Hyperexcitability of Sensory Neurons in Fragile X Mouse Model

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.

AAV-PHP.S-mediated delivery of reporters to cranial ganglion sensory neurons

ABSTRACTBecause of their ease of use and low risk containment, Adeno-Associated Virus vectors are indispensable tools for much of neuroscience. Yet AAVs have been used relatively little to study the identities and connectivity of peripheral sensory neurons because methods to selectively target particular receptive fields or neuron types have been limited. The introduction of the AAV-PHP.S capsid with selective tropism for peripheral neurons (Chan et al., 2017) offered a solution, which we further elaborate here. We demonstrate using AAV-PHP.S with GFP or mScarlet reporters, that all cranial sensory ganglia, i.e. for cranial nerves V, VII, IX and X, are targeted. Pseudounipolar neurons of both somatic and visceral origin, but not satellite glia, are efficiently transduced rapidly and express the gene of interest within 1 week of injection. Fluorescent reporter proteins are transported into the central and peripheral axons of these sensory neurons, permitting visualization of terminals at high resolution, and/or in intact, cleared brain using light sheet microscopy. By combining a Cre-dependent reporter with the AAV-PHP.S capsid, we confirmed expression in a cell-type dependent manner for both anatomical and targeted functional analyses. The AAV-PHP.S capsid will be a powerful tool for mapping the receptive fields and circuits of molecular subtypes of many somatosensory, gustatory and visceral sensory neurons.SIGNIFICANCE STATEMENTAAV vectors have become an essential tool for visualizing, manipulating, and recoding from neurons of the central nervous system. However, the technology is not widely used for peripheral neurons because of several technical limitations. The AAV-PHP.S synthetic capsid, which targets peripheral neurons, was recently introduced (Chan et al., 2017). Here, we establish key parameters for using this virus, including which cells are transduced, the timing of expression in central and peripheral terminals, distant from neuronal somata, and the effectiveness of Cre-dependent constructs for cell type selective expression. This permits the use of AAV for constructing detailed anatomic and functional maps of the projections of molecular subtypes of peripheral sensory neurons.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

Dietary capsaicin normalizes CGRP peptidergic DRG neurons in experimental diabetic peripheral neuropathy

Diabetic sensory neuropathy leads to impairment of peripheral sensory nerves and downregulation of calcitonin gene-related peptide (CGRP) in a functionally specific subset of peripheral sensory neurons mediating pain. Whether CGRP plays a neuroprotective role in peripheral sensory nerve is unclear. We evaluated alterations in noxious thermal sensation and downregulation of CGRP in the 8 weeks after induction of diabetes in rats. We supplemented capsaicin in the diet of the animals to upregulate CGRP and reversed the downregulation of the neuropeptide in the dorsal root ganglion (DRG) neurons dissociated from the diabetic animals, via gene transfection and exogenous CGRP, to test disease-preventing and disease-limiting effects of CGRP. Significant preservation of the nociceptive sensation, CGRP in spinal cord and DRG neurons, and number of CGRP-expressing neurons was found in the diabetic animals given capsaicin. Improvement in the survival of the neurons and the outgrowth of neurites was achieved in the neurons transfected by LV-CGRP or by exogenous CGRP, paralleling the correction of abnormalities of intracellular reactive oxygen species and mitochondrial transmembrane potentials. The results suggest that downregulation of CGRP impairs viability, regeneration and function of peripheral sensory neurons while capsaicin normalizes the CGRP peptidergic DRG neurons and function of the sensory nerves.