Postganglionic Research Articles

P409 CARDIAC SYMPATHOMIMETIC RESPONSE IN PARKINSON‘S PATIENTS VS HEALTHY ADULTS

Abstract In recent works, it has been observed that cardiac sympathetic innervation contributes to the hemodynamic adaptations of the cardiovascular system, in relation to numerous needs. Orimo et al, through the use of scintigraphy with MIBG, showed in Parkinson‘s patients a marked reduction in myocardial uptake of MIBG corresponding to cardiac sympathomimetic denervation, in relation to the severity of the disease. The aim of this work was to evaluate the morpho–functional variations of the heart at a programmed sympathomimetic stimulus (dopamine test), both in healthy patients and in those suffering from Parkinson‘s disease. Materials and Methods At the University Cardiology “La Sapienza” of Rome, Pontine Polo, 10 patients affected by PD (age 56±5,5, of which 6 M and 4 W) and 10 healthy (54±6,4, of which 6M,4W)were tested in the absence of cardiovascular FR; all patients performed: blood chemistry, ECG, chest x–ray, Eco–TSA, tilt test, echocardio– Dopamine (2D–stress) and MIBG Scintigraphy. 2D–stress echocardiogradia was assessed by t–student of F.acc + EF + left and right diastolic times + GLS + Tapse + PAPS at 10, 20.30.40 µ dopa and their correlation (r–pearson) in the two groups. Discussion The observation of the data in the two groups of patients, in the absence of ongoing and remote cardiological pathology, showed a behavior of the values in the range both in basal conditions and during dopa stimulation. The comparison of the two groups both basal and during progressive dose stimulation that a statistical difference (P < 0.05) is observed on both systolic and diastolic function. The comparison in the two groups in the different stimulus phases showed a parallel growth behavior but with a different speed (Dp / Dv). There is no single explanation for these results: certainly the central role is played by the poor representation of post–ganglionic nerve endings as well as by the significant reduction of cardiac adrenergic receptors in Parkison. Conclusion The small number of patients and the data observed, stimulates researchers to continue the study, to better understand which pathophysiological mechanisms underlie these observations and verify the presence of an inverse relationship between PD and all those cardiological pathologies caused by catecholaminergic hyperstimulus.

Сучасні наукові погляди на склад та будову автономної нервової системи (огляд літератури)

The purpose of the study was to analyze literary sources to summarize modern scientific views on the structure and possible changes in the existing classification of the autonomic nervous system. Results and discussion. According to classical views, the anatomically and functionally autonomic nervous system is divided into sympathetic and parasympathetic parts, and the last one includes cranial and sacral centers. This classification, in addition to ontogenetic, anatomical and physiological justification, has historical roots and is associated with the research of J. Langley. For more than a century, such a classification of the autonomic nervous system was considered commonplace. However, modern views on this issue emphasize the mechanisms of development and molecular mechanisms of these systems. More attention was paid to such elements as gene expression, features of embryogenesis and development, as well as the general functions of neurons localized in these parts of the autonomic nervous system. In recent years, a group of researchers Espinosa-Medina et al. determined the differential genetic features and relationships of sympathetic and parasympathetic preganglionic and postganglionic neurons. The aim of the study was to compare the relationships and genetic characteristics of lumbar and sacral preganglionic neurons with cranial (parasympathetic) and thoracic (sympathetic) neurons. In general, 15 phenotypic and ontogenetic features are given that distinguish the pre- and postganglionic neurons of the cranial part of the parasympathetic part from the sympathetic neurons of the thoracolumbar region. Each of the features, according to Espinosa-Medina et al., proves that the sacral section must be considered as part of the sympathetic, and not the parasympathetic, section of the autonomic nervous system. This assumption is based on the study on mice at 11.5, 13, 165 days of embryonic development of several transcription factors common to both sacral and thoracolumbar preganglionic neurons, but absent in cranial preganglionic neurons. When revising the existing classification, cranial vegetative nervous structures are proposed to be considered parasympathetic, and spinal – sympathetic. However, a number of researchers were critical of the proposed changes to the existing classification. They consider the interpretation of the findings controversial, point to possibly misleading conclusions due to misinterpretations of the evidence, and suggest that the phenotypes of common thoracic and sacral preganglionic neurons may simply share a common spinal identity. Conclusion. In recent years, thanks to the latest research, there has been a rethinking and possible change in the ideas that have existed for a long time about the composition and structure of the autonomic nervous system. The proposed reclassification of the autonomic nervous system involves the assignment of the sacral region to its sympathetic, not parasympathetic part. Such a simplified two-component structure offers a new concept of neurophysiology, as well as the evolution and development of the autonomic nervous system. At the same time, a number of researchers give their arguments in favor of rejecting the proposed radical changes and saving the existing classification. In this regard, further studies are highly demanded, which could finally shed light on this issue

Abnormal Cyclic Nucleotide Signaling at the Outer Mitochondrial Membrane In Sympathetic Neurons During the Early Stages of Hypertension.

Background:Disruption of cyclic nucleotide signaling in sympathetic postganglionic neurons contributes to impaired intracellular calcium handling (Ca2+) and the development of dysautonomia during the early stages of hypertension, although how this occurs is poorly understood. Emerging evidence supports the uncoupling of signalosomes in distinct cellular compartments involving cyclic nucleotide–sensitive PDEs (phosphodiesterases), which may underpin the autonomic phenotype in stellate neurons.Methods:Using a combination of single-cell RNA sequencing together with Forster resonance energy transfer–based sensors to monitor cyclic adenosine 3’,5’-monophosphate, PKA (protein kinase A)-dependent phosphorylation and cGMP (cyclic guanosine 3’,5’-monophosphate), we tested the hypothesis that dysregulation occurs in a sub-family of PDEs in the cytosol and outer mitochondrial membrane of neurons from the stellate ganglion.Results:PDE2A, 6D, 7A, 9A genes were highly expressed in young Wistar neurons and also conserved in neurons from spontaneously hypertensive rats (SHRs). In stellate neurons from prehypertensive SHRs, we found the levels of cyclic adenosine 3’,5’-monophosphate and cGMP at the outer mitochondrial membrane were decreased compared with normal neurons. The reduced cyclic adenosine 3’,5’-monophosphate response was due to the hydrolytic activity of overexpressed PDE2A2 located at the mitochondria. Normal cyclic adenosine 3’,5’-monophosphate levels were re-established by inhibition of PDE2A. There was also a greater PKA-dependent phosphorylation in the cytosol and at the outer mitochondrial membrane in spontaneously hypertensive rat neurons, where this response was regulated by protein phosphatases. The cGMP response was only restored by inhibition of PDE6.Conclusions:When taken together, these results suggest that site-specific inhibition of PDE2A and PDE6D at the outer mitochondrial membrane may provide a therapeutic target to ameliorate cardiac sympathetic impairment during the onset of hypertension.

Evidence for Cholinergic Collateral Projections between Sympathetic Neurons in the Murine Stellate Ganglia

Postganglionic neurons of the stellate ganglia (SG) are an integral component of sympathetic regulation of the heart. SG neurons receive preganglionic cholinergic inputs via T‐2 and send noradrenergic projections to the heart via the inferior cardiac nerve (iCN) to influence cardiac activity. SG neurons are thought to receive simple monosynaptic inputs from preganglionic neurons, but early studies have hinted that cholinergic collateral projections may be present in the SG. In culture, SG neurons form noradrenergic and cholinergic synapses between one another, suggesting that SG neurons express dual neurochemical phenotypes. This study tests the hypothesis that cholinergic collateral projections are present between sympathetic neurons of the intact SG in mice.Whole‐cell patch clamp recordings were made of intact SG neurons from male (N=8) and female (N=6) ChATTH+CreERT2/lox mice with and without tamoxifen treatment (N=7 ChAT KO mice and N=7 control mice, respectively). Tamoxifen treatment in ChATTH+CreERT2/lox mice induced the deletion of choline acetyltransferase (ChAT) in tyrosine hydroxylase positive (i.e. noradrenergic) cells. This strategy blocks putative acetylcholine synthesis in SG neurons while leaving preganglionic cholinergic synapses intact. To search for collaterals, the preganglionic nerve trunk, T‐2, was electrically stimulated to evoke excitatory postsynaptic currents (eEPSCs). The amplitude and jitter (i.e. variability in latency) of eEPSCs were examined. Additionally, the postganglionic nerve trunk, the iCN, was electrically stimulated to evoke retrograde action currents (rAC) and the presence of evoked synaptic currents was assessed. The amplitude and frequency of spontaneous EPSCs (sEPSCs) were also examined.ChAT KO significantly reduced the frequency of sEPSCs compared to control mice (1.21±0.559events/s vs. 0.13±0.048events/s, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) while the amplitude was unaffected (74.4±26.10pA vs. 39.4±10.46pA, P>0.05 from Student’s unpaired t‐test, N=5‐6 cells). This suggests that ChAT KO reduces the presynaptic activity of postganglionic noradrenergic neurons within the SG. In control mice, stimulation of T‐2 evoked high jitter eEPSCs, which was significantly reduced in ChAT KO mice (327.1±92.14μs vs. 39.2±15.69μs, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) Additionally, the proportion of neurons that exhibited evoked synaptic currents following stimulation of the iCN was significantly reduced in ChAT KO mice (3/4 cells vs. 1/9 cells, P<0.05 from χ² test).Chemical transmission at additional synapses accounts for the high jitter events observed in disynaptic pathways. High jitter responses following T‐2 stimulation and evoked synaptic responses after iCN stimulation in control neurons are both consistent with the collateral hypothesis. The loss of high jitter responses and evoked synaptic currents in ChAT KO mice also suggests these collateral projections are cholinergic. This suggests that collateral synaptic activity contributes to the increased frequency of sEPSCs in control mice. Understanding SG neurocircuitry is critical in deciphering changes in sympathetic activity in many pathophysiological conditions such as myocardial infarction and heart failure and may identify novel therapeutic targets for the treatment of autonomic imbalance.

Uncovering Anti‐Arrhythmic Potential of Stellate Ganglion Purinergic Receptors

Cardiovascular disease affects over 30% of people worldwide and is one of the leading causes of death each year. Elevated sympathetic nerve activity is a common feature of cardiovascular disease, contributing to end‐organ damage, morbidity and mortality. Recent findings indicate that short‐circuiting sympathetic nerve overactivity by removal of the stellate ganglion can eradicate arrhythmias, emphasising the need for novel therapeutic targets to correct signalling non‐invasively.We have revealed upregulation of the P2X3 purinergic receptor in stellate ganglia of Spontaneously Hypertensive (SHR) compared to Wistar rats (16 week‐old), via RNASeq transcriptional profiling. We hypothesise that these purinergic receptors within cardiac stellate ganglia play a role in the excessive sympathetic drive to the heart in cardiovascular disease and can initiate cardiac arrhythmias. Thus, we have investigated the functional role of purinergic receptors in the stellate ganglion.Administration of ATP to acutely isolated post‐ganglionic sympathetic neurones from the stellate ganglia of Wistar rats (4‐6 week‐old) evokes a significant increase (Median; 0.21) in [Ca2+]i as measured by Fura‐2AM imaging (n=15, Wilcoxon matched‐pairs test; p<0.0001). This ATP‐induced calcium transient was inhibited by specific P2X3 receptor antagonism with either NF‐10 or AF‐130.Cardiac effects of stellate ganglion purinergic stimulation were investigated in the working heart‐brainstem preparation of Wistar rats (4‐6 week‐old). Increasing doses of ATP (50‐250 µg) delivered via microinjection (50‐250 µL) directly to the right stellate ganglion produced either a tachycardia (18 ± 6 bpm, n=6) or bradycardia (‐24 ± 12 bpm, n=5); these responses were typically found at distinct sites within the ganglion. Preliminary data from SHR suggest that higher doses of ATP (≥250µg) may be arrhythmogenic (n=3).P2X3 purinergic receptors are present in the sympathetic stellate ganglion and contribute to calcium ion flux and cardiac chronotropic regulation. Overexpression of P2X3 receptors is likely to contribute to excessive cardiac sympathetic activity and the development of hypertension and cardiac arrhythmias, making them a promising novel therapeutic target.

PO-650-08 VALUE OF ATROPINE TEST TO ESTIMATE THE CARDIONEUROABLATION RESULT

Vagal innervation keeps an autonomic tone that predominates most of the time. Because of this, the isolated sinus heart rate (HR) is higher than the in-situ sinus rate. Atropine stops vagal action by blocking muscarinic receptors in the heart. Several studies have shown that cardioneuroablation (CNA) also promotes blocking of vagal action by partially eliminating the postganglionic parasympathetic neuron. Thus, when CNA is performed, there is typically an increase in HR. Previously knowing whether HR will increase with CNA is critical information since CNA is not indicated if there is no atropine response.

Role of the P2 × 4 Receptor in Sympathetic-mediated Inflammatory Low Back Pain

The sympathetic nervous system mediates various pain conditions including inflammatory low back pain. To better understand the underlying mechanisms, we developed a surgical procedure named localized microsympathectomy (mSYMPX) in rats and mice, which consists of cutting the grey rami containing postganglionic sympathetic fibers as they enter the spinal nerves near the lumbar dorsal root ganglia (DRG). We reported that mSYMPX attenuated pain behaviors in several preclinical pain models including a mouse model of chemotherapy-induced peripheral neuropathy (CIPN model). Under pathological conditions release of ATP from sympathetic nerve endings locally in the DRG activates purinergic receptor P2 × 4 in macrophages resulting in a more M1/pro-inflammatory polarization. In this study we asked whether the macrophage-enriched P2 × 4 receptors are involved in sympathetic-mediated inflammatory low back pain/radiculopathy produced by intravertebral injection of the immune activator zymosan, causing mechanical hypersensitivity (LID model). Our previous microarray study in rats showed upregulation of the macrophage cell marker Iba1 and purinergic receptor (P2rx4) after LID. As previously reported in rats, we found that mice showed mechanical hypersensitivity assessed by calibrated von Frey filaments and transcriptional upregulation of Iba1 and P2rx4 three days after LID. Interestingly, mSYMPX prevented mechanical hypersensitivity, and also caused significant transcriptional upregulation of Iba1 and a trend towards downregulation of P2rx4 three days after LID. The enhancement of P2 × 4 signaling with the allosteric modulator ivermectin reduced expression of the M2 marker in cultured peritoneal macrophages. Conversely, blocking P2 × 4 signaling with PSB12062 in M1 skewed macrophages mitigated the expression of M1 marker. Furthermore, the mechanical hypersensitivity in the LID model was reversed by intrathecal injection of the P2 × 4 antagonist (PSB12062) three days after LID. These findings suggest P2 × 4 receptor as a potential therapeutic target for managing inflammatory low back pain/radiculopathy. Funded by NS045594 and the HEAL supplement to NINDS grant NS045594 (J-M Z).

Topographical Mapping of Catecholaminergic Axon Distribution and Morphology in the Flat‐Mounts of the Mouse Heart

The sympathetic nervous system plays a crucial role in the control of heart functions. However, the distribution and morphology of sympathetic postganglionic innervation of the heart has not been well elucidated, which has significantly impeded the understanding of the sympathetic neuroanatomical organization and control of the heart. Our goal is to provide a map of the topographical innervation of sympathetic postganglionic neurons in the whole heart of mice. In this study, we used Tyrosine hydroxylase (TH) as a marker for sympathetic axons and applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing and immunohistochemistry, Neurolucida 360 Tracing and integration of the tracing data onto a 3D heart scaffold. We found that 1) Multiple large extrinsic TH‐IR nerve bundles entered the heart and branched out into smaller axons which covered the whole left and right atria and ventricles, 2) These TH‐IR bundles branched out into smaller axon bundles and ramified into individual axons covering the whole left and right atria and ventricles. 3) The TH‐IR axons formed an extensive neural network in the epicardium and axons formed varicose synaptic endings within the cardiac muscle. 4) TH‐IR axons and terminals were densest in the sinoatrial (SA) and atrioventricular (AV) node regions. 5) TH‐IR axons innervated blood vessels and fat cells. 6) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent (SIF) cells were TH‐IR. 7) However, TH‐IR axons do not appear to innervate any cardiac principal neurons and SIF cells. 8) TH‐IR axon innervation will be integrated into a 3D scaffold. Our work will provide a comprehensive topographical map of the catecholaminergic efferent axon distribution, innervation, and morphology of the whole heart at single cell/axon/synapse scale that will be used to create a cardiac sympathetic‐brain atlas. It will also provide an anatomical reference to evaluate the altered sympathetic cardiac control in pathological conditions. The first two authors contributed equally to this work.

Overproduction of reactive oxygen species in cardiac vagal postganglionic neurons contributes to myocardial infarction‐induced lethal ventricular arrhythmias in type 2 diabetes mellitus

Myocardial infarction (MI)‐evoked ventricular arrhythmia is the major cause of mortality in type 2 diabetes mellitus (T2DM) patients. Although intensive glycemic control over time have been noted in T2DM patients, it fails to reduce MI‐related mortality in T2DM patients. Withdrawal of cardiac vagal (parasympathetic) activity is a common complication and is associated with arrhythmia‐related sudden cardiac death in T2DM patients. Our recent study found that T2DM‐reduced cell excitability of CVP neurons contributes to the withdrawal of cardiac vagal activity and further exacerbates acute MI‐evoked fatal ventricular arrhythmias and high mortality rate in T2DM. However, the mechanisms responsible for T2DM‐reduced cell excitability of CVP neurons are unclear. Since reactive oxygen species (ROS) has been reported to modulate calcium (Ca2+) channel activity in peripheral postganglionic neurons, here we tested if and how hydrogen peroxide (H2O2, a type of ROS) inactivates CVP neurons and subsequently contributes to MI‐evoked fatal ventricular arrhythmias in T2DM rats. Rat T2DM was induced by a high‐fat diet plus streptozotocin injection. Adenoviral catalase gene (Ad.CAT) or its vector control (Ad.Empty, 2 μl, 1x1010 pfu/ml) was microinjected into CVP neurons. Local in vivo transfection of Ad.CAT successfully induced overexpression of catalase and attenuated H2O2 overproduction in CVP neurons in T2DM rats. Additionally, transfection of Ad.CAT not only restored protein expression and ion currents of N‐type Ca2+ channels, but also increased cell excitability of CVP neurons in T2DM rats. Using Fluo3/AM with a confocal microscope, we also demonstrated that Ad.CAT gene transfection significantly restored intracellular Ca2+ levels in isolated CVP neurons from T2DM rats. Moreover, data from 24‐hour continuous ECG recording in conscious rats demonstrated that transfection of Ad.CAT into CVP neurons alleviated the heterogeneity of ventricular electrical activity (a critical factor of ventricular arrhythmogenesis). More importantly, Ad.CAT transfection into CVP neurons significantly reduced the cumulative duration of VT/VF induced by MI in conscious T2DM rats (28.3 ± 6.9 sec/hour in the T2DM+AD.CAT group vs. 71.7 ± 7.4 sec/hour in the T2DM group, P<0.05). Ad.Empty gene transfection into CVP neurons had no effects on intracellular H2O2 and Ca2+ levels, protein expression and ion currents of N‐type Ca2+ channels, cell excitability of CVP neurons, and duration of VT/VF induced by MI in T2DM rats. Based on these data, we concluded that overproduction of H2O2 decreased protein expression and activation of N‐type Ca2+ channels and reduced cell excitability of CVP neurons, which further contributed to ventricular arrhythmogenesis in T2DM. Our current study suggests that targeting H2O2‐N‐type Ca2+ channel signaling pathway in CVP neurons is an effective intervention against MI‐evoked fatal ventricular arrhythmias in the T2DM state.

An Exploration of the Sympathetic Component of the Recurrent Laryngeal Nerve

INTRODUCTIONHigh interest in the recurrent laryngeal nerve (RLN) morphology has been dictated by the frequent iatrogenic injures that can result in multifaceted functional losses such as dysphagia, voice alteration, and breathing. Thus, RLN has been extensively studied on the macroscopic and histological levels. According to the literature, RLN is a mixed nerve carrying motor, sensory, and preganglionic parasympathetic fibers to the larynx and displays asymmetry in branching from the vagus nerve. Even though gross anastomoses between RLN and the cervical sympathetic trunk have been observed, the presence of sympathetic postganglionic fibers within RLN have not been addressed. The present study was conducted to determine if catecholaminergic fibers are present within the RLN using immunohistochemical analysis for tyrosine hydroxylase (TH).METHODSRLN samples were obtained from 19 formalin‐preserved adult human cadavers (male n=11, female n=8). Anastomoses with sympathetic structures were noted and documented before obtaining tissue samples for histological examination. Samples were collected one centimeter distal to the RLN branching site. All sections were prepared using Luxol fast blue staining to reveal the presence of myelinated and unmyelinated fibers and TH antibody staining was used to determine the presence of catecholaminergic fibers. Histological slides were then photographed and quantitative analysis of the images was conducted with ImageJ software by measuring of the effective surface area (ESA), TH‐positive, myelinated, and unmyelinated. We reported results as mean ± SEM.RESULTSNeural communications between right RLNs and sympathetic ganglia were observed in two whole‐body donors. No statistically significant differences in the number of the fascicles, ESA, myelinated, and unmyelinated fibers between right and left RLNs were found. TH‐positive fibers were detected bilaterally or unilaterally in 89.5% of examined donors. Bilateral absence of TH activity throughout the effective surface area was observed in two cases. TH‐positive nerve fiber area within the RLNs varied from 0% to 22.14% (Figure 1). The average of TH‐positive fibers on the right was 0.045 ± 0.011 mm2 (4.47% ± 1.1% of ESA) and 0.05 ± 0.015 mm2 (5.03±1.5% of ESA) on the left. A positive correlation between the number of unmyelinated fibers and TH‐positive component was found (P=0.022 and P=0.0026 on the right and left, respectfully). All parameters, such as fascicles, ESA, myelinated, unmyelinated, and TH‐positive fibers, occurred independently of age, gender, or conditions (p>0.05).CONCLUSIONSympathetic postganglionic fibers within RLN nerve exist; however, there is a considerable difference in their number between individuals. However, the origin and functions of this component are ambiguous.SIGNIFICANCEKnowledge of the sympathetic component of RLN could assist in better understanding the physiological effects and adverse responses of vagus nerve stimulation therapy and the sequala of the RLN injury.

The Origin of Sympathetic Postganglionic Fibers in the Cervical Region of the Vagus Nerve

IntroductionThe vagus nerve (VN) is a cranial nerve that is mainly known for its parasympathetic function. Although a communication between the VN and sympathetic ganglia was described as early as 1858 by Henry Gray, the existence of sympathetic fibers within the VN is still under debate. Studies utilizing immunohistochemical verification with anti‐Tyrosine Hydroxylase (TH) antibody have found variable distribution of catecholaminergic activity in the cervical and thoracic trunks of the VN, ranging from 0% to 21.63%. We aimed to elucidate the presence and distribution of the postganglionic sympathetic element within the cervical region of the VN using immunohistochemical analysis with anti‐TH antibody. We evaluated macroscopic neural communications between cervical sympathetic chain ganglia (SCG) and the cervical VN as a potential source of intravagal catecholaminergic fibers.MethodsDissection of the VN, cervical chain ganglia, and any communicating branches was performed bilaterally on 20 fully embalmed cadavers (male n=11, female n=9) using standard dissection tools. All communications were histologically validated to confirm neural tissue and cervical VN samples were collected bilaterally in two locations. Tissue sections were stained with anti‐TH antibody and Luxol Fast Blue. Histological slides were viewed and photographed with a Leica DM 500 compound microscope. All images underwent quantitative analysis with ImageJ software to measure effective surface area (ESA) and TH‐positive areas.ResultsTwenty pairs of VN and SCG with associated neural communications were identified. Interconnections were found in 11 donors (55%), resulting in 13 total neural anastomoses between the VN and either the superior, middle, or inferior cervical ganglia. All measurements are reported as mean ± SEM. When comparing the right and left superior cervical vagus nerve ESA, the right side had a larger ESA than the left (2.27±0.26mm2 and 1.82±0.15mm2; p=0.039). TH Immunoreactivity was detected in all donors bilaterally. We observed TH positive fibers in three distribution patterns (Figure 1). There was a significant interaction of location and gender on the percentage of TH‐positive fibers. Females expressed a higher percentage of TH‐positive fibers on the right side (3.0 ±1.22% in females and 1.2 ±0.37% in males; p=0.044). The average ESA of communications on the right side was 0.15±0.07 mm2 and 0.04±0.009 mm2 on the left (p>0.05). The number of fascicles within each communication varied from one to seven (2.77±0.4956). Most fibers stained positive for TH, occupying 91.86% ±4.1% of the total ESA. Presence or size of communications did not influence number of TH positive fibers within the VN. We identified ganglion cells within the superior and inferior samples of the cervical VN (Figure 2).ConclusionTH‐positivity within the cervical VN and its communications with the SCG suggests a ganglionic sympathetic source. However, identification of ganglion cells within the VN suggests a postganglionic source that warrants further study. Investigating sympathetic activity of the VN and its source, aids in understanding its physiologic role.

Hydrogen Peroxide Scavenging Restores N-Type Calcium Channels in Cardiac Vagal Postganglionic Neurons and Mitigates Myocardial Infarction-Evoked Ventricular Arrhythmias in Type 2 Diabetes Mellitus.

ObjectiveWithdrawal of cardiac vagal activity is associated with ventricular arrhythmia-related high mortality in patients with type 2 diabetes mellitus (T2DM). Our recent study found that reduced cell excitability of cardiac vagal postganglionic (CVP) neurons is involved in cardiac vagal dysfunction and further exacerbates myocardial infarction (MI)-evoked ventricular arrhythmias and mortality in T2DM. However, the mechanisms responsible for T2DM-impaired cell excitability of CVP neurons remain unclear. This study tested if and how elevation of hydrogen peroxide (H2O2) inactivates CVP neurons and contributes to cardiac vagal dysfunction and ventricular arrhythmogenesis in T2DM.Methods and ResultsRat T2DM was induced by a high-fat diet plus streptozotocin injection. Local in vivo transfection of adenoviral catalase gene (Ad.CAT) successfully induced overexpression of catalase and subsequently reduced cytosolic H2O2 levels in CVP neurons in T2DM rats. Ad.CAT restored protein expression and ion currents of N-type Ca2+ channels and increased cell excitability of CVP neurons in T2DM. Ad.CAT normalized T2DM-impaired cardiac vagal activation, vagal control of ventricular function, and heterogeneity of ventricular electrical activity. Additionally, Ad.CAT not only reduced the susceptibility to ventricular arrhythmias, but also suppressed MI-evoked lethal ventricular arrhythmias such as VT/VF in T2DM.ConclusionsWe concluded that endogenous H2O2 elevation inhibited protein expression and activation of N-type Ca2+ channels and reduced cell excitability of CVP neurons, which further contributed to the withdrawal of cardiac vagal activity and ventricular arrhythmogenesis in T2DM. Our current study suggests that the H2O2-N-type Ca2+ channel signaling axis might be an effective therapeutic target to suppress ventricular arrhythmias in T2DM patients with MI.

Multicolor labeling of airway neurons and analysis of parasympathetic heterogeneity

We report subpopulations of airway parasympathetic neurons expressing substance P, neuronal nitric oxide synthase, and tyrosine hydroxylase, highlighting unexplored heterogeneity in this population. These neurotransmitter-specific subpopulations did not form intraganglionic interneurons, but rather, extended outside the ganglia, into the airways, to distant innervation targets. Our experiments demonstrate the utility of multicolor labeling to characterize airway innervation, allowing us to confirm the extensive heterogeneity of postganglionic parasympathetic neurons. These methods will facilitate future investigations of neurophysiology and neural contributions to airway disease.

Interactive effects of apneic and baroreflex stress on neuronal coding strategies in human muscle sympathetic nerve activity

The sympathetic nervous system exhibits patterns of action potential (AP) discharge in human muscle sympathetic nerve activity that suggest coding strategies express reflex specificity. This study explored the interactive effects of baroreceptor unloading using lower body negative pressure (LBNP) and volitional end-expiratory apnea (APN) on sympathetic postganglionic neuronal discharge patterns inferred from the firing patterns of differently sized sympathetic AP clusters. Seven individuals were studied using multiunit microneurography (fibular) and a continuous wavelet approach to quantify AP discharge probability, recruitment, and latency during APN performed under ambient conditions, -10, and -40 mmHg LBNP. Compared with the ambient condition, LBNP increased AP discharge rate at -10 and -40 mmHg and recruited larger previously silent sympathetic neurons at -40 mmHg. Compared with spontaneous breathing, APN increased AP discharge when performed during the ambient condition (Δ351 ± 132 AP/min), -10 mmHg (Δ423 ± 184 AP/min), and -40 mmHg (Δ355 ± 278 AP/min; main effect APN: P < 0.01; LBNP-by-APN interaction: P = 0.55). APN recruited larger previously silent AP clusters during the ambient condition (Δ4 ± 3; P < 0.02) and -10 mmHg (Δ4 ± 3; P < 0.01), but not -40 mmHg (Δ0 ± 2; P = 0.53; LBNP-by-APN: P < 0.01). LBNP did not affect AP latency. However, APN reduced AP latency similarly during all conditions (ambient pressure: Δ-0.04 ± 0.04s, -10 mmHg: Δ-0.03 ± 0.03s, -40 mmHg: Δ-0.03 ± 0.04s; main effect APN: P < 0.01; LBNP-by-APN: P = 0.48). These data indicate that apneic and baroreflex mechanisms appear to additively modify the axonal discharge rate of previously active sympathetic postganglionic neurons and interact to affect recruitment of previously silent sympathetic neurons. Reductions in AP latency due to apneic stress were not impacted by baroreflex unloading.NEW & NOTEWORTHY Discrete physiological stressors differentially affect sympathetic postganglionic neuronal rate-, population-, and temporal-coding strategies. When performing end-expiratory apnea (APN) during graded baroreflex unloading via lower body negative pressure (LBNP), we found: 1) augmented sympathetic axonal firing probability, 2) recruitment of larger and previously silent sympathetic postganglionic neurons at ambient and -10 mmHg, but not -40 mmHg LBNP, and 3) APN reduced axonal discharge latency similarly across all conditions, independent of the level of baroreflex unloading.

The Paraventricular Nucleus of the Hypothalamus in Control of Blood Pressure and Blood Pressure Variability

The paraventricular nucleus (PVN) is a highly organized structure of the hypothalamus that has a key role in regulating cardiovascular and osmotic homeostasis. Functionally, the PVN is divided into autonomic and neuroendocrine (neurosecretory) compartments, both equally important for maintaining blood pressure (BP) and body fluids in the physiological range. Neurosecretory magnocellular neurons (MCNs) of the PVN are the main source of the hormones vasopressin (VP), responsible for water conservation and hydromineral balance, and oxytocin (OT), involved in parturition and milk ejection during lactation. Further, neurosecretory parvocellular neurons (PCNs) take part in modulation of the hypothalamic–pituitary–adrenal axis and stress responses. Additionally, the PVN takes central place in autonomic adjustment of BP to environmental challenges and contributes to its variability (BPV), underpinning the PVN as an autonomic master controller of cardiovascular function. Autonomic PCNs of the PVN modulate sympathetic outflow toward heart, blood vessels and kidneys. These pre-autonomic neurons send projections to the vasomotor nucleus of rostral ventrolateral medulla and to intermediolateral column of the spinal cord, where postganglionic fibers toward target organs arise. Also, PVN PCNs synapse with NTS neurons which are the end-point of baroreceptor primary afferents, thus, enabling the PVN to modify the function of baroreflex. Neuroendocrine and autonomic parts of the PVN are segregated morphologically but they work in concert when the organism is exposed to environmental challenges via somatodendritically released VP and OT by MCNs. The purpose of this overview is to address both neuroendocrine and autonomic PVN roles in BP and BPV regulation.

Frequency-dependent characteristics of nerve-mediated ATP and acetylcholine release from detrusor smooth muscle.

What is the central question of this study? Is the frequency dependence of co-transmitter release from postganglionic nerve fibres different for each transmitter? What is the main finding and its importance? Release of co-transmitters from the parasympathetic supply to detrusor smooth muscle can be independently regulated. This offers a targeted drug model to reduce selectively the release of transmitter associated with human pathologies (ATP) and may also be applicable to other smooth muscle-based disorders of visceral tissues. Nerve-mediated contractions of detrusor smooth muscle are mediated by acetylcholine (ACh) and ATP release in most animals. However, with the normal human bladder, only ACh is a functional transmitter, but in benign pathologies such as overactive bladder (OAB), ATP re-emerges as a secondary transmitter. The selective regulation of ATP release offers a therapeutic approach to manage OAB, in contrast to current primary strategies that target ACh actions. However, the release characteristics of nerve-mediated ACh and ATP are poorly defined and this study aimed to measure the frequency dependence of ACh and ATP release and determine if selective regulation of ATP or ACh was possible. Experiments were carried out in vitro with mouse detrusor with nerve-mediated ATP and ACh release measured simultaneously with tension recording. ATP was released in two frequency-dependent components, both at lower frequencies (mid-range 0.4 and 5.5Hz stimulation) compared to a single compartment release of ACh at 14Hz. Intervention with the phosphodiesterase type-5 inhibitor sildenafil attenuated ATP release, equally from both components, but had no effect on ACh release. These data demonstrate that nerve-mediated ACh and ATP release characteristics are distinct and may be separately manipulated. This offers a potential targeted drug model to manage benign lower urinary tract conditions such as OAB.

Untangling Peripheral Sympathetic Neurocircuits

The sympathetic nervous system plays a critical role in regulating many autonomic functions, including cardiac rhythm. The postganglionic neurons in the sympathetic chain ganglia are essential components that relay sympathetic signals to target tissues and disruption of their activity leads to poor health outcomes. Despite this importance, the neurocircuitry within sympathetic ganglia is poorly understood. Canonically, postganglionic sympathetic neurons are thought to simply be activated by monosynaptic inputs from preganglionic cholinergic neurons of the intermediolateral cell columns of the spinal cord. Early electrophysiological studies of sympathetic ganglia where the peripheral nerve trunks were electrically stimulated identified excitatory cholinergic synaptic events in addition to retrograde action potentials, leading some to speculate that excitatory collateral projections are present. However, this seemed unlikely since sympathetic postganglionic neurons were known to synthesize and release norepinephrine and expression of dual neurochemical phenotypes had not been well recognized. In vitro studies clearly established the capacity of cultured sympathetic neurons to express and release acetylcholine and norepinephrine throughout development and even in pathophysiological conditions. Given this insight, we believe that the canonical view of ganglionic transmission needs to be reevaluated and may provide a mechanistic understanding of autonomic imbalance in disease. Further studies likely will require genetic models manipulating neurochemical phenotypes within sympathetic ganglia to resolve the function of cholinergic collateral projections between postganglionic neurons. In this perspective article, we will discuss the evidence for collateral projections in sympathetic ganglia, determine if current laboratory techniques could address these questions, and discuss potential obstacles and caveats.

Electrophysiological characterization of mouse intracardiac calbindin neurons

Neural control of the heart involves central and peripheral neurons that act interdependently to modulate cardiac parameters such as heart rate, conduction velocity or contractility. Within this cardiac neuronal regulation, the intrinsic cardiac nervous system, which correspond to clusters of neurons found on the dorsal atrial surface of the heart, is receiving growing attention. Indeed, whereas they were initially considered as simple parasympathetic postganglionic neurons, studies conducted over the past 30 years suggested a more complex organization, involving the existence of sensory, local regulatory and motor neurons within intracardiac ganglia.

Segmental anhidrosis and heat intolerance

A 62 year-old woman with no previous medical history presented to the Dermatology department with sweating disorders for several years. She described segmental hyperhidrosis and hyperemia while running. This excessive sweating occurred in the left hemiface, the left half of the trunk, the left upper limb and the right lower limb (Fig. 1A). This was associated with heat intolerance and previous faintness during sport. She did not describe any chest pain before faintness and the cardiac auscultation was normal. The neurological examination revealed a diffuse areflexia in the absence of any sensory or motor disorders. By illuminating the right eye, we observed a delay in ipsilateral pupillary contraction, whereas we observed an absence of the consensual photomotor reflex of the right eye by illuminating the left eye (Adie's tonic pupil, Fig. 1B and C, and Supplementary file 1). However, she did not complain of reduced visual acuity. Ross syndrome is a rare autonomic disorder caused by a selective degenerative process of the postganglionic cholinergic sudomotor neurons [[1]Sommer C. Lindenlaub T. Zillikens D. Toyka K.V. Naumann M. Selective loss of cholinergic sudomotor fibers causes anhidrosis in Ross syndrome.Ann Neurol. 2002; 52: 247-250Google Scholar]. Ross syndrome is diagnosed by the presence of a triad of symptoms associating segmental anhidrosis (with compensatory hyperhidrosis), Adie's tonic pupil and areflexia [[2]Mishra A.K. Kharkongor M. Kuriakose C.K. George A.A. Peter D. Carey R.A.B. et al.Is ross syndrome an autoimmune entity? A case series of 11 patients.Can J Neurol Sci. 2017; 44: 318-321Google Scholar]. The main complaint of patients is generally the segmental compensatory hyperhidrosis, the anhidrosis in contrast is not even noticed. The two other signs must be specifically examined for to make the diagnosis. The deep tendon hyporeflexia or areflexia affects the four limbs, whereas the tonic pupil is observed on the pathological and anhidrotic side of the face. It has been proposed to treat hyperhidrosis with botulinum toxin [[3]Bergmann I. Dauphin M. Naumann M. Flachenecker P. Müllges W. Koltzenburg M. et al.Selective degeneration of sudomotor fibers in Ross syndrome and successful treatment of compensatory hyperhidrosis with botulinum toxin.Muscle Nerve. 1998; 21: 1790-1793Google Scholar], but this treatment may theoretically increase the heat intolerance and the risk of faintness during sport. Even if the disorder is benign, the objective is to avoid any faintness. Clinicians should advise patients with Ross syndrome to wear light clothing for sports and to avoid excessive heat exposure.

Serotonergic Paracrine Targets in the Intestinal Mucosa.

Serotonin functions as a neurotransmitter in the enteric nervous system. Aside from its neurotransmitter role, serotonin also is a paracrine mediatorial signal in the digestive tract. It is a major paracrine signaling molecule in the integrated physiology of several classes of cells in the intestinal mucosa. Paracrine action can be initiation or suppression of activity in populations of cells that make up divergent phenotypic classes. This underlies phenotypic plasticity in single classes and links single classes to other neighboring phenotypic classes, thereby forming a single and higher-order organization in which different categories of function are integrated to work in harmony as a single homeostatic entity at higher levels of physiological organization. Phenotypic classes of cells that are linked by serotonergic paracrine signaling at upper levels of functional organization in the small intestine are (1) enterochromaffin cells; (2) enteric mast cells; (3) spinal sensory afferents; (4) sympathetic postganglionic neurons; (5) enteric neurons.