A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes.

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

High-resolution mapping of sensory fibers at the healthy and post-myocardial infarct whole transgenic hearts.

The sensory nervous system is critical to maintain cardiac function. As opposed to efferent innervation, less is known about cardiac afferents. For this, we mapped the VGLUT2-expressing cardiac afferent fibers of spinal and vagal origin by using the VGLUT2::tdTomato double transgenic mouse as an approach to visualize the whole hearts both at the dorsal and ventral sides. For comparison, we colabeled mixed-sex transgenic hearts with either TUJ1 protein for global cardiac innervation or tyrosine hydroxylase for the sympathetic network at the healthy state or following ischemic injury. Interestingly, the nerve density for global and VGLUT2-expressing afferents was found significantly higher on the dorsal side compared to the ventral side. From the global nerve innervation detected by TUJ1 immunoreactivity, VGLUT2 afferent innervation was detected to be 15-25% of the total network. The detailed characterization of both the atria and the ventricles revealed a remarkable diversity of spinal afferent nerve ending morphologies of flower sprays, intramuscular endings, and end-net branches that innervate distinct anatomical parts of the heart. Using this integrative approach in a chronic myocardial infarct model, we showed a significant increase in hyperinnervation in the form of axonal sprouts for cardiac afferents at the infarct border zone, as well as denervation at distal sites of the ischemic area. The functional and physiological consequences of the abnormal sensory innervation remodeling post-ischemic injury should be further evaluated in future studies regarding their potential contribution to cardiac dysfunction.

Caenorhabditis elegans sine oculis/SIX-type homeobox genes act as homeotic switches to define neuronal subtype identities.

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.

Oxidative DNA Damage and Cisplatin Neurotoxicity Is Exacerbated by Inhibition of OGG1 Glycosylase Activity and APE1 Endonuclease Activity in Sensory Neurons.

Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. Our previous work suggests that the DNA repair activity of APE1/Ref-1, the rate-limiting enzyme of the base excision repair (BER) pathway, is critical for neuroprotection against cisplatin. A specific role for 8-oxoguanine DNA glycosylase-1 (OGG1), the glycosylase that removes the most common oxidative DNA lesion, and putative coordination of OGG1 with APE1/Ref-1 in sensory neurons, has not been investigated. We investigated whether inhibiting OGG1 glycosylase activity with the small molecule inhibitor, TH5487, and/or APE1/Ref-1 endonuclease activity with APE Repair Inhibitor III would alter the neurotoxic effects of cisplatin in sensory neuronal cultures. Sensory neuron function was assessed by calcitonin gene-related peptide (CGRP) release, as a marker of sensitivity and by neurite outgrowth. Cisplatin altered neuropeptide release in an inverse U-shaped fashion, with low concentrations enhancing and higher concentrations diminishing CGRP release. Pretreatment with BER inhibitors exacerbated the functional effects of cisplatin and enhanced 8oxo-dG and adduct lesions in the presence of cisplatin. Our studies demonstrate that inhibition of OGG1 and APE1 endonuclease activity enhances oxidative DNA damage and exacerbates neurotoxicity, thus limiting oxidative DNA damage in sensory neurons that might alleviate cisplatin-induced neuropathy.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

A high-throughput method to deliver targeted optogenetic stimulation to moving C. elegans populations.

We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal's body such as its head or tail; it automatically delivers stimuli triggered upon the animal's behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal's behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior-posterior intensity combinations were measured. The animal's probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal's response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.

Sensory representation and detection mechanisms of gut osmolality change.

Ingested food and water stimulate sensory systems in the oropharyngeal and gastrointestinal areas before absorption1,2. These sensory signals modulate brain appetite circuits in a feed-forward manner3-5. Emerging evidence suggests that osmolality sensing in the gut rapidly inhibits thirst neurons upon water intake. Nevertheless, it remains unclear how peripheral sensory neurons detect visceral osmolality changes, and how they modulate thirst. Here we use optical and electrical recording combined with genetic approaches to visualize osmolality responses from sensory ganglion neurons. Gut hypotonic stimuli activate a dedicated vagal population distinct from mechanical-, hypertonic- or nutrient-sensitive neurons. We demonstrate that hypotonic responses are mediated by vagal afferents innervating the hepatic portal area (HPA), through which most water and nutrients are absorbed. Eliminating sensory inputs from this area selectively abolished hypotonic but not mechanical responses in vagal neurons. Recording from forebrain thirst neurons and behavioural analyses show that HPA-derived osmolality signals are required for feed-forward thirst satiation and drinking termination. Notably, HPA-innervating vagal afferents do not sense osmolality itself. Instead, these responses are mediated partly by vasoactive intestinal peptide secreted after water ingestion. Together, our results reveal visceral hypoosmolality as an important vagal sensory modality, and that intestinal osmolality change is translated into hormonal signals to regulate thirst circuit activity through the HPA pathway.

The impact of SBF2 on taxane-induced peripheral neuropathy.

Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients. In addition to its impact on quality of life, this toxicity may lead to dose reductions or treatment discontinuation, adversely impacting survival outcomes and leading to health disparities in African Americans (AA). Our lab has previously identified deleterious mutations in SET-Binding Factor 2 (SBF2) that significantly associated with severe TIPN in AA patients. Here, we demonstrate the impact of SBF2 on taxane-induced neuronal damage using an ex vivo model of SBF2 knockdown of induced pluripotent stem cell-derived sensory neurons. Knockdown of SBF2 exacerbated paclitaxel changes to cell viability and neurite outgrowth while attenuating paclitaxel-induced sodium current inhibition. Our studies identified paclitaxel-induced expression changes specific to mature sensory neurons and revealed candidate genes involved in the exacerbation of paclitaxel-induced phenotypes accompanying SBF2 knockdown. Overall, these findings provide ex vivo support for the impact of SBF2 on the development of TIPN and shed light on the potential pathways involved.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP.

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair.

Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single-cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPARα signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPARα agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single-cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.

Quantitative, structural and molecular changes in neuroglia of aging mammals: A review.

The neuroglia of the central and peripheral nervous systems undergo numerous changes during normal aging. Astrocytes become hypertrophic and accumulate intermediate filaments. Oligodendrocytes and Schwann cells undergo alterations that are often accompanied by degenerative changes to the myelin sheath. In microglia, proliferation in response to injury, motility of cell processes, ability to migrate to sites of neural injury, and phagocytic and autophagic capabilities are reduced. In sensory ganglia, the number and extent of gaps between perineuronal satellite cells - that leave the surfaces of sensory ganglion neurons directly exposed to basal lamina- increase significantly. The molecular profiles of neuroglia also change in old age, which, in view of the interactions between neurons and neuroglia, have negative consequences for important physiological processes in the nervous system. Since neuroglia actively participate in numerous nervous system processes, it is likely that not only neurons but also neuroglia will prove to be useful targets for interventions to prevent, reverse or slow the behavioral changes and cognitive decline that often accompany senescence.

Quantitative description of the interactions among kinase cascades underlying long-term plasticity of Aplysia sensory neurons.

Kinases play critical roles in synaptic and neuronal changes involved in the formation of memory. However, significant gaps exist in the understanding of how interactions among kinase pathways contribute to the mechanistically distinct temporal domains of memory ranging from short-term memory to long-term memory (LTM). Activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathways are critical for long-term enhancement of neuronal excitability (LTEE) and long-term synaptic facilitation (LTF), essential processes in memory formation. This study provides new insights into how these pathways contribute to the temporal domains of memory, using empirical and computational approaches. Empirical studies of Aplysia sensory neurons identified a positive feedforward loop in which the PKA and ERK pathways converge to regulate RSK, and a negative feedback loop in which p38 MAPK inhibits the activation of ERK and RSK. A computational model incorporated these findings to simulate the dynamics of kinase activity produced by different stimulus protocols and predict the critical roles of kinase interactions in the dynamics of these pathways. These findings may provide insights into the mechanisms underlying aberrant synaptic plasticity observed in genetic disorders such as RASopathies and Coffin-Lowry syndrome.

HoxD transcription factors define monosynaptic sensory-motor specificity in the developing spinal cord.

The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. Here, we show that members of the HoxD family of transcription factors play a crucial role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10 and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.

An imaging analysis protocol to trace, quantify, and model multi-signal neuron morphology.

We describe how to reconstruct and quantify multi-signal neuronal morphology, using the dendritic distributions of microtubules and F-actin in sensory neurons from fly larvae as examples. We then provide a detailed procedure to analyze channel-specific morphometrics from these enhanced reconstructions. To illustrate applications, we demonstrate how to run a cytoskeleton-constrained simulation of dendritic tree generation and explain its validation against experimental data. This protocol is applicable to any species, developmental stage, brain region, cell class, branching process, and signal type. For complete details on the use and execution of this protocol, please refer to Nanda et al. (2020).

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Mini-Review: Mitochondrial dysfunction and chemotherapy-induced neuropathic pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is a somatosensory axonopathy in cancer patients receiving any of a variety of widely-use antitumor agents. CIPN can lead to long-lasting neuropathic pain that limits the dose or length of otherwise life-saving cancer therapy. Accumulating evidence over the last two decades indicates that many chemotherapeutic agents cause mitochondrial injury in the peripheral sensory nerves by disrupting mitochondrial structure and bioenergetics, increasing nitro-oxidative stress and altering mitochondrial transport, fission, fusion and mitophagy. The accumulation of abnormal and dysfunctional mitochondria in sensory neurons are linked to axonal growth defects resulting in the loss of intraepidermal nerve fibers in the hands and feet, increased spontaneous discharge and the sensitization of peripheral sensory neurons that provoke and promote changes in the central nervous system that establish a chronic neuropathic pain state. This has led to the propose mitotoxicity theory of CIPN. Strategies that improve mitochondrial function have shown success in preventing and reversing CIPN in pre-clinical animal models and have begun to show some progress toward translation to the clinic. In this review, we will review the evidence for, the causes and effects of and current strategies to target mitochondrial dysfunction in CIPN.

Building a circuit through correlated spontaneous neuronal activity in the developing vertebrate and invertebrate visual systems.

During the development of the vertebrate nervous systems, genetic programs assemble an immature circuit that is subsequently refined by neuronal activity evoked by external stimuli. However, prior to sensory experience, the intrinsic property of the developing nervous system also triggers correlated network-level neuronal activity, with retinal waves in the developing vertebrate retina being the best documented example. Spontaneous activity has also been found in the visual system of Drosophila Here, we compare the spontaneous activity of the developing visual system between mammalian and Drosophila and suggest that Drosophila is an emerging model for mechanistic and functional studies of correlated spontaneous activity.

MrgprC11+ sensory neurons mediate glabrous skin itch.

Itch arising from glabrous skin (palms and soles) has attracted limited attention within the field due to the lack of methodology. This is despite glabrous itch arising from many medical conditions such as plantar and palmar psoriasis, dyshidrosis, and cholestasis. Therefore, we developed a mouse glabrous skin behavioral assay to investigate the contribution of three previously identified pruriceptive neurons in glabrous skin itch. Our results show that MrgprA3+ and MrgprD+ neurons, although key mediators for hairy skin itch, do not play important roles in glabrous skin itch, demonstrating a mechanistic difference in itch sensation between hairy and glabrous skin. We found that MrgprC11+ neurons are the major mediators for glabrous skin itch. Activation of MrgprC11+ neurons induced glabrous skin itch, while ablation of MrgprC11+ neurons reduced both acute and chronic glabrous skin itch. Our study provides insights into the mechanisms of itch and opens up new avenues for future glabrous skin itch research.