Thalamic reticular impairment underlies attention deficit in Ptchd1Y/− 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.

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Figure 1: Impaired repetitive bursting and SK2 currents in TRN neurons from knockout mice.
Figure 2: Decreased spindles and sleep fragmentation in knockout mice
Figure 3: Reduced sensory-evoked thalamic inhibition in knockout mice.
Figure 4: Knockout mice show attention, locomotor, and learning impairment.
Figure 5: TRN dysfunction explains ADHD-like behaviours in knockout mice.
Figure 6: SK conductance augmentation corrects ADHD-like symptoms in knockout mice.

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Acknowledgements

We thank R. Tang for insightful discussion during the initiation of the project, H. Wang, T. Dalia, E. Kwan, H. Zaniewski for technical support, and J. Vincent for insightful discussion. We thank J. Petravicz and T. Emery from the Sur laboratory for assistance with Ca2+ imaging and A. Heynen from the Bear laboratory for technical advice on the inhibitory avoidance task. We thank all members of the Feng laboratory for their help and support. We thank M. Ball and J. Ball for their insight and inspiration throughout this project. We also thank S.F. Lin and R. Buxton for their support of this research. This work was supported by a grant from Simons Foundation Autism Research Initiative (SFARI Award ID: 307913) to G.F. and M.M.H., NIH grants to G.F. (NIH/NIMH, R01MH097104) and M.M.H. (R01MH107680), and funds from the Poitras Center for Affective Disorders Research and the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard to G.F. M.M.H. is additionally supported by the Brain and Behavior, Sloan, Klingenstein and Feldstein Foundations. M.F.W. is supported by an NIH Ruth L. Kirschstein National Research Service Award (FMH098641A). R.D.W. is supported by the Swiss National Science Foundation.

Author information

M.F.W. and G.F. conceived the genetic studies and designed associated experiments; R.D.W. and M.M.H. conceived the physiologic studies and designed associated experiments. All authors designed the behavioural studies. M.F.W. and R.D.W. collected the data. M.F.W., R.D.W. and L.I.S. analysed the data. M.F.W., R.D.W., M.M.H. and G.F. interpreted the results. M.F.W., M.M.H. and G.F. wrote the paper with input from R.D.W.

Correspondence to Guoping Feng.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Developmental expression pattern of Ptchd1.

a–d, In situ hybridization labelling of Ptchd1 mRNA at P0 (coronal) (a), P15 (coronal) (b), and P35 (coronal and sagittal) (cd) from 3 C57/Bl6 wild-type mice per age. White arrows indicate location of TRN region (scale bars, 1 mm).

Extended Data Figure 2 Generation of Ptchd1-knockout mouse.

a, Schematic describing strategy to create Ptchd1-knockout 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. b, 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. 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 knockout mice. e, qPCR of wild-type and knockout cDNA samples shows removal of full-length Ptchd1 isoform. Het, heterozygous.

Extended Data Figure 3 Burst and spindle phase-locking characteristics of Ptchd1-knockout and wild-type 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 non-rapid eye movement sleep with coloured ticks indicating corresponding individual spikes. A burst was identified as at least 2 spikes with an inter-spike interval of ≤10 ms, preceded by a period of 70 ms silence. Enlarged trace shows the accelerando–decelerando firing pattern characteristic for a TRN burst. b, Firing rate during non-rapid eye movement sleep is comparable between genotypes (n = 89 WT, 80 KO cells from 4 WT, 3 KO mice; P > 0.1, Kolmogorov–Smirnov test). c, Ptchd1-knockout TRN neurons show reduced propensity to generate bursts, even when excluding the 10% of knockout cells with the highest firing rate (n = 89 WT, 72 KO cells from 4 WT, 3 KO mice; P < 0.05, Kolmogorov–Smirnov test). d, Spindle-phase histogram for an example wild-type and knockout neuron. Note, the wild-type neuron shows a preferred phase around the peak (0 degrees) of the spindle oscillation in wild type but not knockout. e, Example local field potential recording (LFP; top) showing the temporal alignment of TRN spikes (bottom) to the preferred phase of the spindle activity (9–15 Hz, middle). f, Ptchd1-knockout mice show reduced phase-locking strength to spindle activity compared to wild-type littermates (n = 89 WT, 80 KO cells from 4 WT, 3 KO mice).

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

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

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

a, Comparable learning curves between wild-type and knockout mice during cued training protocol. b, Intact spatial learning demonstrated in 24 h probe trial. c, Ptchd1-knockout mice show normal reversal learning curve. d, No significant difference between wild-type and knockout mice in 24 h probe trial after reversal learning protocol (n = 10 WT, 10 KO). e, Representative images of wild-type (black) and knockout (red) strides. Forepaw position is represented by green paint and hindpaw position is represented by pink paint (scale bar, 2 cm). Quantification reveals elongated stride length and width (n = 10 WT, 11 KO). f, Knockout mice show marked reductions in grip strength as measured by the hanging wire test (n = 12 WT, 11 KO). g, h, Knockout mice attack intruder mice for a longer duration (g) and with a shorter latency to attack (h) in the resident–intruder test for aggression (n = 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 rank-sum tests (g, h) were used for statistical analysis. Chance performance (25%) represented by dashed grey lines (b, d). Error bars, mean ± s.e.m (a, e); mean (b, d, ef), median (g, h). *P < 0.05; **P < 0.01; ***P < 0.001.

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

a, Ptchd1-knockout mice showed increased locomotor activity (n = 10 WT, 11 KO). b, c, Knockout mice show decreased mean holding time in the hanging wire test (b) (n = 10 WT, 12 KO), but normal motor coordination in the rotarod task (c) (n = 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 knockout mice (n = 10 WT, 12 KO). g–h, Normal sociability (g) and novel social recognition (h) in mixed background Ptchd1-knockout mice (n = 10 WT, 11 KO). i, Knockout mice show impaired associative learning and memory in the inhibitory avoidance task (n = 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, ce, i) were used for statistical analysis. Error bars, mean ± s.e.m.; horizontal bars, mean (b, f–i). *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 7 Normal grooming and social interaction behaviours in Ptchd1-knockout mice.

a, Knockout mice do not show excessive or injurious grooming behaviours (n = 9 WT, 13 KO). b, c, Knockout mice spent comparable amounts of time interacting with stranger mice in the three-chambered social interaction task (b) and display normal social novelty behaviours (c) (n = 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. Horizontal bars, mean. ***P < 0.001.

Extended Data Figure 8 YFP overlap with somatostatin 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 a YFP-bovine growth hormone poly-A tail (BGH) cassette. b, YFP+ cells co-label with anti-GAD67 antibody in TRN and the Purkinje layer of the cerebellum, but not in cortex or striatum. c, YFP+ cells also co-label with anti-somatostatin antibody in TRN, but not in other structures. Arrows denote overlap. Scale bars, 20 μm.

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

a, The progeny of Sst-Cre+ mice crossed to mice showing Cre-dependent expression of the TdTomato fluorescent protein (Sst-Cre+ TdTomato+) show TdTomato+ cells in the TRN at P4. Inset shows magnified image taken with 20× objective. b, c, At P15 (b) and P30 (c), Cre recombinase activity in the TRN of the Sst-Cre+ TdTomato+ mice 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 behaviours.

a, b, Sst-Cre+ Ptchd1Y/fl mice appear normal in the hanging wire (a) (n = 12 Ptchd1Y/+, 11 Ptchd1Y/fl) and resident intruder task (b) (n = 6 Ptchd1Y/+, 6 Ptchd1Y/fl). c–e, Sst-Cre+ Ptchd1Y/fl mice show reductions in sleep bout duration as shown in cumulative probability plot and comparison of medians (d) with no differences in total time spent sleeping when compared to Sst-Cre+:Ptchd1Y/+ littermates (e) (n = 10 Ptchd1Y/+, 10 Ptchd1Y/fl). f, g, 1-EBIO treatment has no effect on performance on the hanging wire (f) or resident intruder task (g) (n = 6 WT veh., 6 WT 1-EBIO, 6 KO veh., 6 KO 1-EBIO). Kolomgorov–Smirnov test (a), Wilcoxon rank-sum 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. Horizontal bars, median (b, c, eg), mean (d, f). *P < 0.05; **P < 0.01; ***P < 0.001.

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Wells, M., Wimmer, R., Schmitt, L. et al. Thalamic reticular impairment underlies attention deficit in Ptchd1Y/− mice. Nature 532, 58–63 (2016) doi:10.1038/nature17427

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