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Anthracyclines are a class of widely prescribed anti-cancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we utilized CUT&Tag to profile RNA polymerase II during anthracycline treatment inDrosophilacells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of elongating RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation impacts chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally-oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of non-canonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the anti-cancer activity of aclarubicin is driven by the effects of nucleosome disruption on RNA polymerase II, chromatin accessibility and DNA structures.

Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that, using the true pairwise distribution, we can define and maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images. Distance distribution correction (DDC) eliminates repeat localizations caused by fluorophore blinking without the need for calibrations. Use of DDC yields accurate and quantifiable single-molecule localization microscopy data.

A 24-year-old Caucasian woman with a history of intravenous drug abuse and multiple hospital admissions for substance abuse related medical problems presented with pneumonia and was discharged home on oral antibiotics. Three days later, her blood culture grew acid fast bacilli, which was subsequently identified as M. abscessus subspecies abscessus. Transthoracic and transesophageal echocardiogram (TTE and TEE respectively) was suggestive of tricuspid valve (TV) endocarditis. CT scan of the chest showed evidence of septic pulmonary emboli, pneumonic consolidation and pleural effusion requiring chest tube placement. BAL and blood cultures grew Mycobacterium abscessus while pleural fluid cultures remained sterile. She was treated with a combination antibiotic therapy and completed a six-week course with resolution of her symptoms and microbiological cure. We present this rare case of M. abscessus native tricuspid valve endocarditis associated with lung infection treated with a short course of combination antibiotic therapy.

Epigenetic mechanisms play essential roles in determining distinct cell fates during the development of multicellular organisms. Histone proteins represent crucial epigenetic components that help specify cell identities. Previous work has demonstrated that during the asymmetric cell division of the Drosophila male germline stem cells (GSCs), histones H3 and H4 are asymmetrically inherited, such that preexisting (old) histones are segregated towards the self-renewing GSC whereas newly synthesized (new) histones are enriched towards the differentiating daughter cell. In order to further understand the molecular mechanisms underlying this striking phenomenon, two key questions must be answered: when and how old and new histones are differentially incorporated by sister chromatids, and how epigenetically distinct sister chromatids are specifically recognized and segregated. Here we discuss recent advances in our understanding of the molecular mechanisms and cellular bases underlying these fundamental and important biological processes responsible for generating two distinct cells through one cell division.

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. Here, we tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. We found unique features that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.

In human, intradermal administration of β-alanine (ALA) and bovine adrenal medulla peptide 8-22 (BAM8-22) evokes the sensation of itch. Currently, it is unknown which human dorsal root ganglion (DRG) neurons express the receptors of these pruritogens, MRGPRD and MRGPRX1 respectively, and which cutaneous afferents these pruritogens activate in primate. In situ hybridization studies revealed that MRGPRD and MRGPRX1 are co-expressed in a subpopulation of TRPV1+ human DRG neurons. In electrophysiological recordings in nonhuman primates (Macaca nemestrina), subtypes of polymodal C-fiber nociceptors are preferentially activated by ALA and BAM8-22, with significant overlap. When pruritogens ALA, BAM8-22 and histamine, that activate different subclasses of C-fiber afferents, are administered in combination, human volunteers report itch and nociceptive sensations similar to those induced by a single pruritogen. Our results provide evidence for differences in pruriceptive processing between primates and rodents, and do not support the spatial contrast theory of coding of itch and pain.

Stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. DuringDrosophilamale germline stem cell (GSC) asymmetric division, preexisting old histones H3 and H4 are enriched in the self-renewed stem daughter cell, whereas the newly synthesized H3 and H4 are enriched in the differentiating daughter cell. However, the biological consequences in the two daughter cells resulting from asymmetric histone inheritance remained to be elucidated. In this work, we track both old and new histones throughout GSC cell cycle using high spatial and temporal resolution microscopy. We find several unique features differentiating old versus new histone-enriched sister chromatids, including nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, an essential component of the pre-replication complex, which subsequently contributes to asynchronous initiation of DNA replication in the two resulting daughter cells. Disruption of asymmetric histone inheritance abolishes both differential Cdc6 association and asynchronous S-phase entry, demonstrating that asymmetric histone acts upstream of these critical events during cell cycle progression. Furthermore, GSC defects are detected under these conditions, indicating a connection between histone inheritance, cell cycle progression and cell fate decision. Together, these studies reveal that cell cycle remodeling as a crucial biological ‘readout’ of asymmetric histone inheritance, which precedes and could lead to other well-known readouts such as differential gene expression. This work also enhances our understanding of asymmetric histone inheritance and epigenetic regulation in other stem cells or asymmetrically dividing cells in multicellular organisms.

The synaptonemal complex (SC) is a zipper-like protein structure that forms between paired homologous chromosomes during meiosis. Hurlock et al. discover two novel SC components, SYP-5 and SYP-6, in C. elegans and investigate their contributions in both limiting and promoting crossover formation., The synaptonemal complex (SC) is a tripartite protein scaffold that forms between homologous chromosomes during meiosis. Although the SC is essential for stable homologue pairing and crossover recombination in diverse eukaryotes, it is unknown how individual components assemble into the highly conserved SC structure. Here we report the biochemical identification of two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans. SYP-5 and SYP-6 are paralogous to each other and play redundant roles in synapsis, providing an explanation for why these genes have evaded previous genetic screens. Superresolution microscopy reveals that they localize between the chromosome axes and span the width of the SC in a head-to-head manner, similar to the orientation of other known transverse filament proteins. Using genetic redundancy and structure–function analyses to truncate C-terminal tails of SYP-5/6, we provide evidence supporting the role of SC in both limiting and promoting crossover formation.

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations void of blinking-artifacts, generating an accurate reconstruction of the underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking-artifact correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications. The simplicity and robustness of DDC will allow it to become the field standard in SMLM imaging, enabling the most accurate reconstruction and quantification of SMLM images to date.

During DNA replication, the genetic information of a cell is copied. Subsequently, identical genetic information is segregated reliably to the two daughter cells through cell division. Meanwhile, DNA replication is intrinsically linked to the process of chromatin duplication, which is required for regulating gene expression and establishing cell identities. Understanding how chromatin is established, maintained or changed during DNA replication represents a fundamental question in biology. Recently, we developed a method to directly visualize chromatin components at individual replication forks undergoing DNA replication. This method builds upon the existing chromatin fiber technique and combines it with cell type–specific chromatin labeling and superresolution microscopy. In this method, a short pulse of nucleoside analog labels replicative regions in the cells of interest. Chromatin fibers are subsequently isolated and attached to a glass slide, after which a laminar flow of lysis buffer extends the lysed chromatin fibers parallel with the direction of the flow. Fibers are then immunostained for different chromatin-associated proteins and mounted for visualization using superresolution microscopy. Replication foci, or ‘bubbles,’ are identified by the presence of the incorporated nucleoside analog. For researchers experienced in molecular biology and superresolution microscopy, this protocol typically takes 2–3 d from sample preparation to data acquisition, with an additional day for data processing and quantification. Chromatin fibers are isolated from replicating tissues or cells and laid on glass slides. Labeled replicating DNA and chromatin-associated proteins are imaged by supperresolution microscopy, and their distribution on sister chromatids is quantified.

SUMMARY Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

SESSION TITLE: Fellows Chest Infections Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: A patient with sarcoidosis presented with persistent shortness of breath after being diagnosed with a virus and suspected sarcoidosis flare He was treated with supportive measures and steroids, but did not improve In a patient receiving immunosuppression to control an autoimmune disease, it is important to have a high suspicion for opportunistic infections CASE PRESENTATION: 51-year-old male with history of sarcoidosis was admitted three times in three weeks with fevers, chills, sweats, non-productive cough, and headaches A computerized tomography (CT) of the chest showed extensive mediastinal/hilar adenopathy and worsening of pre-existing parenchymal lung disease (figure 1) During the first two hospital admissions, the patient tested positive for coronavirus OC43 strain (not COVID-19) and was treated supportively He was also treated with steroids for a possible flare of his sarcoidosis Because of lack of improvement, fungal infection was considered Cultures from bronchoscopy with bronchoalveolar lavage and transbronchial biopsy of the right upper lobe revealed Histoplasma capsulatum (figure 2) Because of persistent headaches, the patient underwent a lumbar puncture which also demonstrated Histoplasma capsulatum in the cerebrospinal fluid He was diagnosed with disseminated histoplasmosis and started on amphotericin B Symptoms resolved and respiratory status returned to baseline He completed about 1 1/2 weeks of amphotericin before being transitioned to itraconazole He completed three months of itraconazole before being switched to posaconazole due to drug intolerances Urine histoplasma antigen was undetectable at 6-month follow-up Immunosuppressive regimen was judiciously decreased to balance controlling his sarcoidosis while limiting risk for reigniting the histoplasmosis and developing future opportunistic infections DISCUSSION: Histoplasmosis is the most prevalent endemic mycosis in the United States, typically found in the soil from bird droppings in the Midwest However, it can be found on the east coast, and patients receiving immunosuppression to control their autoimmune disease are at risk for fungal infections such as histoplasmosis This patient was receiving infliximab, a tumor necrosis factor antagonist that has been associated with histoplasmosis Although discontinuing or at least reducing immunosuppressant agents is standard of care while treating histoplasmosis, it is unclear when to restart them However, a retrospective analysis of 98 cases of histoplasmosis reported that it appears safe to resume immunosuppression, and even tumor necrosis factor antagonist therapy, if antifungal therapy was administered for at least 12 months with an appropriate clinical response CONCLUSIONS: Opportunistic infections must be ruled out in the setting of any immunosuppression, which can further exacerbate the acute illness and vastly increase mortality Reference #1: Kasper DL, Fauci AS, Hauser SL, et al Histoplasmosis Harrison’s Infectious Diseases 3rd Ed 2017;111:996-999 Reference #2: Vergidis P, Avery RK, Wheat LJ, et al Histoplasmosis Complicating Tumor Necrosis Factor-α Blocker Therapy: A Retrospective Analysis of 98 Cases Clinical Infectious Diseases 2015;61(3):409-17 Reference #3: Kobayashi T, Cho C Mediastinal granuloma due to histoplasmosis in a patient on infliximab Cleveland Clinic Journal of Medicine 2019;86(9);579-81 DISCLOSURES: No relevant relationships by Paul Cook, source=Web Response No relevant relationships by Poorvi King, source=Web Response No relevant relationships by Ogugua Obi, source=Web Response No relevant relationships by William Wooten, source=Web Response

One Sentence SummaryDNA replication establishes asymmetric epigenomesSummaryOne of the most fundamental questions in developmental biology concerns how cells with identical genomes differentiate into distinct cell types. One important context for understanding cell fate specification is asymmetric cell division, where the two daughter cells establish different cell fates following a single division. Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell1–5. Here we show that histone H4 is inherited asymmetrically in asymmetrically dividing Drosophila male germline stem cells, similar to H36. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy with the chromatin fiber method, we are able to study histone inheritance patterns on newly replicated chromatin fibers. Using this technique, we find asymmetric inheritance patterns for old and new H3, but symmetric inheritance patterns for old and new H2A on replicating sister chromatids. Furthermore, co-localization studies on isolated chromatin fibers and proximity ligation assays on intact nuclei reveal that old H3 are preferentially incorporated by the leading strand while newly synthesized H3 are enriched on the lagging strand. Finally, using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional DNA replication on germline-derived chromatin fibers and DNA fibers. The unidirectional fork movement coupled with the strand preference of histone incorporation could explain how old and new H3 are asymmetrically incorporated by replicating sister chromatids. In summary, our work demonstrates that the intrinsic asymmetries in DNA replication may help construct sister chromatids enriched with distinct populations of histones. Therefore, these results suggest unappreciated roles for DNA replication in asymmetrically dividing cells in multicellular organisms.

Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. We previously reported that in male Drosophila germline stem cells (GSCs), preexisting (old) histone H3 is segregated to the self-renewed stem cell, whereas the daughter incorporates newly synthesized (new) H3. Here, we show that histone H4 is likewise segregated asymmetrically, while histones H2A and H2B are segregated symmetrically. Using superresolution imaging, we visualize spatially separable old and new H3 distributions in interphase GSC nuclei and on isolated replicating chromatin fibers. Furthermore, using both superresolved chromatin fibers and proximity ligation assay, we demonstrate that old H3 are preferentially retained on the leading strand while new H3 preferentially associate with the lagging strand. Finally, using a dual nucleoside analog incorporation assay, the temporal separation between leading strand and lagging strand synthesis is detectable. Together, these results demonstrate that the spatial and temporal asymmetries inherent to DNA replication may serve to bias histone incorporation, suggesting an unappreciated role for DNA replication in asymmetrically dividing cells.

Background Infective endocarditis of the native heart valves due to M. abscessus has been reported in individuals who inject drugs. Only one case thus far has been reported with survival beyond 4 months after completion of antibiotic therapy. 24-year-old Caucasian woman with a history of intravenous drug use presented with intermittent fevers for 3–4 months. She was having chills, night sweats, and productive cough. She acknowledged using intravenous cocaine and heroin every few days after being discharged from our hospital 6 months previously when she was treated for tricuspid valve endocarditis. Chest radiograph demonstrated right lower lobe airspace disease suggestive of pneumonia. Three days later her blood cultures grew acid-fast bacilli, later identified as M. abscessus. Methods She was empirically treated with azithromycin, amikacin, and imipenem. Transthoracic and transesophageal echocardiograms were suggestive of tricuspid valve endocarditis. CT angiogram of the chest showed filling defects within several large right lower lobe pulmonary arteries and dense right lower lobe consolidation with pleural effusion. The patient underwent bronchoscopy with bronchoalveolar lavage (BAL) and therapeutic thoracoscopy with drainage and thoracostomy tube placement. BAL cultures also grew AFB. Multiple sets of blood cultures drawn after commencement of antibiotic therapy during the hospital course were negative. Drug susceptibilities were available 3 weeks later. The isolate was susceptible to amikacin. Linezolid, imipenem, and cefoxitin exhibited intermediate activity, and TMP/SMX, ciprofloxacin, moxifloxacin, doxycycline, minocycline, and clarithromycin were reported resistant. Inducible erm gene was present. Azithromycin and linezolid were discontinued and tigecycline was added. Results Patient completed 6 weeks of antibiotics from the day of the first negative blood culture. Repeat TTE 1 month after completion of therapy revealed a decrease in tricuspid valve vegetation. Blood culture done 3 months later was sterile. AFB blood cultures done a year later did not report any growth. Conclusion This case opens a debate, if in a selected group of patients, a short course of combination antibiotic therapy is enough to obtain cure. This will require further analysis. Disclosures All authors: No reported disclosures.

SummaryA long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of “old” H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

Asymmetric cell division (ACD) gives rise to two daughter cells with distinct fates. ACD is widely used during development and by many types of adult stem cells during tissue homeostasis and regeneration. ACD can be regulated by extrinsic cues, such as signaling molecules, as well as by intrinsic factors, such as organelles and cortex proteins. The recent discovery of asymmetric histone inheritance during stem cell ACD has revealed another intrinsic mechanism by which ACD produces two distinct daughters. In this review, we will discuss these findings in the context of cell cycle regulation, as well as other studies of ACD, to begin understanding the underlying mechanisms and biological relevance of this phenomenon.

Objective: Persons with type 2 diabetes have increased incidence of hyperuricemia and gout. The hypothesis that Urate transporter 1 (URAT1) levels are increased in type 2 diabetic Zucker rats and this is responsible for elevation of uric acid was tested. Methods: Male 12-week-old obese Zucker rats were compared to non-diabetic lean counterparts. Plasma glucose, uric acid and creatinine were measured. URAT1 protein levels in the renal cortex and medulla were determined by Western blot. Immunohistochemistry was used to determine the location of URAT1 inrenal tubules. Results: Plasma glucose and uric acid levels were higher in the diabetic rats. There was no difference in plasma createnine. URAT1 antibody-positive bands of 27, 31, 50, 62 and 70 kDa were observed in cortex. A similar pattern was observed in medulla with addition of a 44 kDa band. No differences were observed in URAT1 proteins in the cortex between obese and lean rats. In the medulla, expression of the 44 and 50 kDa proteins was higher in lean rats. Expression of 27, 50, 62 kDa URAT1 proteins in the cortex was higher than in the medulla, while expression of the 70 kDa URAT1 was higher in medulla than in cortex. Localization of URAT1 did not differ between groups and included tubules in both cortex and medulla. Conclusions: In male Zucker rats, URAT1 protein expression was observed in both kidney cortex and medulla. Uric acid elevation in the obese group was associated with decreases in the 44 and 50 kDa URAT1 proteins in renal medulla.

OBJECT Percutaneous treatments for trigeminal neuralgia are safe, simple, and effective for achieving good pain control. Procedural risks could be minimized by using noninvasive imaging techniques to improve the placement of the radiofrequency thermocoagulation probe into the trigeminal ganglion. Positioning of a probe is crucial to maximize pain relief and to minimize unwanted side effects, such as denervation in unaffected areas. This investigation examined the use of laser speckle imaging during probe placement in an animal model. METHODS This preclinical safety study used nonhuman primates, Macaca nemestrina (pigtail monkeys), to examine whether real-time imaging of blood flow in the face during the positioning of a coagulation probe could monitor the location and guide the positioning of the probe within the trigeminal ganglion. RESULTS Data from 6 experiments in 3 pigtail monkeys support the hypothesis that laser imaging is safe and improves the accuracy of probe placement. CONCLUSIONS Noninvasive laser speckle imaging can be performed safely in nonhuman primates. Because improved probe placement may reduce morbidity associated with percutaneous rhizotomies, efficacy trials of laser speckle imaging should be conducted in humans.

In primates, C-fibre polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, an MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically insensitive C-fibres (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin-responsive neurons in primate dorsal root ganglion. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs, and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibres likely corresponding to murine MrgprD-expressing, non-peptidergic nociceptive afferents.

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.