/+, 0.411 s, Ptchd1^Y/−, 1.41 s, p = 0.002; maximum escape speed; Ptchd1^Y/+, 69.1 cm s⁻¹, Ptchd1^Y/−, 54.2 cm s⁻¹, p = 0.005). (E) Average total looms triggered per genotype across 5 days of testing (Ptchd1^Y/+, 5.44 looms; Ptchd1^Y/−, 28.8 looms, P = 0.004). (F) Average total shelter exits across the 5 test days (Ptchd1^Y/+, 15; Ptchd1^Y/−, 53, P = 0.025). (G) Ethogram of shelter exits during the prestimulus acclimatisation, first and last trial of the LER paradigm. Each row represents one animal, filled and open dots represent exits that crossed into the threat zone or not, respectively. (H) Relationship between the number of shelter exits and the average reaction time per animal (Ptchd1^Y/+, p = 0.126; Ptchd1^Y/−, r = 0.76, p = 0.012, Pearson’s correlation). (I-P) Same as (A-H) but for Cul3. (I) Cul3^+/+ (top, n = 10, 61 trials) and Cul3^+/− (bottom, n = 10, 112 trials). Bottom, distribution of reaction times (p < 0.001, two-way KS test). (J) Cul3^+/+, 0.892, Cul3^+/−, 0.347, p < 0.001, two-way KS test). (K) Reaction time; Cul3^+/+, 0.464 s, Cul3^+/−, 1.11 s, P < 0.001. (I) Maximum escape speed; Cul3^+/+, 62.7 cm s⁻¹, Cul3^+/−, 46.8 cm s⁻¹, P < 0.001). (M) (Cul3^+/+, 5.60 looms; Cul3^+/−, 12.9 looms, P = 0.023). (N) (Cul3^+/+, 47; Cul3^+/−, 89, P = 0.006). (P) (Cul3^+/+, p = 0.078; Cul3^+/−, p = 0.571, Pearson’s correlation). Box-and-whisker plots show median, IQR, and range. Shaded areas represent SEM. Lines are shaded areas, mean ± SEM, respectively. P-values are Wilcoxon’s test, p-values: two-sample Kolmogorov–Smirnov test, unless specified. The data underlying this figure can be found in S3 Data.

Fig 4. Activation of deep SC neurons recapitulates delayed LER in Setd5^+/− animals.

(A) Timeline of the experimental protocol for optogenetic activation of the dmSC. (B) Video frame during an optogenetics trial. (C) Confocal micrograph of AAV-ChR2 expression in dmSC and optic fibre location reconstruction, scale bar: 200 μm. (D) Raster plot of mouse speed in response to optogenetic activation, sorted by laser intensity and (E) mean speed responses at increasing laser intensities for Setd5^+/+ (n = 4, 312 trials). (F, G) As (D, E) but for Setd5^+/− mice (n = 4, 291 trials). Blue-shaded areas represent the laser stimulation. (H) Subplots of trials in (D, F) showing the immediate change in speed upon light activation at different laser intensities for Setd5^+/+ (n = 4, paired t tests) and Setd5^+/− (n = 4, paired Wilcoxon’s tests) optogenetics trials. S_at is the mean speed of the animal ±50 ms of laser onset, and S_im is the mean speed of the animal 300–800 ms after laser onset. (I) Proportion of trials at different laser intensities that either show an increase (white), decrease (black), or no change (grey) in speed upon light activation for Setd5^+/+ (top) and Setd5^+/− (bottom). 0.01 mW mm⁻²: 35 trials, p = 0.649; 0.5 mW mm⁻²: 111 trials, p = 0.193; 5 mW mm⁻²: 50 trials, p < 0.001; 10 mW mm⁻²: 49 trials, p < 0.001; 15 mW mm⁻²: 61 trials, p = 0.004; 20 mW mm⁻²: 49 trials, p < 0.001, X² test of independence. (J) Schematic of the behavioural divergence between genotypes with increasing stimulus intensity (laser or loom). (K) Summary of mean ± SEM of reaction time to the LER paradigm at different stimulus contrasts (p = 0.021 for the interaction between genotype and contrast, Setd5^+/+, n = 13, 68 trials; Setd5^+/−, n = 13, 59 trials, repeated measures ANOVA. p = 0.018 for 98% contrast, with multiple comparisons and Bonferroni correction. Setd5^+/+, 24 trials; Setd5^+/−, 22 trials). (l) Proportion of trials at different contrast looms that show an increase (white), decrease (black), or no change (grey) in speed upon light activation (20%: p = 0.698; 50%, p = 0.026; 98%, p = 0.021, X² test of independence). The data underlying this figure can be found in S4 Data.

Fig 5. Setd5^+/− dPAG cells are hypoexcitable.

(A) Schematic of the experimental approach, in vitro patch clamp recordings (top) and micrograph of VGluT2⁺ dmSC projections to the dPAG infected with AAV9-ires-DIO-ChR2 (green) with dPAG biocytin filled and recorded cells (red and arrow heads, scale bar: 100 μm). (B) Top, whole-cell voltage clamp traces of example Setd5^+/+ and Setd5^+/− cells (black and red, respectively) responding to 10 Hz light stimulation (blue ticks). Amplitude (middle, p = 0.078) and relative EPSC amplitude (bottom, p = 0.565) of responses to sequential light pulses in a 10-Hz train. (C) Intrinsic properties of Setd5^+/+ (n = 6, 11 cells) and Setd5^+/− (n = 7, 14 cells) dPAG cells. Input resistance (P = 0.756, top left), membrane constant tau (P = 0.436, top right), membrane capacitance (P > 0.995, bottom left) and resting membrane potential (P = 0.213, bottom right). (D) Summary of the relationship between current injection and action potential firing for putative glutamatergic cells showing a strong reduction in firing (p < 0.001). Inset, representative example traces to a 50-pA current injection. Grey area indicates the current injection values that significantly differ between Setd5^+/+ cells (black) and Setd5^+/− cells (red) found by a multiple comparisons analysis with Tukey correction. (E) Average shape and (F) phase plane analysis of the action potentials generated in the rheobase sweep (Setd5^+/+, 13 cells, 42 spikes; Setd5^+/−, 14 cells, 72 spikes). (G) Summary of the relationship between current injection and action potential firing for all Setd5^+/+ cells (black, n = 18) and Setd5^+/− cells (red, n = 20) before and after (Setd5^+/+: blue, n = 12 cells; Setd5^+/−: purple, n = 13 cells) application of α-Dendrotoxin (α-DTX, 100 nM, p > 0.995 and p = 0.0147 for the effect of α-DTX on Setd5^+/+ and Setd5^+/− firing, respectively). Inset, representative example traces from Setd5^+/+ cells (blue) and Setd5^+/− (purple) cells after α-DTX application to a 50-pA current injection. (H) Effect of α-DTX on firing in response to 120 pA current injection. Before α-DTX (Setd5^+/+ versus Setd5^+/−; p = 0.0017), effect of α-DTX on Setd5^+/+ (before versus after α-DTX; P > 0.995) and Setd5^+/− (before versus after α-DTX; P < 0.001, Setd5^+/+ before versus Setd5^+/− after α-DTX; P > 0.995). Multiple comparison analysis after rm-ANOVA with Tukey correction. (I) α-DTX-sensitive current densities in Setd5^+/+ and Setd5^+/− dPAG neurons (p < 0.001). Inset, example of α-DTX-sensitive traces (Setd5^+/+: black, n = 7 cells; Setd5^+/−: red, n = 6 cells). Grey area indicates the current values that significantly differ between Setd5^+/+ cells (black) and Setd5^+/− cells (red) found by a multiple comparisons analysis with Tukey correction. (J) Action potential shape and (K) phase plane analysis of the action potentials generated in the rheobase current in Setd5^+/+ cells (without α-DTX, black, n = 19; with α-DTX, blue, n = 12), Setd5^+/− cells (without α-DTX, red, n = 21; with αDTX, purple, n = 13). The data underlying this figure can be found in S5 Data.

Fig 6. Protein levels of Kv channels are not changed in Setd5^+/− dPAG cells.

(A) Volcano plot for differential protein levels between adult Setd5^+/+ and Setd5^+/− mice (n = 6 samples per genotype, cyan dots represent proteins annotated as ion-channels and purple dots represent Kv1.1, Kv1.2, and Kv1.6. Lines and shaded areas, mean ± SEM, respectively. Box-and-whisker plots show the median, IQR, and range. P-values are Wilcoxon’s rank sum test. p-values are two-way repeated measures ANOVA. Volcano plot horizontal dashed line represents the significance threshold (P-value < 0.1) from a two-sided moderated t test, while the vertical dashed lines indicate fold change values greater or lower than 0.4 between Setd5^+/+ and Setd5^+/−. (B, C) Tissue-specific western blots of Kv1.1 protein content in the dorsal periaqueductal grey (dPAG) for Setd5^+/+ and Setd5^+/− mice and (C) their quantification (dPAG: Setd5^+/+, 0.259; Setd5^+/−, 0.157, P = 0.090). (D) Schematic of SC and PAG regions of interest. (E, F) Antibody staining for Kv1.1 in (E) Setd5^+/+ and (F) Setd5^+/− (E, 55 μm projection; F, 30 μm projection). Arrowheads indicate somas stained for Kv1.1. Scale bar: D: 200 μm; E: 50 μm; F: 100 μm. P-values are two-tailed Wilcoxon’s signed-rank test. The data underlying this figure can be found in S6 and S7 Data.

Fig 7. Pharmacological Kv channel block rescues delayed LER and place avoidance.

(A) Timeline of in vivo α-DTX cannula experiments. (B) Confocal micrograph of a coronal section of the location of the cannula above the dPAG as well as the expression of neurobiotin that was infused into the cannula at the end of the experiments. Scale bar: 500 μm. (C) Raster plots of mouse speed in response to the looming stimuli for Setd5^+/+ before (top, n = 6, 10 trials) and after (bottom, n = 6, 11 trials) infusion of α-DTX (500 nL, 500 nM) sorted by reaction time. (D) As for (C) but for Setd5^+/− before (top, n = 6, 15 trials) and after (bottom, n = 6, 13 trials) animals after infusion of α-DTX (500 nL, 500 nM). (E) Effect of α-DTX on reaction time. Before α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.017), effect of α-DTX on Setd5^+/+ (saline versus α-DTX; p = 0.104) and Setd5^+/− (saline versus α-DTX; p = 0.014) and after α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.571). (F) Effect of α-DTX on escape vigour. Before α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.052), effect of α-DTX on Setd5^+/+ (saline versus α-DTX; p = 0.760) and Setd5^+/− (saline versus α-DTX; p = 0.649) and after α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.075). (G) Shelter exit behaviour during the first LER trial when the mice are injected with saline (Setd5^+/+, black, top left; Setd5^+/−, red, bottom left) or α-DTX (Setd5^+/+, blue, top right; Setd5^+/−, purple, bottom right). Each row represents 1 animal, filled and open dots represent exits crossed into the threat zone or not, respectively. (H) Effect of α-DTX on shelter exits. Before α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.012), effect of α-DTX on Setd5^+/+ (saline versus α-DTX; p = 0.038) and Setd5^+/− (saline versus α-DTX; p = 0.003) and after α-DTX (Setd5^+/+ versus Setd5^+/−; P = 0.495). Markers represent the average values across all trials for individual animals. P-values: Wilcoxon’s test, p-values: paired t test. The data underlying this figure can be found in S8 Data.