Preganglionic Research Articles

Optogenetic stimulation of cholinergic neuronal endings in the celiac‐superior mesenteric ganglion complex suppresses inflammation in murine endotoxemia

The vagus nerve plays a key role in the regulation of inflammation within the inflammatory reflex (Annu Rev Immunol, 2018, 36:783). In this major physiological neuroimmunoregulatory mechanism, efferent vagus nerve cholinergic axons interact with catecholaminergic neurons of the splenic nerve in the celiac‐superior mesenteric ganglion (CSMG) complex(Proc Natl Acad Sci USA, 2020, 117(47):29803).Previous studies utilizing electrical and optogenetic neuromodulation have demonstrated that vagus nerve cholinergic signalling suppresses pro‐inflammatory cytokine levels during endotoxemia and other inflammatory conditions. However, the specific role of cholinergic axonal terminals in the CSMG complex in this regulation remained enigmatic. To provide insight, we investigated the effect of selective optogenetic stimulation of cholinergic neuronal endings in the CSMG complex on cytokine and chemokine levels during murine endotoxemia. We used transgenic female 10–14‐week‐old mice expressing channelrhodopsin 2 coupled with yellow fluorescent protein on cholinergic, i.e., choline acetyltransferase positive neurons (ChAT‐ChR2‐eYFP). Anesthetized mice were subjected to abdominal surgery and the CSMG complex was localized medial to the upper pole left kidney and along the branch of the abdominal aorta, above the left renal vein. Blue laser light stimulation at 473nm was delivered in square pulses for 10mins at 20Hz, 25% duty cycle through an optic fiber cannula placed in contact with the CSMG complex. 30mins following optogenetic stimulation or sham stimulation, lipopolysaccharide (0.25mg/kg) was injected intraperitoneally and 90mins later mice were euthanized and blood, and spleen were collected and processed for cytokine and chemokines. Optogenetic stimulation of the CSMG complex in ChAT‐ChR2 mice (n=14) suppressed serum pro‐inflammatory cytokines and chemokines compared with sham stimulation (n=13), including TNF(401.7 ± 102.9 pg/ml vs 766.1 ± 207.2 pg/ml; P<0.0001) and MIP‐1α (6,523 ± 3,041 pg/ml vs 10,672 ± 3,915 pg/ml, P=0.0066), while the serum levels of the anti‐inflammatory cytokine IL‐10 were significantly increased (30,779 ± 10,035 pg/ml vs 22,979 ± 9,669 pg/ml, P=0.0427). There was also a significant reduction in splenic TNF levels in the stimulated vs the sham group(P=0.0023). Of note, optogenetic stimulation of the CSMG complex in littermate mice that do not express ChR2 on cholinergic neurons did not alter serum and splenic cytokine levels. These results provide the first experimental evidence that selective stimulation of cholinergic neuronal endings in the CSMG complex attenuates cytokine and chemokine levels. Future studies examining the specific contribution of vagal and sympathetic preganglionic cholinergic neurons in the CSMG complex in the regulation of immune responses will be instrumental for developing new targeted therapeutic anti‐inflammatory approaches.

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.

Exercise Training and Chemogenetic Activation of the Dorsal Motor Nucleus of the Vagus Rescue the Changes in the Hepatic Vein Glucose Concentration by Central Action of Insulin in SHR

The Dorsal Motor Nucleus of the Vagus (DMNV) is the main parasympathetic output from CNS as part of the brain‐liver axis that participates on glucose homeostasis. Previously, we have shown that icv injection of insulin elicited a decrease the hepatic vein glucose concentration (HVGC) of Wistar, but not in SHR. Here we aimed to investigate whether the deficiency of central action of insulin to control the HVGC of SHR is due to a dysfunction in the cholinergic neurotransmission in the DMNV and which putative neurotransmitter could play a role in this circuitry. To this, we used in vivo and in vitro studies using chemogenetic tools, and the whole‐cell patch‐clamp recordings. Ethical approval: CEUA/ICB‐USP #8336131119). IN VIVO STUDIES: Male SHR (12 weeks old) received an injection of DREADD (LVV‐Gq‐hM3q‐PRSx8‐eGFP) to selectively activate de DMNV neurons with clozapine‐N‐oxide (CNO). After 10 days of CNO treatment, the animals received icv injection of insulin and the HVGC was monitored in freely moving rats. IN VITRO STUDIES: to identify liver‐projecting DMVN neurons, age‐matched (12 weeks old) of Wistar and sedentary and exercised trained SHR (SHRt) received, 5 days prior to the experiments, injections of Fluorogold (FG) on the liver parenchyma. At the day of the electrophysiological studies, the FG‐labeled DMVN neurons was visualized and recorded in whole‐cell patch‐clamp mode, before and after application of oxytocin (OT) in the bath. The results of in vivo studies have shown that in the DREADD group the icv insulin was able to reduce the HVGC when compared to the control group (Control: 113,6±14,9 mg/dL n=5; vs DREADD: 74,2±6,16mg/dL; p≤0,05). As for the in vitro studies, the application of OT in liver projecting‐DMNV depolarized the resting membrane potential (Basal: −59.4± 0.7 mV vs OT: −56.2 ± 1.2 mV, n = 10; t = 2.403, df = 9; P = 0.0397) and increased the firing frequency (baseline: 1.9±0.8 vs OT: 3.0 ± 0.9 Hz, n=10; P = 0.002) in Wistar, but not in sedentary SHR. Interestingly, in SHR trained for 4 weeks, the OT caused depolarization of resting membrane potential (baseline: −61.3 ± 1.3 vs OT: −59.6 ± 1.5 mV, n=18; P=0.0177) and an increase in the firing frequency (baseline: 3.9±0.7 vs OT: 4.7±0.6 Hz, n=18, P=0.0012). As conclusion, the selective activation of parasympathetic preganglionic neurons of the DMNV rescues the changes in the HVGC elicited by central action of insulin, which used to be defective in SHR, indicating that the circuitry involving DMNV‐liver pathway is somehow dysfunctional, which could be due to the autonomic unbalance observed in this strain. Moreover, OT elicits similar changes in the electrophysiological properties of liver‐projecting DMNV neurons of Wistar and exercise‐trained but not sedentary SHR. These results indicate that exercise training can restore the sensitivity of liver‐projecting DMNV neurones of exercise trained SHR to OT.

Long‐term neuroprosthetic hemotherapy treats autonomic dysreflexia after spinal cord injury

Disrupted descending supraspinal control results in a hyper‐excitable sympathetic nervous system connectome after spinal cord injury (SCI). A condition termed “autonomic dysreflexia” (AD) results, which is characterized by severe elevations in blood pressure that are life‐threatening. There are no effective therapies for AD. Long‐term epidural stimulation below the level of the SCI can promote functionally‐relevant plasticity of volitional motor circuits, and this paradigm may prevent the hyper‐excitable sympathetic connectome after SCI.Here we studied the potential rehabilitative effect of long‐term epidural stimulation, as well as the mechanisms that are responsible for the development of AD. For this experiment, we compared SCI rats that underwent 30 minutes of epidural stimulation 5x/week for 6 weeks to SCI rats receiving normal care. Long‐term epidural stimulation abrogated AD induced by colorectal distension (p<0.0001; Figure 1). Viral tracing of ascending propriospinal neurons was also performed in these groups. Long‐term epidural stimulation normalized the sympathetic connectome as the axons and synapses were significantly reduced compared to SCI rats (p=0.0021; p=0.0127). We next performed chemogenetic silencing of excitatory and inhibitory interneurons, as well as sympathetic preganglionic neurons, in the lower thoracic spinal segments while experimentally inducing AD. Preliminary data indicate that AD is, at least in part, mediated by excitatory interneuronal connections to sympathetic preganglionic neurons.Long‐term epidural stimulation may be an effective hemotherapy for preventing AD after SCI. This project may provide direction for basic and clinical research to develop treatments for sympathetic nervous system dysfunction after SCI, and thereby improve health and patient quality of life over the long term.

Intrathecally administered substance P activated the spinal defecation center and enhanced colorectal motility in anesthetized rats.

Noxious stimuli on the colorectum cause colorectal contractions through activation of descending monoaminergic pathways projecting from the supraspinal defecation center to the spinal defecation center. Since it is known that substance P is involved in the response to peripheral noxious stimuli in the spinal cord, we investigated the effects of intrathecally administered substance P at L6-S1 levels on colorectal motility in rats that were anesthetized with α-chloralose and ketamine. Intrathecally administered substance P enhanced colorectal motility, even after transection of the thoracic spinal cord at the T4 level. Severing the pelvic nerves, but not the colonic nerves, abolished substance P enhanced colorectal motility. In the spinal cord at L6-S1 levels, expression of mRNA coding neurokinin (NK) 1-3 receptors was detected by RT-PCR. Immunohistological experiments revealed that preganglionic neurons of the pelvic nerves express NK1 receptors, whereas expression of NK2 receptors was not found. In addition, substance P-containing fibers densely innervated around the preganglionic neurons expressing NK1 receptors. An intrathecally administered NK1 receptor antagonist (spantide) attenuated capsaicin-induced colorectal contractions. These results suggest that the colokinetic action of substance P is mediated by the NK1 receptor in the spinal defecation center. Our findings indicate that substance P may function as a neurotransmitter in the spinal defecation center.NEW & NOTEWORTHY We found that intrathecally administered substance P enhanced colorectal motility in anesthetized rats. Neurokinin (NK) 1 receptors, but not NK2 receptors, were detected in preganglionic neurons of the pelvic nerves. Blockade of NK1 receptors in the spinal cord attenuated the enhanced colorectal motility in response to intracolonic noxious stimuli. The findings indicate that substance P may function as a neurotransmitter in the spinal reflex pathway controlling defecation.

ACE2, Circumventricular Organs and the Hypothalamus, and COVID-19.

The SARS-CoV-2 virus gains entry to cells by binding to angiotensin-converting enzyme 2 (ACE2). Since circumventricular organs and parts of the hypothalamus lack a blood–brain barrier, and immunohistochemical studies demonstrate that ACE2 is highly expressed in circumventricular organs which are intimately connected to the hypothalamus, and the hypothalamus itself, these might be easy entry points for SARS-CoV-2 into the brain via the circulation. High ACE2 protein expression is found in the subfornical organ, area postrema, and the paraventricular nucleus of the hypothalamus (PVH). The subfornical organ and PVH are parts of a circuit to regulate osmolarity in the blood, through the secretion of anti-diuretic hormone into the posterior pituitary. The PVH is also the stress response centre in the brain. It controls not only pre-ganglionic sympathetic neurons, but is also a source of corticotropin-releasing hormone, that induces the secretion of adrenocorticotropic hormone from the anterior pituitary. It is proposed that the function of ACE2 in the circumventricular organs and the PVH could be diminished by binding with SARS-CoV-2, thus leading to a reduction in the ACE2/Ang (1–7)/Mas receptor (MasR) signalling axis, that modulates ACE/Ang II/AT1R signalling. This could result in increased presympathetic activity/neuroendocrine secretion from the PVH, and effects on the hypothalamic–pituitary–adrenal axis activity. Besides the bloodstream, the hypothalamus might also be affected by SARS-CoV-2 via transneuronal spread along the olfactory/limbic pathways. Exploring potential therapeutic pathways to prevent or attenuate neurological symptoms of COVID-19, including drugs which modulate ACE signalling, remains an important area of unmet medical need.

The Oculomotor Nerve: Anatomy and Pathology

The oculomotor nerve is the third cranial nerve, exiting the brainstem in the medial border of the cerebral peduncle, from where it crosses straight to the superior orbital fissure. It is a purely motor nerve responsible for the innervation of all the extraocular muscles, except the superior oblique and lateral rectus muscles. It also has parasympathetic pre-ganglionic fibers, responsible for the innervation of sphincter pupillae and ciliary muscles. Magnetic resonance imaging (MRI) is the best imaging exam to evaluate patients with clinical signs of third cranial nerve palsy. The oculomotor nerve can be affected by several diseases, such as congenital malformations, trauma, inflammatory or infectious diseases, vascular disorders, and neoplasms. This article aims to review the oculomotor nerve anatomy, discuss the best MRI techniques to evaluate each nerve segment, and demonstrate the imaging aspect of the diseases that most commonly affect it.

Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia.

As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species–dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

Excessive sacral sweating caused by autonomic dysreflexia after traumatic brainstem injury

Abstract Rationale: Autonomic dysreflexia is a release phenomenon of spinal sympathetic preganglionic neurons from supraspinal inhibition, possibly of the lower brainstem origin. It is most frequently observed in patients with severe spinal lesions above the mid-thoracic cord. Theoretically, brainstem lesions may cause autonomic dysreflexia; however, such reports are rare. The author reports a case of autonomic dysreflexia restricted to the sacral area that appeared after brainstem lesions due to a traffic accident. Patient concerns: A 36-year-old man suffered from traumatic subarachnoid hemorrhage and hematomas in the lower brainstem. After he became alert and ambulant, he presented with neurological deficits suggestive of right-sided brainstem lesions, which were visualized on magnetic resonance images. He also complained of excessive sweating in the sacral area whenever he had a desire to void. Diagnoses: On the basis of neurological and radiological findings, and results of the thermal sweating test and sweating induced by bladder or rectal distension, his sacral hyperhidrosis was attributed to autonomic dysreflexia. Interventions: Baclofen (15 mg per day) was administered for more than three months, and the sacral hyperhidrosis gradually subsided. Outcomes: After three months, the patient returned home with minor gait disturbances and sacral sweating. Lessons: This may be the first report of excessive sweating due to autonomic dysreflexia caused by traumatic brainstem lesions restricted to the sacral territory. These observations suggest possible somatotopic arrangements, even in the supraspinal inhibitory pathways. Baclofen may be one of the choices for similar cases.

Spinal Reflex Control of Arterial Blood Pressure: The Role of TRP Channels and Their Endogenous Eicosanoid Modulators.

The spinal cord is an important integrative center for blood pressure control. Spinal sensory fibers send projections to sympathetic preganglionic neurons of the thoracic spinal cord and drive sympathetically-mediated increases in blood pressure. While these reflexes responses occur in able-bodied individuals, they are exaggerated following interruption of descending control – such as occurs following spinal cord injury. Similar reflex control of blood pressure may exist in disease states, other than spinal cord injury, where there is altered input to sympathetic preganglionic neurons. This review primarily focuses on mechanisms wherein visceral afferent information traveling via spinal nerves influences sympathetic nerve activity and blood pressure. There is an abundance of evidence for the widespread presence of this spinal reflex arch originating from virtually every visceral organ and thus having a substantial role in blood pressure control. Additionally, this review highlights specific endogenous eicosanoid species, which modulate the activity of afferent fibers involved in this reflex, through their interactions with transient receptor potential (TRP) cation channels.

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.

Toward rebalancing blood pressure instability after spinal cord injury with spinal cord electrical stimulation: A mini review and critique of the evolving literature.

High-level spinal cord injury commonly leads to blood pressure instability. This manifests clinically as orthostatic hypotension (OH), where blood pressure can drop to the point of loss of consciousness, and autonomic dysreflexia (AD), where systolic blood pressure can climb to over 300 mmHg in response to an unperceived noxious stimulus. These blood pressure fluctuations can occur multiple times a day, contributing to increased vessel shear stress and heightened risk of cardiovascular disease. The pathophysiology of both of these conditions is rooted in impairments in regulation of spinal cord sympathetic preganglionic neurons, which control blood pressure by mediating vascular resistance and catecholamine release. Recently, spinal cord electrical stimulation has provided evidence that it may modulate these blood pressure imbalances. Early proposed mechanisms suggest activation of spinal cord dorsal horn neurons that ultimately act upon the sympathetic preganglionic neuronal pathways. For OH, spinal cord stimulation likely induces local activation of these neurons to generate baseline sympathetic tone and accompanying vasoconstriction. The mechanisms for spinal stimulation regulating AD are less clear, though some suggest it activates inhibitory circuits to dampen the overactive sympathetic response. While questions remain, spinal cord electrical stimulation is an intriguing new modality that may restore blood pressure regulation following spinal cord injury.

Rostral ventrolateral medulla, retropontine region and autonomic regulations

The rostral half of the ventrolateral medulla (RVLM) and adjacent ventrolateral retropontine region (henceforth RVLMRP) have been divided into various sectors by neuroscientists interested in breathing or autonomic regulations. The RVLMRP regulates respiration, glycemia, vigilance and inflammation, in addition to blood pressure. It contains interoceptors that respond to acidification, hypoxia and intracranial pressure and its rostral end contains the retrotrapezoid nucleus (RTN) which is the main central respiratory chemoreceptor. Acid detection by the RTN is an intrinsic property of the principal neurons that is enhanced by paracrine influences from surrounding astrocytes and CO2-dependent vascular constriction. RTN mediates the hypercapnic ventilatory response via complex projections to the respiratory pattern generator (CPG). The RVLM contributes to autonomic response patterns via differential recruitment of several subtypes of adrenergic (C1) and non-adrenergic neurons that directly innervate sympathetic and parasympathetic preganglionic neurons. The RVLM also innervates many brainstem and hypothalamic nuclei that contribute, albeit less directly, to autonomic responses. All lower brainstem noradrenergic clusters including the locus coeruleus are among these targets. Sympathetic tone to the circulatory system is regulated by subsets of presympathetic RVLM neurons whose activity is continuously restrained by the baroreceptors and modulated by the respiratory CPG. The inhibitory input from baroreceptors and the excitatory input from the respiratory CPG originate from neurons located in or close to the rhythm generating region of the respiratory CPG (preBötzinger complex).

Kv3 Channels Contribute to the Excitability of Subpopulations of Spinal Cord Neurons in Lamina VII.

Autonomic parasympathetic preganglionic neurons (PGNs) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be because of recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within Lamina VII, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphologic analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast-firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Intrauterine L-NAME Exposure Weakens the Development of Sympathetic Innervation and Induces the Remodeling of Arterial Vessels in Two-Week-Old Rats.

Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1–2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/β-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Stress and central autonomic network.

The central autonomic network (CAN) plays a critical role in the stress response, which is triggered by challenges on the homeostasis (physiological stressors) or unpleasant social or environmental situations. This review focuses on the role of areas of the CAN including the insular and anterior cingulate cortices, extended amygdala, hypothalamus, periaqueductal gray and locus coeruleus in the stress response. These areas are interconnected and affect sympathetic or parasympathetic output via their influence on premotor or preganglionic autonomic neurons in the lower brainstem and spinal cord. The insula integrates multiple inputs to create a sense of the physiological state of the body, whereas the anterior cingulate initiates predictive visceromotor commands. The amygdala and bed nucleus of the stria terminalis provide automatic emotional tagging and trigger automatic survival responses to threat via their outputs to the hypothalamus, periaqueductal gray, and lower brainstem. Several regions of the hypothalamus, including the paraventricular nucleus, dorsomedial nucleus and lateral hypothalamic area participate in different patterns of stress response according to the type of stimulus and projections to premotor and preganglionic autonomic neurons. The periaqueductal gray initiates different patterns of autonomic, pain modulatory, and motor responses, including the "fight or flight" or "playing dead" responses. The locus coeruleus promotes emotional learning in the amygdala associated with states of anxiety. Neurons of the C1 area of the rostral ventrolateral medulla elicit sympathoexcitatory responses to internal stressors such as hypoxia and inflammation. The ventromedial medulla, including the nucleus raphe pallidus, initiates sympathoexcitatory responses to social and other external stressors.

Innervated adrenomedullary microphysiological system to model nicotine and opioid exposure.

Transition to extrauterine life results in a surge of catecholamines necessary for increased cardiovascular, respiratory, and metabolic activity. Mechanisms mediating adrenomedullary catecholamine release are poorly understood. Important mechanistic insight is provided by newborns delivered by cesarean section or subjected to prenatal nicotine or opioid exposure, demonstrating impaired release of adrenomedullary catecholamines. To investigate mechanisms regulating adrenomedullary innervation, we developed compartmentalized 3D microphysiological systems (MPS) by exploiting GelPins, capillary pressure barriers between cell-laden hydrogels. The MPS comprises discrete cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, we demonstrate that adrenal chromaffin innervation plays a critical role in hypoxia-mediated catecholamine release. Opioids and nicotine were shown to affect adrenal chromaffin cell response to a reduced oxygen environment, but neurogenic control mechanisms remained intact. GelPin containing MPS represent an inexpensive and highly adaptable approach to study innervated organ systems and improve drug screening platforms.

Regional Targeting of Bladder and Urethra Afferents in the Lumbosacral Spinal Cord of Male and Female Rats: A Multiscale Analysis.

Sensorimotor circuits of the lumbosacral spinal cord are required for lower urinary tract (LUT) regulation as well as being engaged in pelvic pain states. To date, no molecular markers have been identified to enable specific visualization of LUT afferents, which are embedded within spinal cord segments that also subserve somatic functions. Moreover, previous studies have not fully investigated the patterning within or across spinal segments, compared afferent innervation of the bladder and urethra, or explored possible structural sex differences in these pathways. We have addressed these questions in adult Sprague Dawley rats, using intramural microinjection of the tract tracer, B subunit of cholera toxin (CTB). Afferent distribution was analyzed within individual sections and 3D reconstructions from sections across four spinal cord segments (L5-S2), and in cleared intact spinal cord viewed with light sheet microscopy. Simultaneous mapping of preganglionic neurons showed their location throughout S1 but restricted to the caudal half of L6. Afferents from both LUT regions extended from L5 to S2, even where preganglionic motor pathways were absent. In L6 and S1, most afferents were associated with the sacral preganglionic nucleus (SPN) and sacral dorsal commissural nucleus (SDCom), with very few in the superficial laminae of the dorsal horn. Spinal innervation patterns by bladder and urethra afferents were remarkably similar, likewise the patterning in male and female rats. In conclusion, microscale to macroscale mapping has identified distinct features of LUT afferent projections to the lumbosacral cord and provided a new anatomic approach for future studies on plasticity, injury responses, and modeling of these pathways.

Protein Kinase C-Dependent Effects of Neurosteroids on Synaptic GABAA Receptor Inhibition Require the δ-Subunit.

The dorsal motor nucleus of the vagus (DMV) contains preganglionic motor neurons important for interpreting sensory input from the periphery, integrating that information, and coding the appropriate parasympathetic (vagal) output to target organs. Despite the critical role of hormonal regulation of vagal motor output, few studies examine the role of neurosteroids in the regulation of the DMV. Of the few examinations, no studies have investigated the potential impact of allopregnanolone (Allo), a neuroactive progesterone-derivative, in the regulation of neurotransmission on the DMV. Since DMV neuronal function is tightly regulated by GABAA receptor activity and Allo is an endogenous GABAA receptor ligand, the present study used in vitro whole cell patch clamp to investigate whether Allo alters GABAergic neurotransmission to DMV neurons. Although Allo did not influence GABAergic neurotransmission during initial application (5–20 min), a TTX-insensitive prolongment of decay time and increase in frequency of GABAergic currents was established after Allo was removed from the bath for at least 30 min (LtAllo). Inhibition of protein kinase C (PKC) abolished these effects, suggesting that PKC is largely required to mediate Allo-induced inhibition of the DMV. Using mice that lack the δ-subunit of the GABAA receptor, we further confirmed that PKC-dependent activity of LtAllo required this subunit. Allo also potentiated GABAA receptor activity after a repeated application of δ-subunit agonist, suggesting that the presence of Allo encodes stronger δ-subunit-mediated inhibition over time. Using current clamp recording, we demonstrated that LtAllo-induced inhibition is sufficient to decrease action potential firing and excitability within DMV neurons. We conclude that the effects of LtAllo on GABAergic inhibition are dependent on δ-subunit and PKC activation. Taken together, DMV neurons can undergo long lasting Allo-dependent GABAA receptor plasticity.