Peripheral Mechanosensory Neuron Dysfunction Underlies Tactile and Behavioral Deficits in Mouse Models of ASDs

Abstract

Patients with autism spectrum disorders (ASDs) commonly experience aberrant tactile sensitivity, yet the neural alterations underlying somatosensory dysfunction and the extent to which tactile deficits contribute to ASD characteristics are unknown. We report that mice harboring mutations in Mecp2, Gabrb3, Shank3, and Fmr1 genes associated with ASDs in humans exhibit altered tactile discrimination and hypersensitivity to gentle touch. Deletion of Mecp2 or Gabrb3 in peripheral somatosensory neurons causes mechanosensory dysfunction through loss of GABAA receptor-mediated presynaptic inhibition of inputs to the CNS. Remarkably, tactile defects resulting from Mecp2 or Gabrb3 deletion in somatosensory neurons during development, but not in adulthood, cause social interaction deficits and anxiety-like behavior. Restoring Mecp2 expression exclusively in the somatosensory neurons of Mecp2-null mice rescues tactile sensitivity, anxiety-like behavior, and social interaction deficits, but not lethality, memory, or motor deficits. Thus, mechanosensory processing defects contribute to anxiety-like behavior and social interaction deficits in ASD mouse models. PAPERCLIP.

Figure 1. ASD Mouse Models Exhibit Aberrant Innocuous Touch Sensitivity

(A) Image showing a smooth textured object (left) and a rough textured object (right) used in the textured NORT. (B) Protocol for the three NORT assays. (C–E) Discrimination indices for textured NORT (C), 5-min control NORT (D), and 1-hr control NORT (E). A positive value indicates a preference for the novel object, compared to the familiar object. *p < 0.05. (F) Diagram for the tactile PPI assay. (G) Response to a light air puff (0.9 PSI, 50 ms) applied to the back hairy skin, in either naive mice or mice in which the back hairy skin was shaved and lidocaine was locally applied to block cutaneous sensory neuron activation. Responses are expressed as a percent of startle response to a 125-dB noise. *p < 0.01. (H) Percent inhibition of the startle response to a 125-dB noise (pulse), when the startle noise was preceded by a light air puff (prepulse) at multiple interstimulus intervals (ISIs) between the prepulse and the pulse. (Two-way ANOVA: p < 0.001, F[1,65] = 51.27.) Post hoc Bonferroni test: *p < 0.05. (I) Magnitude of startle response to a 125-dB noise in mutant mice and control littermates. *p < 0.001. (J) Percent inhibition of the startle response to a 125-dB noise, when the startle noise is preceded by a light air puff (250-ms ISI). *p < 0.05. (K) Response to a light air puff stimulus alone. Responses are expressed as a percent of startle response to a 125-dB noise. *p < 0.05.

Figure 2. Mecp2 Expression in Primary Somatosensory Neurons Is Required for Normal Tactile Behaviors

(A) IHC images of dorsal root ganglion (DRG), transverse SC, or trunk primary somatosensory cortex (brain) showing MECP2 protein expression in control mice or mice with conditional deletion of Mecp2 generated by crossing an Mecp2-floxed mouse line to various Cre recombinase mouse lines, as indicated. IB4 labels lamina IIi of the dorsal horn. (B) Kaplan-Meier curves showing the percentage of mutant mice in each line surviving up to 40 weeks of age. (C–E) Discrimination indices for textured NORT (C), 5-min control NORT (D), and 1-hr control NORT (E) in mutant mice and control littermates. *p < 0.05. (F) Magnitude of startle response to a 125-dB noise. *p < 0.05. (G) Percent inhibition of the startle response to a 125-dB noise, when the startle noise was preceded by a light air puff (250-ms ISI). *p < 0.05. (H) Response to a light air puff alone. *p < 0.05.

Figure 3. Sensory Neuron Deletion of Mecp2 Causes a Decrease in GABRB3 Puncta Associated with Sensory Neuron Terminals in the Spinal Cord, and Gabrb3 in Primary Somatosensory Neurons Is Required for Tactile Sensitivity

(A) IHC images of spinal cord (SC) dorsal horn lamina III from Ai34 (Rosa26^{LSL–Synaptophysin–tdTomato}) mice crossed to Ret^CreERT2, TrkC^CreERT2, or TrkB^CreERT2 mice to label Aβ RAI-LTMRs, Aβ Field-, and Aβ SAI-LTMRs or Aδ-LTMRs, respectively. Sections were immunostained for tdTOMATO (TOMATO) and GABRB3 to mark the presence of GABRB3 puncta in proximity to Aβ- and Aδ-LTMR presynaptic terminals. (B) IHC of SC dorsal horn lamina III from Mecp2^−/y mutant mice and control littermates co-labeled for vGLUT1 (presynaptic terminals for Aβ and Aδ LTMRs) and GABRB3 to mark the presence of GABRB3 puncta at Aβ and Aδ LTMR presynaptic terminals. (C) Quantification of vGLUT1+ puncta co-labeled with GABRB3, relative to the total number of vGLUT1+ puncta visualized per image. *p < 0.001. (D) IHC of SC dorsal horn lamina III from Advillin^Cre; Mecp2^f/y mutant mice and control littermates co-labeled for vGLUT1 and GABRB3 to mark the presence of GABRB3 puncta at Aβ and Aδ LTMR presynaptic terminals. (E) Quantification of vGLUT1+ puncta co-labeled with GABRB3, relative to the total number of vGLUT1+ puncta visualized per image. *p < 0.001. (F) Discrimination index for textured NORT. *p < 0.05. For Advillin^Cre group: (one-way ANOVA: p < 0.001, F[2,29] = 7.287.) Post hoc Bonferroni’s test: *p < 0.05. (G and H) Discrimination indices for 5-min control NORT (G) and 1-hr control NORT (H). *p < 0.05. (I) Magnitude of startle response to a 125-dB noise. (J) Percent inhibition of the startle response to a 125-dB noise, when the startle noise was preceded by a light air puff (250-ms ISI). *p < 0.05. For Advillin^Cre group: (one-way ANOVA: p < 0.001, F[2,33] = 7.238.) Post hoc Tukey’s test: *p < 0.05. (K) Response to a light air puff alone. *p < 0.05. For Advillin^Cre group: (one-way ANOVA: p < 0.05, F[2,33] = 4.821.) Post hoc Tukey’s test:*p < 0.05.

Figure 4. Primary Somatosensory Neuron Deletion of Either Mecp2 or Gabrb3 Induces A-Fiber Synapse Hyperexcitability

(A) Diagram depicting whole-cell patch clamp configuration for recording from PSDC projection neurons, while stimulating dorsal roots. (B–D) Representative quantal EPSC (qEPSC) traces from SC slices of control mice (B), Advillin^Cre; Mecp2^f/y, (C) or Advillin^Cre; Gabrb3^f/+ (D) mutant mice. (E) Mean frequency of qEPSCs recorded from SC slices of mutant or control mice. (One-way ANOVA: p < 0.0001, F[2, 15]= 20.8.) Post hoc Tukey’s test: *p<0.001. (F) Cumulative probability for inter-event interval of qEPSCs recorded from SC slices of control or mutant mice. (Kruskal-Wallis test: p < 0.0001, H = 864.1.) Post hoc Dunn’s test: all three groups are significantly different, p < 0.0001. (G and H) Mean amplitude (G) and cumulative probability (H) of qEPSCs recorded from SC slices of mutant or control mice.

Figure 5. Primary Somatosensory Neuron Deletion of Either Mecp2 or Gabrb3 Leads to Loss of Presynaptic Inhibition in the Spinal Cord Dorsal Horn

(A) Recording configuration schematic, with the location of three recording electrodes and one stimulating electrode. (B) In control mice, bicuculline inhibits the low-threshold evoked dorsal root potential (DRP), while facilitating high-threshold DRPs. Each trace represents an average of ten sweeps, low-pass filtered at 1 kHz. 4T and 10T denote stimulation intensities that are 4× or 10× the threshold of afferent volley recruitment, respectively. (C) Representative traces for evoked DRP recordings at varying stimulus intensities from mutant or control mice. (D) Quantification of average peak amplitude for evoked DRP recordings at varying stimulus intensities. (Two-way ANOVA: p < 0.0001, F[2,100] = 45.23.) Post hoc Holm-Sidak’s test: *p < 0.05. (E) Quantification of total integral for evoked DRP recordings at varying stimulus intensities from mutant or control mice. (Two-way ANOVA: p < 0.0001, F[2,100] = 44.58.) Post hoc Holm-Sidak’s test: *p < 0.05. (F) Sample traces of ventral root reflexes recorded with maximum intensity stimulation, both alone and with bicuculline. Integral windows are marked to include monosynaptic, short latency polysynaptic, and long latency polysynaptic responses. (G) Quantification for the percent change in integral for reflexes at maximum intensity stimulation.

Figure 6. Primary Somatosensory Neuron Deletion of Either Mecp2 or Gabrb3 Leads to Deficits in Cognitive and Social Behaviors

(A) Representative activity traces in the open field (OF) test for Mecp2^−/y and Advillin^Cre; Mecp2^f/y mutant mice and control littermates. (B) Time spent in the center of the OF chamber. *p < 0.05. For Advillin^Cre; Gabrb3 floxed group: (one-way ANOVA: p < 0.05, F[2,36] = 4.325.) Post hoc Bonferroni’s test: *p < 0.05. (C) Total distance traveled in the OF chamber. *p < 0.05. For Advillin^Cre; Gabrb3 floxed group: (one-way ANOVA: p < 0.05, F[2,36] = 3.499.) Post hoc Bonferroni’s test: *p < 0.05. (D) Percent decrease in startle response to a 125-dB noise during a 30-min tactile PPI session, when comparing the first five startle responses to the last five responses to a 125-dB noise. *p < 0.05. For Advillin^Cre; Gabrb3 floxed group: (one-way ANOVA: p < 0.001, F[2,38] = 5.354.) Post hoc Bonferroni’s test: *p < 0.05. (E) Percent of time spent in the open arms of the EPM. *p < 0.05. (F) Representative heat maps of activity in the three-chamber social interaction test during the “Sociability” (top) and “Social Novelty Preference” (panels) portions of the assay for control, Mecp2^−/y, and Advillin^Cre; Mecp2^f/y mice. (G) Preference index for the percentage of time spent investigating the novel mouse in the “Sociability” portion of the three-chamber social interaction test. *p < 0.05. For Advillin^Cre; Gabrb3 floxed group: (one-way ANOVA: p < 0.0001, F[2,40] = 11.17.) Post hoc Bonferroni’s test: *p < 0.001. (H) Preference index for the percentage of time spent investigating the novel mouse in the “Social Novelty Preference” portion of the three-chamber social interaction test. *p < 0.05. For Advillin^Cre; Gabrb3 floxed group: (one-way ANOVA: p < 0.05, F[2,40] = 3.882.) Post hoc Bonferroni’s test: *p < 0.05. (I) Win percentage in the tube dominance test for mutant mice or control littermates. *p < 0.05.

Figure 7. Mecp2 Expression Exclusively in Primary Somatosensory Neurons Is Sufficient for Normal Tactile Sensitivity, GABRB3 Expression, PSI, and Certain Cognitive Behaviors

(A) IHC images of DRG, transverse SC, or primary somatosensory cortex (brain) showing MECP2 protein expression in control, Mecp2-null (Mecp2^STOP/y),or mice in which Mecp2 is expressed only in primary somatosensory neurons (Advillin^Cre; Mecp2^STOP/y). (B) IHC images of lamina III of the SC dorsal horn co-labeled for vGLUT1 and GABRB3 to visualize GABRB3 puncta associated with presynaptic terminals of Aβ-and Aδ-LTMRs. (C) Quantification of vGLUT1+ puncta co-labeled with GABRB3, relative to the total number ofvGLUT1+ puncta visualized per image. Percent of GABRB3 puncta in images from mutant mice relative to control values are indicated in white text within bars. (One-way ANOVA: p < 0.0001, F[2,7] = 44.97.) Post hoc Tukey’s test: *p < 0.05. (D) Discrimination index for textured NORT. (One-way ANOVA: p < 0.05, F[2,62] = 4.322.) Post hoc Tukey’s test: *p < 0.05. (E) Percent of inhibition of the startle response to a 125-dB noise when the startle noise is preceded by a light air puff (250-ms ISI). (One-way ANOVA: p < 0.05, F[2,68] = 5.392.) Post hoc Holm-Sidak’s test: *p < 0.05. (F) Response to a light air puff alone. (One-way ANOVA: p < 0.0001, F[2,68] = 14.47.) Post hoc Tukey’s test: *p < 0.0001. (G) Representative traces for evoked DRP recordings at varying stimulus intensities from mutant mice and control littermates. (H) Peak amplitude for evoked DRP recordings at varying stimulus intensities. (Two-way ANOVA: p < 0.0001, F[2,40] = 16.12.) Post hoc uncorrected Fisher’s LSD test: *p < 0.05. (I) Percent of decrease in startle response to a 125-dB noise during a 30-min tactile PPI session when comparing the first five startle responses to the last five responses to a 125-dB noise. (One-way ANOVA: p < 0.05, F[2,37] = 4.919.) Post hoc Holm-Sidak’s test: *p < 0.05. (J) Representative traces of activity in the OF test. (K) Time spent in the center of the OF chamber. (One-way ANOVA: p < 0.05, F[2,35] = 4.539.) Post hoc Holm-Sidak’s test: *p < 0.05; ^#, p < 0.10. (L) Total distance traveled in the OF chamber. (One-way ANOVA: p < 0.001, F[2,35] = 6.836.) Post hoc Holm-Sidak’s test: *p < 0.05. (M) Percentage of time spent in the open arms of the EPM. (One-way ANOVA: p < 0.01, F[2, 27] = 7.463.) Post hoc Tukey’s test: *p < 0.01. (N) Preference index for the “Sociability” portion of the three-chamber social interaction test. (One-way ANOVA: p < 0.05, F[2,42] = 4.141.) Post hoc Tukey’s test:*p < 0.05. (O) Preference index for the “Social Novelty Preference” portion of the three-chamber social interaction test. (One-way ANOVA: p < 0.0001, F[2, 42] =11.18.) Post hoc Tukey’s test: *p < 0.001. (P) Win percentage in the tube dominance test. *p < 0.05.