Biology subjects

Albers, K. M.

Publications and source records attributed to Albers, K. M..

5 recordsLinked to original sources

bioRxiv2024

Peripheral opioid tolerance involves skin keratinocytes and platelet-derived growth factor type B signaling

Opioid analgesic tolerance drives dose escalation which hampers the therapeutic utility of opioids by increasing centrally mediated deleterious side-effects, including respiratory depression or addiction. Peripheral opioid delivery provides a safer, effective alternative to systemic delivery by avoiding centrally mediated opioid side-effects. However, tolerance still occurs peripherally via mechanisms that remain unknown. Centrally, activation of the mu-opioid receptor (MOPr) by opioids induces release of platelet-derived growth factor-B (PDGF-B); and inhibition of PDGF receptor beta (PDGFR{beta}) prevents opioid tolerance. In the periphery, MOPr and PDGF-B are expressed in keratinocytes, and PDGFR{beta} is expressed in peripheral sensory neurons (PSNs), which are involved in tolerance. Previous studies showed that optogenetic stimulation of keratinocytes modulates PSNs via release of keratinocyte-derived factors. Thus, we hypothesized that mechanisms of peripheral opioid tolerance involve keratinocytes and PDGFR{beta} signaling. Using behavioral pharmacology, optogenetics and imaging in mice, we found that selective inhibition of peripheral PDGFR{beta} prevents peripheral morphine tolerance caused by repeated intraplantar (i.pl.) morphine injections. In addition, we show that PDGF-B is both necessary and sufficient to cause peripheral morphine tolerance. Repeated peripheral morphine injections lead to an increase in PDGF-B mRNA in MOPr-expressing keratinocytes and induce changes in the biophysical properties of keratinocytes as measured by patch-clamp electrophysiology. In parallel, we discovered that repeated optogenetic activation of keratinocytes is sufficient to induce peripheral morphine tolerance in a PDGF-B/PDGFR{beta}-dependent manner. Together, we show a novel epithelial-neuronal communication mechanism that incorporates keratinocytes and PDGF-B/PDGFR{beta} to mediate peripheral opioid tolerance, opening the door to safer, more effective pain therapeutics. Significance StatementPeripheral opioids are a safer alternative to systemic opioids. However, peripheral tolerance leads to reduction of analgesia over time, hampering clinical use of peripheral opioids. Here, we highlight a novel epithelial-neuronal communication mechanism that mediates peripheral tolerance. We discovered that intraplantar (i.pl.) morphine injections in mice cause peripheral tolerance via release of platelet-derived growth factor type B (PDGF-B) and activation of platelet-derived growth factor beta (PDGFR{beta}). We find that morphine i.pl. increases PDGF-B in mu-opioid receptor-expressing keratinocytes, which could be released to activate PDGFR{beta} in cutaneous nociceptor endings to mediate peripheral tolerance. Moreover, we show that photostimulation of keratinocytes is sufficient to cause peripheral tolerance in a PDGF-B/PDGFR{beta}-manner. Thus, keratinocytes and PDGF-B are new promising targets for peripheral opioid tolerance.

neuroscience↗

bioRxiv2020

Unique molecular characteristics of visceral afferents arising from different levels of the neuraxis: location of afferent somata predicts function and stimulus detection modalities

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 StatementVisceral 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.

neuroscience↗

bioRxiv2020

Optogenetic inhibition of colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease

Visceral pain is a prevalent symptom of inflammatory bowel disease (IBD) that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium (DSS) model of inflammatory bowel disease (IBD). Inhibition of the colon epithelium significantly decreased DSS-induced hypersensitivity and was comparable to inhibition of ExPANS. Together these results reveal the potential of targeting the colon epithelium for treatment of pain.

neuroscience↗

bioRxiv2020

Extracellular ATP released from Candida albicans activates non-peptidergic neurons to augment host defense

Intestinal microbes release ATP to modulate local immune responses. Herein we demonstrates that Candida albicans, an opportunistic commensal fungus, also modulates immune responses via secretion of ATP. We found that ATP secretion from C. albicans varied between standard laboratory strains. A survey of eighty-nine clinical isolates revealed heterogeneity in ATP secretion, independent of growth kinetics and intracellular ATP levels. Isolates from blood released less ATP than commensals, suggesting that ATP secretion assists with commensalism. To confirm this, cohorts of mice were infected with strains matched for origin, and intracellular ATP concentration, but high or low extracellular ATP. In all cases fungal burden was inversely correlated with ATP secretion. Mice lacking P2RX7, the key ATP receptor expressed by immune cells in the skin, showed no alteration in fungal burden. Rather, treatments with a P2RX2/3 antagonist result in increased fungal burden. P2RX2/3 is expressed by non-peptidergic neurons that terminate in the epidermis. Cultured sensory neurons flux Ca2+ when exposed to supernatant from heat-killed C. albicans (HKCA), and these non-peptidergic fibers are the dominant subset that respond to HKCA. Ca2+ flux, but not CGRP-release, can be abrogated by pretreatment of HKCA supernatant with apyrase. To determine whether non-peptidergic neurons participate in host defense, we generated MRGPRD-DTR mice. Infection in these mice resulted in increased CFU only for those C. albicans strains with high ATP secretion. Taken together, our findings indicate that C. albicans releases ATP, which is recognized by non-peptidergic nerves in the skin resulting in augmented anti-Candida immune responses. Author SummaryBacterial release of ATP has been shown to modulate immune responses. Candida albicans displays heterogeneity in ATP release among laboratory strains and commensal clinical isolates release more ATP than invasive isolates. C. albicans strains with high ATP secretion show lower fungal burden following epicutaneous infection. Mice lacking P2RX7, the key ATP receptor expressed by immune cells, showed no alteration in fungal burden. In contrast, treatment with P2RX2/3 antagonists resulted in increased fungal burden. P2RX3 is expressed by a subset of non-peptidergic neurons that terminate in the epidermis. These non-peptidergic fibers are the predominant responders when cultured sensory neurons are exposed to heat-killed C. albicans in vitro. Mice lacking non-peptidergic neurons have increased infection when exposed to high but not low ATP-secreting isolates of C. albicans. Taken together, our findings indicate that C. albicans releases ATP which is recognized by non-peptidergic nerves in the skin resulting in augmented anti-Candida immune responses. Bullet pointsO_LIATP released from heat killed C. albicans activates non-peptidergic sensory neurons C_LIO_LILive C. albicans clinical isolates release variable amounts of ATP C_LIO_LIElevated levels of ATP released by C. albicans correlates with reduced infectivity in vivo C_LIO_LIMRGPRD-expressing cutaneous neurons are required for defense against ATP-secreting C. albicans C_LI

immunology↗

bioRxiv2019

Single cell q-PCR derived expression profiles of identified sensory neurons

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/DRG/ 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 qPCR techniques and a limited set of genes, we can identify transcriptionally distinct groups. We have also used calcium imaging and single cell qPCR 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. Significance statementExpression profiles of unidentified sensory neurons have been recently studied using RNASeq techniques. Here, we utilize a multifactorial approach to target identified cutaneous and muscle afferents to examine expression and functional levels of specific high priority candidate genes using ex vivo electrophysiology, Ca2+ imaging, and single cell qPCR. Using this methodology, we were able to identify specific groups of neurons with distinct functional properties that corresponded to unique transcriptional profiles. This represents the first attempt to relate neuronal phenotype with levels of gene expression in single identified afferents and highlights the importance of combining functional analysis with transcriptomics.

neuroscience↗