Specific innervation of guinea-pig superior cervical ganglion cells by preganglionic fibres arising from different levels of the spinal cord.

1. The synaptic contribution of preganglionic nerve fibres arising from the last cervical (C8) and the first seven thoracic spinal cord segments (T1-T7) to neurones of the guinea-pig superior cervical ganglion has been studied by means of intracellular recording during ventral root stimulation in vitro. 2. The majority of neurones received innervation from the middle segments (T2 and T3) of the length of spinal cord from which preganglionic fibres derive; an intermediate number of ganglion cells were innervated by fibres from the segments adjacent to these (T1, T4, and T5), and relatively few neurones by fibres from the most rostral and caudal segments supplying innervation to the ganglion (C8, T6 and T7). 3. Each neurone received preganglionic terminals from multiple thoracic segments (range 1-7, mean = 4-0). The estimated minimum number of preganglionic fibres contacting each neurone was 10, on average. 4. As a rule, the spinal segments innervating a neurone were contiguous. Thus we rarely encountered neurones innervated by segments located both rostrally and caudally to a segment which failed to provide innervation. 5. Neurones tended to be innervated predominantly by axons arising from a single spinal segment, with adjacent segments contributing a synaptic influence that diminished as a function of their distance from the dominant segment. All segments provided dominant innervation to at least some neurones. 6. Stimulating the ventral roots of C8-T7 in vivo showed that the axons arising from each segment produced a characteristic pattern of peripheral effects. Thus different populations of neurones in the superior cervical ganglion of the guinea-pig are innervated by preganglionic axons from different levels of the spinal cord, as originally suggested by Langley (1892) for the cat, dog, and rabbit. 7. On the basis of our in vitro studies we conclude that underlying the specificity of innervation of neurones of the superior cervical ganglion that can be inferred from in vivo experiments is a tendency for individual neurones to be innervated in a systematically graded fashion by a contiguous subset of the eight spinal segments which provide innervation to the ganglion.     

Adrenergic re-innervation of smooth muscle of nictitating membrane by preganglionic sympathetic fibres

1. The preganglionic and post-ganglionic trunks of the cervical sympathetic nerve were joined in an end-to-end anastomosis after excision of the superior cervical ganglion in the cat.2. Seventy-five days after the anastomosis the diameter of the pupil was nearly normal and there was almost complete recovery of the prolapsed palpebra and of the nictitating membrane. The contraction of the nictitating membrane, induced by electrical stimulation, caudally to the point of anastomosis, showed that the smooth muscle of the nictitating membrane had been re-innervated.3. Neither hexamethonium nor nicotine had any marked effect on the contraction of the nictitating membrane. Severing the regenerated nerve trunk produced a degeneration contraction. These results are strong evidence that the denervated membranes were re-innervated by true cholinergic preganglionic fibres.4. Our pharmacological studies indicated that in the re-innervated preparations neuromuscular transmission was adrenergic in the sense that it was blocked by phentolamine and not by atropine. These results were confirmed by the histochemical-fluorescence studies which showed that the endings of the regenerated axons contained high concentrations of catecholamines.5. Electron microscopy showed that the regenerated terminals contained none of the small dense-core vesicles, considered to be typical of adrenergic nerve endings, but contained clear synaptic vesicles and an unusually great number of large granular vesicles.6. Our results suggest that the denervated nictitating membranes were re-innervated by cholinergic presynaptic sympathetic fibres that had been modified so that they could release catecholamines in addition to, or instead of, acetylcholine.     

Re-innervation of guinea-pig superior cervical ganglion cells by preganglionic fibres arising from different levels of the spinal cord

1. The ability of preganglionic axons to re-establish their normal pattern of synaptic connexions with superior cervical ganglion cells has been studied after section of the cervical sympathetic trunk.

2. In vivo stimulation of the last cervical (C8) and the first seven thoracic ventral roots (T1—T7) 3-4 months after section of the trunk produced end-organ responses similar to those observed in normal animals.

3. The pattern of innervation of individual neurones, determined by intracellular recording of synaptic potentials 4-9 months after cutting the sympathetic trunk, was also similar to that observed in normal neurones. Both normal and re-innervated ganglion cells were contacted by pre-ganglionic axons arising from C8 to T7, and each neurone was usually innervated by a contiguous subset of these segments.

4. Re-innervated neurones, as normal cells, were typically dominated by the innervation from a particular spinal cord segment, with the adjacent segments contributing a synaptic influence that decreased as a function of distance from the dominant segment. This was true whether the amplitude of the post-synaptic potential, or the estimated number of contributing axons, was used as the criterion of segmental dominance.

5. Re-innervated neurones, however, showed some abnormalities. The average number of ventral roots contributing innervation to each neurone was reduced from 4·1 to 3·0, and discontinuities in the sequence of innervating segments were more frequent than in normal neurones. Moreover, fewer preganglionic axons contacted each neurone after regeneration.

6. A further difference between normal and re-innervated neurones during the period covered by these experiments was that axons from the more caudal spinal cord segments were less successful in re-establishing contacts with ganglion cells than those from the rostral segments. The more caudal the position of the preganglionic neurones, the more pronounced was this relative deficiency.

7. Although anomalies of ganglion cell innervation were apparent, the basis for the restoration of normal functional effects appears to be the re-establishment of a pattern of innervation of individual neurones similar to that observed in normal ganglia.

     

Competitive and non-competitive re-innervation of mammalian sympathetic neurones by native and foreign fibres.

The ability of native (sympathetic preganglionic) and foreign (vagal) nerve fibres to re-innervate neurones of the guinea-pig superior cervical ganglion, either alone or in competition with each other, has been studied by means of intracellular recording and electron microscopy. 1. Native fibres make synaptic contacts with nearly all ganglion cells within one month of cervical trunk section; within 6 months the degree of innervation, judged by measurement of excitatory post-synaptic potential (e.p.s.p.) amplitude and electron microscopical synapse counts, approaches normal. However, even after 15 months innervation was weaker than in normal control ganglia. 2. Vagal fibres are less successful during re-innervation. Although a similar number of foreign fibres grown into denervated ganglia and make contact with nearly all ganglion cells within a month, after 6-12 months e.p.s.p. amplitudes in response to foreign nerve stimulation remain relatively small, and counts of synapses are only about 60% as great as in ganglia re-innervated with the native nerve. 3. When both native and foreign fibres are allowed to re-innervate ganglion cells simultaneously, about half the neurones in the ganglion receive synapses from both sources after 1 month. The proportion of dually invervated cells remains roughly constant for at least 14 months. Neither set of preganglionic fibres dominates or displaces the other, although neurones generally are re-innervated more effectively by native than foreign fibres, as is true during non-competitive re-innervation. 4. Thus during re-innervation of mammalian sympathetic neurones native fibres are preferred to foreign ones only in the sense that roughly the same number of native fibres form many more synapses on ganglion cells than do vagal axons. A foreign synapse, once formed, is as stable as a native one, and shows no tendency to be replaced by native terminals. These findings are discussed in relation to other evidence which has suggested specificity and selectivity during re-innervation of mammalian autonomic neurones.     

The number and distribution of sympathetic neurons that innervate the rat pineal gland

The sympathetic innervation of the rat pineal gland was examined using a variety of anatomical techniques. Following the injection of horseradish peroxidase into the pineal gland, approximately 250 labeled neurons were found in the ipsilateral superior cervical ganglion. No labeled neurons were found in the middle or inferior cervical ganglia. In animals whose left internal carotid nerve was lesioned prior to the injection of peroxidase, an average of only three labeled neurons was found in the ipsilateral superior cervical ganglion. These data suggest that most, if not all, of the sympathetic neurons innervating the pineal gland exit from the superior cervical ganglia via the internal carotid nerves. The distribution of sympathetic neurons innervating the pineal gland was similar, though slightly more rostrally placed, than the distribution of the entire population of superior cervical ganglion neurons which project into the internal carotid nerve. Both the small number of neurons innervating the pineal gland and their wide distribution in the rostral part of the superior cervical ganglion indicate that their study at the level of the ganglion would be difficult.Sympathetic axons reach the pineal gland via the nervi conarii. Electron microscopic studies indicate that in each nervus conarii there are about 440 axons which make contact with the surface of the pineal gland. In certain cases, bundles of axons from the left and right nervi conarii were found to fuse. Additional evidence for the intermingling of axons from the two nervi conarii was seen in orthograde transport studies with horseradish peroxidase.     

Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells

Summary Beginning prenatally and during the first week after birth, there is normally a loss of axons in rat cervical sympathetic trunk. To test the hypothesis that this spontaneous axonal loss represents a natural process whereby an excessive number of immature preganglionic axons in the cervical sympathetic trunk adapts to the neuronal population in the superior cervical ganglion, the number of nerve cells in the superior cervical ganglion was reduced in newborn rats by administration of nerve growth factor antiserum, 6-hydroxy-dopamine or postganglionic axotomy. Quantitative ultrastructural studies of these animals at later stages of development revealed that, with each method, the number of preganglionic axons and Schwann cells was reduced to nearly one-third of normal. These findings indicate that the superior cervical ganglion plays an important role in the development of the cervical sympathetic trunk. Removal of ganglionic cells causes a retrograde loss of preganglionic fibres. This process probably represents an exaggeration of the normal mechanism for elimination of redundant axons. Because the changes in axonal numbers are associated with similar reductions in the number of Schwann cells, it can also be concluded that postnatal Schwann cell proliferation is influenced by axonal populations.     

The reorganization of synaptic connexions in the rat submandibular ganglion during post-natal development.

1. The innervation of neurones in the submandibular ganglion of neonatal and adult rats has been studied with intracellular recording, and light and electron microscopy. 2. Intracellular recordings from neurones in isolated ganglia from adult animals showed that about 75% of the ganglion cells are innervated by a single preganglionic fibre. 3. However multiple steps in the post-synaptic potential (about five on average) were elicited in ganglion cells from neonatal animals by graded stimulation of the preganglionic nerve. The same result was obtained when the preganglionic fibres were stimulated at their emergence from the brainstem, indicating that neonatal neurones are innervated by several different preganglionic nerve cells. 4. The number of preganglionic fibres innervating individual ganglion cells gradually decreased during the first few weeks of life, and by about 5 weeks each ganglion cell was generally contacted by a single preganglionic axon. 5. Synapses were made on short protuberances in the immediate vicinity of the neuronal cell bodies in both neonatal and adult ganglia as shown by staining presynaptic boutons with the zinc-iodide osmium method, injection of horseradish peroxidase into ganglion cells, and electron microscopical examination. 6. Electron microscopical counts of synaptic profiles per ganglion cell perimeter showed that the number of synaptic contacts made on ganglion cells actually increased during the first few post-natal weeks, when the number of axons innervating each neurone was decreasing. 7. These results show that in the rat submandibular ganglion there is a reorganization of neuronal connexions during the first few weeks of life which results in a transition from multiple to generally single innervation of ganglion cells.     

Presence of neurons with GABA-like immunoreactivity in the superior cervical ganglion of the rat

Using antibodies raised against gamma-aminobutyric acid (GABA)-glutaraldehyde complexes, we have found neurons with GABA-like immunoreactivity in the superior cervical ganglion of adult rats. The processes of these neurons formed pericellular networks around the principal ganglion cells. Electron microscopy revealed that the immunoreactive dendrites were innervated by non-reactive axon terminals which formed asymmetrical synapses and probably originated from the preganglionic nerve. Axons with GABA-like immunoreactivity, especially axonal varicosities filled with synaptic vesicles, were found in direct apposition to principal ganglion cells. The GABA-positive axons and axon varicosities persisted in experimentally decentralized (deafferented) ganglia, suggesting that the perikarya of the immunoreactive neurons were intrinsic to the superior cervical ganglion. Taken together with data on inhibitory effects of GABA in sympathetic ganglia, these findings suggest that the superior cervical ganglion of rats contains a subpopulation of inhibitory interneurons which is GABAergic. This would indicate that GABAergic neurons do not only occur in the central but also in the peripheral nervous system.     

Re-innervation of twitch and slow muscle fibres of the frog after crushing the motor nerves.

1. The conduction velocities of individual motor axons innervating twitch and slow muscle fibres of the frog were determined by intracellular recording of junctional potentials elicited by stimulating the motor nerves at two different points. 2. In normal pyriformis muscles twitch and slow fibres were found to be innervated by two distinct populations of motor axons. Twitch fibre axons conducted at 10-18-7 m/sec, while the conduction velocities of slow fibre axons ranged from 0-5 to 5 m/sec (at 7-9 degrees C). The thresholds for electrical stimulation were significantly lower in the fast than in the slow axons population. 3. Following denervation by crushing the sciatic nerve fast axons which re-innervated the muscle had lower conduction velocities than normal but could still be identified. These lower conduction velocities were measured proximal to the site of the crush and did not recover over a period of 446 days. 4. Fast motor axons regenerated more quickly than slow axons and re-innervated twitch as well as slow muscle fibres non-selectively. About 1 month later slow axons re-established synaptic contacts with slow (and some twitch) muscle fibres. Simultaneous re-innervation by fast and slow motor axons was occasionally observed in slow muscle fibres. Finally, the slow muscle fibres were innervated by slow axons only, while synapses of fast axons could no longer be found in this type of muscle fibre. 5. Action potentials were observed in denervated as well as in re-innervated slow muscle fibres; they disappeared as re-innervation progressed. 6. It is concluded that non-selective re-innervation of slow muscle fibres is present in the frog; it is, however, a transient phenomenon followed by restoration of the original innervation pattern.     

Pre and postganglionic innervation of rat adrenal gland by fluorescent tract tracer – Fast blue

IntroductionTo search for pre and postganglionic neurons innervating the adrenal gland by injecting retrograde tract tracer fast blue in the adrenal medulla.MethodsThe motor innervation of rat adrenal gland was studied by a fluorescent tract tracer fast blue. 5 μl of 2% aqueous suspension of fast blue was injected into left adrenal gland. After a survival period of 4–5 days, spinal cord, sympathetic ganglia, suprarenal ganglion, coeliac ganglion and left adrenal gland were dissected out and 15 μm thick plastic sections (JB4 Polysciences) were examined under a fluorescent microscope.ResultsRetrogradely labeled preganglionic neurons were observed in the ipsilateral intermediolateral column of spinal cord from T3 to L2 spinal segments with maximum concentration of labeled neurons from T6 to T11. The labeled neurons were multipolar, spherical or fusiform in shape with transverse diameter 10–20 μm and vertical diameter varying from 12 to 30 μm. Postganglionic labeled neurons were also observed in the left suprarenal ganglion and left sympathetic ganglia (T5 –L2) with maximum concentration from T6 to L1. Labeled neurons varied from 12 to 30 μm in diameter and were randomly distributed throughout the ganglion.DiscussionThe preganglionic neurons from T3 to L2 spinal segments and postganglionic nerve fibers from ipsilateral sympathetic ganglia (T5 –L2) and suprarenal ganglion supplying the adrenal gland might be responsible for the hormone release by regulating blood flow and also by directly innervating the parenchymal cells.     

[Met5]enkephalin-Arg6-Phe7- and [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibres and neurons in the superior cervical ganglion of the rat

[Met⁵]enkephalin-Arg⁶-Phe⁷- (MEAP-) and [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸- (MEAGL-) immunoreactivity was studied by indirect immunohistochemistry in the superior cervical ganglion of the rat with specific antisera produced in rabbits against the corresponding synthetic opioid peptides. Several MEAP- and a few MEAGL-immunoreactive principal nerve cells were observed in the ganglion, while the small intensely fluorescent cells appeared as non-reactive. The superior cervical ganglion also contained dense networks of MEAP- and MEAGL-immunoreactive nerve fibres, which often formed basket-like structures around the principal nerve cells and small intensely fluorescent cells. After ligation of the preganglionic nerve trunk with simultaneous transection of the main postganglionic trunks, a distinct accumulation of both MEAP- and MEAGL- immunoreactivity was observed on both sides of the ligature. Ligation of the preganglionic nerve trunk caused a marked decrease in the number of both MEAP- and MEAGL-immunoreactive nerve fibres in the ganglion. Ligation of the main postganglionic nerve trunks with simultaneous preganglionic nerve division resulted in accumulation of MEAP- and MEAGL-immunoreactive material on the ganglionic side of the ligature in both the external and internal carotid nerve. After division of both the pre- and postganglionic nerve trunks, some immunoreactive nerve fibres and principal nerve cells were still observed in the ganglion. A few immunoreactive neurons and nerve fibres were also observed in the ganglion stellatum. A large number of MEAP- and MEAGL-immunoreactive nerve fibres was detected in the spinal cord at the levels C6-Th6. A few neurons in the intermediolateral cell column of the spinal cord at levels C8-Thl showed MEAP- but not MEAGL-immunoreactivity. The cultured superior cervical ganglion contained a few MEAP-immunoreactive neurons, and the fibre outgrowth showed immunoreactivity both to MEAP and MEAGL. In electron microscopy, MEAGL-immunoreactivity in the superior cervical ganglion was localized in nerve fibres containing neurotubules and in principal nerve cells.The present results demonstrate that the rat superior cervical ganglion contains both extrinsic and intrinsic MEAP- and MEAGL-immunoreactive nerve fibres. Most of these fibres are of preganglionic origin. Both the principal nerve and small intensely fluorescent cells are often surrounded by MEAP- or MEAGL-immunoreactive nerve fibres and may receive innervation by these fibres. Several ganglionic neurons projecting to the sympathetic target tissues show MEAP- and/or MEAGL-immunoreactivity. Since MEAP- and MEAGL sequences are included in the proenkephalin A molecule, but not in the other opioid precursor molecules, it is evident that a neuronal system containing proenkephalin-A-derived enkephalins, is present in the rat superior cervical ganglion.     

gamma-Aminobutyric acid enables synaptogenesis in the intact superior cervical ganglion of the adult rat

Local gamma-aminobutyric acid (GABA) application into the intact superior cervical ganglion (SCG) of the adult rat allows active innervation of a surgically implanted hypoglossal nerve in addition to the normal nerve supply of the ganglion. In GABA-treated SCG of the adult rat, action potentials could be obtained on stimulation of both the preganglionic nerve trunk and the implanted hypoglossal nerve. Both action potentials were reversibly sensitive to hexamethonium bromide indicating new cholinergic synapses established between axons in the hypoglossal nerve and principal sympathetic neurons. If GABA treatment of the ganglion was omitted, the double innervation did not develop after hypoglossal nerve implantation.     

Nicotine-induced fos-like immunoreactivity in rat sympathetic ganglia and adrenal medulla

The expression of c-fos proteins (Fos) in principal sympathetic neurons and adrenal chromaffin cells was studied immunocytochemically after a single s.c. injection of nicotine (2mg/kg). One and 2 h after injection most of the adrenal chromaffin cells showed nuclear Fos-like immunoreactivity (Fos-IR). In the superior cervical ganglion more than 80% of the neuronal nuclei were Fos-IR 2-5 h after nicotine administration, whereas only 50-60% of the neurons in the hypogastric ganglion were immunoreactive at these time points. The results suggest that nicotinic receptor mediates signal transduction, which induces c-fos expression both in sympathetic neurons and in adrenal chromaffin cells in vivo.     

Preganglionic fibres of the adrenal medulla and cervical sympathetic ganglia: differential involvement during experimental American trypanosomiasis in rats

The acute phase of experimental Chagas' disease in rats is associated with early lesions of the post-ganglionic sympathetic nerve terminals of the heart, the varicosities being the target. In the superior cervical and stellate ganglia the preganglionic fibres showed no signs of lesion in the course of experimental Chagas' disease. In the adrenal medulla, however, the acute phase of the Trypanosoma cruzi infection induced a clear rarefaction of the acetylcholinesterase-positive nerve fibres (20 and 32 days after infection). Recovery of the normal pattern occurred in most animals by day 125 after infection. At days 20, 32 and 46 after infection, electron-microscopic studies demonstrated the occurrence of damage in cholinergic nerve terminals contacting the chromaffin cells. The signs of damage included dense bodies, clumps of synaptic vesicles and filaments, rarefaction of all organelles, vacuoles and irregular contour. The ultrastructural peculiarities of the sympathetic ganglia may explain the ganglionar microenvironment protective against the hazardous factors elicited by acute Chagas' disease.     

Sympathetic neurons in lower cervical ganglia send axons through the superior cervical ganglion

In addition to the preganglionic axons which innervate the superior cervical ganglia, the cervical sympathetic trunks of the rat have been shown to contain axons of ganglionic neurons. Following the application of horseradish peroxidase to the cut cervical sympathetic trunk just caudal to the superior cervical ganglion, a population of approximately 300 labeled neurons was found in the inferior and middle cervical ganglia. The labeled neurons were localized primarily in the more rostral regions of these ganglia. The axons of most of these neurons entered the superior cervical ganglion, passed through it, and left via the external carotid nerve.The relevance of these observations to physiological studies on the cervical sympathetic nervous system is discussed     

Neonatal nerve growth factor treatment alters the preganglionic innervation pattern of rat superior cervical ganglion.

We treated rat pups with nerve growth factor (10 micrograms/animal/day s.c.) over postnatal days 1-7. Subsequent adult neuron numbers and tyrosine hydroxylase content in superior cervical ganglion were normal, but preganglionic inputs, as gauged from ganglionic choline acetyltransferase, were reduced. In parallel, intraganglionic axon terminals containing calcitonin gene-related peptide, but not those containing substance P, were increased in number. We postulate that neonatal nerve growth factor stimulates sprouting of ingrowing axons that have entered the ganglion soon after birth and that this represses subsequent establishment of cholinergic preganglionic synapses.     

Viability of adrenal chromaffin cells in the superior cervical ganglion of young adult and aged rats

Adrenal medullary tissue was autotransplanted to the superior cervical ganglion (SCG) of aged (26 months old) and young adult (3 months old) rats. Four and 20 weeks after operation, the viability of the transplants was evaluated using the formaldehyde-induced fluorescence (FIF) technique and tyrosine hydroxylase (TH) immunocytochemistry. Four weeks postgrafting, the transplant consisted of a densely-packed group of intensively fluorescent chromaffin cells in both age groups. The cells showed strong TH immunoreactivity and some of them were elongated, but only a few displayed short processes. At 20 weeks, most of the cells were spindle shaped and sent out fluorescent processes and a few of them were transformed toward ganglion-like cells. The results suggest that both young and old adrenal chromaffin cells are able to survive, produce neuronal processes and transform toward a neuronal phenotype in the rat superior cervical ganglion.     

Calcitonin gene-related peptide-like immunoreactivity in spinal cord and superior cervical ganglion of the djungarian hamster (Phodopus sungorus)

The indirect immunofluorescent method was employed to investigate the distribution of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the spinal cord and superior cervical ganglion of the Djungarian hamster Phodopus sungorus. In cross-sections of the spinal cord, immunoreactive fibres and terminals were found in laminae 1 and 2 in high density, in the dorsolateral (Lissauer's) tract, in ventral and lateral horns, and in the area surrounding the central canal. A few CGRP-LI perikarya were seen in the ventral but not the dorsal horn. CGRP-LI was further observed in preganglionic sympathetic neurons which were labelled by retrograde axonal transport of fluoro-gold (FG) following injection of the substance unilaterally into the superior cervical ganglion. Preganglionic sympathetic neurons (PSN) were localized ipsilateral to the injection site mainly in the intermediolateral nucleus and the lateral funiculus of the upper thoracic segments. Most PSN exhibited CGRP-LI. Immunoreactive PSN were not seen contralaterally to the site of FG application nor in animals that did not receive injections. When the preganglionic fibres were ligated 4 days before perfusion. CGRP-LI cell bodies were found in preganglionic sympathetic neurons similar to the situation seen upon FG treatment.

In the superior cervical ganglia of untreated hamsters, immunoreactive fibres were seen to enter the ganglion in which they terminated at non-immunoreactive principal ganglion cells.

The present study, the first in a hamster species, describes the widespread distribution of CGRP in the spinal cord of P. sungorus and supports the view that considerable interspecies differences exist in occurrence and location of this neuropeptide.     

Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities.

Physiological studies have established that preganglionic sympathetic nerve fibers innervating the rat superior cervical ganglion release a second transmitter, in addition to acetylcholine. Based on pharmacological and histochemical investigations, possible candidates for this non-cholinergic neurotransmitter include vasoactive intestinal peptide and peptide histidine isoleucine amide. For example, previous immunohistochemical studies have demonstrated that antisera raised against both of these peptides stain neural processes in the rat preganglionic cervical sympathetic trunk and in the superior cervical ganglion. In the present study, it was found that, when the cervical sympathetic trunk was ligated, vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities built up on both sides of the ligature. In addition, examination of the thoracic spinal cord in colchicine-treated animals revealed vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivies in neuronal cell bodies in the intermediolateral cell column and in the region of the lateral funiculus adjacent to it. In a second group of animals in which retrograde tracing techniques were used, these two regions of the spinal cord were shown to contain most of the cell bodies of the preganglionic neurons that project to the superior cervical ganglion. Smaller numbers of retrogradely labeled neurons were found dorsal to the central canal and in the nucleus intercalatus. When either vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunostaining and retrograde labeling were examined in the same animals, double-labeled neurons were found in the intermediolateral cell column and in the lateral funiculus. These data demonstrate that vasoactive intestinal peptide- and peptide histidine amide-like immunoreactivities are present in certain of the preganglionic neurons that project to the superior cervical ganglion, supporting the hypothesis that vasoactive intestinal peptide and peptide histidine isoleucine amide are released in the ganglion when these preganglionic neurons are activated.