Ablation of myocardial autonomic ganglion plexus in the treatment of bradyarrhythmia A one-arm interventional study.

OBJECTIVES: To study the complications and effectiveness of the treatment of chronic arrhythmias with cardiac Ganglion Plexus (GP) ablation, and to explore the value of the treatment of chronic arrhythmias with GP ablation. METHODS: This study was a one-arm interventional study of patients from the first hospital of Xinjiang Medical University and the People's Hospital of Xuancheng City admitted (09/2018-08/2021) because of bradyarrhythmia. The left atrium was modeled using the Carto3 mapping system. The ablation endpoint was the absence of a vagal response under anatomically localized and high-frequency stimulation guidance. Postoperative routine follow-up was conducted. Holter data at 3-, 6-, and 12-months were recorded. RESULTS: Fifty patients (25 male, mean age 33.16 ± 7.89 years) were induced vagal response by either LSGP, LIGP, RAGP, or RIGP. The heart rate was stable at 76 bpm, SNRT 1.092s. DC, DR, HR, SDNN, RMSSD values were lower than that before ablation. AC, SSR, TH values were higher than those before ablation, mean heart rate and the slowest heart rate were significantly increased. There were significant differences in follow-up data between the preoperative and postoperative periods (all p < 0.05). All the patients were successfully ablated, and their blood pressure decreased significantly. No complications such as vascular damage, vascular embolism and pericardial effusion occurred. CONCLUSIONS: Left Atrial GP ablation has good long-term clinical results and can be used as a treatment option for patients with bradyarrhythmia.

Immunological and therapeutic insights in autoimmune autonomic ganglionopathy: What is the position of apheresis in immunotherapy?

Autoimmune autonomic ganglionopathy (AAG) is characterized by various autonomic and extra-autonomic symptoms and is caused by autoantibodies against nicotinic acetylcholine receptors present in the autonomic ganglia (ganglionic acetylcholine receptor, gAChR), requiring immediate and aggressive intervention to prevent the exacerbation of symptoms. However, there is currently no internationally accepted standard of care for the immunotherapy of AAG, including apheresis. Although the rationale for the use of plasma exchange (PLEX) in AAG is strong, whereby pathogenic gAChR antibodies are removed, its overall impact on patient outcomes is not well-established. Based on previous case reports and small case series studies, we provide a comprehensive overview of the challenges and uncertainties surrounding the use of PLEX for the management of AAG and provide current practice recommendations to guide treatment decisions.

Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

[Autoimmune Autonomic Ganglionopathy and Acute Autonomic Sensory Neuropathy].

Autoimmune autonomic ganglionopathy (AAG) and acute autonomic sensory neuropathy (AASN) are immune-mediated neuropathies that affect the autonomic and/or dorsal root ganglia. Autoantibodies against the nicotinic ganglionic acetylcholine receptor (gAChR) detected in the sera of patients with AAG play a key role in the pathogenesis of this condition. Notably, gAChR antibodies are not detected in the sera of patients with AASN. Currently, AAG and AASN are not considered to be on the same spectrum with regard to disease concept based on clinical symptoms and laboratory findings. However, extra-autonomic brain symptoms (including psychiatric symptoms and personality changes) and endocrine disorders occur in both diseases, which suggests shared pathophysiology between the two conditions.

Cardioneuroablation for vasovagal syncope: insights on patients' selection, centre settings, procedural workflow and endpoints-results from an European Heart Rhythm Association survey.

AIMS: Cardioneuroablation (CNA) is a catheter-based intervention for recurrent vasovagal syncope (VVS) that consists in the modulation of the parasympathetic cardiac autonomic nervous system. This survey aims to provide a comprehensive overview of current CNA utilization in Europe. METHODS AND RESULTS: A total of 202 participants from 40 different countries replied to the survey. Half of the respondents have performed a CNA during the last 12 months, reflecting that it is considered a treatment option of a subset of patients. Seventy-one per cent of respondents adopt an approach targeting ganglionated plexuses (GPs) systematically in both the right atrium (RA) and left atrium (LA). The second most common strategy (16%) involves LA GP ablation only after no response following RA ablation. The procedural endpoint is frequently an increase in heart rate. Ganglionated plexus localization predominantly relies on an anatomical approach (90%) and electrogram analysis (59%). Less utilized methods include pre-procedural imaging (20%), high-frequency stimulation (17%), and spectral analysis (10%). Post-CNA, anticoagulation or antiplatelet therapy is prescribed, with only 11% of the respondents discharging patients without such medication. Cardioneuroablation is perceived as effective (80% of respondents) and safe (71% estimated <1% rate of procedure-related complications). Half view CNA emerging as a first-line therapy in the near future. CONCLUSION: This survey offers a snapshot of the current implementation of CNA in Europe. The results show high expectations for the future of CNA, but important heterogeneity exists regarding indications, procedural workflow, and endpoints of CNA. Ongoing efforts are essential to standardize procedural protocols and peri-procedural patient management.

Autoimmune Autonomic Neuropathy: From Pathogenesis to Diagnosis.

Autoimmune autonomic ganglionopathy (AAG) is a disease of autonomic failure caused by ganglionic acetylcholine receptor (gAChR) autoantibodies. Although the detection of autoantibodies is important for distinguishing the disease from other neuropathies that present with autonomic dysfunction, other factors are important for accurate diagnosis. Here, we provide a comprehensive review of the clinical features of AAG, highlighting differences in clinical course, clinical presentation, and laboratory findings from other neuropathies presenting with autonomic symptoms. The first step in diagnosing AAG is careful history taking, which should reveal whether the mode of onset is acute or chronic, followed by an examination of the time course of disease progression, including the presentation of autonomic and extra-autonomic symptoms. AAG is a neuropathy that should be differentiated from other neuropathies when the patient presents with autonomic dysfunction. Immune-mediated neuropathies, such as acute autonomic sensory neuropathy, are sometimes difficult to differentiate, and therefore, differences in clinical and laboratory findings should be well understood. Other non-neuropathic conditions, such as postural orthostatic tachycardia syndrome, chronic fatigue syndrome, and long COVID, also present with symptoms similar to those of AAG. Although often challenging, efforts should be made to differentiate among the disease candidates.

Liver Cancer with Overlapping Myasthenia Gravis, Myocarditis, Seronegative Autoimmune Autonomic Ganglionopathy, and Myositis Symptoms Induced by Atezolizumab.

An 83-year-old man with hepatocellular carcinoma developed muscle weakness, ptosis, and dyspnea 3 weeks after receiving atezolizumab. Soon after, mechanical ventilation was initiated, which was followed by marked blood pressure spikes. The levels of creatine kinase and troponin-I were significantly elevated, and acetylcholine receptor antibodies were positive. The patient was diagnosed with immune checkpoint inhibitor (ICI)-induced myositis, myasthenia gravis (MG), myocarditis, and suspected autoimmune autonomic ganglionopathy (AAG). After immunotherapy, the serum markers and blood pressure normalized, and he was weaned from the ventilator after five months. To our knowledge, this is the first reported case of AAG secondary to ICI-induced myositis, MG, and myocarditis.

Pulsed field ablation of the right superior pulmonary vein prevents vagal responses via anterior right ganglionated plexus modulation.

BACKGROUND: Pulsed field ablation (PFA) is selective for the myocardium. However, vagal responses and reversible effects on ganglionated plexi (GP) are observed during pulmonary vein isolation (PVI). Anterior-right GP ablation has been proven to effectively prevent vagal responses during radiofrequency-based PVI. OBJECTIVE: The purpose of this study was to test the hypothesis that PFA-induced transient anterior-right GP modulation when targeting the right superior pulmonary vein (RSPV) before any other pulmonary veins (PVs) may effectively prevent intraprocedural vagal responses. METHODS: Eighty consecutive paroxysmal atrial fibrillation patients undergoing PVI with PFA were prospectively included. In the first 40 patients, PVI was performed first targeting the left superior pulmonary vein (LSPV-first group). In the last 40 patients, RSPV was targeted first, followed by left PVs and right inferior PV (RSPV-first group). Heart rate (HR) and extracardiac vagal stimulation (ECVS) were evaluated at baseline, during PVI, and postablation to assess GP modulation. RESULTS: Vagal responses occurred in 31 patients (78%) in the LSPV-first group and 5 (13%) in the RSPV-first group (P <.001). Temporary pacing was needed in 14 patients (35%) in the LSPV-first group and 3 (8%) in the RSPV-first group (P = .003). RSPV isolation was associated with similar acute HR increase in the 2 groups (13 ± 11 bpm vs 15 ± 12 bpm; P = .3). No significant residual changes in HR or ECVS response were documented in both groups at the end of the procedure compared to baseline (all P >.05). CONCLUSION: PVI with PFA frequently induced vagal responses when initiated from the LSPV. Nevertheless, an RSPV-first approach promoted transient HR increase and reduced vagal response occurrence.

[A man in his fifties with near-syncope episodes and persistent dizziness]. / En mann i 50-årene med nærbesvimelser og vedvarende svimmelhet.

BACKGROUND: A previously healthy male patient in his fifties presented with subacute onset of severe, diffuse dysautonomia with orthostatic hypotension as the main symptom. A lengthy interdisciplinary workup revealed a rare condition. CASE PRESENTATION: Over the course of a year, the patient was twice admitted to the local department of internal medicine because of severe hypotension. Testing showed severe orthostatic hypotension with normal cardiac function tests and no apparent underlying cause. On referral to neurological examination, symptoms of a broader autonomic dysfunction were discovered, with symptoms of xerostomia, irregular bowel habits, anhidrosis and erectile dysfunction. The neurological examination was normal, except for bilateral mydriatic pupils. The patient was tested for ganglionic acetylcholine receptor (gAChR) antibodies. A strong positive result confirmed the diagnosis of autoimmune autonomic ganglionopathy. There were no signs of underlying malignancy. The patient received induction treatment with intravenous immunoglobulin and later maintenance treatment with rituximab, resulting in significant clinical improvement. INTERPRETATION: Autoimmune autonomic ganglionopathy is a rare but likely underdiagnosed condition, which may cause limited or widespread autonomic failure. Approximately half of the patients have ganglionic acetylcholine receptor antibodies in serum. It is important to diagnose the condition as it can cause high morbidity and mortality, but responds to immunotherapy.

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

Postganglionic 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, because of 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 a 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.NEW & NOTEWORTHY The functional activity and information processing within autonomic ganglia is mostly unknown because of technical and physical constraints in recording electrophysiological signals from these neurons in vivo. Here, we use a micro-endoscopic approach to measure in vivo functional activity patterns from a population of autonomic neurons controlling bladder function for the first time. This approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.

Functional Significance of Angiotensin Receptor Type 2 in the Neuroplasticity of Autonomic Ganglia in (mRen2)27 Transgenic Hypertensive Rats.

ABSTRACT: The over-expression of Ren -2 d gene in (mRen2)27 rats leads to development of hypertension mediated by the renin-angiotensin-system axis and exaggerated sympathetic nerve activity. Exogenously applied angiotensin II (AngII) on the superior cervical ganglion evokes ganglionic compound action potentials (gCAP) and ganglionic long-term potentiation (gLTP). We studied the functional role of angiotensin receptors and expression of reactive oxygen species marker, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) proteins in AngII-induced postganglionic transmission. Bath-applied AngII revealed that the indices of ganglionic transmission, synaptic strength of gCAP, and decay time for gLTP are remarkably prolonged in (mRen2)27 rats and were abolished by an angiotensin receptor blocker (ARB), suggesting postganglionic AngII Type 1 (AT 1 ) receptor localization and mediation. Receptor density for AT 1 was similar in (mRen2)27 and control animals, and quantitative reverse transcription polymerase chain reaction revealed that it is consistent with the mRNA profile. Furthermore, immunocytochemistry analysis showed similar AT 1 receptor distribution and signals. However, assessment of Type 2 (AT 2 ), Ang-(1-7)-MAS and NOX4-specific proteins showed that AT 2 receptor protein expression was 4-fold lower, consistent with a low mRNA profile. MAS receptor expression was 10-fold lower and NOX4 protein was 2-fold lower. Despite similarity in the densities of AT 1 receptor, the low levels of the components of the protective arm of the renin-angiotensin system at the ganglia may contribute to the differential superior cervical ganglion sensitivity to AngII. The lower NOX4 affects reactive oxygen species balance and possibly results in activation of downstream pathways to promote increased sympathetic nerve activity. We speculate that the significant diminution in AT 2, MAS, and NOX4 protein expressions may play an indirect role in the alteration and efficacy of gCAP and gLTP in hypertension.

Emx1-Cre Is Expressed in Peripheral Autonomic Ganglia That Regulate Central Cardiorespiratory Functions.

The Emx1-IRES-Cre transgenic mouse is commonly used to direct genetic recombination in forebrain excitatory neurons. However, the original study reported that Emx1-Cre is also expressed embryonically in peripheral autonomic ganglia, which could potentially affect the interpretation of targeted circuitry contributing to systemic phenotypes. Here, we report that Emx1-Cre is expressed in the afferent vagus nerve system involved in autonomic cardiorespiratory regulatory pathways. Our imaging studies revealed expression of Emx1-Cre driven tdtomato fluorescence in the afferent vagus nerve innervating the dorsal medulla of brainstem, cell bodies in the nodose ganglion, and their potential target structures at the carotid bifurcation such as the carotid sinus and the superior cervical ganglion (SCG). Photostimulation of the afferent terminals in the nucleus tractus solitarius (NTS) in vitro using Emx1-Cre driven ChR2 reliably evoked EPSCs in the postsynaptic neurons with electrophysiological characteristics consistent with the vagus afferent nerves. In addition, optogenetic stimulation targeting the Emx1-Cre expressing structures identified in this study, such as vagus nerve, carotid bifurcation, and the dorsal medulla surface transiently depressed cardiorespiratory rate in urethane anesthetized mice in vivo Together, our study demonstrates that Emx1-IRES-Cre is expressed in the key peripheral autonomic nerve system and can modulate cardiorespiratory function independently of forebrain expression. These results raise caution when interpreting systemic phenotypes of Emx1-IRES-Cre conditional recombinant mice, and also suggest the utility of this line to investigate modulators of the afferent vagal system.

[Over 5 years follow-up of three cases of autoimmune autonomic ganglionopathy].

We report the clinical course of three cases of anti-ganglionic acetylcholine receptor (gAChR) antibody positive auto-immune autonomic ganglionopathy (AAG) that have been followed for over 5 years. In all three cases, the symptoms improved by acute treatment, but ultimately relapsed. The first case was a female in her 20s who had a chronic history of photophobia, constipation and amenorrhea. The symptoms almost disappeared by plasma exchange, and menstruation resumed. During the course, it relapsed once after a cold. There was no recurrence of AAG during the two pregnancies. The second case was a male in his 60s who visited a hospital for the acute onset of orthostatic hypotension (OH) and psychological symptoms (infantilization and psychogenic pseudosyncope). Although IVIg was effective, it recurred frequently and was difficult to treat. However, all the symptoms disappeared eight years after the onset without any particular reasons. The third case was a female in her 80s who had a chronic history of OH. Acute treatment was effective, but AAG recurred repeatedly. Additionally, it was difficult to judge relapse because of the residual sequelae. During the course, cerebral hemorrhage due to supine hypertension or short-time blood pressure variability and femoral neck fracture caused by OH occurred. She eventually became a wheelchair. This report is clinically important because there are few reports of long-term follow-up of AAG.

Severe treatment-refractory antibody positive autoimmune autonomic ganglionopathy after mRNA COVID19 vaccination.

BACKGROUND: COVID-19 vaccine-associated peripheral and central neuroimmunological disorders have been well described. We present the case of a 56 year old male who developed α3-ganglionic AChR antibody positive Autoimmune Autonomic Ganglionopathy (AAG) after completion of a two-dose course of mRNA (Comirnaty) vaccination for COVID19. RESULTS: A previously hypertensive 56 year old male presented with the subacute onset of severe constipation, urinary retention, erectile dysfunction, sudomotor failure, sicca symptoms, non-reactive pupils and severe orthostatic hypotension shortly after receiving the second dose of an mRNA vaccine against COVID19. Autonomic testing revealed severe cardiovagal, adrenergic and sudomotor impairment, and tonic 'half-mast' pupils with evidence of sympathetic and parasympathetic denervation. Pathological α3-ganglionic ACHR antibodies were positive in serum as detected by a new flow cytometric immunomodulation assay. Malignancy was excluded. The patient was diagnosed with severe, treatment-refractory acute AAG. CONCLUSIONS: While autonomic dysfunction has been previously reported post-COVID19 vaccination, to our knowledge this is the first reported case of antibody-positive AAG in this setting. The severity of this case is in marked contrast to the existing literature on idiopathic antibody-positive autoimmune pandysautonomia.

Paraneoplastic autoimmune autonomic ganglionopathy as the first symptom of bladder cancer: a case report and review of literature.

BACKGROUND: Autoimmune autonomic ganglionopathy is a rare, immune-mediated disorder associated with anti-ganglionic α3-subunit nicotinic acetylcholine receptor (anti-α3gAChR) antibodies, which bind to acetylcholine receptor in autonomic ganglia (parasympathetic and sympathetic) leading to autonomic failure. This disorder is mostly associated with viral infections, but it can also be associated with systemic malignancies. Here, we report the case of a paraneoplastic autonomic ganglionopathy as the first symptom of bladder cancer. METHOD: Case report. RESULTS: A 47-year-old man, without medical history of interest, stated to the emergency department for progressive blurry vision with eye and mouth dryness, constipation, and dizziness upon standing for the last 2 weeks. Orthostatic hypotension was demonstrated by a drop in 13.3 mmHg mean blood pressure (BP) from supine (100/60 mmHg) to 45° reclining sitting position (80/50 mmHg). Blood tests, chest X-ray, brain MRI, and electroneuronography were unremarkable. Electrochemical skin conductance was reduced. Serological examination was positive for anti-α3gAChR antibodies. A full-body CT scan revealed a bladder tumor, which was treated by transurethral bladder resection. The pathologic study demonstrated a low-grade non-muscle-invasive bladder urothelial carcinoma. After tumor resection, and treatment with intravenous immunoglobulins and corticoids, a gradually improvement was observed. Today, the patient remains asymptomatic. CONCLUSION: Subacute panautonomic failure can be the first symptom for systemic malignancies. This case reports a paraneoplastic autonomic ganglionopathy as the first symptom of bladder cancer. This case highlights the importance of a systemic study to rule out the presence of cancer when autoimmune autonomic ganglionopathy is present.

Novel Cell-Based Assay for Alpha-3 Nicotinic Receptor Antibodies Detects Antibodies Exclusively in Autoimmune Autonomic Ganglionopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies against α3-subunit-containing nicotinic acetylcholine receptors (α3-nAChRs), usually measured by radioimmunoprecipitation assay (RIPA), are detected in patients with autoimmune autonomic ganglionopathy (AAG). However, low α3-nAChR antibody levels are frequently detected in other neurologic diseases with questionable significance. Our objective was to develop a method for the selective detection of the potentially pathogenic α3-nAChR antibodies, seemingly present only in patients with AAG. METHODS: The study involved sera from 55 patients from Greece, suspected for autonomic failure, and 13 patients from Italy diagnosed with autonomic failure, positive for α3-nAChR antibodies by RIPA. In addition, sera from 52 patients with Ca2+ channel or Hu antibodies and from 2,628 controls with various neuroimmune diseases were included. A sensitive live cell-based assay (CBA) with α3-nAChR-transfected cells was developed to detect antibodies against the cell-exposed α3-nAChR domain. RESULTS: Twenty-five patients were found α3-nAChR antibody positive by RIPA. Fifteen of 25 patients were also CBA positive. Of interest, all 15 CBA-positive patients had AAG, whereas all 10 CBA-negative patients had other neurologic diseases. RIPA antibody levels of the CBA-negative sera were low, although our CBA could detect dilutions of AAG sera corresponding to equally low RIPA antibody levels. No serum bound to control-transfected cells, and none of the 2,628 controls was α3-CBA positive. DISCUSSION: This study showed that in contrast to the established RIPA for α3-nAChR antibodies, which at low levels is of moderate disease specificity, our CBA seems AAG specific, while at least equally sensitive with the RIPA. This study provides Class II evidence that α3-nAChR CBA is a specific assay for AAG. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that an α3-nAChR cell-based assay is a more specific assay for AAG than the standard RIPA.

Autoimmune autonomic ganglionopathy: Ganglionic acetylcholine receptor autoantibodies.

Autoimmune Autonomic Ganglionopathy (AAG) is a rare immune-mediated disease of the autonomic nervous system. The incidence of AAG is unknown and diagnosis is often difficult due to the multicompartmental nature of the autonomic nervous system - sympathetic, parasympathetic and enteric components - with variable severity and number of components affected. Diagnostic confidence is increased when ganglionic acetylcholine receptor (gnACHR) autoantibodies are detected. Three gnACHR autoantibody diagnostic assays have been described (two binding assays, one receptor immunomodulation assay), but cross-validation between assays is limited. The prevalence of gnACHR autoantibodies in AAG is not known, with application of different clinical and laboratory criteria in the few studies of AAG cohorts and large retrospective laboratory studies of positive gnACHR autoantibodies lacking adequate clinical characterisation. Furthermore, the rate of unexpected gnACHR autoantibody positivity in conditions without overt autonomic dysfunction (false positive results) adds to the complexity of their interpretation. We review the pathophysiology of gnACHR autoantibodies and assays for their detection, with immunomodulation and high titer radioimmunoprecipitation results likely offering better AAG disease identification.

Evaluation of commercially available antibodies and fluorescent conotoxins for the detection of surface ganglionic acetylcholine receptor on the neuroblastoma cell line, IMR-32 by flow cytometry.

Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017). We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use. We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.