Biology subjects

Gereau, R. W.

Publications and source records attributed to Gereau, R. W..

18 recordsLinked to original sources

bioRxiv2026

PixelPaws: a low-cost, open-source integrated platform for automated scoring of mouse behaviors

Behavioral scoring is central to preclinical pain research but is typically performed manually, which is inherently low-throughput and subject to variability between observers. Automated alternatives exist, but cost and programming expertise limit adoption beyond the laboratories that build them. Here we describe PixelPaws, an open-source platform combining a near-infrared imaging enclosure built from consumer parts for under $300, multi-camera acquisition software, a bundled pose-estimation network, and eight classifiers in a graphical user interface. We evaluated the platform across standard assays of pain, itch, and opioid withdrawal. Licking, scratching, and jumping classifiers were validated against trained human observers by leave-one-session-out evaluation. In the formalin assay, classifier-scored licking followed the expected biphasic time course and was reduced dose-dependently by oxycodone, with a phase 2 potency estimate comparable to blinded manual scoring. Chloroquine roughly tripled scratching, and naloxone precipitated a burst of jumping in oxycodone-treated mice. From the same recordings we derived a set of paw contour metrics. Contour intensity fell after formalin and tracked the oxycodone dose series, and contour area shifted toward symmetry after oxycodone in nerve-injured mice. Behavioral sequencing revealed distinct changes in the transi

neuroscience↗

bioRxiv2025

Single nucleus multiomic atlas of human dorsal root ganglia reveals the contribution of non-neuronal cell types to pain

Sensory neurons residing in dorsal root ganglia (DRG) transmit sensory information such as pain, itch, touch, pressure and bodily position to the central nervous system. The activity of sensory neurons is regulated by non-neuronal cell types in the DRG, including satellite glial cells (SGCs), immune cells and fibroblasts. Dysregulated gene expression in DRG cells contributes to sensory nervous system disorders such as chronic pain. Understanding the genetic underpinnings of these conditions requires dissecting transcriptional regulation in human tissue. In this study, we profiled transcriptomic and chromatin accessibility landscapes from postmortem human DRG (hDRG) samples at single-nucleus level. We demonstrate that sequencing depth significantly impacts downstream analysis, with deeper sequencing yielding more detected cells and features, improved data integration, refined clustering and annotation, and more accurate scientific interpretations. We identified nine major cell types, defined their molecular signatures, and mapped cis-regulatory landscapes. Integration of gene expression with chromatin accessibility enabled peak-gene association and transcriptional network analyses, revealing transcription factors, their target genes, regulatory elements and potential partners that cooperatively drive cell-type-specific gene expression programs. This integrative approach identified cell types, genes, and cis-regulatory regions potentially driving pain conditions. Our unbiased genome-wide analysis not only recovered known pain-related genes but also highlighted novel candidate genes and regulatory regions implicated in pain mechanisms. Importantly, our results demonstrate that non-neuronal cells, including endothelial cells, fibroblasts, macrophages, and SGCs, play critical roles in pain pathogenesis and should be investigated as therapeutic targets. One Sentence SummaryOur work provides a comprehensive single-nucleus multi-omic atlas of human dorsal root ganglia, uncovering novel cell-type-specific regulatory mechanisms and candidate therapeutic targets for pain, thereby directly advancing translational insights into human sensory disorders and chronic pain pathogenesis.

neuroscience↗

bioRxiv2025

A multi-omic atlas of human autonomic and sensory ganglia implicates cell types in peripheral neuropathies

The human peripheral nervous system (PNS) consists of many ganglia including sympathetic ganglia (SG) and dorsal root ganglia (DRG). These ganglia house the cell bodies of diverse PNS neurons that transmit autonomic and sensory signals, as well as a much larger number of non-neuronal cells. However, the molecular and cellular diversity of these human PNS cell types and their implications in human disease 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 cell-type-specific transcriptional and epigenomic features are similar between human SG and DRG, we identify key differences between SG and DRG cell types. These differences highlight the distinct molecular and cellular mechanisms underlying their specific functions. We also profiled key genomic regulatory networks that govern cell-type-specific gene expression in these peripheral ganglia. Moreover, by mapping the expression and chromatin accessibility of disease-associated genes in human SG and DRG, we identify cell types 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.

neuroscience↗

bioRxiv2025

Central Amygdala Neuronal Ensembles Coordinate Visceral Pain and Its Affective Behaviors

Visceral pain and its affective component associated with cystitis remain poorly understood. Here, we delineate the role of central amygdala (CeA) neuronal ensembles in encoding and modulating cystitis-induced bladder pain and its affective components. Utilizing a multidisciplinary approach combining behavioral assays, optogenetic manipulations, whole-cell electrophysiology, and activity-dependent genetic labeling, we identified functionally discrete CeA subpopulations that are selectively recruited during bladder inflammation. Bidirectional optogenetic modulation of these ensembles produced opposing effects on nocifensive and anxiety-like behaviors, indicating their causal involvement. Single-nucleus RNA sequencing of FosTRAP-labeled neurons revealed distinct transcriptional signatures associated with inflammatory activation. Integrating electrophysiological and transcriptomic data, we demonstrate that cystitis drives cell type-specific adaptations in CeA circuits. These findings provide mechanistic insight and uncover a molecularly and functionally defined CeA ensemble that orchestrates the sensory and affective dimensions of visceral pain.

neuroscience↗

bioRxiv2025

Expansion of OSMR expression and signaling in the human dorsal root ganglion links OSM to neuropathic pain

RNA sequencing studies on human dorsal root ganglion (hDRG) from patients suffering from neuropathic pain show upregulation of OSM, linking this IL-6 family cytokine to pain disorders. In mice, however, OSM signaling causes itch behaviors through a direct effect on its cognate receptor expressed uniquely by pruriceptive sensory neurons. We hypothesized that an expansion in function of OSM-OSM receptor (OSMR) in sensory disorders in humans could be explained by species differences in receptor expression and signaling. Our in situ hybridization and immunohistochemical findings demonstrate broad expression of OSMR in DRG nociceptors and afferent fibers innervating the superficial and deep skin of humans. In patch-clamp electrophysiology, OSM directly activates human sensory neurons engaging MAPK signaling to promote action potential firing. Using CRISPR editing we show that OSM activation of MAPK signaling is dependent on OSMR and not LIFR in hDRG. Bulk, single-nuclei, and single-cell RNA-seq of OSM-treated hDRG cultures reveal expansive similarities in the transcriptomic signature observed in pain DRGs from neuropathic patients, indicating that OSM alone can orchestrate transcriptomic signatures associated with pain. We conclude that OSM-OSMR signaling via MAPKs is a critical signaling factor for DRG plasticity that may underlie neuropathic pain in patients.

neuroscience↗

bioRxiv2025

Human sensory neurons exhibit cell-type-specific, pain-associated differences in intrinsic excitability and expression of SCN9A and SCN10A

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 donors 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. TeaserMapping electrical properties to transcriptional profiles of human sensory neurons offers new insights into pain neurobiology

neuroscience↗

bioRxiv2025

Inhibitory effects of dopamine agonists on pain-responsive neurons in the central nucleus of the amygdala

The central nucleus of the amygdala (CeA) is a heterogenous region of primarily GABAergic neurons that contributes to numerous behaviors, including fear learning, feeding, reward, and pain. Dopaminergic inputs to the CeA have been shown to regulate many of these behaviors, but how dopamine exerts these effects at the cellular level has not been well characterized. We used the Targeted Recombination in Active Populations (TRAP) mouse line to fluorescently label pain-responsive CeA neurons, and then targeted these cells for patch-clamp recordings in acute slices to test the effects of dopamine agonists. The D1 agonist SKF-38393 and D2 agonist quinpirole both had inhibitory effects, reducing the input resistance and evoked firing and increasing rheobase of labeled CeA neurons. Both agents also inhibited the NMDA component of excitatory postsynaptic currents (EPSCs) evoked by basolateral amygdala (BLA) stimulation, but did not affect the AMPA component. D1 activation, but not D2, also appeared to have a presynaptic effect, increasing the frequency of spontaneous EPSCs. These results provide new insights into how dopamine regulates activity within pain-responsive CeA networks. NEW & NOTEWORTHYDopamine is known to regulate activity within the central amygdala (CeA), an important region for central pain processing. However, its effects at the cellular level have not been well characterized. We targeted pain-responsive CeA neurons for patch-clamp recordings to examine the cellular and synaptic effects of D1 and D2 agonists. Activation of either D1 or D2 receptors induced inhibitory effects, suggesting dopamine signaling in CeA dampens pain-related activity and could be a target for analgesics.

neuroscience↗

bioRxiv2024

An Autonomous Implantable Device for the Prevention of Death from Opioid Overdose

Opioid overdose accounts for nearly 75,000 deaths per year in the United States, representing a leading cause of mortality amongst the prime working age population (25-54 years). At overdose levels, opioid-induced respiratory depression becomes fatal without timely administration of the rescue drug naloxone. Currently, overdose survival relies entirely on bystander intervention, requiring a nearby person to discover and identify the overdosed individual, and have immediate access to naloxone to administer. Government efforts have focused on providing naloxone in abundance but do not address the equally critical component for overdose rescue: a willing and informed bystander. To address this unmet need, we developed the Naloximeter: a class of life-saving implantable devices that autonomously detect and treat overdose, with the ability to simultaneously contact first-responders. We present three Naloximeter platforms, for both fundamental research and clinical translation, all equipped with optical sensors, drug delivery mechanisms, and a supporting ecosystem of technology to counteract opioid-induced respiratory depression. In small and large animal studies, the Naloximeter rescues from otherwise fatal opioid overdose within minutes. This work introduces life-changing, clinically translatable technologies that broadly benefit a susceptible population recovering from opioid use disorder.

bioengineering↗

bioRxiv2023

Intrinsic Homeostatic Plasticity in Mouse and Human Sensory Neurons

In response to changes in activity induced by environmental cues, neurons in the central nervous system undergo homeostatic plasticity to sustain overall network function during abrupt changes in synaptic strengths. Homeostatic plasticity involves changes in synaptic scaling and regulation of intrinsic excitability. Increases in spontaneous firing and excitability of sensory neurons are evident in some forms of chronic pain in animal models and human patients. However, whether mechanisms of homeostatic plasticity are engaged in sensory neurons under normal conditions or altered after chronic pain is unknown. Here, we showed that sustained depolarization induced by 30mM KCl induces a compensatory decrease in the excitability in mouse and human sensory neurons. Moreover, voltage-gated sodium currents are robustly reduced in mouse sensory neurons contributing to the overall decrease in neuronal excitability. Decreased efficacy of these homeostatic mechanisms could potentially contribute to the development of the pathophysiology of chronic pain.

neuroscience↗

bioRxiv2023

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether BK receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, while prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donors history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute BK-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting BK signaling as a potential therapeutic target for treating pain in humans.

neuroscience↗

bioRxiv2021

Paw placement during walking is altered by analgesic doses of opioids and post-surgical injury in mice

Hind paw-directed assays are commonly used to study the analgesic effects of opioids in mice. However, opioid-induced hyper-locomotion can obscure results of such assays. We aimed to overcome this potential confound by using gait analysis to observe hind paw usage during walking in mice. We measured changes in paw print area following induction of post-surgical pain (using the paw incision model) and treatment with oxycodone. Paw incision surgery reduced the paw print area of the injured hind paw as the mice avoided placing the incised section of the paw on the floor. Surprisingly, oxycodone caused a tiptoe-like gait in mice, resulting in a reduced paw print area in both hind paws. Further investigation of this opioid-induced phenotype revealed that analgesic doses of oxycodone or morphine dose-dependently reduced hind paw print area in uninjured mice. The gait changes were not dependent on opioid-induced increases in locomotor activity; speed and paw print area had no correlation in opioid-treated mice, and other analgesic compounds that alter locomotor activity did not affect paw print area. Unfortunately, the opioid-induced "tiptoe" gait phenotype prevented gait analysis from being a viable metric for demonstrating opioid analgesia in injured mice. However, this work reveals an important, previously uncharacterized effect of treatment with analgesic doses of opioids on paw placement. Our characterization of how opioids affect gait has important implications for the use of mice to study opioid pharmacology and suggests that scientists should use caution when using hind paw-directed nociceptive assays to test opioid analgesia in mice.

pharmacology and toxicology↗

bioRxiv2021

Profiling the molecular signature of Satellite Glial Cells at the single cell level reveals high similarities between rodent and human

Peripheral sensory neurons located in dorsal root ganglia relay sensory information from the peripheral tissue to the brain. Satellite glial cells (SGC) are unique glial cells that form an envelope completely surrounding each sensory neuron soma. This organization allows for close bi-directional communication between the neuron and it surrounding glial coat. Morphological and molecular changes in SGC have been observed in multiple pathological conditions such as inflammation, chemotherapy-induced neuropathy, viral infection and nerve injuries. There is evidence that changes in SGC contribute to chronic pain by augmenting neuronal activity in various rodent pain models. SGC also play a critical role in axon regeneration. Whether findings made in rodent model systems are relevant to human physiology have not been investigated. Here we present a detailed characterization of the transcriptional profile of SGC in mouse, rat and human at the single cell level. Our findings suggest that key features of SGC in rodent models are conserved in human. Our study provides the potential to leverage rodent SGC properties and identify potential targets in humans for the treatment of nerve injuries and alleviation of painful conditions.

neuroscience↗

bioRxiv2021

A photoswitchable GPCR-based opsin for presynaptic silencing

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has been far more difficult to achieve. Instead of relying on unpredictable ion manipulations or slow photoactivatable toxins at axon terminals, we took a different approach to leverage the natural ability of inhibitory presynaptic GPCRs to silence synaptic transmission. Here we characterize parapinopsin (PPO), a photoswitchable non-visual opsin from lamprey pineal gland that couples to Gi/o-signaling cascades. PPO can be rapidly activated by pulsed blue light, switched off with amber light, and is effective for repeated or prolonged inhibition. We developed viral vectors for cell-specific expression of PPO, which traffics very effectively in numerous neuron types. At presynaptic terminals, PPO can silence glutamate release and suppress dopamine-dependent reward and cocaine place preference behaviors in vivo. PPO immediately fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition, and has broader utility for achieving spatiotemporal control of inhibitory GPCR signaling cascades in other biological and pharmacological applications.

neuroscience↗

bioRxiv2021

Cellular, Circuit and Transcriptional Framework for Modulation of Itch in the Central Amygdala

Itch is an unpleasant sensation that elicits robust scratching and active avoidance. However, the identity of the cells and neural circuits that organize this information remains elusive. Here we show the necessity and sufficiency of itch-activated neurons in the central amygdala (CeA) for both itch sensation and active avoidance. Further, we show that itch-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of itch-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send a critical projection to the periaqueductal gray to mediate modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice.

neuroscience↗

bioRxiv2020

Extended amygdala-parabrachial circuits alter threat assessment to regulate feeding

An animals evolutionary success depends on the ability to seek and consume foods while avoiding environmental threats. However, how evolutionarily conserved threat detection circuits modulate feeding is unknown. In mammals, feeding and threat assessment are strongly influenced by the parabrachial nucleus (PBN), a structure that responds to threats and inhibits feeding. Here, we report that the PBN receives dense inputs from the bed nucleus of the stria terminalis (BNST), an extended amygdala structure that encodes affective information. Using a series of complementary approaches, we identify opposing BNST-PBN circuits that modulate a genetically-defined population of PBN neurons to control feeding. This previously unrecognized neural circuit integrates threat assessment with the intrinsic drive to eat.

neuroscience↗

bioRxiv2018

Cell type-specific modulation of sensory and affective components of itch in the periaqueductal gray

Itch is a distinct aversive sensation that elicits a strong urge to scratch. Despite recent advances in our understanding of the peripheral basis of itch, we know very little regarding how central neural circuits modulate acute and chronic itch processing. Here we establish the causal contributions of defined periaqueductal gray (PAG) neuronal populations in itch modulation. Chemogenetic manipulations demonstrate bidirectional modulation of scratching by neurons in the PAG. Fiber photometry studies show that activity of GABAergic and glutamatergic neurons in the PAG is modulated in an opposing manner during chloroquine-evoked scratching. Furthermore, activation of PAG GABAergic neurons or inhibition of glutamatergic neurons resulted in attenuation of scratching in both acute and chronic pruritis. Surprisingly, PAG GABAergic neurons, but not glutamatergic neurons, seem to encode the aversive component of itch. Thus, the PAG represents a neuromodulatory hub that regulates both the sensory and affective aspects of acute and chronic itch.

neuroscience↗

bioRxiv2018

Pharmacokinetics, Side Effects, and Anti-Hyperalgesic Efficacy of The Mglu5 Antagonist Fenobam.

Metabotropic glutamate receptor 5 (mGlu5) has been shown to modulate nociception in animals, but no mGlu5 antagonists have been developed commercially as analgesics. The mGlu5 antagonist fenobam [N-(3-chlorophenyl)-N-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] was originally evaluated for development as a non-benzodiazepine anxiolytic. Fenobam is analgesic in numerous mouse pain models, acting exclusively via mGlu5 blockade. Furthermore, fenobam showed no signs of analgesic tolerance with up to two weeks of daily dosing in mice. Analgesic effects of fenobam in humans have not been reported. The purpose of this investigation was to evaluate fenobam pharmacokinetics and analgesic effects in humans. We first evaluated single-dose oral fenobam disposition in a parallel-group dose-escalation study in healthy volunteers. A second investigation tested the analgesic effects of fenobam in an established experimental human pain model of cutaneous sensitization utilizing capsaicin cream and heat, in a double-blind placebo-controlled study. The primary outcome measure was the area of hyperalgesia and allodynia around the area applied with heat/capsaicin. Secondary outcome measures included nociception, measured as pain rating on a visual analog scale, heat-pain detection threshold, and effects on cognition and mood. Fenobam plasma exposures showed considerable inter-individual variability, and were not linear with dose. Fenobam reduced sensitization vs placebo at a single time-point (peak plasma concentration); we found no other difference between fenobam and placebo. Our results suggest highly variable fenobam disposition, and minimal analgesic effects at the dose tested. We suggest that future studies testing analgesic effects of mGlu5 blockade are warranted, but such studies should employ molecules with improved pharmacokinetic profiles.

neuroscience↗

bioRxiv2017

Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-A member-1 (TRPA1). Mechanical and cold pain hypersensitivity that are characteristic of neuropathic conditions can be attenuated by chemogenetic depletion of peripheral macrophages and AT2R-null hematopoietic cell transplantation. Our findings show no AT2R expression in mouse or human sensory neurons, rather AT2R expression and activation in macrophages triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on sensory neurons. Our study defines the precise neuro-immune crosstalk underlying nociceptor sensitization at the site of nerve injury. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic neuropathic pain, and therefore identifies multiple analgesic targets.

neuroscience↗