Primary Sensory Neurons Research Articles (Page 1)

Nociceptive afferents and dorsal horn neurons undergo significant functional changes in pathological pain conditions. The structural remodeling of synapses of C afferents, which may contribute to the long-term maintenance of these changes, is not well understood. To investigate this issue, we used quantitative immuno-electron microscopy with serial sections to examine the structural changes of calcitonin gene-related peptide (CGRP)-immunopositive (+) and isolectin-B4+ (IB4+) axon terminals (boutons) and their pre- and postsynaptic elements in the rat medullary dorsal horn (MDH, trigeminal caudal nucleus). The study was conducted at 4 days (CFA 4-day) and 21 days (CFA 21-day) following complete Freund's adjuvant (CFA) injection into the vibrissa pad of the male Sprague-Dawley rats, when thermal hyperalgesia was severe and had recovered, respectively. The ultrastructural parameters correlated with synaptic strength (bouton volume, mitochondrial volume, docked vesicle number, postsynaptic density area, dendritic spine number and size) in CGRP+ and IB4+ boutons and their postsynaptic dendrites increased significantly in the CFA 4-day group compared to control. The fraction of IB4+ boutons receiving axoaxonic synapses and the number of GAD65/67+ boutons involved in pre- and postsynaptic inhibition decreased significantly in the CFA 4-day group compared to control; these changes were restored to control levels in the CFA 21-day group. These structural changes in the C afferents and their pre- and postsynaptic elements in the MDH following inflammation may provide the morphological basis for the development and long-term maintenance of craniofacial inflammatory pain.Significance statement Two distinct classes of primary sensory neurons with unmyelinated (C) afferent fibers are involved in the development of hypersensitivity following inflammation: peptidergic, which express calcitonin gene-related peptide (CGRP) and nonpeptidergic, which express isolectin B4 (IB4). In a model of inflammatory pain, we demonstrate: 1) extensive reversible structural changes in the CGRP+ and IB4+ afferent terminals in the medullary dorsal horn, which correlate with the indicators of synaptic strength, bouton volume, mitochondrial volume, number of docked vesicles, area of postsynaptic density, and number and size of postsynaptic dendritic spines; 2) reversible loss of inhibitory boutons that underlie the presynaptic inhibition of IB4+ afferents. These changes may serve as the morphological basis for the development and persistence of craniofacial inflammatory pain.

Impaired synaptic inhibition by GABA and glycine contributes to excitatory-inhibitory imbalance in the spinal cord associated with chronic neuropathic pain; however, the underlying mechanisms remain unclear. Here, we investigated how GABAergic and glycinergic inputs regulate synaptic N-methyl-d-aspartate receptor (NMDAR) activity in excitatory and inhibitory neurons of the spinal dorsal horn in male and female mice. Vesicular glutamate transporter 2 (VGluT2)-expressing excitatory neurons and vesicular γ-aminobutyric acid transporter (VGAT)-expressing inhibitory neurons exhibited comparable mixed GABAergic and glycinergic inhibitory postsynaptic currents. Blockade of GABAA receptors with gabazine or glycine receptors with strychnine potentiated NMDAR-mediated miniature excitatory postsynaptic current (mEPSC) frequency, the amplitude of EPSCs monosynaptically evoked from the dorsal root, and puff NMDA currents in VGluT2, but not VGAT, neurons. These effects were abolished by silencing neuronal activity with tetrodotoxin or in Cacna2d1 knock-out (KO) mice. In mice with conditional Grin1 KO in primary sensory neurons (Grin1-cKO), gabazine and strychnine did not affect mEPSC frequency but still enhanced puff NMDA currents in dorsal horn neurons. Furthermore, intrathecal gabazine- or strychnine-induced nociceptive hypersensitivity was diminished by Grin1-cKO, Cacna2d1 KO, or α2δ-1 C-terminus peptide. Additionally, blocking metabotropic glutamate receptor 5 (mGluR5) prevents gabazine- and strychnine-induced increases in NMDAR-mediated mEPSC frequency, evoked EPSCs, and puff NMDA currents in VGluT2 neurons as well as nociceptive hypersensitivity. Our findings reveal that GABAergic and glycinergic inhibition tonically suppresses both presynaptic and postsynaptic NMDAR activity at primary afferent→excitatory neuron synapses. α2δ-1 and mGluR5 are essential for disinhibition-induced nociceptive hypersensitivity and synaptic NMDAR hyperactivity in the spinal cord.

Differential expression of TRPV1 and TRPM8 in the mouse trigeminal ganglion and spinal dorsal root ganglion.

Biologically calibrated and quantitative spiciness measurement based on a TRPV1-Responsive biosensing platform.

Genetic Deletion of TRPM8 Channels Restores Microvascular Function and Mitigates Chronic Kidney Disease Progression.

Primary afferent neurons detect sensory stimuli in the periphery and transmit this information to the dorsal horn of the spinal cord, where it is processed by excitatory and inhibitory controls before being sent to the brain. Our understanding of the synaptic architecture of these spinal circuits in the rodent has been massively advanced using antibodies raised against scaffolding proteins Homer1 and gephyrin, which anchor glutamate and GABA receptors to the membrane, respectively. Few studies have attempted to visualize spinal cord synapses in human tissue, partly due to a lack of high-quality tissue with low postmortem intervals. In this study, we reveal both excitatory and inhibitory synapses at a high resolution in human lumbar spinal cord tissue using Homer1 and gephyrin immunolabeling and show that the basic organization of these proteins within the dorsal horn is similar to that in the rodent. Homer1+ puncta are highly colocalized with ionotropic glutamate receptors, and over 75% are in contact with a presynaptic axon terminal containing the vesicular glutamate transporter 2 (VGluT2). Similarly, most gephyrin+ profiles are coextensive with the GABAA-α2 subunit but fewer than 10% colocalize with Homer1+ puncta, confirming the specificity of these markers. Finally, we use Homer1 immunolabeling to demonstrate that primary afferents can form complex synaptic arrangements in human spinal cord. We conclude that these antibodies can be used as reliable tools for the study of human synaptic circuitry, and we have used them to reveal insight into the spinal connections underlying somatosensation that can be expanded upon in future studies.

Genome-wide association studies (GWAS) with multiple human populations have uncovered single nucleotide variants of the CD2AP gene locus that are associated with Alzheimers Disease (AD) risk. However, the role of CD2AP in AD pathogenesis remains unknown. In adult PNS neurons, previous work demonstrated that CD2AP functions as a docking-scaffold/adaptor protein coordinator of nerve growth factor (NGF) trophic signaling and RAB5-mediated endocytosis. In the adult CNS, whereas CD2AP is robustly expressed in non-neuronal cells and neurovasculature, neuronal expression is restricted and poorly characterized. In this study using publicly available single cell/nucleus RNA sequencing, we observed that CD2AP mRNA is enriched in a population of TrkA-expressing cholinergic neurons of the adult mouse basal forebrain. Immunohistology using brain tissue from adult choline acetyltransferase (ChAT) GFP reporter mice, confirmed enrichment of CD2AP protein in cholinergic projections, and in neuron soma in the diagonal band of Broca where it co-localized with RAB5. Together with previous studies from NGF-responsive PNS primary sensory neurons, these observations indicate that CD2AP may play a role in retrograde trophic signaling in NGF-responsive CNS cholinergic neurons. In addition, we observed that CD2AP expression in these soma was increased in aged mice (18-month-old), concomitant with a reduction of co-localization with RAB5, suggesting a potential role for CD2AP in aging-associated changes in retrograde trophic signaling in these neurons. Basal forebrain cholinergic neurons project to the hippocampus and cortex, are required for learning and memory, are critical for brain health during aging, and disruption of RAB5 mediated endocytosis in these neurons is central to the pathogenesis of Alzheimers disease (AD). Future studies are therefore warranted to determine if CD2AP risk variants impact endocytosis and trophic signaling in cholinergic neurons in healthy aging and/or AD.

In vivo voltage imaging is a powerful tool for monitoring action potentials and dynamic electrical events in heterogeneous sensory neurons enabling the deciphering of rapid somatosensory information processing. Virus-driven expression of genetically encoded voltage indicator (GEVI) suffers from inconsistent expression levels and offers a limited time window for optimal voltage imaging. Here, we generated and characterized a knock-in mouse line with Pirt-driven expression of Marina, a positively tuned GEVI, in primary sensory neurons. Pirt-Marina mice enable optical reporting of touch, itch, and nociceptive sensations in vivo and distinct action potential patterns in the trigeminal and dorsal root ganglion neurons. Notably, Pirt-Marina mice display robust fluorescence signals in response to mechanical, thermal, or chemical stimuli, allowing visualization of transformations in sensory coding following inflammation and injury. This Pirt-Marina mouse line provides optical access to dynamic neuronal activity and plasticity in the peripheral nervous system (PNS) with high temporal accuracy, fidelity, and reliability.

The nuclear mitogen- and stress-activated kinases (MSKs) play a critical role in the development and persistence of pain after tissue injury. Here, we ascertained the MSK isoform, the cells and mechanisms, which mediate MSKs' pronociceptive function. Nocifensive behaviour evoked by subcutaneous formalin injection into the paw was quantified in wild type (WT), MSK1 and MSK2 global knock out (MSK1-/- and MSK2-/-) mice, and a month after injecting adeno-associated viral vector carrying short-hairpin (sh) RNA directed towards the MSK1-encoding gene Rps6ka5 mRNA or scrambled shRNA into the sciatic nerve of WT mice. Rps6ka5 expression in nociceptors was ascertained by analysing publicly available single cell and single nucleus RNA sequencing datasets on primary sensory neurons and reverse transcription polymerase chain reaction on dorsal root ganglia (DRG). Mitogen- and stress-activated kinase 1 expression was verified by immunofluorescent staining on DRG sections. MSK1-/- but not MSK2-/- mice exhibited significantly attenuated evoked nocifensive behaviour specifically in the second but not the first phase of the formalin test. Downregulating Rps6ka5 in nociceptors by the viral vector tool attenuated formalin-induced pain behaviour to the same extent as observed in MSK1-/- animals. Rps6ka5 expression was found in DRG and various transcriptionally defined groups of nociceptive primary sensory neurons (nociceptors). Immunofluorescence confirmed the presence of MSK1 predominantly in peptidergic nociceptors. MSK1 constitutes the principal MSK isoform, which is critically important for regulating cellular components that enable the transient activation of a specific subpopulation of nociceptors by formalin.

Opioids relieve pain by activating μ-opioid receptors (MORs), which inhibit communication between pain-sensing neurons (nociceptors) and the spinal cord. However, prolonged opioid use can paradoxically lead to increased pain sensitivity (hyperalgesia) and reduced analgesic efficacy (tolerance), partly because of the activation of NMDA-type glutamate receptors (NMDARs) at the central terminals of primary sensory neurons in the spinal cord. Here, we identified a critical role for the G protein Gαq in this paradox. Pharmacological inhibition of Gαq in rats reversed morphine-induced increases in NMDAR phosphorylation, synaptic trafficking, and activity at sensory neuron terminals and reduced morphine-induced excitatory nociceptive input to spinal dorsal horn neurons. Morphine enhanced Gαq coupling specifically to metabotropic glutamate receptor 5 (mGluR5) dimers in the spinal cord. Furthermore, targeted knockdown of Gαq in dorsal root ganglion neurons in mice normalized NMDAR-related changes and prevented NMDAR-mediated synaptic potentiation triggered by MOR activation. In addition, either pharmacological or genetic disruption of Gαq signaling enhanced morphine's analgesic effects while reducing hyperalgesia and tolerance. These findings reveal that Gαq signaling contributes to opioid-induced NMDAR hyperactivity at nociceptor central terminals by promoting MOR-mGluR5 cross-talk. Targeting this pathway may improve the safety and efficacy of opioid-based pain management.

Impaired plasma membrane calcium ATPase activity and mitochondrial dysfunction contribute to calcium dysregulation in Fabry disease-related painful neuropathy.

Activation of cannabinoid CB1 receptors suppresses HCN channels function in dorsal root ganglion neurons of rats.

Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy characterized by sensory dysfunction and muscle weakness, manifesting in the most distal limbs first and progressing more proximal. Over a hundred genes are currently linked to CMT with enrichment for activities in myelination, axon transport, and protein synthesis. Mutations in tRNA synthetases cause dominantly inherited forms of CMT and animal models with CMT-linked mutations in these enzymes display defects in neuronal protein synthesis. Rescuing protein synthesis in CMT mutant neurons could offer exciting therapeutic options beyond symptom management. To address this need, we expressed CMT-linked variants in tyrosyl tRNA synthetase (YARS-CMT) in primary sensory neurons and evaluated impacts on protein synthesis and cell viability. YARS-CMT expression reduced protein synthesis in these neurons prior to the onset of caspase-dependent axon degeneration and cell death. To determine how YARS-CMT expression affects axon outgrowth, we dissociated and replated these neurons to stimulate axon regeneration. To our surprise, axonal regrowth occurred normally in replated YARS-CMT neurons. Moreover, replating YARS-CMT neurons rescued protein synthesis. Inhibiting mTOR suppressed rescue of protein synthesis after replating, consistent with its significant role in protein synthesis during axon regeneration. These discoveries identify new avenues for augmenting protein synthesis in diseased neurons and restoring protein synthesis in CMT or other neurological disorders.

SUMMARYBoth sensory and non-sensory brain regions receive mixed inputs from single neurons which require decomposition and integration before proceeding through a processing hierarchy. Whether mixed input signals are used in biological neural networks to derive pure single neuron representations, or distributed as new population representations from mixed single neurons, is not clear. In this study, we measured the distribution of single neuron hue and luminance tuning in the dorsolateral geniculate nucleus (dLGN) and primary visual cortex (V1) of mice, as well as the information about and structure of hue and luminance representations in populations of hundred of simultaneously sampled neurons. We compare single neuron and population encoding to null models expected for random integration and extraction of pure categorical single neuron representation. Using both univariate and multivariate regression techniques, we consistently noted that tuning for hue and luminance, rather than clustering into categorical response structures, formed uniform distributions. While the distribution of single neuron selectivity varied across the thalamocortical circuit, we found no evidence of categorical tuning organization emerging in the hierarchy. Nevertheless, populations contained complete information, in either high-dimensional linear representations or low-dimensional non-linear representations. In summary, we find that as early as primary sensory cortex and thalamus single neurons that have mixed selectivity for hue and luminance form a high dimensional representation of those variables, which can be non-linearly embedded in multiple separable representations.

Alloknesis refers to itch caused by normally non itch-inducing stimuli, particularly light mechanical stimuli, such as contacts with clothes or other human bodies. This symptom occurs in patients suffering from chronic itch. While it has been mainly described in patients with atopic dermatitis, it is probably present in numerous other conditions and it could induce a severe burden. Until now, it is mainly diagnosed using Von Frey filaments and validated questionnaires are lacking. Alloknesis differs from mechanical pruritus in that it is linked to sensitization to pruritus and therefore occurs in pathological conditions, whereas mechanical pruritus (triggered by the presence of insects on the skin, for example) is a physiological phenomenon. While the role of central sensitization to pruritus in alloknesis is still poorly understood, the role of peripheral sensitization is becoming clearer. Interactions between low-threshold mechanoreceptors (LTMRs) and spinal interneurons are especially involved. Both the mechanical labelled pathway and the polymodal pathway have been shown to contribute to mechanical alloknesis. The mechanical labelled pathway comprises dedicated primary sensory neurons, spinal interneurons, and projection neurons that are functionally distinct from those involved in chemical itch. The polymodal pathway relies on a subset of primary sensory neurons traditionally associated with chemical itch, which can also transduce light mechanical stimuli through the activation of the mechanosensitive ion channel PIEZO1. Both converge onto the gastrin-releasing peptide (GRP) - GRP receptor (GRPR) chemical itch pathway in the spinal cord. Alloknesis is largely unknown to healthcare professionals and even more so to patients, and is not actively investigated. The objective of reducing alloknesis should be considered a therapeutic goal. To date, it has not been investigated in clinical trials. A novel research domain is emerging concerning this symptom, which exerts a substantial impact on the daily lives of numerous patients.

Head movements are detected and signalled to primary sensory neurons by vestibular types I and II hair cells. Signal transmission involves glutamate exocytosis from hair cells, which is triggered by Ca2+ inflow through voltage‐gated CaV1.3 Ca2+ channels. In a previous study on mice, we reported a Ca2+‐dependent exocytosis in both hair cell types, measured as a sustained change in cell membrane capacitance (ΔCm) following cell depolarization, which was significantly smaller in type I than in type II hair cells. By contrast, only type I hair cells showed a large transient ΔCm, which was still present in CaV1.3−/− mouse type I hair cells. Here we investigated the nature of this transient ΔCm. We found that it was unaffected by 10 mm intracellular EGTA, which blocked most of the sustained exocytosis in these cells, demonstrating its insensitivity to intracellular Ca2+. Moreover the amplitude of the transient ΔCm correlated with the degree of activation of the low‐voltage activated outward rectifying K+ conductance, GK,L, expressed by type I, but not type II hair cells. Finally the sign and kinetics of the transient ΔCm changed based on voltage steps activating or deactivating GK,L. These findings are consistent with the transient ΔCm arising from the mobilization of charges during the gating of K,L channels, while excluding fast transient neurotransmitter exocytosis. Its large amplitude can be explained by the high resistance of the calyceal synaptic cleft since it was significantly reduced in Caspr−/− mice, which show a significantly larger synaptic cleft compared to wild type mice.Key pointsVestibular type I and type II hair cells signal head movement to the central nervous system.Signal transmission from both hair cell types relies on Ca2+‐dependent glutamate exocytosis, measured here as a sustained change in cell membrane capacitance (ΔCm). Type I hair cells exhibit also a large transient ΔCm, whose nature has not been elucidated.In this study we found that the transient ΔCm does not involve exocytosis, but it is generated by the gating of the low‐voltage activated outward rectifying K+ conductance, specifically expressed in type I hair cells.Transient ΔCm analysis (also carried out in mice lacking the core protein of the septate‐like junction) conclusively demonstrates that type I hair cells, like type II ones, do not elicit a transient release of neurotransmitter.Knowledge of the basic mechanisms of vestibular signalling is crucial in the study of pharmacological treatment for vestibular disorders and in the drug side effects targeted there.

Potentiation of acid-sensing ion channel currents by EGF/EGFR signaling in rat dorsal root ganglion neurons.

Vascular dysfunction causes vascular pain (VP), the most common clinical manifestation of prevalent vascular diseases, while the mechanisms remain elusive. Mouse models are developed by mimicking peripheral vascular diseases and combining multiple strategies to demonstrate that primary sensory neuronal endothelin A receptor (ETAR) mediates experimental and clinical VP through an endothelial-neural axis. Endothelial cells (ECs), but not macrophages or smooth muscle cells (SMCs), release endothelin-1 (ET-1) and directly activate primary sensory neurons by binding to ETAR on sensory neurons, resulting in VP. Mice that underwent vessel ligation exhibit long-lasting mechanical hyperalgesia, but no heat hyperalgesia or cold allodynia, without inflammatory cell infiltration into or gliosis in the nervous system. These mice also display a moderate decrease in blood perfusion in the affected hindpaws, without evident ischemic injury or tissue necrosis after vessel ligation. Activating ECs with optogenetics or chemogenetics elicits spontaneous pain-like behaviors and lasting mechanical hyperalgesia. Blocking the increased endothelial ET-1/neural ETAR signal reduces pain-like behaviors caused by vessel ligation or activation of ECs. Treatment with oral bosentan alleviates VP in clinical patients with tourniquet-induced VP. These findings suggest that targeting the endothelial-neural axis and ET-1/ETAR pathway may represent therapeutic strategies for VP.

Primary somatosensory neurons, glial cells in the peripheral ganglia, and neural circuits in the spinal cord function as dynamic network circuits that transmit information to the brain. Although a variety of methods and techniques have been used to study individual neurons or tissue explants, the number of neurons that can be monitored is limited. Imaging intact primary sensory neurons, such as those in the dorsal root ganglion and trigeminal ganglia, and the spinal cord in vivo using fluorescent calcium markers helps overcome the limitations of previous methods and techniques by allowing researchers to monitor tens to thousands of cells simultaneously. This allows researchers to conduct experiments to elucidate somatosensory mechanisms and responses to axonal injury that were previously difficult or impossible to observe. Using this approach, researchers have studied dynamic neural network circuits, connectivity, responses to soft and deep touch, heat, cold, chemicals, inflammation, and injury, and they have repeatedly imaged individual neurons over long periods of time. Approaches include using calcium-sensitive fluorescent dyes and genetically encoded markers, performing terminal exposure surgeries, using chambers designed to monitor large numbers of cells or repeatedly imaging small numbers of cells, and imaging animals with or without anesthesia. This review discusses the advantages and disadvantages of in vivo calcium imaging for studying somatosensory and axonal injury in peripheral sensory ganglia and the dorsal spinal cord, as well as anticipated future directions.

Mosquitoes detect sound with their antennae, which transmit vibrations to mechanosensory neurons in Johnston's organ. However, their auditory system is exposed to low-frequency noise such as convective and thermal noise, as well as noise induced by flight, which could impair sensitivity. High-pass filters (HPFs) may mitigate this issue by suppressing low-frequency interference before it is transformed into neuronal signals. We investigated HPF mechanisms in Culex pipiens mosquitoes by analyzing the phase-frequency characteristics of the primary sensory neurons in the Johnston's organ. Electrophysiological recordings from male and female mosquitoes revealed phase shifts consistent with high-pass filtering. Initial modeling suggested a single HPF; however, experimentally obtained phase shifts exceeding -90° required revising the model to include two serially connected HPFs. The results showed that male mosquitoes exhibit stronger low-frequency suppression (~32 dB at 10 Hz) compared to females (~21 dB), with some female neurons showing negligible filtering. The estimated delay in signal transmission was ~7 ms for both sexes. These findings suggest that HPFs enhance noise immunity, particularly in males, whose auditory sensitivity is critical for mating. The diversity in female neuronal tuning may reflect broader auditory functions in addition to mating, such as host detection. This study provides indirect evidence for HPFs in mosquito hearing and highlights sex-specific adaptations in auditory processing. The proposed dual-HPF model improves our understanding of how mosquitoes maintain high auditory sensitivity in noisy environments.