Biology subjects

Griffith, T. N.

Publications and source records attributed to Griffith, T. N..

5 recordsLinked to original sources

bioRxiv2026

Intersectional CRISPR-Cas9 genetic targeting reveals acute role of Nav1.1 in proprioceptive behavior and function

Proprioceptors, a specialized subset of mechanosensory neurons that relay sensory feedback from muscles and tendons, are required for precise, goal-directed movement. Like all neurons, proprioceptors rely on voltage-gated ion channels to generate and transmit electrical signals. Investigating ion channel function in proprioceptors in vivo is technically challenging because current approaches to selectively target proprioceptors require the generation of triple transgenic models and creation of both loxP- and Frt-flanked alleles. To facilitate selective targeting of genes within proprioceptors, we employed an intersectional cell-specific gene editing approach that leverages CRISPR/Cas9 and sensory-neuron selective viral capsids. This approach combines single-guide RNA (sgRNA) delivery in sensory neuron-selective adeno-associated viral (AAV) capsids in mice with parvalbumin-driven Cas9 expression. We tested this approach by targeting the voltage-gated sodium (NaV) channel NaV1.1. Targeting NaV1.1 using this approach led to significant motor coordination deficits as early as 3 weeks following sgRNA delivery. Furthermore, whole-cell current clamp recordings from transduced proprioceptors revealed NaV1.1 is required for maintaining short-duration action potentials, which would support high-frequency firing typically observed in proprioceptors. Collectively, this study establishes a versatile platform for precise spatiotemporal gene manipulation in otherwise hard-to-access sensory neuron populations, while also providing evidence that NaV1.1 is essential for proprioceptor function in adulthood. Significance StatementProprioceptors are sensory neurons that relay information about muscle length and force to enable coordinated movement and motor reflexes. Investigating how ion channels contribute to proprioceptor function has been limited by the lack of straightforward and selective genetic tools, which can also confound interpretation of behavioral phenotypes. Here, we developed an intersectional CRISPR/Cas9 strategy that combines sensory-neuron specific viral delivery of sgRNAs with spatially restricted Cas9 expression in mice. Using this method we targeted the voltage-gated sodium channel, NaV1.1, which led to persistent motor coordination deficits and impaired proprioceptor action potential waveform, establishing a direct, cell-autonomous role for NaV1.1 in proprioceptor function. Thus, our approach provides a flexible platform for spatially and temporally precise gene manipulation in proprioceptors.

neuroscience↗

bioRxiv2025

Activity-driven proprioceptive synaptic refinement in the developing spinal cord by complement signaling mechanisms

Proprioceptive group Ia afferents detect muscle stretch to guide effortless and purposeful movement and make monosynaptic connections with spinal a-motor neurons to mediate reflexes, such as the stretch reflex. It is thought that proprioceptive Ia afferents target motor neurons of the same spinal segment; yet, how this specificity, if any, is established during early development is unknown. Using ex vivo spinal cord electrophysiology preparations from neonatal mice of both sexes, we identified a developmental period during which proprioceptive la afferents evoke both segmental and intersegmental responses at monosynaptic latencies. We provide anatomical evidence that motor neurons in the lumbar segment 4 (L4) receive direct input from proprioceptive Ia afferents in L5 during early postnatal development. Intersegmental responses (L4/L5) were prominent at postnatal days (P) 4-7 but were virtually absent by P11-13. To test the role of proprioceptor activity on segmental specification, we analyzed NaV1.6 conditional knockout mice (NaV1.6cKO), in which proprioceptor signaling is impaired, and found that intersegmental responses persist up to P11-13 but were absent in age-matched floxed controls. We predict this is due to impaired activation of complement signaling pathways, as NaV1.6cKO mice showed reduced C1qA expression in the ventral spinal cord at P9. Consistent with this, C1qA knockout mice also retain intersegmental responses at P11-13. Collectively, these findings identify an important postnatal window during which segmental specificity of proprioceptive circuits emerges and suggest that proprioceptor activity induces C1qA-mediated elimination of excessive intersegmental connectivity. Key points summaryO_LIDuring the first ten days of postnatal development, the spinal monosynaptic reflex arc possesses intersegmental proprioceptive afferent projections onto motor neurons of an adjacent segment. C_LIO_LIThese exuberant Ia-motoneuron projections are accompanied by evocable intersegmental reflex responses. In normal conditions, the intersegmental response is lost by postnatal day 11. Conversely, in a mouse model of impaired proprioceptive signaling, the intersegmental response persists. C_LIO_LIWe find that impaired proprioceptive signaling through the monosynaptic reflex arc leads to low expression levels of the complement signaling protein, C1qA, near ChAT-positive motor neurons, whereas microglia recruitment is unaffected. This was consistent with the persistence of robust intersegmental responses in C1qA knockout mice. C_LIO_LICollectively, our results show that proprioceptor activity induces complement cascade signaling to prune exuberant intersegmental synapses in the spinal cord formed during early development, resulting in segmental restriction of the monosynaptic reflex arc by the end of the second postnatal week. C_LI

neuroscience↗

bioRxiv2024

Differential encoding of mammalian proprioception by voltage-gated sodium channels

Animals that require purposeful movement for survival are endowed with mechanosensory neurons called proprioceptors that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we have identified distinct and nonredundant roles for two voltage-gated sodium channels (NaVs), NaV1.1 and NaV1.6, in mammalian proprioception. Deletion of NaV1.6 in somatosensory neurons (NaV1.6cKO mice) causes severe motor deficits accompanied by complete loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target NaV1.1 (NaV1.1cKO). In NaV1.6cKO animals, loss of proprioceptive feedback caused non-cell- autonomous impairments in proprioceptor end-organs and skeletal muscle that were absent in NaV1.1cKO mice. We attribute the differential contribution of NaV1.1 and NaV1.6 in proprioceptor function to distinct cellular localization patterns. Collectively, these data provide the first evidence that NaV subtypes uniquely shape neurotransmission within a somatosensory modality. TeaserVoltage gated sodium channels differentially encode mammalian proprioception via distinct cellular localization patterns.

neuroscience↗

bioRxiv2022

Computational design of peptides to target Nav1.7 channel with high potency and selectivity for the treatment of pain

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1,000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally-designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

biophysics↗

bioRxiv2019

Tetrodotoxin-sensitive sodium channels mediate action potential firing and excitability in menthol-sensitive Vglut3-lineage sensory neurons

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaVs transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV currents readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.\n\nSignificance StatementSomatosensensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

neuroscience↗