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How thermal, mechanical and chemical stimuli applied to the skin are transduced into signals transmitted by peripheral neurons to the CNS is an area of intense study. Several studies indicate that transduction mechanisms are intrinsic to cutaneous neurons and that epidermal keratinocytes only modulate this transduction. Using mice expressing channelrhodopsin (ChR2) in keratinocytes we show that blue light activation of the epidermis alone can produce action potentials (APs) in multiple types of cutaneous sensory neurons including SA1, A-HTMR, CM, CH, CMC, CMH and CMHC fiber types. In loss of function studies, yellow light stimulation of keratinocytes that express halorhodopsin reduced AP generation in response to naturalistic stimuli. These findings support the idea that intrinsic sensory transduction mechanisms in epidermal keratinocytes can directly elicit AP firing in nociceptive as well as tactile sensory afferents and suggest a significantly expanded role for the epidermis in sensory processing. When a person touches a hot saucepan, nerve cells in the skin send a message to the brain that causes the person to pull away quickly. Similar messages alert the brain when the skin comes in contact with an object that is cold or causes pain. These nerve cells also help to transmit information about other sensations like holding a ball. Scientists believe that skin cells may release messages that influence how the nerves in the skin respond to sensations. But it is difficult to distinguish the respective roles of skin cells and nerve cells in experiments because these cells often appear to react at the same time. Researchers have discovered that a technique called optogenetics, which originally developed to study the brain, can help. Optogenetics uses genetic engineering to create skin cells that respond to light instead of touch. Baumbauer, DeBerry, Adelman et al. genetically engineered mice to express a light-sensitive protein in their skin cells. When these skin cells were exposed to light, the mice pulled away just like they would if they were responding to painful contact. This behavior coincided with electrical signals in the nerve cells even though the nerve cells themselves were not light sensitive. In further experiments, mice were genetically engineered to express another protein in their skin cells that prevents the neurons from being able to generate electrical signals. When these skin cells were exposed to light, the surrounding nerve cells produced fewer electrical signals. Together, the experiments show that skin cells are able to directly trigger electrical signals in nerve cells. Baumbauer, DeBerry, Adelman et al.'s findings may help researchers to understand why some patients with particular inflammatory conditions are in pain due to overactive nerve cells.

Sensory neurons are chemically and functionally heterogeneous, and this heterogeneity has been examined extensively over the last several decades. These studies have employed a variety of different methodologies, including anatomical, electrophysiological, and molecular approaches. Recent studies using next-generation sequencing techniques have examined the transcriptome of single sensory neurons. Although these reports have provided a wealth of exciting new information on the heterogeneity of sensory neurons, correlation with functional types is lacking. Here, we employed retrograde tracing of cutaneous and muscle afferents to examine the variety of mRNA expression profiles of individual, target-specific sensory neurons. In addition, we used an ex vivo skin/nerve/dorsal root ganglion/spinal cord preparation to record and characterize the functional response properties of individual cutaneous sensory neurons that were then intracellularly labeled with fluorescent dyes, recovered from dissociated cultures, and analyzed for gene expression. We found that by using single-cell quantitative polymerase chain reaction techniques and a set of 28 genes, we can identify transcriptionally distinct groups. We have also used calcium imaging and single-cell quantitative polymerase chain reaction to determine the correlation between levels of mRNA expression and functional protein expression and how functional properties correlated with the different transcriptional groups. These studies show that although transcriptomics does map to functional types, within any one functional subgroup, there are highly variable patterns of gene expression. Thus, studies that rely on the expression pattern of one or a few genes as a stand in for physiological experiments, runs a high risk of data misinterpretation with respect to function.

Primary sensory neurons are responsible for cutaneous somatosensory transduction. These neurons can transduce mechanical force, temperature, and chemical sensitivity, which we perceive as pressure, position, heat, cold, itch, and pain. It has long been recognized that different afferents are specific for distinct modalities, but it has not been clear if afferent sensitivities represent a continuum or unique populations of afferents with common properties. I found that mouse dorsal root ganglion afferents can be clearly divided into groups by their transcriptional expression and that these groups share common modality sensitivities and functional properties. Further investigation revealed that the levels of some individual transduction channels may be an even more accurate way to track modality sensitivity, giving a potential molecular signature for function in murine afferents. These findings imply that recognized histological projection bands in the spinal cord (Substance P, CGRP, IB4, etc.) reflect subpopulations of afferents with unique properties that are projecting to unique areas of the spinal cord. This is relevant to sensory coding models, but may also be very important for the development of neuropathic pain after nerve injury. Nerve injury and regeneration alters mRNA and protein expression, and causes parallel changes in afferent properties. These functional changes in afferents could cause unusual activity in corresponding spinal cord circuits (e.g. a spinal cord circuit that is normally mechanically insensitive would receive mechanical input). I investigated this possibility and discovered population-specific regulation of transcripts. This included a mechanotransducer, Piezo2, which is upregulated in the small peptidergic subpopulation. Subsequent knockdown using Piezo2 siRNA reduced the number of mechanically sensitive afferents. These findings suggest that Piezo2 is necessary for mechanotransduction in injured afferents and could be responsible for induced neuropathic pain.

Abstract Visceral organs receive neural innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in the mouse. Profiling of TL and LS bladder afferents was also done. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single cell RT-qPCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function or neuroimmune interaction. A total of 132 colon afferents (from NG, TL and LS) and 128 bladder afferents (from TL and LS) were analyzed. Retrograde labeling from the colon showed NG and TL afferents innervate proximal and distal regions of the colon whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon DRG (TL or LS) neurons. These results indicate that populations of primary visceral afferents are functionally “tuned” to detect and interact with the internal environment and that information from all levels is integrated at higher (CNS) levels, not only for regulation of homeostatic functions, but for conscious visceral sensations including pain. Significance Statement Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain is mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. Transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation is the result of convergence, and not segregation, of sensory input. Competing Interest Statement The authors have declared no competing interest. Footnotes * Supported by NINDS NS023725, NS096705 (HRK), NIAMS AR069951 (KMA), NIDDK DK124955 (BMD) * Abbreviations DRG dorsal root ganglia NG nodose ganglia RT-qPCR real-time quantitative polymerase chain reaction TL thoracolumbar LS lumbosacral

ABSTRACT Most cutaneous C-fibers, including both peptidergic and non-peptidergic subtypes are presumed to be nociceptors and respond to noxious input in a graded manner. However, mechanically sensitive, non-peptidergic C-fibers also respond to mechanical input in the innocuous range, and so the degree to which they contribute to nociception remains unclear. To address this gap, we investigated the function of non-peptidergic afferents using the MrgprdCre allele. In real time place aversion studies, we found that low frequency optogenetic activation of MrgrpdCre lineage neurons was not aversive in naïve mice, but became aversive after spared nerve injury (SNI). To address the underlying mechanisms of this allodynia, we recorded from lamina I spinoparabrachial (SPB) neurons using the semi-intact ex vivo preparation. Following SNI, innocuous brushing of the skin gave rise to abnormal activity in lamina I SPB neurons, consisting of an increase in the proportion of recorded neurons that responded with excitatory post synaptic potentials or action potentials. This increase was likely due, at least in part, to an increase in the proportion of lamina I (LI) SPB neurons that received input upon optogenetic activation of MrgprdCre lineage neurons. Intriguingly, in SPB neurons there was a significant increase in the EPSC latency from MrgprdCre lineage input following SNI, consistent with the possibility that the greater activation post SNI could be due to the recruitment of a new polysynaptic circuit. Together, our findings suggest MrgprdCre lineage neurons can provide mechanical input to the dorsal horn that is non-noxious before injury but becomes noxious afterwards due the engagement of a previously silent polysynaptic circuit in the dorsal horn. Competing Interest Statement The authors have declared no competing interest.

The human rights dimensions of climate change have been largely ignored until recently. This study ventures beyond the immediate threat to many rights posed by global warming - to health, food, lives and livelihoods - to inquire into the possible future rights implications of policies such as emissions trading and forestation regimes.

Small intestinal histologic abnormalities in celiac disease include atrophy of the intestinal villi, hypertrophy of the crypts and lymphocytic infiltration of intraepithelial spaces and lamina propria. These findings are central to diagnosis and their severity and change over time are valuable to monitor disease course and response to therapy. Subjective methods to grade celiac disease histological severity include the Marsh-Oberhuber and Corazza-Villanacci systems. Quantitative histology uses villus height (Vh), crypt depth (Cd), and intra-epithelial lymphocyte count (per 100 enterocytes) to provide objective measures of histologic changes including Vh:Cd ratio. Here we examine the available literature regarding these methodologies and support the use of quantitative histology as the preferred method for accurately and reproducibly demonstrating change of relevant histologic end points over time. We also propose a Quantitative-Mucosal Algorithmic Rules for Scoring Histology (Q-MARSH) system to partially align quantitative histology results with the traditional Marsh, Marsh-Oberhuber, and Corazza-Villanacci systems. Q-MARSH can provide a standardized, objective, and quantitative histology scoring system for use as a clinical or research application.

SignificanceSensory processing depends upon the integration of widely distributed neural assemblies. During every day listening, our ears receive different information (due to interaural time and amplitude differences) and it is known that both hemispheres extract different acoustic features. Nonetheless, acoustic features belonging to the same source become integrated. It has been suggested that the brain overcomes this “binding problem” by synchronization of oscillatory activity across the relevant regions. Here we probe interhemispheric oscillatory synchronization as a mechanism for acoustic feature binding using bihemispheric transcranial alternating current stimulation. Concurrent functional MRI reveals that antiphase stimulation of auditory areas changes effective connectivity between these areas, and that this change in connectivity predicts perceptual integration of dichotic stimuli. Brain connectivity plays a major role in the encoding, transfer, and integration of sensory information. Interregional synchronization of neural oscillations in the γ-frequency band has been suggested as a key mechanism underlying perceptual integration. In a recent study, we found evidence for this hypothesis showing that the modulation of interhemispheric oscillatory synchrony by means of bihemispheric high-density transcranial alternating current stimulation (HD-TACS) affects binaural integration of dichotic acoustic features. Here, we aimed to establish a direct link between oscillatory synchrony, effective brain connectivity, and binaural integration. We experimentally manipulated oscillatory synchrony (using bihemispheric γ-TACS with different interhemispheric phase lags) and assessed the effect on effective brain connectivity and binaural integration (as measured with functional MRI and a dichotic listening task, respectively). We found that TACS reduced intrahemispheric connectivity within the auditory cortices and antiphase (interhemispheric phase lag 180°) TACS modulated connectivity between the two auditory cortices. Importantly, the changes in intra- and interhemispheric connectivity induced by TACS were correlated with changes in perceptual integration. Our results indicate that γ-band synchronization between the two auditory cortices plays a functional role in binaural integration, supporting the proposed role of interregional oscillatory synchrony in perceptual integration.

Cardiovascular disease remains the leading cause of mortality in the United States, and cardiac arrhythmia underlies the majority of these deaths. Here, we report a new mechanism for congenital human cardiac arrhythmia due to defects in the regulation of the primary cardiac Na v channel, Na v 1.5 ( SCN5A ), by a family of signaling molecules termed fibroblast growth factor homologous factors (FHFs). Individuals harboring SCN5A variants that affect Na v 1.5/FHF interactions display atrial and ventricular phenotypes, syncope, and sudden cardiac death. The human variant results in aberrant Na v 1.5 inactivation, causing prolonged action potential duration and afterdepolarizations in murine myocytes, thereby providing a rationale for the human arrhythmia. Na v channels are essential for metazoan membrane depolarization, and Na v channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Na v channelopathies are primarily caused by variants that directly affect Na v channel permeability or gating. However, a new class of human Na v channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Na v channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Na v channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Na v channel Na v 1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Na v 1.5 for FHFs. Consistent with these data, Na v 1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Na v 1.5 p.H1849R as a gain-of-function for I Na by altering steady-state inactivation and slowing the rate of Na v 1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Na v 1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Na v channelopathy based on altered Na v 1.5 association with FHF proteins.

Brain connectivity plays a major role in the encoding, transfer, and integration of sensory information. Interregional synchronization of neural oscillations in the γ-frequency band has been suggested as a key mechanism underlying perceptual integration. In a recent study, we found evidence for this hypothesis showing that the modulation of interhemispheric oscillatory synchrony by means of bihemispheric high-density transcranial alternating current stimulation (HD-TACS) affects binaural integration of dichotic acoustic features. Here, we aimed to establish a direct link between oscillatory synchrony, effective brain connectivity, and binaural integration. We experimentally manipulated oscillatory synchrony (using bihemispheric γ-TACS with different interhemispheric phase lags) and assessed the effect on effective brain connectivity and binaural integration (as measured with functional MRI and a dichotic listening task, respectively). We found that TACS reduced intrahemispheric connectivity within the auditory cortices and antiphase (interhemispheric phase lag 180°) TACS modulated connectivity between the two auditory cortices. Importantly, the changes in intra- and interhemispheric connectivity induced by TACS were correlated with changes in perceptual integration. Our results indicate that γ-band synchronization between the two auditory cortices plays a functional role in binaural integration, supporting the proposed role of interregional oscillatory synchrony in perceptual integration.