GABA in the control of sympathetic preganglionic neurons.

1. Amino acid neurotransmitters are critical for controlling the activity of most central neurons, including sympathetic preganglionic neurons (SPN), the spinal cord neurons involved in controlling blood pressure and other autonomic functions. 2. In studies reviewed here, SPN were identified either by retrograde tracing from a peripheral target (superior cervical ganglion or adrenal medulla) or by detection of immunoreactivity for choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme that is a marker for all SPN, in intact or completely transected rat spinal cord. 3. Postembedding immunogold labelling on ultrathin sections was then used to detect GABA and sometimes glutamate in nerve terminals on SPN or near them in the neuropil of the lateral horn. 4. In some cases, the terminals were prelabelled to show an anterograde tracer or immunoreactivity for ChAT or neuropeptide Y. 5. This anatomical work has provided information that is helpful in understanding how SPN are influenced by their GABAergic innervation. 6. Immunogold studies showed that the proportion of input provided by GABAergic terminals varies between different groups of SPN. For some groups, this input may be preferentially targeted to cell bodies. 7. Anterograde tracing demonstrated that supraspinal as well as intraspinal GABAergic neurons innervate SPN and investigations on completely transected cord suggested that supraspinal neurons may provide a surprisingly large proportion of the GABAergic terminals that contact SPN. 8. The double-labelling studies in which other amino acids, ChAT or neuropeptide Y were localized along with GABA indicate that GABAergic terminals contain other neurochemicals that could modulate the actions of GABA, depending on the complement of receptors that are present pre- and post-synaptically. 9. Taken together, these data indicate that GABAergic transmission to SPN may be much more complicated than suggested by the currently available electrophysiological studies.

Axons of sacral preganglionic neurons in the cat: I. Origin, initial segment, and myelination.

Parasympathetic preganglionic neurons in the cat sacral spinal cord innervate intraspinal neurons and pelvic target organs. Retrograde tracing studies have revealed little of the morphology of their axons including their origin, initial segments, or their myelin, due to methodological limitations. Intracellular labeling of single neurons with neurobiotin or HRP has overcome these problems. Axons were studied in 24 preganglionic neurons. In 21 neurons the axon originated as a branch of a dendrite, without a detectable axon hillock, at distances from the soma ranging from 10 to 110 microm (average 34.1 microm ). In 3 neurons the axon was derived from the soma. Initial segments, present in all cells, ranged from 15 to 40 microm (average 26.8 microm). Nearly all axons followed the initial segment with unmyelinated segments that varied between 59 to 630 microm, followed by myelin and nodes of Ranvier. Internodal distances were variable and relatively short (average 93 microm). Axonal diameters measured over the intraspinal course in 18 axons averaged 1.3 microm (range 0.6-2.4 microm) and were relatively constant compared with other neurons. Spine-like protrusions were observed on the initial segments of 12 cells. Axon collaterals originated from unmyelinated sections and nodes of Ranvier. Antidromic action potentials showing initial segment, soma-dendritic inflections, did not differentiate between soma-derived and dendrite-derived axons. The data suggest that axons originating from a dendrite are the normal structure of preganglionic neurons in the lateral sacral parasympathetic nucleus. It is proposed that the particular structure of these axons may be part of a timing mechanism that coordinates preganglionic neurons with other spinal neurons involved in target organ reflexes.

Differential distribution of nerve terminals immunoreactive for substance P and cholecystokinin in the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer.

Immunoreactivity for substance P and cholecystokinin-8 was examined in the nerve fibers in the central autonomic nucleus, a cell column for sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Substance P-immunoreactive fibers were distributed throughout the entire rostrocaudal extent, but were more abundant in the caudal part of the column, where substance P-immunoreactive varicosities sometimes made contacts with the sympathetic preganglionic neurons. Cholecystokinin-8-immunoreactive fibers were found almost entirely in the rostral part of the column, where a dense network of varicosities was in close apposition to a considerable number of the sympathetic preganglionic neurons. Double labeling immunohistochemistry showed that substance P fibers and cholecystokin-8 fibers were entirely different, and distinct from serotonin-immunoreactive fibers. By using immunoelectron microscopy, synaptic specialization was sometimes observed between the dendrites of preganglionic neurons and varicosities immunoreactive for substance P and cholecystokinin-8. Substance P- and cholecystokinin-8 fibers were seen from the descending trigeminal tract, through the dorsolateral funiculus and the ventral portion of the dorsal horn, to the central autonomic nucleus. After colchicine treatment, substance P-immunoreactive perikarya were found in the cranial and spinal sensory ganglia. These results suggest that the sympathetic preganglionic neurons of the filefish receive innervation by substance P fibers and cholecystokinin fibers, and that the former might be of primary sensory origin. Topographical distribution of cholecystokinin-8-immunoreactive terminals in the central autonomic nucleus along the rostrocaudal extent might underlie the differential regulation of sympathetic activity via a distinct population of sympathetic preganglionic neurons.

Effect of pulmonary C-fibre afferent stimulation on cardiac vagal neurones in the nucleus ambiguus in anaesthetized cats.

It has been demonstrated previously that the vagal bradycardia evoked by activation of pulmonary C-fibres is not respiratory modulated. Experiments were carried out in alpha-chloralose anaesthetized cats to determine if these cardiac vagal preganglionic neurones (CVPNs) in the nucleus ambiguus (NA), which have respiratory modulated activity, can be activated when pulmonary C-fibre afferents are stimulated by right atrial injections of phenylbiguanide (PBG). Eleven CVPNs with B-fibre axons in the right cardiac vagal branches were identified and found to be localized within or ventrolateral to the nucleus ambiguus. Ionophoretic application of a high current of dl-homocysteic acid (DLH) induced a vagally mediated bradycardia and hypotension in six of eight sites from which CVPNs were recorded. The activity of B-fibre CVPNs, whether spontaneous (n = 4) or induced by ionophoresis of DLH (n = 7) was respiratory modulated, firing perferentially during post-inspiration and stage 2 expiration. This activity also correlated with the rising phase of the arterial blood pressure wave consistent with these CVPNs receiving an arterial baroreceptor input. Right atrial injections of PBG excited nine of eleven CVPNs tested. In eight of these activated neurones the onset latency of the excitation was within the pulmonary circulation time, consistent with being activated only by pulmonary C-fibre afferents. In two neurones the PBG-evoked excitation still occurred when central inspiratory drive was inhibited, as indicated by the disappearance of phrenic nerve activity. In conclusion, B-fibre respiratory modulated CVPNs can be activated following stimulation of pulmonary C-fibre afferents.

Reelin controls position of autonomic neurons in the spinal cord.

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell-cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157-172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055-1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Expression of Fos protein in adrenal preganglionic neurons following chemical stimulation of the rostral ventrolateral medulla of the rat.

The ventrolateral medulla is known to be involved in the regulation of arterial blood pressure, especially via its connections with sympathetic preganglionic neurons (SPNs) mainly located in the intermediolateral nucleus of the spinal cord. It has been shown that stimulation of the rostral part of the ventrolateral medulla (RVLM) elicits a release of catecholamines from the adrenal medulla. The aim of the present study was to demonstrate the existence of a functional pathway between the RVLM and adrenal SPNs using the combination of a retrograde tract tracing technique (cholera toxin B subunit) with the immunohistochemical detection of Fos protein following the chemical stimulation of RVLM. The data obtained showed that: (1) chemical stimulation of the RVLM induced Fos immunoreactivity in the intermediolateral nucleus and particularly in SPNs projecting to the adrenal medulla; (2) along the thoracic segments T2-T12, 26.1% of retrogradely identified adrenal SPNs were Fos-immunoreactive with the greatest percentage (30.9%) in the T8 segment. These results favored a functional control of the RVLM on adrenal SPNs which may contribute to a substantial activation of the cardiovascular system via the release of adrenal catecholamines.

Distinct localization and target specificity of galanin-immunoreactive sympathetic preganglionic neurons of a teleost, the filefish Stephanolepis cirrhifer.

Immunoreactivity for galanin was examined in the sympathetic preganglionic neurons in the spinal cord, adrenal glands, sympathetic ganglia, and some sensory ganglia of the filefish Stephanolepis cirrhifer. Galanin-immunoreactive neurons were found only in the rostral part, but not in the caudal part of the central autonomic nucleus (a column of sympathetic preganglionic neurons of teleosts). Many galanin-immunoreactive nerve terminals were found in contact with neurons in the celiac ganglia and the cranial sympathetic ganglia on both sides of the body. Most neurons encircled by galanin-immunoreactive nerve fibers were negative for tyrosine hydroxylase. Galanin-immunoreactive nerve fibers were very sparse in the spinal sympathetic paravertebral ganglia. No galanin-immunoreactive nerve fibers were found in the adrenal glands. No sensory neurons of the trigeminal, vagal, or spinal dorsal root ganglia were positive for galanin-immunoreactivity. These results suggest that galanin-immunoreactive sympathetic preganglionic neurons have distinct segmental localization and might project specifically to a population of non-adrenergic sympathetic postganglionic neurons in the celiac and cranial sympathetic ganglia.

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study.

Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris-projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene-related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP-immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons.

CNS innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus.

The CNS cell groups that project to vagal preganglionic neurons which innervate the most distal part of the airways were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the lung parenchyma of C8 spinal rats and after 5 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic cells (first-order neurons) were seen mainly in the ventral medulla oblongata: the compact portion of the nucleus ambiguus and the area ventral to it. Occasionally, a few labeled cells were seen within the rostral part of the dorsal vagal nucleus. This labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin beta-subunit (CT-b) injections in the lung parenchyma. PRV transneuronally labeled neurons (second-order and/or presumed third-order neurons) were found throughout the CNS with the characteristic labeling in the brainstem. Labeled neurons were identified along and just beneath the ventral medullary surface, and in nearby areas: the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei, as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Several nucleus tractus solitarius (nTS) regions contained labeled cells including the commissural, medial, and ventrolateral nTS subnuclei. The A5 cell group and a small number of locus coeruleus neurons were also labeled. PRV-infected neurons were present in the Kölliker-Fuse and Barrington's nuclei. In the mesencephalon, neurons within the ventral periventricular gray matter were labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei, and within the amygdaloid complex. In summary, the parasympathetic preganglionic neurons that innervate the peripheral airways are controlled by networks of lower brainstem and suprapontine neurons that lie in the same regions known to be involved in central regulation of autonomic functions.

Endogenous lectin (RL-29) expression of the autonomic preganglionic neurons in the rat spinal cord.

Rat spinal neurons expressing lectin RL-29 are visualized immunohistochemically. RL-29 immunoreactive (RL-29 IR) neurons are found in the lateral parts of laminae V-VII, designated as the intermediolateral cell column (IML) in the thoracic cord, the sacral parasympathetic nucleus (SPN) in the lumbosacral cord, and the dorsomedial nucleus (DMN) of the ventral horn. The majority of RL-29 IR neurons in the SPN are also labeled by a retrograde tracer DAPI applied to the cut L6-S1 ventral roots. These data indicate that the majority of RL-29 IR neurons in the SPN are autonomic preganglionic neurons, thus suggesting that RL-29 can be a useful tool in marking this subpopulation of neurons. In addition, the presence of previously described RL-29 IR primary afferent fibers and terminals in the dorsal parts of the cord are confirmed.

Glutamate receptor subunits associated with rat sympathetic preganglionic neurons.

The purpose of this study was to determine what subunits of the glutamate (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)) receptor are expressed by sympathetic preganglionic neurons in the spinal cord of adult rats. Preganglionic neurons were retrogradely labelled with Fluorogold, double-labelled with choline acetyltransferase immunofluorescence, and examined with confocal microscopy for evidence of immunoreactivity for GluR1, GluR2, GluR2/3 and GluR4 subunits. Quantitative analysis revealed that 92, 63 and 85% of preganglionic cells in the T8 segment express GluR1, GluR2 and GluR2/3 subunits, respectively. Cells were not immunoreactive for the GluR4 subunit. This evidence is consistent with the idea that most sympathetic preganglionic neurons form heteromeric AMPA receptors. Cells with GluR2 subunits will assemble receptors which are impermeable to calcium ions and may be resistant to excitotoxic cell death.

Identification of CNS neurons innervating the rat prostate: a transneuronal tracing study using pseudorabies virus.

The spinal and brain neurons that innervate the rat prostate were identified using the transneuronal tracing technique. Three groups of rats were prepared: (1) nerve intact, (2) bilateral pelvic nerve cut and right hypogastric nerve cut and (3) bilateral hypogastric nerve cut and right pelvic nerve cut. Pseudorabies virus (PRV) was injected into the ventral prostate on the left side. After 2-4 days, the rats were perfused transcardially under deep anesthesia and the spinal cord and brain removed. PRV-labelled cells were identified using immunohistochemistry. After 3 days survival, sympathetic and parasympathetic preganglionic neurons were labelled with PRV. In addition, spinal interneurons were found in the dorsal gray commissure (DGC) of T13-S1. Rats with only one hypogastric nerve intact resulted in spinal labelling of sympathetic preganglionic neurons in the DGC and ipsilateral intermediolateral cell column (IML). In addition, many spinal interneurons were found from L1 to L6 in the medial gray. Rats with only one pelvic nerve intact displayed PRV-labelled cells in the parasympathetic preganglionic nucleus ipsilateral to the injection site. Spinal interneurons were present in the region of the IML and in the medial cord. In the brain, areas predominately labelled with PRV included the nucleus gigantocellularis and paragigantocellularis, raphe magnus, raphe pallidus, A5, Barrington's nucleus, central gray, ventral tegmental area, the paraventricular nucleus of the hypothalamus, lateral hypothalamus and medial preoptic area. These data demonstrate the sympathetic and parasympathetic spinal circuits and demonstrate the overlap of supraspinal innervation of the spinal interneurons.

Differential vulnerability of autonomic and somatic motor neurons to N-methyl-D-aspartate-induced excitotoxicity.

Two closely-related subsets of spinal motor neurons are differentially vulnerable in the degenerative neurological disease amyotrophic lateral sclerosis. Autonomic motor neurons (i.e. preganglionic sympathetic neurons) survive in this disorder, whereas most spinal somatic motor neurons do not. The present study was undertaken in order to begin to understand the phenotypic differences between the two motor neuronal subsets which might contribute to this differential survival. Organotypic slice cultures of postnatal rat thoracic spinal cord were maintained in defined medium for one to 12 days in the presence or absence of N-methyl-D-aspartate or its antagonist, D-amino-phosphonopentanoic acid. Autonomic motor neurons that were stained for either nicotinamide adenine dinucleotide phosphate reduced diaphorase or choline acetyltransferase only were both able to tolerate 50 microM N-methyl-D-aspartate treatment for over seven days in culture with no apparent adverse effects. In contrast, cultures maintained for only one day in medium containing 50 microM N-methyl-D-aspartate showed a dramatic and highly significant decrease in the numbers of neurofilament-positive somatic motor neurons, as well as nicotinamide adenine dinucleotide phosphate reduced diaphorase-positive interneurons. These N-methyl-D-aspartate-induced effects were dose-dependent and blockable. The results of this investigation indicated that autonomic motor neurons and somatic motor neurons were differentially susceptible to N-methyl-D-aspartate-induced excitotoxicity, and that the resistance of autonomic motor neurons to this insult appeared to be independent of the nicotinamide adenine dinucleotide phosphate reduced diaphorase phenotype.

Periaqueductal gray matter projection to vagal preganglionic neurons and the nucleus tractus solitarius.

The periaqueductal gray matter (PAG) has been implicated in a variety of different functions, including autonomic regulation. Chemical stimulation of the lateral PAG produces hypertension and tachycardia while activation of the ventrolateral PAG produces the opposite effect. While these effects are the result of alterations in sympathetic activity, little is known about whether the PAG can modulate vagal functions as well. The anterograde axonal tracing method using the plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) was used to determine whether both of the lateral and ventrolateral PAG columns project to vagal preganglionic neurons and/or to the nucleus tractus solitarius (NTS). Highly restricted PHA-L injections were made in all four PAG columns throughout their rostrocaudal extent in rats. Labeled fibers were visualized by immunohistochemistry and studied in relationship with choline acetyltransferase (ChAT) immunostained parasympathetic preganglionic neurons of the dorsal motor vagal nucleus (DMV) and nucleus ambiguous (NA). The lateral PAG projects to the lateral DMV and to the caudal part of the external NA. The ventrolateral PAG innervates the same regions and also projects to the rostral part of the external NA -- a site that contains cardiac parasympathetic preganglionic neurons. Both the lateral and ventrolateral PAG project to the NTS in a similar fashion innervating the medial, ventrolateral and commissural subnuclei. In summary, the lateral and ventrolateral PAG have similar patterns of innervation of the NTS and DMV, but their projection to the NA is different: the rostral external NA receives innervation only from the ventrolateral PAG and the lateral PAG innervates the caudal part.

Electrophysiological evidence for multiple glycinergic inputs to neonatal rat sympathetic preganglionic neurons in vitro.

The time pattern of glycinergic inhibitory postsynaptic currents (IPSCs) in sympathetic preganglionic neurons was studied in thin transverse spinal cord slices of neonatal (1-10 days postnatal) rats by means of the patchclamp technique. Three time patterns could be distinguished: (i) large events [mostly > 400 pA (30-36 degrees C)] occurring at regular intervals, (ii) small events occurring at irregular intervals, and (iii) small events occurring in transient (1.5-10 s), high-frequency (> 15 Hz) bursts of synaptic activity. The large regular events had uniform kinetics which was consistent with the idea of a proximal site of origin for all of these events. They were reversibly inhibited in amplitude and frequency by extracellular application of a high concentration of acetylcholine (200 microM) or the specific nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium iodide (DMPP; 1 mM), but unaffected by glutamate (100 microM). IPSCs occurring in bursts had slower and less uniform kinetics, suggesting a more diverse site of origin. The frequency of events decreased during a burst. Similar bursts could be induced by extracellular application of glutamate receptor agonists. These results indicate that sympathetic pregnanglionic neurons in a thin, transverse spinal cord slice receive at least two different glycinergic inputs.

Cytochrome oxidase staining in the major pelvic ganglion of the male rat.

Cytochrome oxidase staining was used as a marker of metabolic activity in neural elements in the rat major pelvic ganglion. Many neurons in the ventral pole of the ganglion have little cytochrome oxidase activity, while neurons in other locations show gradations in staining intensity. Punctate staining around principal neurons may represent preganglionic terminals, since it was greatly reduced after denervation of the ganglion. Image analysis was used to compare neuronal size to staining intensity. There was a negative correlation between cell size and staining intensity; the largest neurons were only lightly stained for cytochrome oxidase, while the medium and the small neurons showed a full range of metabolic activity. To study metabolic activity of an identified neuronal population, the seminal vesicles were injected with a retrograde tracer. The largest seminal vesicles neurons (1500 to 3200 microns2) had low enzyme activity, whereas the majority of neurons to this organ were smaller with gradations in staining. These results are indicative of the metabolic activity of the autonomic innervation to various pelvic tissues. Cytochrome oxidase histochemistry should prove valuable in assessing the demands placed on autonomic ganglia in differing functional and dysfunctional states.

Vagal preganglionic projections to the enteric nervous system characterized with Phaseolus vulgaris-leucoagglutinin.

The patterns and extent of vagal preganglionic divergence and convergence within the gastrointestinal tract of the rat were characterized with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Three weeks after tracer was iontophoretically injected into two to four sites within the dorsal motor nucleus of the vagus, wholemounts of perfused gut organs (stomach, duodenum, cecum) were prepared, counterstained with Cuprolinic blue, and processed for PHA-L using the avidin biotin complex with diaminobenzidine. Controls included animals injected with PHA-L after intracranial deafferentations. Well-positioned injections labeled an extremely dense and intricate network of varicose efferent axons throughout the gastric myenteric plexus (including that of the fundus). Individual fibers collateralized extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervated subsets of neurons within ganglia. Most enteric neurons were contacted by varicosities of more than one vagal fiber. The patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resembled the organization in the stomach in many aspects, but the projections in each organ had distinctive characteristics, and label was less dense in the intestines than in the stomach. Vagal preganglionic fibers directly innervated submucosal ganglia, although sparsely. Brainstem injections of PHA-L retrogradely labeled a few myenteric neurons in the corpus, fundus, and duodenum: These "gastrobulbar" and "duodenobulbar" neurons received reciprocal vagal preganglionic innervation. Finally, the PHA-L that spread to the nucleus of the solitary tract occasionally produced transganglionic labeling of afferent intramuscular arrays (gastric fundus). The results of this paper provide strong evidence that the traditional "command neuron" or "mother cell" hypotheses of vagal-enteric organization should be abandoned for an integrative neural network model.

Topographic relationship of neurotensin-containing axon terminals with cardiac and noncardiac principal ganglion cells in the stellate ganglia of the cat.

The pattern of association between neurotensin (NT)-immunoreactive (NTIR) preganglionic nerve terminals and cardiac and noncardiac neurons in the stellate ganglion of the cat is analyzed, based on the finding of an excitatory modulation effect of exogenous NT on cardiac functions. For this purpose, NT-containing terminals were labeled by immunohistochemistry, and ganglion cells were detected by retrograde labeling of cardiac and vertebral nerves to identify cardiac and noncardiac neurons. To determine a possible regional localization of NTIR terminals and ganglion cells, the ganglia were divided into four areas: caudal, dorsomedial, cranial, and ventromedial, related to the two major afferent nerves (thoracic white rami 3 [T3WR] and 2 [T2WR]) and the two efferent nerves (vertebral and cardiac). NTIR terminals were widespread in the complete ganglion tissue; they covered practically all the regions explored, although two clusters of high concentration of NTIR terminals were detected in the cranial and caudal areas. By retrograde labelling it was found that cardiac cells were arranged around the exit of the cardiac nerve and that the vertebral neurons were extended from the exit of the vertebral nerve to the entrance of T3WR. The finding of association of NTIR terminals with cardiac neurons may account for the cardioregulatory effect of NT; however, since the presence of NTIR terminals close to the noncardiac neurons is notorious, other regulatory functions of NT must be considered.

Spinal and vagal projections to the sympathetic trunk of the wrasse, Halichoeres poecilopterus.

The sympathetic preganglionic neurons of the teleost, Halichoeres poecilopterus, were identified by retrograde axonal tracing. After horseradish peroxidase was applied to the sympathetic trunk, labeled neurons were found at the caudalmost level of the medulla, in the spinal cord near the fourth spinal nerve root (rostral spinal group), and in the spinal cord from rostral to the sixth spinal nerve root to caudal to the tenth spinal nerve root (caudal spinal group). The rostral spinal group has three cell columns segregated mediolaterally from the central gray zone to the lateral funiculus. Labeled neurons were found predominantly on the side ipsilateral to the application. In the caudal spinal group, labeled neurons were found bilaterally in the central gray zone. This condition is different from that previously reported in the puffer fish and filefish. The labeling in the medulla suggests that the preganglionic neurons in the brainstem may send fibers to the sympathetic trunk of this fish, although their peripheral targets are unknown.

Voltage-dependent potassium currents of sympathetic preganglionic neurons in neonatal rat spinal cord thin slices.

Voltage-dependent potassium currents were analyzed in the visually identified sympathetic preganglionic neurons (SPNs) of neonatal rat spinal cord thin slices by the whole-cell patch-clamp technique. Some of the SPNs were identified by the presence of retrogradely transported fluorescent dye, DiI, injected into the superior cervical ganglion several days prior to experimentation. In a tetrodotoxin (TTX)-containing solution, a step depolarization from the holding potential of -72 mV generated a slow outward current that was suppressed by tetraethylammonium (TEA) and by Ca(2+)-free/2.5 ImM Co2+ solution. Ca(2+)-dependent current consisted of a transient and a sustained components. In a Ca(2+)-free (substituted with Mg2+) solution with TTX and TEA, a step depolarization from a hyperpolarized potential evoked a transient outward current that was blocked by 4-aminopyridine (4-AP). A step hyperpolarization evoked a voltage-dependent inward current, the conductance of which was dependent not only on the membrane potential, but also on the extracellular K+ concentration. Tail current analyses revealed that all of these currents were carried by K+ ions. These results indicate that SPN possesses at least five types of voltage-dependent K+ current, including the delayed rectifier current (IK), Ca(2+)-dependent transient current (IC), Ca(2+)-dependent sustained current (IAHP), A-current (IA) and inward rectifying current (Iu), which may be targets of putative transmitters released from various descending and segmental inputs impinging upon the SPN.