Bryan A. Copits

Plasticity within somatosensory circuits enables adaptations to environmental changes, however these can become maladaptive following injury and lead to chronic pain states. Presynaptic neurexins (Nrxns) are key organizers of synapse function that bind to a wide variety of postsynaptic proteins in a splice-isoform dependent manner to regulate synaptic connectivity, transmission and plasticity. Here we observed that NRXN alternative splicing at splice site 4 (SS4), an exon critical for synaptic function and long-term potentiation at synapses in the central nervous system, is plastic in human sensory neurons of the dorsal root ganglia (DRG). Taking a reverse translational approach, we used mouse models to understand what might regulate these splicing changes in DRG. We observed a shift to transcripts lacking this alternatively spliced exon for Nrxn1 and Nrxn3 in response to depolarization, but not sensitization with PGE ₂ . Alternative splicing at SS4 was not altered by acute inflammatory pain but was reduced by central axotomy in vivo . We found that Nrxn alternative splicing patterns in DRG are dynamic during nervous system development, and vary between neurons of the DRG, spinal cord, and cortex in adult animals. Lastly, we observed that exon use at this site is conserved between species for Nrxn1α, but significantly different for all other isoforms. Thus, Nrxn alternative splicing at SS4 is plastic in human and mouse DRG neurons, which may influence somatosensory circuit wiring and synaptic function. Significance statement: The neurexin family of adhesion molecules act as key organizers of synaptic connectivity and function. These molecules undergo extensive alternative slicing to generate thousands of isoforms that can influence their interactions with diverse families of postsynaptic adhesion molecules. We found that alternative splicing at one of these key sites is altered in human somatosensory obtained from organ donors with damaged spinal grey matter. We then took a reverse translational approach in mouse model organisms to understand if Nrxn alternative splicing is plastic in peripheral somatosensory neurons of the dorsal root ganglia.

Peripheral nerve injury induces long-lasting changes in sensory neurons that contribute to the development of neuropathic pain. Although the histamine H₃ receptor (H₃R) is best known for regulating neurotransmitter release in the central nervous system, its expression and role in dorsal root ganglia (DRG) remain poorly understood. In this study, we examined the expression, cellular distribution, and functional role of H₃R in DRG and implications in neuropathic pain. Sciatic nerve injury increased membrane-associated H₃R protein expression in the DRG, suggesting enhanced receptor trafficking or stabilization at the neuronal membrane. Single-cell transcriptomic analysis revealed that H₃R mRNA expression in DRG is predominantly restricted to peptidergic sensory neurons and C-LTMRs rodents. Fluorescence imaging and transcriptomic analysis also suggest that the majority of H3R co-express with TrkA ⁺ (a NGF receptor) sensory neuron populations. These findings identify a subset of NGF-responsive nociceptors in which H₃R may directly influence injury-induced sensitization. Pain behavior assessment demonstrated a paradoxical role of an H3R inverse agonist (GSK334429), which reduces mechanical hypersensitivity caused by chronic constriction injury (CCI) with no change in thermal pain. Patch-clamp recordings show that the GSK334429 attenuates NGF-induced hyperexcitability in DRG neuronal cultures. Overall, our work suggests a contributing role of H3R in modality-specific effects on sensory processing through its enrichment in specific neuronal populations in DRG. Graphical

Descending projections from the brain to the spinal cord can regulate painful stimulus processing and are modulated by endogenous and exogenous opioids. We investigated the role of mu opioid receptors (MORs) in GABAergic vs. glutamatergic neurons of the rostral ventral medulla (RVM) in a mouse model of chronic neuropathic pain. We found that activating glutamatergic and GABAergic neurons in the RVM both result in antinociception at baseline, but glutamatergic neurons enhance pain responses after nerve injury. We then interrogated the role of RVM MOR signaling on neuropathic pain by using CRISPR/Cas9 to delete MOR in glutamatergic or GABAergic RVM neurons. We found that MOR knockout in glutamatergic and GABAergic RVM neurons precipitates early neuropathic pain onset with no effect on chronic pain intensity. These results suggest that RVM MOR signaling modulates hypersensitivity in the early phase of injury, but chronic neuropathic pain is largely independent of mu opioid receptor signaling.

Patients with chronic neuropathic pain typically experience affective symptoms that drive reduced quality of life and negatively impact pain management. Mesolimbic dopamine is necessary for reward valuation and learning, and the existence of a hypodopaminergic state has been proposed to underlie these affective symptoms of chronic pain. However, direct functional evidence for this hypodopaminergic state is lacking, and the mechanisms underlying its emergence over the acute to chronic pain transition are unknown. Here, we find a selective deficit in the ability of mesolimbic dopamine neurons to sustain burst firing, which is apparent uniquely at chronic timepoints following neuropathic injury. As a result, animals are unable to sustain effortful pursuit of rewards under conditions of high effort or time costs. Convergent biophysical modeling and experimental electrophysiology establish that in a spared nerve injury (SNI) model of chronic neuropathic pain, calcium-activated, small-conductance potassium (SK) channel function is impaired, resulting in lower peak firing and earlier entry into depolarization block of mesolimbic dopamine neurons. Critically, dopamine dependent reward learning, formation of cue-reward associations and locomotor activity remain intact, arguing against the interpretation of a generalized hypodopaminergic state. These results elucidate a circuit-level basis for selective motivational deficits emerging in chronic neuropathic pain.

The human peripheral nervous system (PNS) consists of many ganglia, including sympathetic ganglia (SG) and dorsal root ganglia (DRG), that house the cell bodies of many constituent neuron types and non-neuronal cells of the PNS. However, the molecular and cellular diversity of these human PNS cell types and their implications in human diseases remain elusive. By generating an integrated single-cell multi-omic atlas of human SG and DRG, we provide comprehensive transcriptional and epigenomic landscapes of various cell types in these peripheral ganglia. While the major cell types and their transcriptional and epigenomic features are similar between human SG and DRG, we identify key transcriptional and epigenomic differences between SG and DRG cell types, highlighting the distinct molecular mechanisms underlying their specific functions. Moreover, by mapping the expression and chromatin accessibility of disease-associated genes in human SG and DRG cell types, we identify cellular and molecular mechanisms that may underlie various peripheral neuropathies. This atlas serves as a valuable resource for understanding the intricate cell-type-specific molecules and interactions in the human PNS and their implications in human health and diseases.

Corticotropin-releasing hormone (CRH) is a peptide associated with stress and anxiety that acts as a potent modulator throughout the nervous system. The thalamic reticular nucleus (TRN) displays high expression of the CRH receptor 1 (CRHR1), but whether CRH modulates key TRN functions, such as sleep spindle rhythmogenesis, remained unexplored. Combining polysomnographic and photometric recordings in mice, we show that CRH release in TRN during non-rapid-eye movement sleep (NREMS) oscillates with a ~50-s periodicity, anti-correlating with sleep spindle dynamics. Optogenetic manipulations of CRH release in TRN modulated NREMS fragmentation through microarousals with corresponding changes in sigma and delta power. In ex-vivo recordings, CRHR1 activation decreased the propensity of TRN neurons to fire calcium bursts. CRHR1 knockdown in parvalbumin TRN neurons prevented the effects of CRH on NREMS and TRN bursting. Thus, CRHR1 impacts NREMS by modulating thalamic excitability, providing a potential target to stabilize sleep impairments associated with stress and anxiety.

Despite major advances in pain science, the approval of novel therapeutics has been slow. A major cause for the lack of new analgesics may be fundamental biological differences between humans and model organisms used in preclinical research. Large-scale transcriptional profiling efforts on human dorsal root ganglia (hDRG) have now identified at least 22 distinct neuronal subtypes; however, a significant knowledge gap exists in ascribing functional phenotypes to these diverse neuronal populations. In this study, we use Patch-seq recordings in hDRG to link electrical properties to transcriptionally defined cell types. First, through unbiased clustering of electrophysiological properties from 228 hDRG neurons, we identify three electrophysiological subtypes (E-types). Next, we show that E-types can be mapped onto specific transcriptional classes (T-types) of hDRG neurons. We find that donor’s pain history is associated with E-type-specific differences in electrical properties, some of which may be associated with higher expression of voltage-gated sodium channels, NaV1.7 and NaV1.8. These results highlight the importance of using multimodal profiling to better understand human sensory neuron biology and may help reveal novel therapeutic targets driving chronic pain. Teaser: Mapping electrical properties to transcriptional profiles of human sensory neurons offers new insights into pain neurobiology

Introduction: Autoantibodies to the Fibroblast Growth Factor Receptor 3 (FGFR3-AAbs) have been associated with idiopathic sensory-predominant neuropathy. The pathogenicity of FGFR3-AAbs in this disorder is unknown. Pathogenic mechanisms of autoantibodies in other dysimmune neuropathies commonly involve their direct binding to antigens on either neurons or glia. The expression of FGFR3 in the human peripheral nervous system is unknown. Therefore, as an initial step toward clarifying the pathogenicity of FGFR3-AAbs, we characterized the expression of FGFR3 in nerve (hNerve), dorsal root ganglia (hDRG) and spinal cord (hSC) in human. Methods: FGFR3 mRNA was assayed via in situ hybridization (ISH) on post-mortem sections of hNerve, hDRG and hSC, and by re-analysis of RNA-sequencing data from hDRG. FGFR3 protein was assayed in these tissues using capillary electrophoretic immunoassays (CEIA) with several validated anti-FGFR3 antibodies. Results: FGFR3 mRNA was not detected in hNerve or hDRG but was abundant in hSC by ISH. FGFR3 protein was absent from hNerve and hDRG by CEIA but was moderately expressed in hSC. Discussion: A direct pathogenic mechanism of FGFR3-AAbs in sensory neuropathy would require the expression of FGFR3 in either neurons or non-neuronal cells in nerve or DRG. Using multiple methods, we did not detect FGFR3 expression at the mRNA or protein levels in these tissues. Given the absence of FGFR3 from hNerve and hDRG, it is improbable that FGFR3-AAbs cause direct damage to the neural components involved in neuropathy and thus are unlikely to be pathogenic, although indirect mechanisms via non-neural cells cannot be excluded.

G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signalling required for cells is likely supplemented by signalling regulators such as non-GPCR GEFs and guanine nucleotide dissociation inhibitors (GDIs). Activators of G-protein signalling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure–function relationship or signalling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3’s G-protein regulatory motif, to understand its GDI activity and induce standalone Gβγ signalling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signalling pathways and triggering GPCR-independent Gβγ signalling in cells and in vivo .

We recently developed an approach for cell type–specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from Streptococcus pyogenes (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.