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

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Cell. Author manuscript; available in PMC 2017 August 23. Published in final edited form as: Cell. 2016 July 14; 166(2): 299–313. doi:10.1016/j.cell.2016.05.033.

Peripheral Mechanosensory Neuron Dysfunction Underlies Tactile and Behavioral Deficits in Mouse Models of ASDs Lauren L. Orefice1, Amanda L. Zimmerman1, Anda M. Chirila1, Steven J. Sleboda1, Joshua P. Head1, and David D. Ginty1,* 1Department

of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA

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SUMMARY

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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.

Graphical abstract

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*

Correspondence: [email protected]. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.cell.2016.05.033. An audio PaperClip is available at http://dx.doi.org/10.1016/j.cell.2016.05.033#mmc3. AUTHOR CONTRIBUTIONS L.L.O. and D.D.G. conceived the study. L.L.O. developed the textured NORT and tactile PPI assays, performed the behavioral analyses with assistance from S.J.S. and J.P.H., and executed the IHC with assistance from J.P.H. A.M.C. performed the SC slice electrophysiology experiments, and A.L.Z. performed the SC DRP experiments. L.L.O. and D.D.G. wrote the paper, with input from A.L.Z. and A.M.C. and editing provided by S.J.S. and J.P.H.

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INTRODUCTION

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Autism spectrum disorders (ASDs) are a highly prevalent class of neurodevelopmental disorders characterized by impairments in social communication and interactions, as well as restricted and repetitive behaviors. Strikingly, 95% of individuals with ASDs also exhibit aberrant reactivity to sensory stimuli, including tactile stimuli. Indeed, a majority of ASD patients (60.9%) report altered tactile sensitivity in both glabrous (smooth) and hairy skin (Tomchek and Dunn, 2007) and increased sensitivity to vibration and thermal pain (Blakemore et al., 2006; Cascio et al., 2008). As with idiopathic or non-syndromic ASDs, pervasive developmental disorders that cause syndromic forms of ASDs are also associated with altered somatosensation (Tomchek and Dunn, 2007). For example, tactile hypersensitivity is common in patients with Rett Syndrome, which is caused by mutations in the X-linked methyl-CpG-binding protein 2 (Mecp2) gene (Badr et al., 1987; Amir et al., 1999). Similarly, abnormalities in tactile perception are observed in patients with fragile X syndrome, which is highly associated with ASDs and caused by mutations in Fmr1 (Rogers et al., 2003). Moreover, an inverse correlation exists between the presence of ASD traits in human subjects and their neural responses to C-low-threshold mechanoreceptor-targeted affective touch (Voos et al., 2013).

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Although abnormalities in touch perception are commonly reported in ASDs, the underlying neural mechanisms are unknown. The first step leading to normal touch perception is the activation of low-threshold mechanosensory neurons (LTMRs) with highly specialized endings in the skin. LTMRs respond to innocuous mechanical stimuli and mediate perception of object shape, texture, skin stroking, skin indentation, hair movement, and vibration (Abraira and Ginty, 2013). As with all mammalian somatosensory neurons, cutaneous LTMRs are pseudo-unipolar neurons with one peripheral axonal branch that innervates the skin and another branch that innervates the CNS. While LTMR central projections terminate in a somatotopic manner within the spinal cord (SC) dorsal horn,

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forming synaptic contacts onto both locally projecting interneurons and postsynaptic dorsal column projection neurons (PSDCs), a large subset of myelinated LTMRs also send an axonal branch via the dorsal column that terminates in the dorsal column nuclei (DCN) of the brainstem. Thus, the SC dorsal horn and DCN are initial sites of integration and processing of innocuous touch information then conveyed to higher brain centers. In principle, LTMRs, the SC dorsal horn, DCN, thalamus, and cortex represent potential loci of dysfunction underlying impairments in touch perception in ASD patients.

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The great majority of ASD research has focused on brain-specific mechanisms and circuits, with little attention to potential contributions of the peripheral nervous system and SC to ASD phenotypes. Systemic virally mediated replacement of Mecp2 in Mecp2 hemizygous (Mecp2−/y) male mice effectively rescues behavioral deficits relevant to some ASD phenotypes. In contrast, intracranial viral delivery of Mecp2 only mildly improves behavioral phenotypes (Garg et al., 2013). These findings prompted us to investigate the role of peripheral nervous system or SC deficiencies caused by the disruption of Mecp2 or other ASD-associated genes in cutaneous tactile sensitivity. Moreover, as early childhood tactile experiences are critical for the acquisition of normal social behavior and communication skills in humans and rodents (Hertenstein et al., 2006), we hypothesized that tactile processing deficits in ASDs contribute to aberrant cognitive and social behaviors.

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In the present study we have used a range of mouse ASD genetic models combined with behavioral testing, synaptic analyses, and electrophysiology to define both the etiology of aberrant tactile sensitivity in ASDs and the contribution of somatosensory dysfunction to the expression of ASD-like traits. Our findings reveal a SC locus of mechanosensory neuron synaptic dysfunction underlying aberrant tactile perception in ASDs and a contribution of tactile processing deficiency during development to anxiety-like behavior and social interaction deficits in adulthood.

RESULTS ASD Mouse Models Exhibit Aberrant Innocuous Touch Sensitivity

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We asked whether mouse models of syndromic and non-syndromic forms of ASDs exhibit deficits in texture discrimination and tactile sensitivity. We examined Mecp2-null mice, as well as mice with an arginine-to-cysteine missense mutation in Mecp2 (Mecp2R306C), a common mutation found in human RTT patients (Lyst et al., 2013). Mice harboring mutations in Shank3 and Fmr1, which in humans are associated with forms of ASDs and fragile X syndrome, respectively (Peça et al., 2011; Spencer et al., 2005), were also analyzed. Thus, six-week-old male Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mice and control littermates were subjected to tactile-based tasks to assess mechanosensory behaviors and sensitivity. To assess glabrous skin tactile discrimination abilities in mice, we developed a texturespecific novel object recognition test (textured NORT), utilizing 4-cm-long cubes that differ only in texture (rough or smooth; Figures 1A, 1B, and S1; see the Experimental Procedures). While control mice preferentially explored the cube with novel texture in this assay, Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mice did not (Figure 1C). The deficits are


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specific for textured NORT, and not a general lack of novelty-seeking behavior, as mutant mice performed comparably to control mice on a control NORT in which objects differed in color and shape, but not in texture, when the retention period was 5 min (Figure 1D). Moreover, the amount of time spent investigating objects during NORT did not differ between mutants and control littermates (Figure S2A). This indicates that mutant mice did not exhibit an aversion to the objects, and they did not avoid tactile exploration. Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mice did not show a preference for novel colored/ shaped objects when the retention period was increased to 1 hr (Figure 1E). This is consistent with previous studies demonstrating that mice with mutations in these genes have learning/memory deficits (Arnett et al., 2014; Garg et al., 2013; Wang et al., 2011). Thus, four distinct ASD/RTT mouse models exhibit impairments in glabrous skin-based texture discrimination.

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Next, we asked whether Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mice exhibit abnormalities in hairy skin sensitivity and/or sensorimotor gating using a novel tactile prepulse inhibition (tactile PPI) assay. For tactile PPI, the “prepulse” is a light air puff (0.9 PSI) applied to back hairy skin, and this prepulse is followed by a broadband white noise acoustic startle “pulse” of 125 decibels (dB) to elicit an acoustic startle reflex. Air puffs were administered to the backs of mice at various times (inter-stimulus intervals [ISIs]) prior to the acoustic startle pulse to assess both hairy skin sensitivity and sensorimotor gating (Figure 1F). We have found that a light air puff prepulse robustly reduces the magnitude of an acoustic startle response in control mice (Figures 1G and 1H). Tactile PPI is mediated by cutaneous sensory neuron detection of the tactile prepulse because it is abolished when back hairy skin is pretreated with lidocaine to silence cutaneous sensory nerve fibers or when the air puff stimulus is pointed away from the mouse (Figures 1G, 1H, S1I, and S1J).

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Interestingly, Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mice all exhibited enhanced tactile PPI responses, compared to control littermates (Figure 1 J). For comparison, all ASD and control mice were also tested on an acoustic version of PPI, with varying acoustic prepulse intensities. Mecp2−/y and Mecp2R306C mice exhibited an increase in acoustic PPI at a prepulse intensity 15 dB above background, while the other mutant lines tested showed no acoustic PPI deficits (Figure S2B). Mecp2−/y and Mecp2R306C mice also reacted significantly less to the 125-dB startle noise, indicative of motor impairments (Figure 1I). Tactile PPI alterations in the mutants are, at least in part, due to hypersensitivity to the air puff prepulse stimulus itself, as Mecp2−/y, Mecp2R306C, Shank3B+/−, and Fmr1−/y mutant mice displayed significantly increased responses to the air puff alone (Figure 1K), but not to an acoustic prepulse alone (Figure S2C), compared to control littermates.

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Somatosensory Neuron Deletion of Mecp2 in Either Development or Adulthood Leads to Aberrant Tactile Sensitivity We sought to identify the cellular locus of tactile sensory dysfunction in Mecp2 mutants using a conditional Mecp2 allele. Crossing Mecp2 floxed (Mecp2f/y) mice (Guy et al., 2001) with mice expressing Cre recombinase in specific cell types allowed for Mecp2 deletion and loss of MECP2 protein in either excitatory neurons in the forebrain (Emx1Cre; Mecp2f/y) (Gorski et al., 2002), all cells caudal to cervical level 2 (Cdx2Cre; Mecp2f/y) (Akyol et al.,


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2008), or all DRG and trigeminal somatosensory neurons (AdvillinCre; Mecp2f/y) (Hasegawa et al., 2007) (Figure 2A). Deletion of Mecp2 in cells below the neck (Cdx2Cre; Mecp2f/y) recapitulated the reduced lifespan observed in Mecp2−/y and Mecp2R306C mutant mice; the majority of these mice did not survive beyond 16 weeks (Figure 2B), likely due to respiratory and/or cardiovascular deficits. Similar to Mecp2−/y mice, Cdx2Cre; Mecp2f/y mutants also displayed abnormal breathing patterns, decreased respiratory tidal volume and rate, and decreased CO2 expiration as measured by plethysmography (Figures S3E–S3H). Cdx2Cre; Mecp2f/y mutants also exhibited reduced body weight, a hindlimb-clasping phenotype, and motor deficits as measured using an accelerating rotarod, but not reduced brain size (Figures S3D, S3I, and S3J). In contrast, mice lacking Mecp2 exclusively in somatosensory neurons (AdvillinCre; Mecp2f/y) exhibited normal body weight, brain size, hindlimb extension, motor performance, respiration (Figures S3D–S3J), and lifespan (Figure 2B). Thus, Mecp2 deletion in primary somatosensory neurons does not lead to overt RTT phenotypes.

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To ask whether Mecp2 in excitatory neurons of the forebrain, SC, and/or peripheral somatosensory neurons contribute to tactile perception deficits, we next subjected Emx1Cre; Mecp2f/y, Cdx2Cre; Mecp2f/y, and AdvillinCre; Mecp2f/y mice to textured and control NORT, as well as tactile and acoustic PPI. Strikingly, Cdx2Cre; Mecp2f/y and AdvillinCre; Mecp2f/y mice, but not Emx1Cre; Mecp2f/y mice, had a significant deficit in textured NORT, even though both conditional mutants were able to discriminate between objects that differ in color and shape in both the 5-min and 1-hr retention period tests (Figures 2C–2E). Females with heterozygous deletion of Mecp2 in somatosensory neurons (AdvillinCre; Mecp2f/+) also exhibited deficits in textured NORT (Figure 2C). We did not observe any differences in object exploration time in any condition or genotype tested (Figure S3A). Similarly, deletion of Mecp2 in neurons below the neck or in primary somatosensory neurons led to tactile hypersensitivity in hairy skin, as measured by tactile PPI (Figure 2G). These changes are, at least in part, due to hypersensitivity to tactile stimuli applied to hairy skin because Cdx2Cre; Mecp2f/y, AdvillinCre; Mecp2f/y, and AdvillinCre; Mecp2f/+ mutants exhibited enhanced responses to air puff alone (Figure 2H), but not to an acoustic prepulse (Figure S3C), compared to control littermates. As expected, Cdx2Cre; Mecp2f/y mice showed a significant reduction in startle amplitude compared to control littermates (Figure 2F). Mice with deletion of Mecp2 in excitatory forebrain neurons (Emx1Cre; Mecp2f/y) performed similarly to control littermates on the tactile PPI assay and did not show increased responsiveness to air puff stimuli alone (Figures 2G and 2H).

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We also addressed whether Mecp2 expression in primary somatosensory neurons is required in adulthood for normal tactile discrimination and sensitivity. For this, Mecp2f/y mice were crossed to an AdvillinCreERT2 mouse line (Lau et al., 2011), enabling excision of floxed alleles in primary somatosensory neurons of adult mice following treatment with tamoxifen. Administration of 1 mg tamoxifen per day for 5 days beginning at P28 resulted in deletion of Mecp2 in >90% of DRG neurons, whereas Mecp2 expression in the SC and brain was unaltered (Figure 2A). Interestingly, adult deletion of Mecp2 in primary somatosensory neurons (in either males or heterozygous females) recapitulated the tactile behavioral deficits observed in the developmental Mecp2 mutants; both textured NORT and tactile PPI


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were impaired (Figures 2C–2H). Thus, Mecp2 expression in primary somatosensory neurons is necessary for both normal glabrous skin tactile discrimination and hairy skin sensitivity. Sensory Neuron Deletion of Mecp2 Leads to a Decrease in GABRB3 in the Dorsal Horn, and Gabrb3 Expression in Sensory Neurons Is Required for Tactile Sensitivity


Multiple etiologies of ASDs exist, and one common observation among ASD animal models is a deficit in GABAergic signaling (Braat and Kooy, 2015). Indeed, mutations in the GABAA receptor subunit β3 (Gabrb3) gene are associated with ASDs in humans, and mice harboring a Gabrb3 mutation (Gabrb3+/−) exhibit social behavior deficits, hypersensitivity to both thermal and mechanical stimuli, and sensorimotor impairments (DeLorey et al., 2008, 2011). Therefore, we asked whether GABRB3 is localized to synapses between LTMR subtypes and their postsynaptic partners in the SC dorsal horn. Previous work has shown that the β3 subunit of the GABAA receptor is associated with sensory terminals in the SC, both during development and in adulthood (Zeilhofer et al., 2012). Furthermore, the β3 subunit is an obligate component of GABAA receptors in DRG neurons, as it is the predominant β subunit expressed in DRG neurons in adulthood (Ma et al., 1993). We have found that GABRB3-containing GABAA receptors are indeed localized to presynaptic compartments of Aβ- and Aδ-LTMR central terminals in the dorsal horn (Figure 3A). GABRB3 puncta are also found on presynaptic primary sensory neuron terminals associated with PSDC projection neurons (data not shown). The finding that GABRB3 is associated with LTMR central terminals is consistent with previous work showing that GABAA receptors containing GABRB3 modulate sensory input to the SC (Chen et al., 2014). SC sections from Cdx2Cre; Gabrb3f/f mutant mice revealed a 75% loss in the number of GABRB3 puncta in the dorsal horn, compared to controls (Figures S4A and S4B). GABRB3 puncta associated with axonal fibers emanating from DRG sensory neurons were completely absent in AdvillinCre; Gabrb3f/f mice (Figures S4C and S4D), demonstrating both antibody specificity and primary somatosensory neurons as the cellular source of most dorsal horn GABRB3 puncta.

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Mecp2 deficiency leads to decreased expression of Gabrb3 in brains of both humans and mice (Samaco et al., 2005). To determine whether tactile perception abnormalities in Mecp2 mutants are associated with a loss of GABRB3-containing GABAA receptors on primary somatosensory neuron terminals in the dorsal horn, GABRB3 immunohistochemistry (IHC) was performed using SC sections of Mecp2 mutant mice and control littermates. Mecp2-null mice exhibited >80% reduction in GABRB3 puncta associated with vGlut1+ terminals in the dorsal horn (Figures 3B and 3C). Strikingly, AdvillinCre; Mecp2f/y mutant mice exhibited ~70% loss of GABRB3 puncta associated with vGlut1 + terminals in the dorsal horn (Figures 3D and 3E), indicating a reduction in GABRB3-containing GABAA receptors on presynaptic terminals of primary somatosensory neurons. Our findings suggested a model in which tactile perception abnormalities in Mecp2 mutants arise due to a reduction in presynaptic GABAA receptors on LTMR terminals in the dorsal horn and thus a lack of presynaptic inhibition (PSI) of LTMR inputs to the CNS. If this is the case, then deletion of the ASD-associated gene Gabrb3 should recapitulate the Mecp2 mutant tactile behavior phenotype. Indeed, as with Mecp2 mutants, Gabrb3+/− mice


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exhibited deficits in both glabrous skin tactile discrimination and hairy skin sensitivity (Figures 3F–3K). Next, we generated AdvillinCre; Gabrb3f/+, AdvillinCre; Gabrb3f/f, and AdvillinCreERT2; Gabrb3f/+ mutant mice, using a Gabrb3 floxed mouse line (Chen et al., 2014), to ask whether reduced Gabrb3 expression selectively in somatosensory neurons during development and in adulthood leads to tactile response abnormalities in adults. AdvillinCre; Gabrb3f/+, AdvillinCre; Gabrb3f/f, and AdvillinCreERT2; Gabrb3f/+ mice exhibited deficits in textured NORT, while no impairments were observed in control NORT assays (Figures 3F–3H and S4E). Moreover, Gabrb3 conditional mutant mice from each group exhibited enhanced responses to air puff stimuli alone and enhanced tactile PPI, but were not different from control littermates in acoustic PPI performance (Figures 3I–3K, S4F, and S4G). Thus, mice lacking Gabrb3 in primary somatosensory neurons phenocopy mice lacking Mecp2 in somatosensory neurons, implicating a functional link between these ASDassociated genes.

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Sensory Neuron Deletion of Either Mecp2 or Gabrb3 Induces A-Fiber Synapse Hyperexcitability


Our findings led us to hypothesize that the loss of GABAA receptor-dependent PSI of LTMR inputs in the dorsal horn causes excessive LTMR excitatory drive onto postsynaptic SC neurons and thus increases responsiveness to tactile stimuli in both Mecp2 and Gabrb3 mutant mice. This hypothesis was tested using both SC slice (Figure 4) and intact isolated SC (Figure 5) electrophysiological measurements. We recorded from retrogradely labeled PSDC projection neurons in SC slices with dorsal roots attached, enabling direct electrical stimulation of the roots to evoke postsynaptic responses in PSDCs (Figure 4A). To selectively monitor quantal events from primary somatosensory neurons onto PSDCs, as opposed to global miniature excitatory postsynaptic currents (mEPSCs) that reflect both primary afferent and other, non-primary afferent synaptic inputs, we replaced calcium in the extracellular recording solution with strontium and evoked asynchronous quantal EPSCs (qEPSCs) from stimulated sensory afferents (Chirila et al., 2014; Oliet et al., 1996). Using this approach, we reliably triggered qEPSCs in PSDCs following dorsal root stimulation at an intensity that selectively activates A-fibers (100 µA, 0.1 ms) (Torsney and MacDermott, 2006). Strikingly, qEPSC frequency, but not amplitude or decay time, was increased in slices from AdvillinCre; Mecp2f/y mutants compared to slices from age-matched controls (Figures 4B–4H; data not shown). These results indicate that enhanced presynaptic release probability, and not a change in the number and/or biophysical properties of postsynaptic glutamate receptors, accounts for increased synaptic transmission between mechanosensory afferents lacking Mecp2 and PSDCs. A similar finding of enhanced presynaptic release probability was observed in recordings from slices obtained from AdvillinCre; Gabrb3f/+ mice compared with controls (Figures 4B–4H; data not shown). These findings suggest a model in which neurotransmitter release probability is significantly enhanced at A-fiber synapses in the SC dorsal horn of mice lacking Mecp2 due to a loss of GABA-mediated PSI at these terminals.


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Sensory Neuron Deletion of Either Mecp2 or Gabrb3 Leads to Loss of PSI in the SC Dorsal Horn


We tested our model by directly measuring PSI in the dorsal horn of both AdvillinCre; Mecp2f/y and AdvillinCre; Gabrb3f/+ mutant mice using an isolated SC preparation. In the SC dorsal horn, PSI of LTMRs is predominantly mediated through primary afferent depolarization (PAD) (Barker and Nicoll, 1972). PAD is believed to be generated through a trisynaptic circuit in which primary afferents innervate dorsal horn glutamatergic interneurons, which then innervate GABAergic interneurons that form axo-axonic synapses onto primary afferent terminals (Figure 5A). Due to the relatively depolarized chloride reversal potential of primary somatosensory neurons, GABA acting on primary afferent terminals leads to their depolarization, which, in turn, reduces action potential-evoked transmitter release from sensory neuron terminals in the dorsal horn (Rudomin and Schmidt, 1999). PAD can be measured as a back-propagating depolarization of the dorsal root, or dorsal root potential (DRP) (Rudomin and Schmidt, 1999; Russo et al., 2000). Activitydependent PSI can thus be assessed by electrically stimulating one dorsal root and recording a DRP on an adjacent dorsal root (Figure 5A). By stimulating at low intensity (here, defined as ≤4 times the threshold of afferent volley recruitment), a low-threshold DRP was readily elicited in control mice (Figure 5B). This DRP is almost entirely inhibited by the GABAA receptor blocker bicuculline (Figure 5B) (Levy et al., 1971). As stimulus intensity increases, a greater number and variety of afferents are recruited, and DRPs increase in magnitude. At maximal intensity, DRPs are no longer solely GABAA receptor dependent (Figure 5B) (Russo et al., 2000). Evoked DRPs were greatly diminished in both AdvillinCre; Mecp2f/y and AdvillinCre; Gabrb3f/+ mice (Figures 5C–5E). At low threshold stimulus intensity, DRPs in these mice were virtually non-existent, and as stimulus intensity increased, small DRPs were observed. As GABAA receptors on SC interneurons are likely to be unaffected in AdvillinCre; Mecp2f/y and AdvillinCre; Gabrb3f/+ mice, we hypothesized that motor reflexes, as recorded electrophysiologically from ventral roots, would be unchanged. Indeed, motor reflexes were unaffected in both AdvillinCre; Mecp2f/y and AdvillinCre; Gabrb3f/+ mutants (data not shown), and GABAA receptor modulation of these reflexes was comparable to that of control mice (Figures 5F and 5G). These findings indicate that primary somatosensory neuron deletion of either Mecp2 or Gabrb3 increases sensitivity to light touch due to a deficiency of GABAA receptors on somatosensory neuron terminals and a loss of GABAA receptor-mediated PSI. Primary Somatosensory Neuron Deletion of Either Mecp2 or Gabrb3 Leads to Deficits in Cognitive and Mouse Social Behaviors

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Next, we asked whether mice harboring developmental or adult deletions of either Mecp2 or Gabrb3 in primary somatosensory neurons exhibit anxiety-like behavior, deficits in nest building, and/or social interactions. Mutant and control mice were first subjected to a 10-min open-field (OF) test; mice with anxiety-like behavior spend less time exploring the center of the chamber and travel shorter distances (McGill et al., 2006). In agreement with previous reports, Mecp2−/y and Gabrb3+/− mutant mice exhibited a decrease in both the percentage of time spent in the center of the chamber and total distance traveled during the OF test (Figures 6A–6C, S5B, and S5C). Remarkably, mice lacking either Mecp2 or Gabrb3 exclusively in primary somatosensory neurons during development (AdvillinCre; Mecp2f/y, Cell. Author manuscript; available in PMC 2017 August 23.

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AdvillinCre; Gabrb3f/f, and AdvillinCre; Gabrb3f/+ mice), but not mice in which Mecp2 or Gabrb3 was deleted in somatosensory neurons in adulthood (AdvillinCreERT2; Mecp2f/y or AdvillinCreERT2; Gabrb3f/+), also exhibited anxiety-like behavior compared to control littermates (Figures 6A–6C and S5A–S5C). Whether ASD mouse models exhibit abnormal habituation to aversive stimuli, which is a behavior associated with anxiety in both humans and rodents (Campbell et al., 2014), was also measured. In normal humans and mice, the startle response to loud acoustic stimuli decreases over time, whereas humans and rodents with anxiety exhibit less habituation to aversive startle stimuli (Campbell et al., 2014; Glowa et al., 1992). To assess habituation to acoustic startle noises, we performed acoustic startle trials before and after the PPI trials described above. Across genotypes, control mice exhibited ~30% reduction in the acoustic startle response at the end of the session compared to responses measured at the beginning of the session (Figures 6D and S5D). Interestingly, AdvillinCre; Mecp2f/y and AdvillinCre; Gabrb3f/f, as well as germline Mecp2 and Gabrb3 mutants, failed to habituate to the acoustic startle noise (Figures 6D and S5D). In contrast, mice in which either Mecp2 or Gabrb3 was deleted in primary somatosensory neurons in adulthood (AdvillinCreERT2; Mecp2f/y or AdvillinCreERT2; Gabrb3f/+) exhibited normal decreases in their startle responses over time (Figure 6D). Next, we tested a subset of the mouse lines on an additional measure of anxiety-like behavior in rodents, the elevated plus maze (EPM) (McGill et al., 2006). Mecp2R306C and AdvillinCre; Mecp2f/y mutant mice spent significantly less time in the open arms of the EPM compared to littermate controls, indicative of increased anxiety-like behavior in these mice (Figure 6E). Conversely, Emx1Cre; Mecp2f/y and AdvillinCreERT2; Mecp2f/y exhibited normal behavior in this test (Figure 6E). Thus, primary somatosensory neuron deletion of either Mecp2 or Gabrb3 during development, but not in adulthood, leads to anxiety-like behavior in mice.

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Next, we assessed nest building, a socially relevant tactile behavior of adult rodents in which both Mecp2 and Gabrb3 mutant mice display deficits (DeLorey et al., 2008; Garg et al., 2013). Adult mice were placed in individual cages and provided nestlets 30 min before the start of the dark cycle. 14 to 16 hours later, nest construction was scored as previously described using a five-point scale (Deacon, 2006). Mice with either embryonic or adult deletion of Mecp2 or Gabrb3 in primary somatosensory neurons showed impairments in nest building behavior, compared to control littermates, although these nesting deficits were not as severe as those of Mecp2−/y and Mecp2R306C mutant mice (Figures S5E and S5F).

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We also implemented the three-chamber social interaction test, which is used to assess both sociability and social recognition/ preference in rodents, and ASD mouse models displayed altered behaviors in this assay (Silverman et al., 2010). Control mice with normal social preferences spent more time exploring a novel mouse in both the sociability and social novelty preference tests. In contrast, several ASD mouse models, including Gabrb3+/− mice, displayed a reduced amount of time exploring the novel mouse in this assay (DeLorey et al., 2008). Thus, as predicted, we observed that Mecp2−/y, Mecp2R306C, and Gabrb3+/− mice exhibit impairments in both sessions of this test, as they did not prefer a novel mouse compared to an empty cup (sociability test), and they did not prefer a novel mouse to a familiar mouse (social novelty preference test) (Figures 6F–6H and S5G–S5I; Table S1). Mice with primary somatosensory neuron deletion of either Mecp2 (AdvillinCre; Mecp2f/y) or Gabrb3 (AdvillinCre; Gabrb3f/f and AdvillinCre; Gabrb3f/+) displayed comparable deficits Cell. Author manuscript; available in PMC 2017 August 23.

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in both sociability and social novelty preference (Figures 6F–6H; Table S1). On the other hand, mice in which either Mecp2 or Gabrb3 was deleted in adulthood (AdvillinCreERT2; Mecp2f/y or AdvillinCreERT2; Gabrb3f/+) displayed significant preference for the novel mouse during the sociability test, but did not display a preference for a novel mouse compared to a familiar mouse during the social novelty preference test (Figures 6G and 6H; Table S1).

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To further evaluate social interactions, mice were subjected to a tube dominance test, which allowed us to assess social approach/avoidance behavior, as well as evaluate dominance hierarchies in mice. These approaches can be used to assess social impairments in ASD mouse models (Shahbazian et al., 2002; Spencer et al., 2005). We have found that Mecp2−/y, Mecp2R306C, and Gabrb3+/− mutant mice exhibit decreased social dominance and aggression, as they lost the majority of matches against control mice (Figures 6I and S5J). These results are consistent with previous reports indicating that Mecp2-null mice exhibit increased escape and avoidance behaviors in a predator-threat stimulus test (Pearson et al., 2015). Similarly, Cdx2Cre; Mecp2f/y, AdvillinCre; Mecp2f/y, and AdvillinCre; Gabrb3f/f mutant mice were all significantly more submissive and lost the majority of their matches (Figures 6I and S5J). In contrast, mice in which either Mecp2 or Gabrb3 was deleted in somatosensory neurons in adulthood demonstrated increased dominance, as these mice won the majority of their matches (Figure 6I). Mice with deletion of Mecp2 in excitatory forebrain neurons were neither submissive nor dominant in this assay. Taken together, these results indicate that there is a developmental requirement of Mecp2 and Gabrb3 in primary somatosensory neurons for the acquisition of certain cognitive and social behaviors in mice.

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Mecp2 Expression in Primary Somatosensory Neurons Is Sufficient for Normal Tactile Discrimination, PSI, and Certain Cognitive and Social Behaviors

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Whether Mecp2 in primary somatosensory neurons is sufficient for normal tactile function was addressed by restoring Mecp2 expression exclusively in the peripheral somatosensory neurons in a Mecp2-null background. Here, we utilized a Mecp2STOP/y mouse line (Guy et al., 2007), in which Mecp2 is expressed only in cells following Cre-mediated excision of a STOP codon (Figure 7A). Expression of Mecp2 exclusively in primary somatosensory neurons in an otherwise Mecp2-null background (AdvillinCre; Mecp2STOP/y; Figure 7A) rescued the deficits in textured NORT (Figure 7D), hypersensitivity to air puff and alterations in tactile PPI (Figures 7E and 7F) observed in Mecp2-null mutants. We also have found that Mecp2 expression in primary somatosensory neurons alone is sufficient to rescue expression of GABRB3 at presynaptic sensory neuron terminals (Figures 7B and 7C) and normalize GABAA receptor-mediated PSI of somatosensory input to the dorsal horn (Figures 7G and 7H). Remarkably, behavioral analyses of mice with expression of Mecp2 only in primary somatosensory neurons revealed normal levels of anxiety-like behaviors, assessed by their performances in the OF test, habituation to acoustic startle noises, and by EPM, whereas Mecp2STOP/y mutants showed increased anxiety-like behavior in each of these tests (Figures 7I–7M). Although AdvillinCre; Mecp2STOP/y mice displayed lower nest building scores than controls, their values were improved relative to Mecp2STOP/y mutants (Figure S6I).


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AdvillinCre; Mecp2STOP/y mice also performed no differently than controls in either portion of the three-chamber social interaction test (Figures 7N, 7O, and S6J–S6L), and they exhibited neither submissive nor dominant behavior in the tube dominance assay (Figure 7P). However, expression of Mecp2 exclusively in primary somatosensory neurons did not rescue decreased brain size, memory, motor, or acoustic PPI abnormalities (Figure S6). Thus, Mecp2 expression in primary somatosensory neurons in an otherwise Mecp2-null mouse rescues tactile behavioral deficits, deficits in GABRB3 expression associated with LTMR terminals, abnormalities in tactile PPI, anxiety-like behaviors, and a subset of the social behavior deficits observed in Mecp2-null mutants.

DISCUSSION


The majority of ASD research has focused on brain-specific functions of ASD-related genes and their impact on behavior. Here, we report that mice harboring mutations in Mecp2, Gabrb3, Shank3, and Fmr1, genes that when mutated in humans are associated with ASDs, exhibit altered tactile discrimination and hypersensitivity to gentle touch. Deletion of either Mecp2 or Gabrb3 selectively in peripheral somatosensory neurons results in aberrant tactile sensitivity due to a loss of PSI of somatosensory neuron transmission in the SC. Remarkably, tactile processing dysfunction caused by developmental deletion of either Mecp2 or Gabrb3 in mechanosensory neurons leads to social and cognitive behavior deficits that recapitulate some of the core phenotypes of ASDs, including anxiety-like behavior. Moreover, while deletion of Mecp2 or Gabrb3 in adult primary somatosensory neurons also leads to altered tactile sensitivity, it does not cause anxiety-like behavior and results in more modest social behavior deficits. These findings reveal an essential, cell-autonomous requirement for the ASD-associated genes Mecp2 and Gabrb3 for mechanosensory neuron synaptic transmission and tactile sensitivity, and they implicate developmental somatosensory dysfunction in the genesis of anxiety and aberrant social behaviors in patients with ASDs.

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While ablation of Mecp2 or Gabrb3 in primary somatosensory neurons during either development or in adulthood leads to tactile discrimination deficits and hairy skin hypersensitivity, the behavioral deficits resulting from developmental or adult deletion of these genes are strikingly distinct. Developmental ablation of either Mecp2 or Gabrb3 exclusively in primary somatosensory neurons leads to anxiety-like behaviors, reduced sociability, and preference for social novelty and increased sub-missive behavior. In contrast, mice in which either Mecp2 or Gabrb3 was deleted in primary somatosensory neurons in adulthood do not exhibit anxiety-like behavior, their sociability deficits are less pronounced, and they are conversely aggressive in the tube dominance test. Thus, tactile processing defects that manifest during development or in adulthood lead to different behavioral outcomes in adult mice. Our behavioral findings are reminiscent of human, non-human primate, and rodent tactile deprivation studies, which showed that early life experiences and developmental tactile stimulation are essential for proper brain development, cognition, and adult social behaviors. Indeed, children who experienced institutional rearing with low caregiver investment and physical handling exhibited deficits in cognitive function, delayed, or impaired language


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acquisition and social interactions and an increased incidence of anxiety and psychiatric disorders (Frank et al., 1996; Sheridan et al., 2012). The related anxiety-like behavior and social interaction deficits in ASD models and in rodents reared in the absence of normal tactile experiences lead us to speculate the existence of a common mechanistic link between these two conditions. Consistent with this idea, young children with ASDs are typically averse to tactile stimuli and an aversion to nurturing touch has been suggested to impact their development and behavior (Cascio, 2010). On the other hand, environmental enrichment, such as increased maternal licking and grooming in mice, during early postnatal periods can improve behavioral outcomes in ASD models (Lonetti et al., 2010).

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We speculate that aberrant PSI and thus defective gain control in the SC and brainstem renders ASD patients incapable of properly interpreting the physical world. Thus, loss of PSI of LTMR inputs to the CNS may lead to misinterpreted or overwhelming tactile sensations during normal physical exploration, which may manifest as anxiety and withdrawal from social situations that are normally associated with unpredictable and dynamic physical interactions. It should be emphasized that sensory impairment in ASDs is not restricted to the sense of touch. ASD patients and the associated animal models of these disorders often exhibit visual, auditory, olfactory, and gustatory abnormalities. Therefore, impairments in multiple sensory systems during critical developmental windows may contribute to abnormal language acquisition, cognition, anxiety, and social behaviors. Defining the functional relationships between the initial stages of sensory information processing, anxiety, and social interaction behaviors in the mouse models described here and other ASD models may aid in the development of novel approaches to treat patients with ASDs.

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EXPERIMENTAL PROCEDURES Mouse Lines Mice were group housed in standard housing on a 12-hr light/dark cycle. Mecp2−/y, Mecp2R306C, Mecp2f/y, Mecp2STOP, Shank3B+/−, Fmr1−/y, Gabrb3f/f, EIIaCre, Emx1Cre, Cdx2Cre, AdvillinCre, and AdvillinCreERT2 mouse lines have been previously described, and references are included in the Supplemental Experimental Procedures. For all experiments, experimenters were blinded to genotype. Behavioral Testing

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Male and female mice were of mixed genetic backgrounds (C57BL/6J and 129/SvEv), except for Mecp2-null and Mecp2R306C mice, which were on a C57BL/6J background. Testing began at 6 weeks of age and, in most cases, was completed by 8 weeks of age. The OF test, NORT, startle reflex/PPI, EPM, plethysmography, rotarod, tube dominance test, nest building, and three-chamber social interaction tests are described in the Supplemental Experimental Procedures. Electrophysiological Recordings P14-P19 acute SC slices with dorsal roots attached were used for whole-cell patch clamp recordings of PSDCs. Quantal EPSCs were recorded in ACSF with 4-mM strontium


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replacing Ca2+ using a holding potential of −70 mV and induced by dorsal root stimulation at A-fiber strength (100 µA, 0.1 ms). For DRP measurements, isolated spinal cords with spinal roots attached were prepared. Bipolar glass suction electrodes (inner diameter 80–120 µm) were used to stimulate the T11-L1 dorsal root; recordings were taken from the same root to measure afferent recruitment, from an adjacent dorsal root to measure DRPs, and from ventral roots to measure spinal reflexes. See the Supplemental Experimental Procedures. Histological Analyses IHC of tissue sections were performed using standard procedures (Bai et al., 2015). For GABRB3 puncta analysis, z stack images of SC slices were taken on a Zeiss LSM 700 confocal microscope using a 63× oil-immersion lens (Zeiss; Plan-Apochromat 63×/na 1.40), as described in the Supplemental Experimental Procedures.

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Statistical Analyses All data are expressed as the mean ± SEM. Unless otherwise stated in the figure legend, data were analyzed using Student’s t test. Animal numbers per group are shown in the bars or legends of each panel. See the Supplemental Experimental Procedures.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments Author Manuscript

We thank Chinfei Chen, Patrick Forcelli, Michael Greenberg, Christopher Harvey, Alex Kolodkin, Laura Mamounas, David Paul, and members of the D.D.G. laboratory for discussions and comments on the manuscript. We thank Michael Greenberg, John Wood, and Fan Wang for mouse lines, Ofer Mazor and Pavel Gorelik in the Harvard Research Instrumentation Core, Patrick Forcelli and the Harvard NeuroBehavior Laboratory Core for advice about behavioral analyses, and Ryan Dosumu-Johnson and Susan Dymecki for assistance with plethysmography. This work was supported by a Hearst Postdoctoral Fellowship (to L.L.O.), a SFARI Pilot Award from the Simons Foundation (to D.D.G.), and NIH grants (T32 NS007484-14 to L.L.O.; DE022750 and NS34814 to D.D.G). D.D.G. is an investigator of the Howard Hughes Medical Institute.

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Highlights •

Several ASD mouse models exhibit aberrant tactile sensitivity

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Mecp2 and Gabrb3 function in somatosensory neurons for normal tactile behaviors

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Mecp2 and Gabrb3 function in somatosensory neurons to control presynaptic inhibition

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Developmental tactile abnormalities contribute to behaviorial deficits in adult mice


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In Brief Changes in the brain are thought to underlie behaviors associated with autism, but now evidence from mouse models indicates that deficits in peripheral sensory neurons can contribute to the syndrome.


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Author Manuscript Author Manuscript Author Manuscript Figure 1. ASD Mouse Models Exhibit Aberrant Innocuous Touch Sensitivity

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(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


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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.


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

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(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. Cell. Author manuscript; available in PMC 2017 August 23.

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Author Manuscript Author Manuscript Author Manuscript 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

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(A) IHC images of spinal cord (SC) dorsal horn lamina III from Ai34 (Rosa26LSL–Synaptophysin–tdTomato) mice crossed to RetCreERT2, TrkCCreERT2, or TrkBCreERT2 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 colabeled 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.


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(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 AdvillinCre; Mecp2f/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 AdvillinCre 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 AdvillinCre 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 AdvillinCre group: (one-way ANOVA: p < 0.05, F[2,33] = 4.821.) Post hoc Tukey’s test:*p < 0.05.

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

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(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), AdvillinCre; Mecp2f/y, (C) or AdvillinCre; Gabrb3f/+ (D) mutant mice. (E) Mean frequency of qEPSCs recorded from SC slices of mutant or control mice. (Oneway ANOVA: p < 0.0001, F[2, 15]= 20.8.) Post hoc Tukey’s test: *p

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