Spinal relay neurons for central control of autonomic pathways in a photoperiodic rodent.

Location and distribution of spinal sympathetic preganglionic neurons projecting to the superior cervical ganglion were investigated in a rodent model organism for photoperiodic regulation, the Djungarian hamster (Phodopus sungorus). Upon unilateral injection of Fluoro-Gold into the superior cervical ganglia, retrograde neuronal tracing demonstrated labeled neurons ipsilateral to the injection site. They were seen in spinal segments C8 to Th5 of which the segments Th1 to Th3 contained about 98% of the labeled cells. Neurons were found in the spinal cord predominantly in the intermediolateral nucleus pars principalis and pars funicularis. At the same time, the central autonomic area and the intercalated region contained only very few labeled cells. In the intermediolateral nucleus, cells often were arranged in clusters, of which several were seen in each spinal segment. Selected sections were exposed to antibodies directed against arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or substance P. It was found that about two-thirds of sympathetic preganglionic neurons produced the gaseous neuroactive substance nitric oxide and that few contained small amounts of neuropeptide Y. Fibers of putative supraspinal origin immunopositive for either arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or, in particular, substance P were found in the vicinity of labeled sympathetic preganglionic neurons. These results demonstrate the location of relay neurons for autonomic control of cranial and cardial structures and provide further knowledge on neurochemical properties of sympathetic preganglionic neurons and related structures.

Insulin-responsive autonomic neurons in rat medulla oblongata.

Low blood glucose activates brainstem adrenergic and cholinergic neurons, driving adrenaline secretion from the adrenal medulla and glucagon release from the pancreas. Despite their roles in maintaining glucose homeostasis, the distributions of insulin-responsive adrenergic and cholinergic neurons in the medulla are unknown. We fasted rats overnight and gave them insulin (10 U/kg i.p.) or saline after 2 weeks of handling. Blood samples were collected before injection and before perfusion at 90 min. We immunoperoxidase-stained transverse sections of perfused medulla to show Fos plus either phenylethanolamine N-methyltransferase (PNMT) or choline acetyltransferase (ChAT). Insulin injection lowered blood glucose from 4.9 ± 0.3 mmol/L to 1.7 ± 0.2 mmol/L (mean ± SEM; n = 6); saline injection had no effect. In insulin-treated rats, many PNMT-immunoreactive C1 neurons had Fos-immunoreactive nuclei, with the proportion of activated neurons being highest in the caudal part of the C1 column. In the rostral ventrolateral medulla, 33.3% ± 1.4% (n = 8) of C1 neurons were Fos-positive. Insulin also induced Fos in 47.2% ± 2.0% (n = 5) of dorsal medullary C3 neurons and in some C2 neurons. In the dorsal motor nucleus of the vagus (DMV), insulin evoked Fos in many ChAT-positive neurons. Activated neurons were concentrated in the medial and middle regions of the DMV beneath and just rostral to the area postrema. In control rats, very few C1, C2, or C3 neurons and no DMV neurons were Fos-positive. The high numbers of PNMT-immunoreactive and ChAT-immunoreactive neurons that express Fos after insulin treatment reinforce the importance of these neurons in the central response to a decrease in glucose bioavailability.

Hippocampal modulation of cardiorespiratory function.

Changes in cardiorespiratory control accompany the expression of complex emotions, indicative of limbic brain inputs onto bulbar autonomic pathways. Previous studies have focussed on the role of the prefrontal cortex in autonomic regulation. However, the role of the hippocampus, also important in limbic processing, has not been addressed in detail. Anaesthetised, instrumented rats were used to map the location of hippocampal sites capable of evoking changes in cardiorespiratory control showing that stimulation of discrete regions within the CA1 fields of both the dorsal and ventral hippocampus potently alter breathing and cardiovascular activity. Additionally, tracing of the neuroanatomical tracts and pharmacological inactivation studies were used to demonstrate a role of the basomedial amygdala in hippocampal evoked responses. Collectively, these data support the existence of a hippocampal-amygdala neural circuit capable of modulating bulbar cardiorespiratory control networks and may suggest a role for this circuit in the top-down regulation of breathing and autonomic outflow necessary for the expression of complex emotions.

Neural connection supporting endogenous 5-hydroxytryptamine influence on autonomic activity in medial prefrontal cortex.

5-hydroxytryptamine (5-HT) transmission in the medial prefrontal cortex (mPFC) enhances or suppresses signal outflow to influence emotion-/cognition-based function performances and, putatively, the autonomic responses. The top-down cortical modulation of autonomic activities may be mediated in part through projections from mPFC to brain stem dorsal vagal complex (DVC). The abundant and heterogeneous densities of 5-HT fibers across laminae in mPFC suggest serotonergic innervation of mPFC-DVC projection neurons whereby endogenous 5-HT acts to regulate autonomic activities. The present study investigated the physical relationship between 5-HT fibers and the autonomic-related mPFC neurons by examining and quantitatively characterizing the 5-HT contacts upon retrogradely labeled mPFC-DVC projection neurons in pre- and infra-limbic cortices (PrL/IL) with light and electron microscopies combined with immunocytochemistry for 5-HT and presynaptic vesicle marker synaptophysin (Syn). 5-HT varicosities were observed, under confocal microscope, to form close appositions to or, at ultrastructural level, to form asymmetric axodendritic synapses and direct contacts upon the target neurons. About 16% of the entire 5-HTergic varicosities in lamina V of PrL/IL coexpressed Syn and about 24% of the peri-somatic 5-HTergic swellings demonstrated Syn-immunoreactivity (ir), suggesting a low frequency of putative synapses estimated at optical level. Ultrastructurally, examination of thirty-seven serially cut thin 5-HT boutons closely apposed to the labeled dendritic profiles demonstrated that only three contacts presented with identifiable asymmetric, synaptic membrane specializations. These data provide the first and direct morphological evidence supporting that endogenous 5-HT may be released mainly via direct contacts bearing no identifiable synaptic specializations as well as synapses, targeting autonomic-related mPFC neurons for autonomic regulation.

High density penetrating electrode arrays for autonomic nerves.

Electrode arrays for recording and stimulation in the central nervous system have enabled numerous advances in basic science and therapeutic strategies. In particular, micro-fabricated arrays with precision size and spacing offer the benefit of accessing single neurons and permit mapping of neuronal function. Similar advances are envisioned toward understanding the autonomic nervous system and developing therapies based on its modulation, but appropriate electrode arrays are lacking. Here, we present for the first time, a multi-channel electrode array suitable for penetration of peripheral nerves having diameters as small as 0.1mm, and demonstrate performance in vivo. These arrays have the potential to access multiple discrete nerve fibers in small nerves. We fabricated and characterized five-channel arrays and obtained preliminary recordings of activity when penetrating rat carotid sinus nerve. The electrodes were constructed using hybrid microfabrication processes. The individual electrode shafts are as small as 0.01mm in diameter and at its tip each has a defined site that is addressable via a standard electronic connector. In addition to acute in vivo results, we evaluate the device by electrochemical impedance spectroscopy. Having established the fabrication method, our next steps are to incorporate the arrays into an implantable configuration for chronic studies, and here we further describe concepts for such a device.

Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung.

Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line.

Sex and estrogen affect the distribution of urocortin-1 immunoreactivity in brainstem autonomic nuclei of the rat.

Urocortin-1 (UCN-1), a neuropeptide closely related to the hypothalamic hormone corticotropin-releasing factor, has been associated with stress, feeding behaviors, cardiovascular control, and to exhibit functional gender differences. This study was done to investigate whether estrogen (E; 17ß-estradiol) treatment (9 weeks) altered UCN-1 immunoreactivity in brainstem autonomic nuclei in female Wistar rats. Experiments were done in age matched adult males (controls), females (intact), and ovariectomized (OVX) only and OVX+E (30pg/ml plasma) treated females. All animals received intracerebroventricular injections of colchicine and were then perfused transcardially with Zamboni's fixative. Coronal brainstem sections (40µm) were cut and processed immunohistochemically for UCN-1. In males, moderate UCN-1 fiber labeling was found in the nucleus of the solitary tract (NTS) and throughout the rostral ventral lateral medulla (RVLM). Additionally, a few UCN-1 immunoreactive neurons were observed in hypoglossal nucleus (XII), facial nucleus (FN) and nucleus ambiguus (Amb). In intact females and OVX+E females, fewer UCN-1 labeled fibers were found within NTS compared to males. In contrast, the RVLM was more densely innervated in the female cases. Furthermore, in both intact and OVX+E females UCN-1 labeled neurons were found not only within Amb, FN and XII, but also within NTS, RVLM and nucleus raphé pallidus (RP). In OVX only animals, moderate to dense UCN-1 fiber labeling was observed in the NTS complex and throughout RVLM compared to males and the other female groups. However, in contrast to all other groups, UCN-1 labeled neurons were found in greater number within Amb, FN, NTS, dorsal motor nucleus of the vagus, XII, RVLM, magnocellular reticular nucleus and RP. These data not only suggest that sex differences exist in the distribution of UCN-1 within brainstem autonomic areas, but that circulating level of E may play an important role with regards to the function of these UCN-1 neurons during stress responses.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

The anterior cingulate cortex may enhance inhibition of lateral prefrontal cortex via m2 cholinergic receptors at dual synaptic sites.

The anterior cingulate cortex (ACC) and dorsolateral prefrontal cortices (DLPFC) share robust excitatory connections. However, during rapid eye movement (REM) sleep, when cortical activity is dominated by acetylcholine, the ACC is activated but DLPFC is suppressed. Using pathway tracing and electron microscopy in nonhuman primates (Macaca mulatta), we tested the hypothesis that the opposite states may reflect specific modulation by acetylcholine through strategic synaptic localization of muscarinic m2 receptors, which inhibit neurotransmitter release presynaptically, but are thought to be excitatory postsynaptically. In the ACC pathway to DLPFC (area 32 to area 9), m2 receptors predominated in ACC axon terminals and in more than half of the targeted dendrites of presumed inhibitory neurons, suggesting inhibitory cholinergic influence. In contrast, in a pathway linking the DLPFC area 46 to DLPFC area 9, postsynaptic m2 receptors predominated in targeted spines of presumed excitatory neurons, consistent with their mutual activation in working memory. These novel findings suggest that presynaptic and postsynaptic specificity of m2 cholinergic receptors may help explain the differential engagement of ACC and DLPFC areas in REM sleep for memory consolidation and synergism in awake states for cognitive control.

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.

Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats.

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO(2) from and the delivery of O(2) to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n=12) that ran on a treadmill and a no-exercise group (n=7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kölliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise.

Efferent projections of C3 adrenergic neurons in the rat central nervous system.

C3 neurons constitute one of three known adrenergic nuclei in the rat central nervous system (CNS). While the adrenergic C1 cell group has been extensively characterized both physiologically and anatomically, the C3 nucleus has remained relatively obscure. This study employed a lentiviral tracing technique that expresses green fluorescent protein behind a promoter selective to noradrenergic and adrenergic neurons. Microinjection of this virus into the C3 nucleus enabled the selective tracing of C3 efferents throughout the rat CNS, thus revealing the anatomical framework of C3 projections. C3 terminal fields were observed in over 40 different CNS nuclei, spanning all levels of the spinal cord, as well as various medullary, mesencephalic, hypothalamic, thalamic, and telencephalic nuclei. The highest densities of C3 axon varicosities were observed in Lamina X and the intermediolateral cell column of the thoracic spinal cord, as well as the dorsomedial medulla (both commissural and medial nuclei of the solitary tract, area postrema, and the dorsal motor nucleus of the vagus), ventrolateral periaqueductal gray, dorsal parabrachial nucleus, periventricular and rhomboid nuclei of the thalamus, and paraventricular and periventricular nuclei of the hypothalamus. In addition, moderate and sparse projections were observed in many catecholaminergic and serotonergic nuclei, as well as the area anterior and ventral to the third ventricle, Lamina X of the cervical, lumbar, and sacral spinal cord, and various hypothalamic and telencephalic nuclei. The anatomical map of C3 projections detailed in this survey hopes to lay the first steps toward developing a functional framework for this nucleus.

Immunohistochemical characteristics of the nerve fibres of sow retractor clitoridis muscle.

The occurrence of several biologically active neuropeptides (calcitonine gene-related peptide, leu-enkephaline, neuropeptide Y, substance P, and vasoactive intestinal peptide) or nitric oxide-synthesizing enzymes (neuronal nitric oxide synthase), tyrosine hydroxylase, vesicular acetylcholine transporter, and their co-localization with tyrosine hydroxylase were investigated by immunohistochemistry in the retractor clitoridis muscle of slaughtered sows. Single immunolabelling revealed that tyrosine hydroxylase and neuropeptide Y immunoreactive nerve fibres were the most numerous, followed by the neuronal nitric oxide synthase and calcitonine gene-related peptide immunoreactive ones, the vasoactive intestinal peptide, substance P and leu-enkephaline immunoreactive nerve fibres were few and vesicular acetylcholine transporter immunoreactivity were observed only in single fibres. Double immunolabelling revealed the only co-localization of tyrosyne hydroxylase with neuropeptide Y. The most reliable labelling of nerve fibres of the retractor clitoridis muscle was observed around blood vessels, followed by non-vascular smooth muscles. The present data indicate that the sow retractor clitoridis muscle receives nerve fibres that exhibit different chemical codes and, likely, differences in their chemical coding depend on the target-structure.

Pancreatic neuronal melanocortin-4 receptor modulates serum insulin levels independent of leptin receptor.

The leptin-regulated melanocortin (MC) system modulates energy homeostasis and hypothalamic MC neuronal circuits regulate insulin secretion. We therefore hypothesized that MC system components were present in the pancreas. In order to determine the veracity of the hypothesis, we examined c-Fos, melanocortin-4 receptor (Mc4r), and alpha-melanocyte-stimulating hormone (α-MSH) expression levels in nondiabetic (intact leptin receptor signaling) and Zucker diabetic fatty (ZDF; leptin receptor deficiency) rats. We infused rats via the third ventricle with the α-MSH analog Nle4, D-Phe7-α-MSH (NDP-MSH), a Mc4r agonist. Subsequently, both hypothalamic and pancreatic c-Fos and Mc4r mRNAs were upregulated. Likewise, immunohistochemical analysis showed that an increased Mc4r and α-MSH expression in nerves surrounding the pancreatic vasculature and islets. Increases in c-Fos, α-MSH, and Mc4r expression were independent of leptin receptor function. Conversely, serum insulin was significantly reduced by NDP-MSH treatment, an effect which was reversed by the Mc4r specific blocker HS014. Finally, proopiomelanocortin (POMC) mRNA, the precursor of α-MSH, was detected by RT-PCR in pancreatic tissue homogenates. These findings suggest that pancreatic Mc4r and autonomic neurons participate in a communication pathway between the central MC system and pancreatic islets to regulate insulin secretion.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats.

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Functional asymmetry in the descending cardiovascular pathways from dorsomedial hypothalamic nucleus.

Neurons in the dorsomedial hypothalamus (DMH) play a key role in mediating tachycardia elicited by emotional stress. DMH activation by microinjections of the GABA(A) antagonist evokes tachycardia and physiological changes typically seen in experimental stress. DMH inhibition abolishes the tachycardia evoked by stress. Based on anatomic evidences for lateralization in the pathways from DMH, we investigated a possible inter-hemispheric difference in DMH-evoked cardiovascular responses. In anesthetized rats we compared changes in heart rate (HR), renal sympathetic activity (RSNA), mesenteric blood flow (MBF) and tail vascular conductance produced by activation of right (R) and left (L) sides of the DMH. We also evaluated the tachycardia produced by air jet stress after inhibition of R or L DMH. There were always greater increases in RSNA when bicuculline was injected ipsilaterally to the side where these parameters were recorded (average DeltaRSNA: L=+50% and R=+26%; P<0.05). Compared to pre-injection values, right DMH activation caused pronounced decrease (0.87+/-0.1% vs. 0.4+/-0.11%/mm Hg; P<0.05), whereas bicuculline methiodide (BMI) into left DMH produced no significant changes (0.95+/-0.09% vs. 1.04+/-0.25%/mm Hg) in tail vascular conductance. R or L DMH disinhibition produced decreases in MBF, but no differences in the range of these changes were observed. Activation of the right DMH caused greater tachycardia compared to the left DMH activation (average DeltaHR: R=+92 bpm; L=+48 bpm; P<0.05). Tachycardia evoked by air jet stress was smallest after right DMH inhibition (average DeltaHR: R=+57 bpm and L=+134 bpm; P<0.05). These results indicate that the descending cardiovascular pathways from DMH are predominantly lateralized and the right DMH might exert a prominent control on heart rate changes during emotional stress.

Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

Neurochemistry of the paraventricular nucleus of the hypothalamus: implications for cardiovascular regulation.

The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic and endocrine homeostasis. The PVN integrates specific afferent stimuli to produce an appropriate differential sympathetic output. The neural circuitry and some of the neurochemical substrates within this circuitry are discussed. The PVN has at least three neural circuits to alter sympathetic activity and cardiovascular regulation. These pathways innervate the vasculature and organs such as the heart, kidney and adrenal medulla. The basal level of sympathetic tone at any given time is dependent upon excitatory and inhibitory inputs. Under normal circumstances the sympathetic nervous system is tonically inhibited. This inhibition is dependent upon GABA and nitric oxide such that nitric oxide potentiates local GABAergic synaptic inputs onto the neurones in the PVN. Excitatory neurotransmitters such as glutamate and angiotensin II modify the tonic inhibitory activity. The neurotransmitters oxytocin, vasopressin and dopamine have been shown to affect cardiovascular function. These neurotransmitters are found in neurones of the PVN and within the spinal cord. Oxytocin and vasopressin terminal fibres are closely associated with sympathetic preganglionic neurones (SPNs). Sympathetic preganglionic neurones have been shown to express receptors for oxytocin, vasopressin and dopamine. Oxytocin causes cardioacceleratory and pressor effects that are greatest in the upper thoracic cord while vasopressin cause these effects but more significant in the lower thoracic cord. Dopaminergic effects on the cardiovascular system include inhibitory or excitatory actions attributed to a direct PVN influence or via interneuronal connections to sympathetic preganglionic neurones.

Neurochemistry of bulbospinal presympathetic neurons of the medulla oblongata.

This review focuses on presympathetic neurons in the medulla oblongata including the adrenergic cell groups C1-C3 in the rostral ventrolateral medulla and the serotonergic, GABAergic and glycinergic neurons in the ventromedial medulla. The phenotypes of these neurons including colocalized neuropeptides (e.g., neuropeptide Y, enkephalin, thyrotropin-releasing hormone, substance P) as well as their relative anatomical location are considered in relation to predicting their function in control of sympathetic outflow, in particular the sympathetic outflows controlling blood pressure and thermoregulation. Several explanations are considered for how the neuroeffectors coexisting in these neurons might be functioning, although their exact purpose remains unknown. Although there is abundant data on potential neurotransmitters and neuropeptides contained in the presympathetic neurons, we are still unable to predict function and physiology based solely on the phenotype of these neurons.

Organisation of the catecholaminergic system in the vagal motor nuclei of pigs: a retrograde fluorogold tract tracing study combined with immunohistochemistry of catecholaminergic synthesizing enzymes.

The vagal motor system is involved in the regulation of cardiorespiratory and gastrointestinal functions. Vagal motor neurons are localized near or adjacent to catecholaminergic neurons, but their co-localisation seems species dependent, present in the cat but absent in the rabbit. In pig, a species commonly used as an experimental model in humans brain disorders (sudden infant death syndrome, hypoxia), the relationship is poorly understood. We aimed at describing the distribution of vagal motor neurons and tyrosine hydroxylase-immunoreactive (-ir) neurons by using a double staining method in combination with retrograde tracing of vagal efferent neurons. After fluorogold impregnation of the central part of the sectioned left cervical vagal trunk, two main vagal motor neuronal populations were located in the dorsal motor nucleus of the vagus nerve (DMX) and in the area of the nucleus ambiguus (Amb). Like in the human, the DMX was composed of different subpopulations of neurons with the same morphological characteristics. Immunohistochemistry of catecholaminergic synthesizing enzymes differentiated two main sites containing vagal motor populations: the dorsomedial and the ventrolateral medulla. TH-ir was rarely seen in vagal motor neurons of the DMX, but TH-ir neurons were present around the two main vagal motor neuronal populations that contained TH-ir fibres. The anatomical organisation of the vagal motor and the catecholaminergic neuronal systems are similar to those described in humans and suggest that the involvement of the catecholamines in the control of the vagal motor system may be similar in pigs and in humans.