Robert W. Gereau

Endometriosis is a common yet understudied condition in which sex hormones play a key role in the etiology and pathophysiology of the disease. Sex hormones are dysregulated in endometriosis and are pharmacologically targeted to reduce symptoms including pain, but the relationships between endometriosis-related pain with sex hormone levels and their receptors are not clear. In 60 women previously diagnosed or undergoing laparoscopic surgery for the diagnosis of endometriosis, we collected patient-reported endometriosis/pelvic pain intensity in the last 30 days, pain interference scale, and the Endometriosis Health Profile 30 (EHP30) pain subscale. In a sub-sample of 38 participants, biopsies from control non-lesion pelvic peritoneum and endometrial lesion tissues were collected during surgery to analyze the tissue abundance of the primary receptors for estrogen, as well as androgen, progesterone, and prolactin receptors. Serum was collected to analyze systemic sex hormone levels including estradiol, prolactin, progesterone, and testosterone. Between tissues, we found elevated transcript levels for estrogen receptor alpha ( ESR1 ) in lesions, but no difference in estrogen receptor beta ( ESR2 ) transcript levels between tissue samples were found. Relating tissue receptor transcript levels to clinical pain revealed that only ESR1 and PGR in lesions were negatively associated with endometriosis/pelvic pain intensity, however, following false discovery rate correction the association with ESR1 was no longer statistically significant. No other tissue or systemic factors were associated with clinical pain outcomes. Together, these data demonstrate unique alterations in receptor abundance in lesions yet select transcript levels only weakly relate to pain in endometriosis. One Sentence Summary: Endometriosis-associated pain is largely unrelated to serum sex hormone levels and receptor expression levels in tissue.

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 transitions between behaviors after naloxone-precipitated withdrawal and formalin administration. Oxycodone suppressed formalin-evoked licking without restoring behavioral sequencing toward the vehicle state. These data demonstrate that an integrated, low-cost, open-source platform can make automated behavioral phenotyping accessible without requiring users to build a custom computational pipeline.

Endometriosis is a prevalent condition characterized by chronic pelvic pain that is frequently refractory to treatment. While the mechanisms underlying this pain remain poorly defined, clinical evidence often indicates that lesion innervation, but not disease stage (e.g. number and depth of lesions), correlate with pelvic pain severity. However, characterization of lesion-innervating neurons is incomplete, revealing an opportunity to identify novel, disease-modifying therapeutics. Here, we coupled functional analyses of lesion-innervating neurons in a mouse model with concurrent identification and characterization of lesion-innervating neurons from pain-phenotyped endometriosis patients. Following the confirmation of abdominal-directed pain-like behaviors in the mouse model, electrophysiological analysis revealed that lesion-innervating dorsal root ganglion (DRG) neurons are hyperexcitable compared to matched controls. These neurons are predominantly small-diameter and bind Isolectin B4, an established marker of the GDNF Family Ligand receptor, Ret. GDNF is concentrated within the stromal layer of both mouse and human lesions, adjacent to axons expressing the GDNF co-receptor, GFRα1. Critically, clinical pain correlates with lesion GDNF level, axonal density, and neuronal GFRα1 levels. These data provide evidence that endometrial lesions may recruit the Ret-positive subpopulation of nociceptors where they become sensitized and increase patient pain.

Summary/Abstract Sensory neurons play well defined roles in the regulation of intestinal motility, digestion, and interoception, but transcriptional dissection of intrinsic and extrinsic sensory neurons innervating the intestines has been challenging because these cells share many genetic markers. However, we cross-referenced transcriptional profiles of intrinsic and extrinsic intestinal neurons and the cells that surround them and identified Pdyn, the gene encoding Prodynorphin, as a marker of putative sensory enteric neurons of the mouse intestines. In a Pdyn lineage-reporter mouse, we identified labeled cells in the myenteric and submucosal plexuses of the large intestine, in contrast to their sparse presence in the dorsal root or nodose ganglia. In dissociated cell culture, these neurons mostly display a phasic firing pattern, discharging one or two action potentials at the onset of a depolarizing current pulse, followed by a prompt cessation of firing despite continued current injection, as assessed by whole-cell patch-clamp recordings. Optogenetic activation of Pdyn -lineage neurons propels stool in ex vivo colons, and in untethered and mobile mice, optogenetic stimulation of the proximal colon induces freezing behaviors and orbital tightening, suggesting interoceptive behaviors, without significant stool output differences. Together, these findings suggest that activation of Pdyn -lineage enteric neurons regulates motility and visceral interoception.

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

Summary: 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.

Dopamine 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.

Spinal cord stimulation (SCS) for chronic pain management is an invasive therapy involving surgical implantation of electrodes into spinal epidural space. While the clinical value and mechanistic action of the therapy is debated considerably in recent years, preclinical chronic studies employing rodent models can provide invaluable insights regarding the balance between efficacy and complications as well as mechanistic understanding of SCS therapy. However, current rodent compatible devices require tethered power delivery or bulky batteries, severely limiting the ability to probe long‐term efficacy of SCS therapy. This work introduces a tether‐free, small‐footprint, fully implantable, battery‐free SCS device compatible with rodent models, capable of delivering electrical stimulation to the spinal cord at a wide range of frequency, amplitude, and period via wireless communication adjustable on‐demand without direct interaction with the animal. The presented device features capabilities of clinical SCS devices, with materials and processes amendable to scalable fabrication at a cost suitable for one‐time use enabling high N studies. In this proof of concept, the implantable device serves to assess therapeutic efficacy of various clinically relevant SCS paradigms in alleviating neuropathic pain. This technology offers chronic stability and the potential to serve as the foundation for future research into the development of SCS therapeutic systems.