Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium-binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K-immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K-immunoreactive preganglionic neurons also had co-localised nitric oxide synthase, although a population of preganglionic neurons in the mid- to low thoracic intermediolateral nucleus expressed only calbindin D28K-immunoreactivity. Retrograde-tracing studies showed that calbindin D28K-immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K-immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K-immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K-immunoreactive, and most of these lacked neuropeptide Y-immunoreactivity. Calbindin D28K-immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K-immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y-immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K-immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde-tracer injections into a range of peripheral tissues. Calbindin D28K-immunoreactive nerve terminals, with co-localised nitric oxide synthase-immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997. © 1997 Wiley-Liss, Inc.     

Responses of sympathetic postganglionic neurons to peripheral nerve stimulation in pigeon (Columba livia).

Reflex changes in heart rate and arterial blood pressure can be elicited in pigeons with high cervical transection by stimulation of brachial or lumbosacral peripheral and spinal nerves. This extends the phenomenon of spinally mediated, somatosympathetic reflexes to another vertebrate class. In a preliminary attempt to explore the spinal circuitry mediating these reflexes, the responses of single sympathetic postganglionic neurons were studied during spinal and peripheral nerve stimulation. With stimulation and recording at the same spinal segment, calculation of the central delay suggests the segmental reflex circuitry may be relatively simple, possibly trisynaptic. As the distance between stimulating and recording sites increases, postganglionic neuronal responsiveness decreases and becomes more variable. However, there is clear evidence that lumbosacral afferents can activate postganglionic neurons at brachial levels, indicating an effective propriospinal circuitry for somatosympathetic reflexes. Experiments on birds with intact spinal cords demonstrate that these spino-spinal pathways are also functional in the intact animal. While the segmental reflex is not different in the intact bird, the propriospinal pathways do behave somewhat differently, possible suggesting tonic central control.     

Causalgia in the upper limbs following neck trauma: existence of widespread dysfunction of sympathetic postganglionic fibers

The functions of autonomic nervous system were systematically evaluated in a case of causalgia in the upper limbs after neck trauma. A 14-year-old boy had had hard blow on his nucha in a rough fight. After one week, a sustained burning pain, swelling and skin color change developed in the left upper limb. These symptoms also appeared in the right upper limb after 6 weeks. The physical examinations disclosed edema, reddish moist skin, and atrophic nail in the upper limbs. The neurological examinations showed a radiating pain to the upper limbs caused by the neck movement or pressure on the supraclavicular fossae, weakness of the upper limbs and left lower limb, and loss of sensation in the 5th to 8th cervical and first thoracic dermatomal segments. Deep tendon reflexes were diminished in the upper limbs and exaggerated in the lower limbs. Neither Horner syndrome nor sphincter disturbance was observed. He was diagnosed as being the cervicothoracic radiculopathy and cervical myelopathy due to the mechanical force. The burning pain disappeared with oral administration of guanethidine. On the examinations of the autonomic functions, the sweating response to the thermal stimulation was absent above the 5th thoracic dermatomal segment. The sweating response to the intradermal acetylcholine was decreased in the second and third thoracic dermatomal segments. The systolic hypotension with increasing pulse rate occurred on standing. The reactive elevation of the blood pressure to the intravenous tyramine was absent. The excessive elevation of the systolic blood pressure was induced by the subcutaneous injection of epinephrine. These results indicated the dysfunction of the sympathetic postganglionic sudomotor and vasomotor fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The origin of postganglionic and sensory axons in the cervical sympathetic trunk of the cat: A horseradish peroxidase study

The composition of the cervical sympathetic trunk (CST) in the cat is still not completely understood. The present study investigates, by the horseradish peroxidase (HRP) method of tracing neuronal connections, the presence of postganglionic and sensory neurons projecting via the CST. Following sympathectomy at the midcervical level and the application of HRP crystals to the cut ends of the CST which had been isolated from the surrounding by a 1.5% solution of agar-agar, labelled neurons were seen in the superior cervical (SCG), stellate (SG), inferior vagal ganglia (IVG), and spinal ganglia C₈–T₈. The maximum number of labelled nuerons was 536 in the SCG, 460 in the SG, 180 in the IVG, and 104 in spinal ganglia C₈–T₈.     

Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in autonomic ganglia, which innervate and control the activity of most visceral organs. By combining ultrastructural, immunocytochemical, and pharmacological analyses, we characterized the nAChR subtypes in the rat superior cervical ganglion (SCG) and the effect of pre- and postganglionic nerve crush on their number in the ganglion and their distribution at the intraganglionic synapses. Binding with radioactive nicotinic ligands, immunoprecipitation, and immunolocalization experiments revealed the presence of different nAChR subtypes: those containing the alpha3 subunit associated with beta4 and/or beta2 subunits that bind 3H-Epibatidine with high affinity, and those containing the alpha7 subunit that bind 125I-alphaBungarotoxin. After postganglionic nerve crush, the number of nicotinic receptors and immunopositive intraganglionic synapses for each nAChR subunit strongly decreased. Both the number of nAChRs and immunoreactivity recovered 26 days after injury, when regenerating postganglionic fibers had reinnervated the peripheral target organs, as shown by the restoration of tyrosine hydroxylase immunoreactivity in the iris. This observation and the lack of any effect of preganglionic nerve crush on the number of nicotinic receptors suggest that the peripheral targets affect the organization of intraganglionic synapses in adult SCG.     

Organization of the sympathetic postganglionic innervation of the rat heart

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.     

Transmission of impulses from pre- to postganglionic vasoconstrictor and sudomotor neurons

Transmission of impulses of pre- to postganglionic neurons supplying skeletal muscle and skin of the cat's hindlimb and tail was investigated. The objective of the study was to determine whether these postganglionic neurons can be influenced from the preganglionic side by non-nicotinic synaptic mechanisms in the lumbar sympathetic chain ganglia. The activity of the postganglionic neurons was recorded from their axons being isolated from peripheral skin and muscle nerves. (1) Vasoconstrictor neurons can be activated by muscarinic action of released acetylcholine and by a non-cholinergic synaptic mechanism. This type of non-nicotinic excitation of postganglionic vasoconstrictor neurons requires the activation of thin, probably unmyelinated preganglionic axons and considerable summation. Postganglionic sudomotor and pilomotor neurons cannot be activated in this way. (2) Ongoing activity in postganglionic vasoconstrictor neurons, but not in sudomotor neurons, can be enhanced for up to 60 min by brief trains of stimuli applied to the preganglionic site. Also this enhancement requires the activation of thin preganglionic axons. (3) Stimulation of thin preganglionic axons leads to an activation of muscle vasoconstrictor neurons via non-nicotinic synaptic mechanisms in the ganglia after complete block of nicotine transmission. (4) Postganglionic vasoconstrictor neurons and sudomotor neurons may be inhibited by a catecholaminergic autogenic mechanism in the ganglia. (5) The results indicate that integration may take place in the sympathetic chain ganglia by other than divergent and convergent processes. In this integration muscarinic actions of released acetylcholine and non-cholinergic synaptic mechanisms may be involved.     

Spinal segmental preganglionic outflow to cervical sympathetic trunk and postganglionic cardiac sympathetic nerves

The aim of this study was to verify in which spinal cord segments the preganglionic neurones projecting to the cervical sympathetic trunk or converging onto the somata of the postganglionic cardiac sympathetic neurones are located in cats. The thoracic white rami T1 to T5 were electrically stimulated and the evoked responses were recorded in the cervical sympathetic trunks and postganglionic cardiac nerves. The responses were mostly evoked by electrical stimulation of group B preganglionic fibres. The maximum amplitude of evoked responses in the cervical sympathetic trunk was obtained when the T2 white ramus was stimulated and decreased gradually when followed by the stimulation of T1, T3, T4 and T5 white rami. In most cases the maximum amplitude of evoked responses in the left inferior cardiac nerve, right inferior cardiac nerve and left middle cardiac nerve was obtained when the T3 white ramus was stimulated. The size of the responses decreased when more cranial and caudal white rami were stimulated. It was found that the somata of the postganglionic neurones of the right and left inferior cardiac nerves were placed in the right and left stellate ganglion, respectively. Somata of the postganglionic neurones with axons in the left middle cardiac nerve were mainly located in the left middle cervical ganglion and some in the left stellate ganglion.     

Avian sympathetic cardiac fibers and their cells of origin: anatomical and electrophysiological characteristics

The sympathetic cardiac innervation of the pigeon was investigated to describe certain anatomical and physiological properties of the cardiac nerve fibers and their postganglionic cells of origin. The compound action potential of the right cardiac nerve has two major components, one conducting at 2.0–5.6 m/sec with no chronotropic effect on the heart and the other conducting at 0.4–2.0 m/sec with a cardioacceleratory effect. Postganglionic neurons responding antidromically to cardiac nerve stimulation were then studied in ganglion 14 which contains most cells of origin of the cardiac fibers. These neurons have refractory periods of approximately 4 msec, following frequencies of< 4Hz, and axons conducting at 0.4–2.0 m/sec; this conduction velocity range corresponds to the slower compound action potential component. Electron microscopy of the cardiac nerve revealed unmyelinated fibers ranging in diameter from 0.2 to 1.2 μm and a population of myelinated fibers 1.0–3.6 μm in diameter. The unmyelinated fibers account for the slower compound action potential component and are largely postganglionic cardioaccelerator axons. The myelinated fibers account for the faster compound action potential component which has no chronotropic effect and is not reflected in postganglionic antidromic latencies; it is suggested that these myelinated fibers are cardiac sympathetic afferents. This study thus establishes electrophysiological criteria for identifying cardiac postganglionic neurons and describes the anatomical basis of these criteria.     

The ratio of pre- to postganglionic neurons and related issues in the autonomic nervous system

The motor outflow of the autonomic nervous system (ANS) is differentiated into two major divisions, parasympathetic (PSNS) and sympathetic (SNS). Both are organized hierarchically into pre- and postganglionic levels, but classically the two divisions have been assumed to differ in their ratios of pre- to postganglionic neurons. The PSNS has been characterized as having lower (‘one-to-few’) ratios, whereas the SNS has been described as possessing higher (‘one-to-many’) ratios. These patterns have been assumed to measure differing divergences of the outflows. In this review, a ratio of pre- to postganglionic neurons is called a ratio index, and the idea that the PSNS and SNS have characteristically different ratio indexes and divergences is called the ratio rule. The putative differences in the ratio indexes of the two divisions — as well as Fulton's influential proposal that they form one of the bases of contrasting functional capacities of the PSNS and SNS — have been widely accepted for nearly three quarters of a century. A survey of the original observations yielding the concept of the ratio rule as well as the more recent estimates of pre- and postganglionic numbers, however, challenges both the generality and the adequacy of the ratio rule and indexes. The originally formulated differences between the PSNS and SNS represent an overgeneralization since they were based on observations of only two ganglia, the ciliary ganglion in the PSNS and the superior cervical ganglion in the SNS. Furthermore, these original estimates were based on limited samples and were subject to a number of couting artifacts. A survey of the literature suggests that ratio indexes vary much more within each ANS division than they do between the two divisions. When ganglia other than the ciliary and superior cervical are examined, the two divisions of the ANS have broad, largely overlapping ranges of ratio indexes. Additionally, other PSNS-SNS pairs can be found in which the relative sizes of their respective indexes are completely contrary to the ratio rule. For a given ganglion, there are substantial differences in the ratio index between species, between individuals of the same species, and between stages of development in the same species. Furthermore, both divisions of the ANS have wide and largely overlapping ranges of physiological effects varying from specific to diffuse, from local to widespread. Finally, the ratio index measure ignores the degree of convergence found in different ganglia, and it is insensitive to the fact that many ganglia have multiple functionally distinct motor neuron pools, each with separate inputs varying in their degrees of divergence and/or convergence. Thus ratio indexes do not differentiate the PSNS from the SNS, and conclusions based on such putative distinctions are questionable.     

Localization of sympathetic postganglionic neurons innervating mesenteric artery and vein in rats

Physiological and histochemical studies have demonstrated the control and innervation of sympathetic nerves to the artery and vein vessels of splanchnic circulation. In our laboratory, we first used the technique of retrograde transport of horseradish peroxidase to identify the origin of sympathetic neurons innervating the mesenteric vein. In this study, double fluorescence staining technique was used for a simultaneous localization of the sympathetic postganglionic neurons supplying the mesenteric artery and vein in rats. First-order branches of mesenteric artery (A) and vein (V) in the vicinity of ileo-cecal junction were isolated for application of fluorescent dyes (Fast Blue, FB and Diamidino Yellow, DY). The application of FB and DY on A and V was alternated in the next animal to minimize the difference in dye uptake. The animal was allowed to recover for 6-7 days assuring a complete uptake of FB and DY into the cytoplasm and nucleus, respectively. The number of FB, DY and double staining neurons in the prevertebral and paravertebral ganglia were counted under a fluorescent microscope after animal fixation and serial frozen section (30 microm) of the sympathetic ganglia. Our study revealed the following findings: (1) Distribution of the fluorescence-staining neurons in the sympathetic ganglia was as follows: right celiac ganglion (39%), superior mesenteric ganglion (30%), left celiac ganglion (26%), inferior mesenteric ganglion (1%) and paravertebral ganglia (4%). (2) Double staining neurons that dually innervate A and V amounted to 54% of total staining neurons. There were 41% neurons singly innervating A and 5% innervating V. (3) The ratio of neurons supplying the A and V ranged from 1.41 to 1.75 (average 1.61). (4) There was no distinct topographical distribution with respect to the neuron location innervating A and V. The distribution of neurons appeared in a scattering pattern.     

Localization of sympathetic postganglionic neurons of physiologically identified cardiac nerves in the dog

Cardiac nerves were identified physiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labelphysiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labelphysiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labeled cells were found in the T3 to T6 paravertebral ganglia or in the ganglia contralateral to the nerve injected. following injections into specific cardiac nerves, retrograde labeling was widespread within the middle cervical ganglion, and the distributions of labeled neurons from different nerves overlapped considerably. In the middle cervical ganglion there was little or no regional grouping of cells projecting to specific cardiac nerves. within the stellate ganglion, however, te cardiac-sympathetic cells were clustered primarily at the cranial pole near toe origin of the ventral and dorsal ansae. Mediastinal ganglia and ganglia located in cardiac nerves were frequently as heavily labeled as the ipsilateral stellate ganglion. The occurrence of heavy labeling in mediastinal and cardiac nerve ganglia indicates that these hitherto unreported ganglia play a significant role in cardiac neural regulation. These data imply that the organization of sympathetic neurons controlling the heart is much more complex than has previously been considered.     

Horseradish peroxidase localization of the sympathetic postganglionic neurons innervating the cat heart

The localization of the sympathetic postganglionic neurons innervating the cat heart has been investigated by using retrograde axonal transport of horseradish peroxidase (HRP). HRP was injected into the subepicardial layers of 4 different cardiac regions. The animals were sacrificed 72-96 h later and fixed by perfusion via the left ventricle. The paravertebral sympathetic ganglia from the superior cervical, middle cervical and stellate ganglia to T10 ganglia were removed and processed for HRP identification. Following injections of HRP into the apex of the heart, the sinoatrial (SA) nodal region and the ventral wall of the right ventricle, we observed that HRP-labeled sympathetic neurons were localized predominantly in the right stellate ganglia, and to a lesser extent, in the right superior and middle cervical ganglia, and left stellate ganglia. Fewer labeled cells were found in the right T4-T6. T8 and T9. After HRP injection into the dorsal wall of the left ventricle, HRP-labeled cells were present mainly in the left stellate ganglia.     

The course of postganglionic sympathetic fibres distributed with the facial nerve in the cat

Electrophysiological techniques have been used to determine whether sympathetic fibres are present in branches of the facial nerve. Compound action potentials could be recorded from all branches of the facial nerve during stimulation of the cervical sympathetic trunk. The course taken by the sympathetic fibres was determined by observing whether nerve sections at different sites blocked these responses. Two pathways were found: one group of fibres leaves the superior cervical ganglion in the internal carotid nerve, joins the auricular branch of the vagus nerve at the jugular foramen and passes via this nerve to the facial nerve, which it joins in the facial canal just central to the stylomastoid foramen. A second, apparently smaller group of fibres travels in the internal carotid nerve, crosses the roof of the tympanic bulla in the tympanic plexus, enters the middle cranial cavity through the foramen lacerum and passes with the greater superficial petrosal nerve to join the facial nerve at the geniculate ganglion.     

Medullary cells of origin of vagal cardioinhibitory fibers in the pigeon. II. Electrical stimulation of the dorsal motor nucleus

Stimulation of the peripheral end of the cervical vagus results in marked bradycardia, a secondary fall in blood pressure, and little change in respiration. Stimulation of the central end results in immediate apnea, a moderate fall in blood pressure, and little change in heart rate. Electrical stimulation of the dorsal medulla produces short-latency bradycardia at points clustering in: (a) lateral dorsal motor nucleus, 0.5–1.0 mm rostral to the obex; (b) solitary tract; and (c) commissural nucleus of Cajal. Apnea frequently accompanies bradycardia except when elicited by stimulation of the dorsal motor nucleus. Hypotension always follows bradycardia, while hypertension accompanies tachycardia. These bradycardia responses are abolished by bilateral vagotomy and severly diminished when the ipsilateral vagus is cut or pharmacologically blocked. Beta-sympathetic blockade has no effect on the occurrence of bradycardia responses. It is concluded that an efferent cardioinhibitory region in the pigeon is localized in the lateral dorsal motor nucleus 0.5–1.0 mm rostral to the obex and that bradycardia elicited from the solitary tract and commissural nucleus result from stimulating afferents. These results correlate nicely with the localization of the cells of origin of vagal cardioinhibitory fibers based on retrograde degeneration experiments, and together the anatomical and physiological findings indicate a concentration of these neurons in the ventrolateral aspect of the dorsal motor nucleus approximately three-quarters of a millimeter rostral to the obex.     

Firing properties of single postganglionic sympathetic neurones recorded in awake human subjects

For over three decades, the technique of microneurography has allowed us to record sympathetic neural outflow directly from postganglionic axons in awake human subjects. But because sympathetic axons are clustered within a nerve fascicle, such recordings have been limited to the analysis of multi-unit neural activity. To improve the information content of intraneural recordings, we developed the single-unit approach, in which focal recordings can be made from a single C-fibre via a high-impedance tungsten microelectrode. In this review, we describe our methodology for analyzing unitary sympathetic activity and discuss the similarities in the firing properties of individual muscle vasoconstrictor, cutaneous vasoconstrictor and sudomotor neurones.