Thalamic reticular impairment underlies attention deficit in Ptchd1(Y/-) mice

Abstract

Developmental disabilities, including attention-deficit hyperactivity disorder (ADHD), intellectual disability (ID), and autism spectrum disorders (ASD), affect one in six children in the USA. Recently, gene mutations in patched domain containing 1 (PTCHD1) have been found in ~1% of patients with ID and ASD. Individuals with PTCHD1 deletion show symptoms of ADHD, sleep disruption, hypotonia, aggression, ASD, and ID. Although PTCHD1 is probably critical for normal development, the connection between its deletion and the ensuing behavioural defects is poorly understood. Here we report that during early post-natal development, mouse Ptchd1 is selectively expressed in the thalamic reticular nucleus (TRN), a group of GABAergic neurons that regulate thalamocortical transmission, sleep rhythms, and attention. Ptchd1 deletion attenuates TRN activity through mechanisms involving small conductance calcium-dependent potassium currents (SK). TRN-restricted deletion of Ptchd1 leads to attention deficits and hyperactivity, both of which are rescued by pharmacological augmentation of SK channel activity. Global Ptchd1 deletion recapitulates learning impairment, hyper-aggression, and motor defects, all of which are insensitive to SK pharmacological targeting and not found in the TRN-restricted deletion mouse. This study maps clinically relevant behavioural phenotypes onto TRN dysfunction in a human disease model, while also identifying molecular and circuit targets for intervention.

Extended Data Figure 1. Developmental expression pattern of Ptchd1

a–d, In situ hybridization labeling of Ptchd1 mRNA at (a) P0 (coronal), (b) P15 (coronal), and (c–d) P35 (coronal and sagittal) from 3 C57/BL6 WT mice per age. White arrows indicate location of TRN region (scale bar = 1 mm).

Extended Data Figure 2. Generation of Ptchd1 KO mouse

a, Diagram depicting the “full-length” and non-functional “Exons 1+3” Ptchd1 isoforms. Genetic ablation of Exon 2 results in the removal of a majority of the transmembrane domains normally present in the endogenous full-length isoform. b, Schematic describing strategy to create Ptchd1 KO mouse. Mice containing targeted allele were crossed to β-Actin Flp mice to remove the Neo cassette and β-Actin Cre mice to excise Exon 2. c, In situ hybridization probes targeting Exon 2 confirm successful genetic ablation of full-length Ptchd1 mRNA (scale bar = 1 mm). d, PCR genotyping confirms deletion of Exon 2 from genome of male KO mice. e, qPCR of WT and KO cDNA samples shows removal of full-length Ptchd1 isoform.

Extended Data Figure 3. Burst and spindle phase locking characteristics of Ptchd1 KO and WT TRN neurons in vivo

a, left, Example of unit clustering for a stereotrode recording. Two units (green and blue) are clearly separated when plotting peak-trough of the two electrodes of the streotrode against each other. right, Spike-wave form of the two clustered units as they appear on the two electrodes of the stereotrode. Raw trace below shows a burst discharge (asterisk) of each unit during NREM sleep with colored ticks indicating corresponding individual spikes. A burst was identified as at least 2 spikes with an ISI ≤10ms preceded by a period of 70 ms silence. Enlarged trace shows the accelerando-deccelerando firing pattern characteristic for a TRN burst. b, Firing rate during NREM sleep is comparable between genotypes (89 WT, 80 KO cells from 4 WT, 3 KO mice; P>0.1 Kolmogorov-Smirnov statistics). c, Ptchd1 KO TRN neurons show reduced propensity to generate bursts, even when excluding the 10% of KO cells with the highest firing rate (89 WT, 72 KO cells from 4 WT, 3 KO mice; P<0.05 Kolmogorov-Smirnov statistics). d, Spindle-phase histogram for an example WT and KO neuron. Note that the WT neuron shows a preferred phase around the peak (0 degrees) of the spindle oscillation in WT but not KO. e, Example LFP recording (top) showing the temporal alignment of TRN spikes (bottom) to the preferred phase of the spindle activity (9–15 Hz, middle). f, Ptchd1 KO mice show reduced phase-locking strength to spindle activity compared to WT littermates (89 WT, 80 KO cells from 4 WT, 3 KO mice).

Extended Data Figure 4. Ptchd1 KO mice have intact sensory responses and rotarod performance

a–c, Normal (a) acoustic startle (20 WT, 20 KO), (b) pre-pulse inhibition (20 WT, 20 KO) and (c) hot plate response in Ptchd1 KO mice (20 WT, 21 KO). d, Ptchd1 KO mice show normal motor coordination on the accelerating rotarod test (19 WT, 20 KO). Two-tailed t-tests (c) and two-way repeated measures ANOVA with Bonferroni post-hoc tests (a–b, d) were used for statistical analysis. Mean ± s.e.m. N.S. = not significant.

Extended Data Figure 5. Intact spatial learning but motor and aggression abnormalities in Ptchd1 KO mice

a, Comparable learning curves between WT and KO mice during cued training protocol. b, Intact spatial learning demonstrated in 24hr probe trial. c, Ptchd1 KO mice show normal reversal learning curve. d, No significant difference between WT and KO mice in 24 hour probe trial after reversal learning protocol (10 WT, 10 KO). e, Representative images of WT (black) and KO (red) strides. Forepaw position is represented by green paint and hindpaw position is represented by pink paint (scale bar = 2cm). Quantification reveals elongated stride length and width (10 WT, 11 KO). f, KO mice show drastic reductions in grip strength as measured by the hanging wire test (12 WT, 11 KO). g–h, KO mice attack intruder mice for a longer duration (g) and with a shorter latency to attack (h) in the resident-intruder test for aggression (10 WT, 10 KO). Two-way repeated measures ANOVA with Bonferroni post-hoc tests (a, c), one-way ANOVA with Bonferroni multiple comparison tests (b, d), two-tailed t-tests (e, f) and Wilcoxon ranksum tests (g, h) were used for statistical analysis. Mean ± s.e.m. (a, e), mean (b, d, e–f), median (g–h). *P<0.05; **P<0.01; ***P<0.001; N.S. = not significant.

Extended Data Figure 6. Hyperactivity, hypotonia, and learning deficits in C57/129 Ptchd1 KO mice

a, Ptchd1 KO mice showed increased locomotor activity (10 WT, 11 KO). b, KOs show decreased mean holding time in the hanging wire test (10 WT, 12 KO) but c, normal motor coordination in the rotarod task (10 WT, 10 KO). d–f, Sensory responses as measured by acoustic startle (d), pre-pulse inhibition (e), and hot plate (f) are also normal in KO mice (10 WT, 12 KO). g–h, Normal sociability (g) and novel social recognition (h) in mixed background Ptchd1 KO mice (10 WT, 11 KO). i, KO mice show impaired associative learning and memory in the inhibitory avoidance task (9 WT, 12 KO). Two-tailed t-tests (b, f), one-way ANOVA with Bonferroni multiple comparison tests (g–h), and two-way repeated measures ANOVA with Bonferroni post-hoc tests (a, c–e, i) were used for statistical analysis. Mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; N.S. = not significant.

Extended Data Figure 7. Normal grooming and social interaction behaviors in Ptchd1 KO mice

a, KO mice do not show excessive or injurious grooming behaviors (9 WT, 13 KO). b–c, KO mice (b) spent comparable amounts of time interacting with stranger mice in the three-chambered social interaction task and (c) display normal social novelty behaviors (10 WT, 11 KO). Two-tailed t-tests (a) and two-way repeated measures ANOVA with Bonferroni post-hoc tests (b–c) were used for statistical analysis. Bar in scatterplot denotes mean. ***P<0.001; N.S. = not significant.

Extended Data Figure 8. YFP overlap with SOM interneuron marker is primarily confined to the TRN in Ptchd1-YFP mice

a, Schematic describing strategy to create Ptchd1-YFP mouse in which Exon 1 was replaced with YFP cassette. b, YFP-positive cells co-label with GAD67 antibody in TRN and the purkinje layer of the cerebellum, but not in cortex or striatum. c, YFP-positive cells also co-label with SOM antibody in TRN, but not in other structures. Arrows denote overlap. (bar = 20 μm).

Extended Data Figure 9. Som-Cre recombinase activity is early and robust in TRN neurons

a, P4 Som-Cre⁺:TdTomato⁺ brains show TdTomato⁺ cells in the TRN. Inset shows magnified image taken with 20X objective. b–c, At (b) P15 and (c) P30, Cre recombinase activity in the TRN of the Som-Cre⁺:TdTomato⁺ brains is robust, as shown by the inset depicting the significant TdTomato overlap with the pan-neuronal marker NeuN.

Extended Data Figure 10. Genetic disruption of Ptchd1 TRN expression affects sleep stability but not grip strength or aggressive behaviors

a–b, Som-Cre⁺:Ptchd1^Y/fl mice appear normal in (a) the hanging wire (12 Ptchd1^Y/+, 11 Ptchd1^Ylfl) and (b) resident intruder task (6 Ptchd1^Y/+, 6 Ptchd1^Ylfl). c–e, Som-Cre⁺:Ptchd1^Y/fl mice show reductions in sleep bout duration as shown in (c) cumulative probability plot and (d) comparison of medians with (e) no differences in total time spent sleeping when compared to Som-Cre⁺:Ptchd1^Y/+ littermates (10 Ptchd1^Y/+, 10 Ptchd1^Ylfl). f–g, 1-EBIO treatment has no effect on performance on (f) the hanging wire or (g) resident intruder task (6 WT-Veh, 6 WT-EBIO, 6 KO-Veh, 6 KO-EBIO). Kolomgorov-Smirnov test (a), Wilcoxon ranksum tests (b–c), two-tailed t-tests (d), and two-way repeated measures ANOVA with Bonferroni post-hoc tests (f), and Kruskal-Wallis with Dunn’s multiple comparisons tests were used for statistical analysis. Median (b–c, e–g), mean (d,f). *P<0.05; **P<0.01; ***P<0.001; N.S. = not significant.

Figure 1. Impaired repetitive bursting and SK2 currents in KO TRN neurons

a–b, Ptchd1 expression (3 WT mice). Bar = 1 mm. c, Representative TRN burst traces (out of 8 WT, 9 KO cells). d, Reduced burst firing in KO TRN neurons (8 WT, 9 KO cells). e, Representative T and SK2 current traces. f, Normal T (g) and reduced SK2 currents (h) in KO cells (8 WT, 9 KO). i, Diminished free [Ca²⁺]_i in KO cells. Representative heat maps show background-corrected intensity (37 WT, 36 KO cells). Wilcoxon ranksum (d, f–h) and two-tailed t-tests (i). Mean ± s.e.m. *P<0.05; ***P<0.001; N.S.=not significant.

Figure 2. Decreased spindles and sleep fragmentation in KO mice

a, Multielectrode implant targeting TRN b, Decreased TRN burst discharge in KOs (89 WT, 80 KO cells from 4 WT, 3 KO mice). c, Reduced spindles in KOs. d–f, KOs display shorter sleep bouts with normal total sleep duration (9 WT, 10 KO). Kolomgorov-Smirnov (b, d) and Wilcoxon ranksum tests (c, e–f). Median (c, e–f). *P<0.05; ***P<0.001; N.S.=not significant.

Figure 3. Reduced sensory-evoked thalamic inhibition in KO mice

a, Schematic of CFP-to-YFP FRET. b, Confocal images of superclomeleon expression in LGN (bar = 100 μm). c, Stimuli delivered to the eye contra-lateral to implanted LGN. d, Example traces of visually-evoked CFP and YFP fluorescence changes. e–g, Reduced LGN inhibition in KOs reflected in peak FRET response (f) and smaller area under the curve (g; AUC) (6 WT, 6 KO mice). h–j, KOs also show decreased facilitation of FRET response (6 WT, 6 KO mice). Wilcoxon ranksum tests (e–j). Mean ± s.e.m. *P<0.05; **P<0.01

Figure 4. KO mice show attention, locomotor, and learning impairment

a, Visual detection task design (8 WT, 9 KO). b, KOs showed comparable baseline performance. c, KOs displayed decreased accuracy in the presence of distractors. d, KOs show increased locomotion in open field (30 WT, 31 KO). e–f, KOs show normal responses to amphetamine (5 WT-Veh, 6 WT-Amph, 5 KO-Veh, 6 KO-Amph). g–h, KOs exhibit decreased fear-induced learning behaviors in (g) contextual and (h) cued fear conditioning tests (10 WT, 11 KO). i, Diminished KO latency to cross in inhibitory avoidance task (24 WT, 23 KO). Wilcoxon ranksum (b–c), two-tailed t (g–h), and two-way RM ANOVA with Bonferroni post-hoc tests (d–f, i). Median (b,c), mean ± s.e.m. (d–e), mean (f–i). *P<0.05; **P<0.01; ***P<0.001; N.S.=not significant.

Figure 5. TRN dysfunction explains ADHD-like behaviors in KO

a, Ptchd1 ablation from TRN (bar = 1 mm). b, Som-Cre⁺:Ptchd1^Y/fl mice recapitulate attention deficits observed in KOs (8 Ptchd1^Y/+, 8 Ptchd1^Ylfl). c–d, Som-Cre⁺:Ptchd1^Y/fl mice are hyperactive (21 Ptchd1^Y/+, 22 Ptchd1^Ylfl). e, Som-Cre⁺:Ptchd1^Y/fl mice show intact ability to form complex association (11 Ptchd1^Y/+, 12 Ptchd1^Ylfl). Wilcoxon ranksum (b), two-tailed t (d), and two-way RM ANOVA with Bonferroni post-hoc tests (c, e). Median (b), mean ± s.e.m (c), mean (de). *P<0.05; **P<0.01; N.S.=not significant.

Figure 6. SK2 enhancement corrects ADHD-like KO symptoms

a, 1-EBIO corrects inhibitory transients in KOs (7 WT-Veh, 7 WT-EBIO, 7 KO-Veh, 7 KO-EBIO). b–c, 1-EBIO improves KO attention performance (7 WT-Veh, 7 WT-EBIO, 7 KO-Veh, 7 KO-EBIO). d–e, 1-EBIO rescues KO hyperactivity (13 WT-Veh, 13 WT-EBIO, 13 KO-Veh, 15 KO-EBIO). f, No 1-EBIO effects on inhibitory avoidance (7 WT-Veh, 8 WT-EBIO, 9 KO-Veh, 10 KO-EBIO). Wilcoxon ranksum (a–c) and two-way RM ANOVA with Bonferroni post-hoc tests (d–f). Mean ± s.e.m. (a, d), mean (e–f) *P<0.05; **P<0.01; ***P<0.001; N.S. = not significant.