Mechanoreceptor signal convergence and transformation in the dorsal horn flexibly shape a diversity of outputs to the brain.

The encoding of touch in the spinal cord dorsal horn (DH) and its influence on tactile representations in the brain are poorly understood. Using a range of mechanical stimuli applied to the skin, large-scale in vivo electrophysiological recordings, and genetic manipulations, here we show that neurons in the mouse spinal cord DH receive convergent inputs from both low- and high-threshold mechanoreceptor subtypes and exhibit one of six functionally distinct mechanical response profiles. Genetic disruption of DH feedforward or feedback inhibitory motifs, comprised of interneurons with distinct mechanical response profiles, revealed an extensively interconnected DH network that enables dynamic, flexible tuning of postsynaptic dorsal column (PSDC) output neurons and dictates how neurons in the primary somatosensory cortex respond to touch. Thus, mechanoreceptor subtype convergence and non-linear transformations at the earliest stage of the somatosensory hierarchy shape how touch of the skin is represented in the brain.

Targeting Peripheral Somatosensory Neurons to Improve Tactile-Related Phenotypes in ASD Models.

Somatosensory over-reactivity is common among patients with autism spectrum disorders (ASDs) and is hypothesized to contribute to core ASD behaviors. However, effective treatments for sensory over-reactivity and ASDs are lacking. We found distinct somatosensory neuron pathophysiological mechanisms underlie tactile abnormalities in different ASD mouse models and contribute to some ASD-related behaviors. Developmental loss of ASD-associated genes Shank3 or Mecp2 in peripheral mechanosensory neurons leads to region-specific brain abnormalities, revealing links between developmental somatosensory over-reactivity and the genesis of aberrant behaviors. Moreover, acute treatment with a peripherally restricted GABAA receptor agonist that acts directly on mechanosensory neurons reduced tactile over-reactivity in six distinct ASD models. Chronic treatment of Mecp2 and Shank3 mutant mice improved body condition, some brain abnormalities, anxiety-like behaviors, and some social impairments but not memory impairments, motor deficits, or overgrooming. Our findings reveal a potential therapeutic strategy targeting peripheral mechanosensory neurons to treat tactile over-reactivity and select ASD-related behaviors.

The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.

The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.

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

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.

Long-term potentiation of glycinergic synapses triggered by interleukin 1ß.

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABAA receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.

Nerve injury disrupts temporal processing in the spinal cord dorsal horn through alterations in PV+ interneurons.

How mechanical allodynia following nerve injury is encoded in patterns of neural activity in the spinal cord dorsal horn (DH) remains incompletely understood. We address this in mice using the spared nerve injury model of neuropathic pain and in vivo electrophysiological recordings. Surprisingly, despite dramatic behavioral over-reactivity to mechanical stimuli following nerve injury, an overall increase in sensitivity or reactivity of DH neurons is not observed. We do, however, observe a marked decrease in correlated neural firing patterns, including the synchrony of mechanical stimulus-evoked firing, across the DH. Alterations in DH temporal firing patterns are recapitulated by silencing DH parvalbumin+ (PV+) interneurons, previously implicated in mechanical allodynia, as are allodynic pain-like behaviors. These findings reveal decorrelated DH network activity, driven by alterations in PV+ interneurons, as a prominent feature of neuropathic pain and suggest restoration of proper temporal activity as a potential therapeutic strategy to treat chronic neuropathic pain.

Loss of interneuron LTD and attenuated pyramidal cell LTP in Trpv1 and Trpv3 KO mice.

TRPV (transient receptor potential, vanilloid) channels are a family of nonselective cation channels that are activated by a wide variety of chemical and physical stimuli. TRPV1 channels are highly expressed in sensory neurons in the peripheral nervous system. However, a number of studies have also reported TRPV channels in the brain, though their functions are less well understood. In the hippocampus, the TRPV1 channel is a novel mediator of long-term depression (LTD) at excitatory synapses on interneurons. Here we tested the role of other TRPV channels in hippocampal synaptic plasticity, using hippocampal slices from Trpv1, Trpv3 and Trpv4 knockout (KO) mice. LTD at excitatory synapses on s. radiatum hippocampal interneurons was attenuated in slices from Trpv3 KO mice (as well as in Trpv1 KO mice as previously reported), but not in slices from Trpv4 KO mice. A previous study found that in hippocampal area CA1, slices from Trpv1 KO mice have reduced tetanus-induced long-term potentiation (LTP) following high-frequency stimulation; here we confirmed this and found a similar reduction in Trpv3 KO mice. We hypothesized that the loss of LTD at the excitatory synapses on local inhibitory interneurons caused the attenuated LTP in the mutants. Consistent with this idea, blocking GABAergic inhibition rescued LTP in slices from Trpv1 KO and Trpv3 KO mice. Our findings suggest a novel role for TRPV3 channels in synaptic plasticity and provide a possible mechanism by which TRPV1 and TRPV3 channels modulate hippocampal output.

Nerve injury disrupts temporal processing in the spinal cord dorsal horn through alterations in PV + interneurons.

How mechanical allodynia following nerve injury is encoded in patterns of neural activity in the spinal cord dorsal horn (DH) is not known. We addressed this using the spared nerve injury model of neuropathic pain and in vivo electrophysiological recordings. Surprisingly, despite dramatic behavioral over-reactivity to mechanical stimuli following nerve injury, an overall increase in sensitivity or reactivity of DH neurons was not observed. We did, however, observe a marked decrease in correlated neural firing patterns, including the synchrony of mechanical stimulus-evoked firing, across the DH. Alterations in DH temporal firing patterns were recapitulated by silencing DH parvalbumin + (PV + ) inhibitory interneurons, previously implicated in mechanical allodynia, as were allodynic pain-like behaviors in mice. These findings reveal decorrelated DH network activity, driven by alterations in PV + interneurons, as a prominent feature of neuropathic pain, and suggest that restoration of proper temporal activity is a potential treatment for chronic neuropathic pain.

γ-Protocadherins control synapse formation and peripheral branching of touch sensory neurons.

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn leads to fewer corticospinal synapses on dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and stepwise assembly of central mechanosensory circuitry.

NMDA receptor activation induces long-term potentiation of glycine synapses.

Of the fast ionotropic synapses, glycinergic synapses are the least well understood, but are vital for the maintenance of inhibitory signaling in the brain and spinal cord. Glycinergic signaling comprises half of the inhibitory signaling in the spinal cord, and glycinergic synapses are likely to regulate local nociceptive processing as well as the transmission to the brain of peripheral nociceptive information. Here we have investigated the rapid and prolonged potentiation of glycinergic synapses in the superficial dorsal horn of young male and female mice after brief activation of NMDA receptors (NMDARs). Glycinergic inhibitory postsynaptic currents (IPSCs) evoked with lamina II-III stimulation in identified GABAergic neurons in lamina II were potentiated by bath-applied Zn2+ and were depressed by the prostaglandin PGE2, consistent with the presence of both GlyRα1- and GlyRα3-containing receptors. NMDA application rapidly potentiated synaptic glycinergic currents. Whole-cell currents evoked by exogenous glycine were also readily potentiated by NMDA, indicating that the potentiation results from altered numbers or conductance of postsynaptic glycine receptors. Repetitive depolarization alone of the postsynaptic GABAergic neuron also potentiated glycinergic synapses, and intracellular EGTA prevented both NMDA-induced and depolarization-induced potentiation of glycinergic IPSCs. Optogenetic activation of trpv1 lineage afferents also triggered NMDAR-dependent potentiation of glycinergic synapses. Our results suggest that during peripheral injury or inflammation, nociceptor firing during injury is likely to potentiate glycinergic synapses on GABAergic neurons. This disinhibition mechanism may be engaged rapidly, altering dorsal horn circuitry to promote the transmission of nociceptive information to the brain.

Three-Dimensional Neural Spheroid Culture: An In Vitro Model for Cortical Studies.

There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.