Autonomic Neurons Research Articles

Pre-Clinical Development of an Adenovirus Vector Based RSV and Shingles Vaccine Candidate.

Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.

Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels.

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100µM), the temperature effect was largely retained. Using econazole (10µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Nrf2 and autonomic dysregulation in chronic heart failure and hypertension.

Redox imbalance plays essential role in the pathogenesis of cardiovascular diseases. Chronic heart failure (CHF) and hypertension are associated with central oxidative stress, which is partly mediated by the downregulation of antioxidant enzymes in the central autonomic neurons that regulate sympathetic outflow, resulting in sympathoexcitation. Antioxidant proteins are partially regulated by the transcriptional factor nuclear factor erythroid 2-related factor 2 (Nrf2). Downregulation of Nrf2 is key to disrupting central redox homeostasis and mediating sympathetic nerve activity in the setting of Chronic heart failure and hypertension. Nrf2, in turn, is regulated by various mechanisms, such as extracellular vesicle-enriched microRNAs derived from several cell types, including heart and skeletal muscle. In this review, we discuss the role of Nrf2 in regulating oxidative stress in the brain and its impact on sympathoexcitation in Chronic heart failure and hypertension. Importantly, we also discuss interorgan communication via extracellular vesicle pathways that mediate central redox imbalance through Nrf2 signaling.

Botulinum neurotoxins: Future innovations.

Botulinum neurotoxins (BoNTs) are multi-domain proteins whose potent and selective actions on nerve endings have led to innovations in both basic and clinical science. The various BoNT domains are responsible for binding to gangliosides and proteins associated with nerve cell membranes, internalization into the cell, and cleavage of one or more SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins necessary for vesicle docking and fusion. Novel modifications to BoNT molecules, such as the creation of chimeras, helped identify the protein domains responsible for various aspects of BoNT action, such as localized effects. Other molecular modifications have been introduced in attempts to increase the specificity of BoNTs for autonomic or sensory neurons, with the ultimate goal of optimizing therapeutic selectivity. This research, in turn, has led to the development of BoNT-based proteins that can target non-SNARE substrates such as phosphatase and tensin homolog (PTEN). Still others are developing different BoNT serotypes, subtypes, or variants that are longer- or shorter-acting or have faster onset for various clinical purposes. New formulations of BoNTs that provide convenience for both patients and physicians are under investigation. Novel clinical uses are being evaluated for onabotulinumtoxinA, including in the prevention of post-operative atrial fibrillation. All these innovations capitalize on the unique properties of BoNTs, which continue to intrigue scientists and clinicians across numerous fields of study.

ASCL1-mediated ferroptosis resistance enhances the progress of castration-resistant prostate cancer to neurosecretory prostate cancer

Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that frequently emerges in castration-resistant prostate cancer (CRPC). NEPC is usually associated with tumor aggression, hormone therapy resistance, and poor clinical outcome. However, the mechanisms underlying the trans-differentiation from CRPC to NEPC have not been elucidated. Achaete-scute complex-like 1 (ASCL1) plays a role in neuronal commitment and differentiation and olfactory and autonomic neuron generation. This study revealed that ASCL1 was regulated by the SRY-box transcription factor 2 (SOX2) and highly expressed in NEPC cells, which was closely related to poor prognosis. Moreover, ASCL1 overexpression significantly enhanced CRPC progression to NEPC by resisting ferroptosis. Mechanically, ferroptosis resistance was mediated by CAMP-responsive element binding protein 1 (CREB1) phosphorylation, promoted by substantially upregulated ASCL1 in NEPC cells. In addition, upregulated SOX2 induced PCa cell differentiation into neuroendocrine tumors by mediating their lineage changes. In conclusion, inhibiting the ferroptosis resistance mediated by ASCL1 could provide a new NEPC therapeutic target and increase patient survival.

Deciphering the Molecular Mechanisms of Autonomic Nervous System Neuron Induction through Integrative Bioinformatics Analysis.

In vitro derivation of human neurons in the autonomic nervous system (ANS) is an important technology, given its regulatory roles in maintaining homeostasis in the human body. Although several induction protocols for autonomic lineages have been reported, the regulatory machinery remains largely undefined, primarily due to the absence of a comprehensive understanding of the molecular mechanism regulating human autonomic induction in vitro. In this study, our objective was to pinpoint key regulatory components using integrated bioinformatics analysis. A protein-protein interaction network construction for the proteins encoded by the differentially expressed genes from our RNA sequencing data, and conducting subsequent module analysis, we identified distinct gene clusters and hub genes involved in the induction of autonomic lineages. Moreover, we analyzed the impact of transcription factor (TF) activity on target gene expression, revealing enhanced autonomic TF activity that could lead to the induction of autonomic lineages. The accuracy of this bioinformatics analysis was corroborated by employing calcium imaging to observe specific responses to certain ANS agonists. This investigation offers novel insights into the regulatory machinery in the generation of neurons in the ANS, which would be valuable for further understanding and precise regulation of autonomic induction and differentiation.

Modulation of dopamine's effect at dopamine 2 receptors (D2R) mediated by association with the ghrelin receptor, GHSR

The typical action of dopamine at the dopamine 2 receptor (D2R) on CNS neurons is inhibition, an effect mediated via Gαi/o through GIRK channels. However, at two sites, autonomic preganglionic neurons in the lumbosacral spinal cord, and in the lateral hypothalamus, dopamine causes neuronal excitation through D2R. At both sites, dopamine neurons that are excited by dopamine express the ghrelin receptor, GHSR. Ghrelin is absent from the lateral hypothalamus and spinal cord, and it has been proposed that GHSR may modulate D2R signaling by the formation of heterodimers.In lumbrosacral spinal cord neurons of the defecation centre, D2R and GHSR agonists applied successively were both excitatory. Antagonism of GHSR at these neurons blocked the excitatory effect of DR2 stimulation, which was also blocked by depletion of intracellular calcium (iCa2+). We further investigated this interaction in recombinant cultured cells. In CHO cells expressing D2R, dopamine agonists had almost no effect on iCa2+, whereas in the presence of GHSR, D2R coupling to iCa2+ was observed in response to nanomolar dopamine. The elevation of iCa2+ by dopamine in D2R/GHSR cells was reduced by either D2R or GHSR antagonism, but the effect of a GHSR agonist was reduced only by GHSR antagonism. D2R coupling to iCa2+ in the presence of GHSR was dependent on both Gαq and Gαi/o, whereas ghrelin agonist coupling was dependent only on Gαq. D2R mediated effects on cAMP were dependent only on Gαi/o and were not effected by GHSR antagonism. D2R and GHSR in the membranes of CHO cells, revealed by fluorescent ligands, moved independently when tracked at high resolution in real time and, using fluorescent lifetime imaging, individual labelled DR2 sites were not in close enough proximity to detect resonance energy transfer (no detectable FRET). Consistent with coupling being via downstream crosstalk, no detectable DR2 dependent iCa2+ was evident in cells expressing the GHSR – A204E mutant, which lacks constitutive activity. The DR2-dependent iCa2+ increase was restored in these cells using subthreshold pre-stimulation with ghrelin. In native neurons of the defecation center, inward currents in response to D2R agonism were blocked by U73122, an inhibitor of PLCβ. Together this data indicates that dopamine-mediated excitation was dependent on GHSR, PLCβ and iCa2+, in both native and recombinant cells. We conclude that co-expression of GHSR causes augmentation of agonist-induced, D2R-mediated PLCβ activation and store Ca2+ release in recombinant cells and in a population of autonomic preganglionic neurons. D2R mediated excitation in D2R/GHSR cells is both Gαi/o and Gαq/11 dependent, but does not require receptor dimerisation. Our findings reveal a novel interaction between GPCRs, in a physiologically relevant system, that does not require direct receptor interaction and has broad implications for recoding of metabotropic neurotransmitter responses via modulation through other GPCRs. NHMRC grant, APP2012657, to SGBF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sympathoexcitation and pressor responses induced by acetate, an ethanol metabolite in the hypothalamic paraventricular nucleus involves activation of NMDA receptors in rats

Alcohol abuse plays a crucial role in the prevalence of cardiovascular and neuronal degenerated diseases. However, the central mechanism that mediates these effects are not fully understood. It has been well know that autonomic hypothalamic paraventricular nucleus (PVN) play an important role in regulating sympathetic outflow and arterial blood pressure (ABP). Moreover, it has been demonstrated that the increased glutamatergic activity among PVN neurons contributed to the sympathoexcitation and development of hypertension. Furthermore, we have reported that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in autonomic central nucleus of amygdala. Combing with the fact that autonomic PVN neurons are abundantly expressed NMDA receptors, we hypothesize that acetate, the metabolic products from alcohol, activates the PVN neurons through the increase in glutamatergic activity and contributes to the sympathoexcitation and development of chronic diseases, such as hypertension. In anesthetized rats, the effect of acetate microinjected in the PVN on the renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) was determined. The PVN acetate microinjection increased sympathoexcitatiory and pressor response in a dose dependent dose-dependent (1.5mm, 0.5mm, 2.0mm, 7.5mm) manner, and 2mm of acetate showed a minimum dose evoked a maximal response. To determine the role of acetate-stimulated glutamatergic receptor activation in driving sympathoexcitatory and pressor responses, either 2mm acetate or pre-treatment of Kynurenic acid (KYN, 7.2mm) ionotropic excitatory amino acid receptor blocker, or D-2-amino-5-phosphopentanoate (AP5, 3.0mm), a N-methyl-D-aspartate (NMDA) receptor antagonist, followed by 2mm acetate were microinjected into the PVN of male Sprague Dawley rats (300-500g, n= 6-9/ group). RSNA and ABP responses were compared among vehicle (saline) and 2mm acetate, pre-treatment of KYN and 2mm acetate, or pre-treatment AP5 and 2mm acetate protocols. 2mm acetate significantly increased RSNA (60-70% baseline, p < 0.001) and mean arterial pressure (MAP, 10-12mmHg, p < 0.05). Non-selective glutamatergic receptor antagonist, KYN significantly blocked the sympathoexcitatory (RSNA, p < 0.05) and pressor response (MAP, p < 0.05) evoked by 2mm acetate in the PVN. Furthermore, selective NMDA receptor antagonist, AP5 significantly attenuates the sympathoexcitatory response induced by PVN 2mm acetate (RSNA, P < 0.05 and MAP, p < 0.01). These data indicate acetate can increase glutamatergic activity and excitability of pre-sympathetic PVN neurons via activation of local NMDA receptors. The metabolism of ethanol to acetate following by alcohol consumption may contribute, in part, to the development of neurogenic hypertension and/or other cardiovascular diseases associated with increased sympathetic outflow. R15HL145655, R15HL150703 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Generation of self-organized autonomic ganglion organoids from fibroblasts

Neural organoids have been shown to serve as powerful tools for studying the mechanism of neural development and diseases as well as for screening drugs and developing cell-based therapeutics. Somatic cells have previously been reprogrammed into scattered autonomic ganglion (AG) neurons but not AG organoids. Here we have identified a combination of triple transcription factors (TFs) Ascl1, Phox2a/b, and Hand2 (APH) capable of efficiently reprogramming mouse fibroblasts into self-organized and networked induced AG (iAG) organoids, and characterized them by immunostaining, qRT-PCR, patch-clamping, and scRNA-seq approaches. The iAG neurons exhibit molecular properties, subtype diversity, and electrophysiological characteristics of autonomic neurons. Moreover, they can integrate into the superior cervical ganglia following transplantation and innervate and control the beating rate of co-cultured ventricular myocytes. Thus, iAG organoids may provide a valuable tool to study the pathogenesis of autonomic nervous system diseases and screen for drugs, as well as a source for cell-based therapies.

Gastrointestinal bleeding in children with familial dysautonomia: a case-control study.

Familial dysautonomia (FD) is a rare inherited autosomal recessive disorder with abnormal somatosensory, enteric, and afferent autonomic neurons. We aimed to define the incidence of gastrointestinal bleeding and its associated risk factors in patients with FD. In this retrospective case-control study, we identified all episodes of gastrointestinal bleeding in patients with FD, occurring over four decades (January 1980-December 2017), using the New York University FD registry. We identified 104 episodes of gastrointestinal bleeding occurring in 60 patients with FD. The estimated incidence rate of gastrointestinal bleeds in the FD population rate was 4.20 episodes per 1000 person-years. We compared the 60 cases with 94 age-matched controls. Bleeding in the upper gastrointestinal tract from gastric and duodenal ulcers occurred most frequently (64 bleeds, 75.6%). Patients were more likely to have a gastrostomy (G)-tube and a Nissen fundoplication [odds ratio (OR) 3.73, 95% confidence interval (CI) 1.303-13.565] than controls. The mean time from G-tube placement to first gastrointestinal bleed was 7.01years. The mean time from Nissen fundoplication to bleed was 7.01years. Cases and controls had similar frequency of intake of nonsteroidal antiinflammatory drugs (NSAID) and selective serotonin reuptake inhibitors (SSRI). The incidence of gastrointestinal bleeding in the pediatric FD population was estimated to be 4.20 per 1000 person-years, 21 times higher than in the general pediatric population (0.2 per 1000 person-years). Patients with FD with a G-tube and a Nissen fundoplication had a higher risk of a subsequent gastrointestinal bleeding.

Phosphorylated α-synuclein in skin Schwann cells: a new biomarker for multiple system atrophy.

Multiple system atrophy (MSA) is characterized by accumulation of phosphorylated α-synuclein (p-syn) as glial cytoplasmic inclusions in the brain and a specific biomarker for this disorder is urgently needed. We aimed at investigating if p-syn can also be detected in skin Remak non-myelinating Schwann cells (RSCs) as Schwann cell cytoplasmic inclusions (SCCi) and may represent a reliable clinical biomarker for MSA. This cross-sectional diagnostic study evaluated skin p-syn in 96 patients: 46 with probable MSA (29 with parkinsonism type MSA and 17 with cerebellar type MSA), 34 with Parkinson's disease (PD) and 16 with dementia with Lewy bodies (DLB). We also included 50 healthy control subjects. Patients were recruited from five different medical centres. P-syn aggregates in skin sections were stained by immunofluorescence, followed by analyses with confocal microscopy and immuno-electron microscopy. All analyses were performed in a blinded fashion. Overall, p-syn aggregates were found in 78% of MSA patients and 100% of patients with PD/DLB, whereas they could not be detected in controls. As for neuronal aggregates 78% of MSA patients were positive for p-syn in somatic neurons, whereas all PD/DLB patients were positive in autonomic neurons. When analysing the presence of p-syn in RSCs, 74% of MSA patients were positive, whereas no such SCCi could be observed in PD/DLB patients. Analyses by immuno-electron microscopy confirmed that SCCi were only found in cases with MSA and thus absent in those with PD/DLB. In conclusion, our findings demonstrate that (i) fibrillar p-syn in RSCs is a pathological hallmark of MSA and may be used as a specific and sensitive disease biomarker; (ii) in Lewy body synucleinopathies (PD/DLB) only neurons contain p-syn deposits; and (iii) the cell-specific deposition of p-syn in the skin thus mirrors that of the brain in many aspects and suggests that non-myelinated glial cells are also involved in the MSA pathogenesis.

OnabotulinumtoxinA effects on trigeminal nociceptors.

OnabotulinumtoxinA (onabotA) is approved globally for prevention of chronic migraine; however, the classical mechanism of action of onabotA in motor and autonomic neurons cannot fully explain the effectiveness of onabotulinumtoxinA in this sensory neurological disease. We sought to explore the direct effects of onabotulinumtoxinA on mouse trigeminal ganglion sensory neurons using an inflammatory soup-based model of sensitization. Primary cultured trigeminal ganglion neurons were pre-treated with inflammatory soup, then treated with onabotulinumtoxinA (2.75 pM). Treated neurons were used to examine transient receptor potential vanilloid subtype 1 and transient receptor potential ankyrin 1 cell-surface expression, calcium influx, and neuropeptide release. We found that onabotulinumtoxinA cleaved synaptosomal-associated protein-25 kDa in cultured trigeminal ganglion neurons; synaptosomal-associated protein-25 kDa cleavage was enhanced by inflammatory soup pre-treatment, suggesting greater uptake of toxin under sensitized conditions. OnabotulinumtoxinA also prevented inflammatory soup-mediated increases in TRPV1 and TRPA1 cell-surface expression, without significantly altering TRPV1 or TRPA1 protein expression in unsensitized conditions. We observed similar inhibitory effects of onabotulinumtoxinA on TRP-mediated calcium influx and TRPV1- and TRPA1-mediated release of calcitonin gene-related peptide and prostaglandin 2 under sensitized, but not unsensitized control, conditions. Our data deepen the understanding of the sensory mechanism of action of onabotulinumtoxinA and support the notion that, once endocytosed, the cytosolic light chain of onabotulinumtoxinA cleaves synaptosomal-associated protein-25 kDa to prevent soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated processes more generally in motor, autonomic, and sensory neurons.

Evaluating the transduction efficiency of systemically delivered AAV vectors in the rat nervous system.

Gene delivery or manipulation with viral vectors is a frequently used tool in basic neuroscience studies. Adeno-associated viruses (AAV) are the most widely used vectors due to their relative safety and long-term efficacy without causing overt immunological complications. Many AAV serotypes have been discovered and engineered that preferentially transduce different populations of neurons. However, efficient targeting of peripheral neurons remains challenging for many researchers, and evaluation of peripheral neuron transduction with AAVs in rats is limited. Here, we aimed to test the efficiency of systemic AAVs to transduce peripheral neurons in rats. We administered AAV9-tdTomato, AAV-PHP.S-tdTomato, or AAV-retro-GFP systemically to neonatal rats via intraperitoneal injection. After 5 weeks, we evaluated expression patterns in peripheral sensory, motor, and autonomic neurons. No significant difference between the serotypes in the transduction of sensory neurons was noted, and all serotypes were more efficient in transducing NF200 + neurons compared to smaller CGRP + neurons. AAV-retro was more efficient at transducing motor neurons compared to other serotypes. Moreover, PHP.S was more efficient at transducing sympathetic neurons, and AAV-retro was more efficient at transducing parasympathetic neurons. These results indicate that specific AAV serotypes target peripheral neuron populations more efficiently than others in the neonatal rat.

Imaging neuro-urodynamics of mouse major pelvic ganglion using a micro-endoscopic approach.

Post-ganglionic neurons of the autonomic nervous system lie outside of the central nervous system and innervate specific target effectors such as organs or glands. The major pelvic ganglion (MPG) is one such ganglion that plays a significant role in controlling bladder function in rodents. However, due to technical and physical constraints in recording electrophysiological signals from these neurons in vivo, the functional neural activity in MPG is mostly unknown. Transgenic animal models expressing genetically encoded calcium indicators now provide opportunities to monitor the activity of populations of neurons in vivo to overcome these challenges related to traditional electrophysiological methods. However, like many peripheral neurons, the MPG is not conducive to conventional fluorescent microscopy techniques, as it is located in the pelvic cavity thus limiting robust optical access by benchtop microscopes. Here, we present an endoscopic approach based on a custom miniscope system (UCLA V3) that allows for effective in vivo monitoring of neural activity in the MPG for the first time. We show that our imaging approach can monitor activity of hundreds of MPG neurons simultaneously during the filling and emptying of the bladder in urethane-anesthetized transgenic mouse line expressing GCaMP6s in cholinergic MPG neurons. By using custom analysis scripts, we isolated the activity of hundreds of individual neurons and show that populations of neurons have distinct phasic activation patterns during sequential bladder filling and voiding events. Our imaging approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.

THE HDL LEVEL IN DIABETIC FOOT ULCER PATIENTS WITH AND WITHOUT AMPUTATION AT DR. SEOTOMO GENERAL ACADEMIC HOSPITAL SURABAYA

Highlights1. Diabetes mellitus is a category of metabolic disease characterized by uncontrolled hyperglycemia.2. This study revealed no statistically significant differences in total cholesterol, TG, LDL, HDL, or LDL/HDL ratio between amputated and non-amputated DFU patients. AbstractBackground: Diabetic foot ulcers (DFU) are microvascular and macrovascular consequences of diabetes and are associated with neuropathy, vascular disease, and immunosuppression. Increased blood glucose levels may result in the glycosylation of proteins in the nerve cells of the foot, leading to ischemia and cellular disturbances that can damage muscular, sensory, and autonomic neurons. Objective: To compare the lipid profiles of amputated and non-amputated DFU patients. Material and Method: This was a cross-sectional, analytic, retrospective research using total sample approach to analyze the medical records of all diabetes patients admitted to Dr. Soetomo General Academic Hospital between February 2018 and December 2020, with or without amputation. Result: The average age of DFU patients at Dr. Soetomo General Academic Hospital was 55.6 9.4 years, and the majority of patients were female (57%). Diabetic foot ulcers were most prevalent in the age group ≥50 years old (74%), amputated DFU patients had larger mean total cholesterol, TG, LDL, and HDL than non-amputated DFU patients, although the difference was not statistically significant. While the mean LDL/HDL ratio was greater in non-amputated DFU patients, the difference was statistically insignificant. The HDL level was low in 84% of amputated DFU patients, whereas in non-amputated DFU patients it was 81%. Conclusion: This research showed no significant variation in total cholesterol, TG, LDL, HDL, or the LDL/HDL ratio between amputated and non-amputated DFU patients. Most DFU patients with and without amputations had low HDL values.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Abstract 15832: Optogenetic Stimulation of Intrinsic Cardiac Autonomic Neurons Reveals the Interaction Between Catecholaminergic and Cholinergic Pathways

Autonomic tone, through the interaction of catecholaminergic and cholinergic pathways, is a powerful modulator of cardiac function. Until recently, selective activation of cardiac autonomic pathways has required administration of exogeneous neurotransmitters or careful placement of electrodes to stimulate specific neural populations. Optogenetics enables broader exploration of autonomic interactions through the selective expression of channelrhodopsin (ChR2) in neural populations. Our objective was to simultaneously activate catecholaminergic and cholinergic pathways, by exogenous perfusion of neurotransmitter and photostimulation of opposing autonomic neurons, to test the hypothesis that simultaneous activation will reveal the fast kinetics of cholinergic modulation of catecholaminergic increases in heart rate (HR). Norepinephrine (NE) and acetylcholine (ACh) were administered to perfused transgenic mouse hearts that expressed ChR2 within sympathetic tyrosine hydroxylase neurons (TH+ChR2) or parasympathetic choline acetyltransferase (ChAT+ChR2) neurons. Intrinsic neurons were photostimulated by illuminating the SA node with a 465nm high power microLED. The ECG was recorded to measure beat-to-beat changes in HR. Moderate doses of exogeneous NE increased HR 26.77±5.61% (n=3) and high doses increased HR 59.74±8.77% (n=3). At all NE doses, photostimulation of ChAT+ChR2 neurons immediately reduced HR to values equivalent to, or lower than, pre-NE HR. Arrhythmias were not observed. In companion studies, moderate doses of exogeneous ACh reduced HR 19.98±4.68% (n=7) and high doses reduced HR 51.31±4.07% (n=7). HR increases caused by TH+ChR2 neuron photostimulation were diminished by exogenous ACh in a dose-dependent manner. Increased dosing of exogeneous ACh, with simultaneous TH+ChR2 neuron photostimulation, dramatically shortened the onset of 2 nd degree AV block and increased its severity. At high concentrations of exogeneous ACh, non-sustained arrhythmias were observed upon the cessation of TH+ChR2 neuron photostimulation. These results provide new insight into the kinetics of intrinsic cardiac autonomic interactions and reveal powerful modulation of catecholaminergic activity by cholinergic activation.

Spinal cord injury is associated with changes in synaptic properties of the mouse major pelvic ganglion.

Spinal cord injury (SCI) has substantial impacts on autonomic function. In part, SCI results in loss of normal autonomic activity that contributes to injury-associated pathology such as neurogenic bladder, bowel, and sexual dysfunction. Yet little is known of the impacts of SCI on peripheral autonomic neurons that directly innervate these target organs. In this study, we measured changes in synaptic properties of neurons of the mouse major pelvic ganglion (MPG) associated with acute and chronic SCI. Our data show that functional and physiological properties of synapses onto MPG neurons are altered after SCI and differ between acute and chronic injury. After acute injury excitatory postsynaptic potentials (EPSPs) show increased rise and decay time constants leading to overall broader and longer EPSPs, whereas in chronic-injured animals EPSPs are reduced in amplitude and show faster rise and decay leading to shorter EPSPs. Synaptic depression and low-pass filtering are also altered in injured animals. Finally, cholinergic currents are smaller in acute-injured animals but larger in chronic-injured animals relative to control animals. These changes in synaptic properties are associated with differences in nicotinic receptor subunit expression as well. MPG CHRNA3 mRNA levels decreased after injury, whereas CHRNA4 mRNAs increased. Furthermore, changes in the correlations of α- and β-subunit mRNAs suggest that nicotinic receptor subtype composition is altered after injury. Taken together, our data demonstrate that peripheral autonomic neurons are fundamentally altered after SCI, suggesting that longer-term therapeutic approaches could target these neurons directly to potentially help ameliorate neurogenic target organ dysfunction.NEW & NOTEWORTHY Spinal cord injury (SCI) has substantial impacts on autonomic function, yet little is known of the impacts of SCI on autonomic neurons that directly innervate effectors impacted by injury. Here we investigated changes at the cellular level associated with SCI in neurons that are "downstream" of the central injury. An understanding of these off-target impacts of SCI ultimately will be critical in the context of effective restoration of function through neuromodulation of pharmacological therapeutic approaches.