Overview of the Anatomy, Physiology, and Pharmacology of the Autonomic Nervous System.

Comprised of the sympathetic nervous system, parasympathetic nervous system, and enteric nervous system, the autonomic nervous system (ANS) provides the neural control of all parts of the body except for skeletal muscles. The ANS has the major responsibility to ensure that the physiological integrity of cells, tissues, and organs throughout the entire body is maintained (homeostasis) in the face of perturbations exerted by both the external and internal environments. Many commonly prescribed drugs, over-the-counter drugs, toxins, and toxicants function by altering transmission within the ANS. Autonomic dysfunction is a signature of many neurological diseases or disorders. Despite the physiological relevance of the ANS, most neuroscience textbooks offer very limited coverage of this portion of the nervous system. This review article provides both historical and current information about the anatomy, physiology, and pharmacology of the sympathetic and parasympathetic divisions of the ANS. The ultimate aim is for this article to be a valuable resource for those interested in learning the basics of these two components of the ANS and to appreciate its importance in both health and disease. Other resources should be consulted for a thorough understanding of the third division of the ANS, the enteric nervous system. © 2016 American Physiological Society. Compr Physiol 6:1239-1278, 2016.

The soma and proximal dendrites of sympathetic preganglionic neurons innervating the major pelvic ganglion in female rats receive predominantly inhibitory inputs.

Sympathetic preganglionic neurons (SPNs) in the intermediolateral (IML) and dorsal commissural nucleus (DCN) of the thoracolumbar segments of the spinal cord contribute to the autonomic control of the pelvic visceral organs. We examined the morphology of these neurons at the light and electron microscopic level and quantified the boutons apposing the soma and proximal dendrites of the SPNs innervating the major pelvic ganglion (MPG) in female rats. The majority of these cells resided in the DCN (61.6±6.2%) and IML (33.2±4.4%) nuclei. Measurements of cell volume and shape revealed no differences between SPNs sampled from the DCN and IML populations. Ultrastructural studies of DCN and IML SPNs revealed that coverage of SPNs by synaptic inputs is sparse, with an average of 11.60±2.41% of the soma membrane and 16.33±6.18% of proximal dendrites apposed by boutons, though some somata exhibited no synaptic coverage. Three distinct types of boutons were found to appose the SPN somata and dendrites. The putatively inhibitory F-type bouton covered a significantly greater percentage of membrane on the soma (8.48±2.12%) and dendrites (12.65±4.34%), than the S-type bouton, a putatively excitatory bouton, which only covered 2.94±0.70% of the somatic and 3.68±2.98% of the dendritic membranes. Boutons with dense-core vesicles were rare. Our results demonstrate that SPNs of the DCN and IML of female rats are similar morphologically, and that synaptic input on these cells, though sparse, is predominantly inhibitory.

Differential synaptic inputs to the cell body and proximal dendrites of preganglionic parasympathetic neurons in the rat conus medullaris.

Preganglionic parasympathetic neurons (PPNs) reside in the intermediolateral (IML) nucleus of the rat lumbosacral spinal cord and contribute to the autonomic control of visceral pelvic organs. PPNs provide the final common pathway for efferent parasympathetic information originating in the spinal cord. We examined the detailed ultrastructure of the type and organization of synaptic inputs to the cell body and proximal dendrites of PPNs in the rat conus medullaris. The PPNs were retrogradely labeled by a systemic administration of the B subunit of cholera toxin conjugated to horseradish peroxidase. We demonstrate four distinct types of synaptic boutons in apposition with PPN somata and proximal dendrites: S-type boutons show clear, spheroid vesicles; F-type boutons show flattened vesicles; dense-cored vesicle-type (DCV-type) boutons show a mixture of clear and dense-cored vesicles; L-type boutons were rare, but large, exhibited clear spheroid vesicles, and were only encountered in apposition with the PPN dendrites in our sample. The membrane surface covered by apposed boutons was markedly higher for the proximal dendrites of PPNs, compared with their somata. The inhibitory synaptic influence was markedly higher over the PPN somata compared with their proximal dendrites, as suggested by the higher proportion of putative inhibitory F-type boutons in apposition with the soma and a higher frequency of S-type boutons per membrane length for the proximal dendrites. Our studies suggest that the synaptic input to PPNs originates from multiple distinct sources and is differentially distributed and integrated over the cell membrane surface.

Dynamics of the remodeling of neural connections in the superior cervical ganglion in rats after dosed compression of the preganglionic trunk.

The aim of the present work was to obtain a quantitative evaluation of post-traumatic regeneration of nerve cells in the superior cervical ganglion (SCG) by measuring the ratio of the number of neurons (N) in the ganglion to the number of preganglionic myelinated fibers (F) in the cervical sympathetic trunk (N/F). This was addressed using light and electron microscopy. Studies were performed using white male rats divided into three groups: intact (aged seven months), age controls (aged 19 months), and experimental - one year after compression of the sympathetic trunk at the base of the SCG performed at age seven months. N/F in intact rats was 1:210. With age, N/F changed to 1:173; the value one year after trauma was 1:745. These data led to the conclusion that normalization of the structure of the neural connections of the ganglion did not occur. The process of post-traumatic regeneration was incomplete and became chronic.

VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord.

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro-Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2-immunoreactive axons and synapses. CTB-immunoreactive SPN and PPN received many close appositions from VGLUT2-immunoreactive axons. VGLUT2-immunoreactive synapses occurred on Fluoro-Gold-labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1-immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1-immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1-immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarke's column. These data indicate that SPN and PPN are densely innervated by VGLUT2-immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1-immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN.

Systemic administration of cholera toxin B subunit conjugated to horseradish peroxidase in the adult rat labels preganglionic autonomic neurons, motoneurons, and select primary afferents for light and electron microscopic studies.

Retrograde and transganglionic labeling techniques are commonly used to visualize subsets of neurons and sensory afferent projections in the nervous system. These methods commonly require anesthesia and surgical methods. However, some tracers can also be administered systemically in awake animals, thus reducing risks associated with anesthesia and surgery and allowing for labeling of neuronal populations that are difficult to label with local tracer injections. Here, we demonstrate in the adult rat that intraperitoneal administration of cholera toxin subunit B conjugated to horseradish peroxidase labels preganglionic autonomic neurons, motoneurons, and the terminal projections of select primary afferents for both light and electron microscopic studies. We demonstrate also that this method can be combined with post-embedding immunogold labeling to detect amino acid transmitters in synaptic boutons.

Enkephalins and functionally specific vagal preganglionic neurons to the heart: ultrastructural studies in the cat.

In cat, distinct populations of vagal preganglionic and postganglionic neurons selectively modulate heart rate, atrioventricular conduction and left ventricular contractility, respectively. Vagal preganglionic neurons to the heart originate in the ventrolateral part of nucleus ambiguus and project to postganglionic neurons in intracardiac ganglia, including the sinoatrial (SA), atrioventricular (AV) and cranioventricular (CV) ganglia, which selectively modulate heart rate, AV conduction and left ventricular contractility, respectively. These ganglia receive projections from separate populations of vagal preganglionic neurons. The neurochemical anatomy and synaptic interactions of afferent neurons which mediate central control of these preganglionic neurons is incompletely understood. Enkephalins cause bradycardia when microinjected into nucleus ambiguus. It is not known if this effect is mediated by direct synapses of enkephalinergic terminals upon vagal preganglionic neurons to the heart. The effects of opioids in nucleus ambiguus upon AV conduction and cardiac contractility have also not been studied. We have tested the hypothesis that enkephalinergic nerve terminals synapse upon vagal preganglionic neurons projecting to the SA, AV and CV ganglia. Electron microscopy was used combining retrograde labeling from the SA, AV or CV ganglion with immunocytochemistry for enkephalins in ventrolateral nucleus ambiguus. Eight percent of axodendritic synapses upon negative chronotropic, and 12% of axodendritic synapses upon negative dromotropic vagal preganglionic neurons were enkephalinergic. Enkephalinergic axodendritic synapses were also present upon negative inotropic vagal preganglionic neurons. Thus enkephalinergic terminals in ventrolateral nucleus ambiguus can modulate not only heart rate but also atrioventricular conduction and left ventricular contractility by directly synapsing upon cardioinhibitory vagal preganglionic neurons.

Plateau potentials and membrane oscillations in parasympathetic preganglionic neurones and intermediolateral neurones in the rat lumbosacral spinal cord.

Whole-cell patch recordings were made from parasympathetic preganglionic neurones (P-PGNs) and unidentified intermediolateral (IML) neurones in thick slices of the lower lumbar and sacral spinal cord of 14- to 21-day-old rats. The P-PGNs and IML neurones examined were similar in terms of soma sizes, input resistance and capacitance, and displayed a sag conductance as well as rebound firing. In the absence of drugs, the neurones responded with either tonic or adapting firing to depolarizing current steps. However, in the presence of the group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), almost half of the neurones displayed accelerating firing rates during the constant current injection, followed by a sustained after-discharge. In the presence of TTX, plateau potentials were observed. The firing changes and plateaux were blocked by nifedipine, an L-type Ca2+ channel blocker, and (S)-(-)-Bay K8644 was able to produce these firing changes and plateaux in the absence of DHPG, demonstrating the involvement of an L-type Ca2+ conductance. Ca2+-activated nonspecific cationic conductances also appear to contribute to the firing changes. A few neurones displayed membrane oscillations and burst firing in the presence of DHPG. The results suggest that the firing characteristics of both P-PGNs and other neurones likely to be involved in caudal spinal reflex control are not static but, rather, quite dynamic and under metabotropic glutamate receptor modulatory control. Such changes in firing patterns may be involved in normal pelvic parasympathetic reflex function during micturition, defaecation and sexual reflexes, and may contribute to the abnormal output patterns seen with loss of descending brainstem input and visceral or perineal sensory disturbances.

A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.

Orexin-immunoreactive inputs to rat sympathetic preganglionic neurons.

Orexin increases blood pressure and orexin-immunoreactive (IR) axons robustly innervate the spinal cord. Seeking anatomical evidence for direct effects of orexin on sympathetic preganglionic neurons (SPN), we used immunohistochemistry to study the relationships between orexin-IR axons and SPN identified by immunoreactivity for choline acetyltransferase (ChAT) or for cholera toxin B retrogradely transported from the superior cervical ganglion (SCG). In the intermediolateral cell column (IML), varicose, orexin-positive axons closely apposed almost all SPN in segments T1 and T2, but appositions were rare in T4-L2. Orexin fibers also apposed ChAT-IR cell bodies in the intercalated nucleus and the central autonomic area from T1 to L2. Orexin-IR synapses were identified ultrastructurally on SPN projecting to the SCG. Since SPN involved in cardiovascular control cluster in the IML of mid- and lower thoracic cord, these findings suggest that orexin affects blood pressure by acting on supraspinal neurons rather than SPN.

Catecholaminergic microcircuitry controlling the output of airway-related vagal preganglionic neurons.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.

Quantitative analysis of the dendrites of sacral preganglionic neurons in the cat.

Quantitative analyses were performed on the dendrites and somata of 25 electrophysiologically identified preganglionic neurons (PGN) obtained from the sacral spinal cord of the cat by intracellular injection of Neurobiotin or horseradish peroxidase. Total dendritic length and surface area were measured for each dendrite. The sizes of the stem dendrites measured at their base were positively correlated with the sizes of the entire tree and numbers of end branches. Total surface area of somata and dendrites averaged 39,138 microm(2); 90.7% of that was from the dendrites. To obtain measurements of the relative contributions of PGN dendrites to specific regions of the spinal cord, the percentage of each dendrite occupying eight spinal cord regions was recorded. Sixty-three percent of the dendrites projected dorsal to their somata, whereas an average of 33.3% of dendrites were located in the white matter, most of them in the lateral and dorsolateral funiculi. The neurons within this sample formed a continuum with some neurons having a large percentage of dendrites in lamina I but little in the white matter, whereas at the other end of the continuum were cells with the reverse configuration. The intermediate neurons had dendrites in both locations. Taken together, these data indicate a heterogeneous population of PGN in the lateral band of the sacral parasympathetic nucleus.

Increased innervation of rat preganglionic sympathetic neurons by substance P containing nerve fibers in response to spinal cord injury.

Substance P (SP) is elevated in the intermediate zone caudal to a spinal cord lesion presumably due to sprouting of intraspinal and primary afferent axons. It is unclear, however, if axon terminals are in direct contact with preganglionic neurons located within the different autonomic subnuclei. Therefore, the innervation of preganglionic sympathetic neurons by SP was quantified using confocal imaging and morphometric image analysis. The number of SP-immunoreactive varicosities apposed to nitric oxide synthase-positive neurons significantly increased bilaterally in all sympathetic areas of segment T2 one week after low cervical hemisection at C6/7. Consequently, direct excitatory effects of SP on preganglionic neurons may play an important role in the dysregulation of arterial blood pressure observed in patients with spinal cord injury at the cervical or upper thoracic level.

Age-dependent changes of electrophysiologic characteristics of the stellate ganglion conducting pathways in kittens.

The conducting pathways of the cat stellate ganglia were examined in newborn, 10- and 20-day-old kittens by recording the evoked responses in the branches of the ganglion following electrical stimulation of its other branches. In the course of postnatal ontogenesis, the average conduction velocity of excitation and the average amplitude of the responses was increased. In newborn and 10-day-old kittens, all fibers on their conduction velocities belong to C-fibers. In 20-day-old kittens, Adelta- and B-fibers also appeared. In this age-group, two subgroups among fibers of the C group were determined according to their conduction velocity of excitation. From 10 days of age, the axon reflex was recorded in the anastomosis and the inferior cardiac nerve following stimulation of the cranial and caudal branches of the subclavian loop. All branches of the stellate ganglia of 20-day-old kittens contain both continuous and synaptically switched fibers.

Synaptic organisation of neuropeptide-containing preganglionic boutons in lumbar sympathetic ganglia of guinea pigs.

Within the lumbar sympathetic ganglia of guinea pigs, the endings of different populations of neuropeptide-containing preganglionic neurons form well-defined pericellular baskets of boutons around target neurons in specific functional pathways. We have used multiple-labelling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to investigate synaptic organisation within pericellular baskets labelled for immunoreactivity to calcitonin gene-related peptide (CGRP), substance P (SP), or the pro-enkephalin-derived peptide, met-enkephalin-arg-gly-leu (MERGL) in relation to their target neurons. Different functional populations of neurons, identified by their neurochemical profile, showed a significant degree of spatial clustering and predicted well the distribution of specific classes of pericellular baskets. Most of the boutons in a basket were completely surrounded by Schwann cell processes and did not form synapses. The synapses that were present were made mostly onto dendrites enclosed by the Schwann cell sheath surrounding the neuron within the basket. These dendrites probably originated from neurochemically similar neighbouring neurons. Nevertheless, some of the boutons in the baskets did form synapses with the cell body or proximal dendrites of the neuron they surrounded. Occasionally, cell bodies received a relatively high number of synapses and close appositions from boutons in a pericellular basket. Synaptic convergence of two immunohistochemically distinct types of preganglionic inputs was found in baskets of SP-immunoreactive or MERGL-immunoreactive, but not CGRP-immunoreactive, boutons. Taken together, our results show that the appearance of pericellular baskets is primarily due to the packing of the target neurons. The grouping of functionally similar classes of neurons with their pathway-specific projections of peptide-containing preganglionic neurons suggests that peptides could exert their effects in relatively well-defined zones within the ganglia.

Localisation of arginine vasopressin V1a receptors on sympatho-adrenal preganglionic neurones.

Vasopressin-containing nerve terminals are present in the spinal cord of several species. This study was designed to determine whether sympatho-adrenal preganglionic neurones (SPN) express vasopressin receptors (VPRs). SPN in the spinal cord were revealed by retrograde labelling of Fluorogold following its unilateral injection into the adrenal medulla of 12-20 day postnatal rats. VPRs were simultaneously visualised in the Fluorogold-labelled slices of spinal cord using a recently developed biotinylated vasopressin receptor antagonist [1-phenylacetyl,2-O-methyl-D-tyrosine,6-arginine,8-arginine,9-lysinam ide(Nepsilon-biotinamidocaproamide)]vasopressin, PhAcAL(Btn)VP. The VPR:PhAcAL(Btn)VP complexes were visualised either with Texas Red-conjugated avidin or with a Vectastain avidin:alkaline phosphatase detection kit. These dual-labelling experiments revealed VPRs to be present in the spinal grey matter and to be particularly dense in the intermediate grey matter and adjacent regions of the ventral horn. Many SPN were associated with receptor-specific labelling of PhAcAL(Btn)VP, thereby demonstrating that VPRs are expressed by these neurones. These VPRs were pharmacologically defined as the V1a subtype. It is concluded that sympatho-adrenal preganglionic neurones express VPRs and that these are of the V1a subtype. The distribution of VPRs is not, however, restricted to these SPN in the spinal cord.

Neuropeptide Y-like immunoreactivity localizes to preganglionic axon terminals in the rhesus monkey ciliary ganglion.

PURPOSE: To characterize neuropeptide distribution in the ciliary ganglion of rhesus monkeys (Macaca mulatta). METHODS: Cryostat tissue sections of fixed rhesus monkey ciliary, pterygopalatine, superior cervical, and trigeminal ganglia were incubated with antisera to neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Antibody binding was visualized by indirect immunofluorescence. RESULTS: NPY-like immunoreactive (LI) nerve terminals surrounded 80% of ciliary ganglion cells, but ciliary ganglion cell somata were unstained. NPY-LI cells were present in the superior cervical ganglion, in which almost all cells were TH- and DBH-LI, and in the pterygopalatine ganglion, in which almost all cells were VIP-LI. Because neither TH, DBH, nor VIP immunoreactivity was detected in nerves contacting ciliary ganglion cells, the NPY-LI input to ciliary neurons does not likely derive from the autonomic ganglia. The trigeminal ganglion, another potential source, had no NPY-LI neurons. CGRP- and SP-LI axons from the nasociliary nerve traversed the ciliary ganglion; a small number of varicose axons were distributed among ganglion cells and rarely surrounded cell somata. Most ciliary ganglion cells were TH-LI, but only a few were DBH-LI. CONCLUSIONS: Based on these patterns of peptide immunoreactivities, the NPY-LI nerve fibers investing ciliary ganglion cells in the rhesus monkey are most likely preganglionic axon terminals of mesencephalic parasympathetic neurons. Although the origin and function of these NPY-LI nerves remains to be established, the present finding adds to the remarkable diversity of neuropeptide immunoreactivity so far identified in preganglionic and postganglionic cells of the ciliary ganglion in different species of birds and mammals, including primates.

Arborization pattern of sympathetic preganglionic axons in the rat superior cervical and stellate ganglia.

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600-700 microns in the rostrocaudal direction, and about 200 microns in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 microns in the rostrocaudal direction and about 400 microns in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.