Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons.

Studies combine the use of the retrograde tracer, fluorogold, and immunocytochemical staining to determine whether superior cervical ganglion (SCG) neurons projecting to the iris or submandibular gland (SMG) in adult male and female rats show distinctive immunoreactivity to somatostatin (SS), vasoactive intestinal polypeptide (VIP), or neuropeptide Y. Overall, more SMG-projecting neurons than eye-projecting neurons contain VIP-like immunoreactivity (VIP-LI), and more eye-projecting neurons than SMG-projecting neurons contain SS-LI and VIP-LI. Thus, postganglionic neurons of the SCG that project to specific target tissues are heterogeneous in their peptide content, and there are differences in the pattern of peptide-immunoreactivity between neurons projecting to these two target tissues. In addition, the results indicate that there may be gender differences in the expression of these neuropeptides.

The allocation of nerve fibres to the anterior eye segment and peripheral ganglia of rats. II. The sympathetic innervation.

The sympathetic innervation of the peripheral ganglia related to the eye, i.e. the trigeminal ganglion, the ciliary ganglion and the pterygopalatine ganglion, and of the anterior eye segment was studied in rats. Selective labelling of sympathetic nerves was obtained by means of injection of [3H]leucine into the superior cervical ganglion. Bundles of sympathetic nerve fibres were found in the trigeminal ganglion and the pterygopalatine ganglion but were absent in the ciliary ganglion. In addition individual sympathetic nerve fibres, which may have contacts with trigeminal ganglion cells, were found between the ganglion cell bodies all over the trigeminal ganglion indicating a sympathetic innervation of this ganglion. In the anterior eye segment, there appeared to be a sympathetic innervation of the ciliary cleft, the ciliary body and the iris. Within the ciliary body sympathetic nerve fibres innervate the central stroma and the stroma of the ciliary processes. Labelled sympathetic nerve fibres were also observed in the stroma of the iris and were most abundant in its periphery. Most sympathetic fibres reach the iris and ciliary body by way of the base of the ciliary body. Only few sympathetic fibres are present in the ciliary cleft. No sympathetic innervation of the cornea was found.

Spinally projecting noradrenergic neurons of the dorsolateral pontine tegmentum: a combined immunocytochemical and retrograde labeling study.

Two to three days following injection of horseradish peroxidase (HRP) into the spinal cords of 5 cats, the animals were sacrificed and perfused, and the brainstems removed and sectioned. The sections were then processed for HRP and, immunocytochemically, for tyrosine hydroxylase (TH). The dorsolateral pontine tegmentum was divided into the locus coeruleus, subcoeruleus and Kölliker-Fuse nucleus; the mean percentage of pontospinal neurons containing TH were found to be 85.5 +/- 2.5 (S.E.M.), 79.6 +/- 5.6 and 87.1 +/- 3.1, respectively. The cell diameters of locus coeruleus cells were also measured.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus.

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labeling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T4-T13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactive phenylethanolamine-N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves.

The innervation of the guinea pig trachea was studied in wholemount preparations stained for acetylcholinesterase, catecholamines, and substance P immunoreactivity and by electron microscopy. The majority of parasympathetic and afferent nerve fibres arrive from the vagus via branches of the recurrent laryngeal nerves. The recurrent laryngeal nerves are composed of several fascicles comprising 600-700 small myelinated fibres (2-5 microns diameter) and about 1,000-2,000 unmyelinated fibres; both components exit from the nerve and project in fine branches to the trachea. A separate component of 200-250 large myelinated fibres (more than 5 microns diameter) runs the full length of the nerve and innervates the striated muscles of the larynx. The recurrent laryngeal nerves are slightly asymmetric in their origin, length, number, and composition of fibres, with the right nerve being shorter but with more numerous and thinner myelinated fibres. At the distal end of the recurrent nerve, a fine branch called the ramus anastomoticus connects it to the superior laryngeal nerve. In the tracheal plexus, there are on average 222 ganglion cells (range 166-327), distributed mostly in small ganglia of 12 or fewer neurons. The ganglionated plexus is situated entirely outside the tracheal wall, overlying the smooth muscle. Ligation experiments show that sympathetic nerve fibres reach the trachea with the recurrent nerves via anastomoses between the sympathetic chain and vagus nerves, or occasionally with recurrent nerves directly, the largest being at the level of the ansa subclavia. There are also perivascular sympathetic nerve plexuses. Substance P immunoreactive fibres enter the trachea from the vagus nerves and by pathways similar to those of sympathetic nerves. There are also paraganglion cells within the recurrent laryngeal nerve that contain catecholamines and are surrounded by substance P immunoreactive fibres. After cervical vagotomy, all the large myelinated fibres of the ipsilateral recurrent laryngeal nerve degenerate and so do all but 10 or 20 small myelinated fibres and all but a few unmyelinated fibres. Degenerating fibres are found within the entire tracheal plexus, indicating bilateral innervation. The small myelinated fibres that survive cervical vagotomy probably represent sympathetic or afferent nerves with their cell bodies located in sympathetic or dorsal root ganglia.

Glucocorticoid receptor-immunoreactivity in C1, C2, and C3 adrenergic neurons that project to the hypothalamus or to the spinal cord in the rat.

A combined retrograde transport-double immunohistochemical staining method was used to determine the extent to which rat liver glucocorticoid receptor-immunoreactivity (GR-ir) is contained within phenylethanolamine-N-methyltransferase (PNMT)-ir neurons that project to the paraventricular nucleus of the hypothalamus (PVH) or the spinal cord. The results confirmed that cells in the C1, C2, and C3 adrenergic cell groups each contribute to the adrenergic innervation of the PVH, and indicated that the great majority of retrogradely labeled neurons in each group (80% overall) also express GR-ir. Following injections in the upper thoracic segments of the spinal cord, the bulk of adrenergic neurons that were retrogradely labeled were found in the C1 cell group, though 31% of the total number PNMT-ir cells that could be retrogradely labeled following spinal injections were localized in the C2 and C3 regions. Of these spinally projecting PNMT-ir neurons, 62% displayed GR-ir. The results suggest all three medullary adrenergic cell groups contribute projections to the spinal cord and/or the PVH, and that the capacity to express the GR phenotype is a common, though perhaps not universal, attribute of PNMT-ir neurons. No pronounced differences in the expression GR-ir were observed in adrenergic neurons as a function of their location or efferent projections. Brainstem adrenergic neurons may play a role in integrating neuronal and hormonal controls of adrenal function via ascending and descending projections.

Aged adrenal medullary tissue survives intraocular grafting, forms nerve fibers and responds to nerve growth factor.

Adrenal medullary tissue from aged (24 months old) and young adult (2 months old) rats was grafted to the anterior chamber of the eye of previously sympathectomized animals. Nerve growth factor (NGF) was administered by weekly bilateral intraocular injections. Five weeks postgrafting, irides were prepared as whole mounts and processed for Falck-Hillarp histochemistry for visualization of catecholamines. NGF appeared to partially prevent the reduction in volume that both old and young grafts underwent. In the presence of NGF, an extensive, dense fiber network, closely resembling the normal adrenergic innervation, was formed in the host irides by grafts from aged donors. The area of outgrowth from aged transplants without NGF treatment was as large as with NGF treatment but less dense. The reinnervation of irides by NGF-treated young adult grafts occupied a similar area as that seen with aged grafts, but the pattern of innervation was irregular, particularly close to the transplants. Transplants from young adult donors without NGF treatment generated a sparse, limited network of nerves in the irides. All grafts were tyrosine hydroxylase-, adrenaline-, and dopamine-beta-hydroxylase-immunoreactive in about the same proportion of cells, but the grafts from the young donors were smaller in size. We concluded that the ability of chromaffin cells to transform toward a neuronal phenotype, produce nerve fibers, and respond to exogenous NGF is maintained in aged adrenals.

Neuronal galanin is widely distributed in the chicken respiratory tract and coexists with multiple neuropeptides.

The mammalian airways are known to be richly innervated by several types of peptide-containing nerve fibers. Galanin-containing fibers are, however, comparatively few. The results of the present immunocytochemical study indicate that the chicken airways receive a notably dense supply of galanin-storing fibers. Other major neuropeptides were neuropeptide Y, vasoactive intestinal peptide and substance P. Nerve fibers containing these peptides were distributed in the trachea, main bronchi, and the lungs. Minor nerve fiber populations contained calcitonin gene-related peptide, enkephalin and gastrin-releasing peptide. In the trachea and main bronchi the majority of peptide-containing nerve fibers was distributed beneath and sometimes also within the epithelium; fibers were fewer in the lamina propria. In the lungs they occurred both in association with the epithelium of small bronchi and in the septa. Adrenergic nerves (using tyrosine hydroxylase as marker) were predominantly distributed in the lamina propria among bundles of smooth muscle and blood vessels. In the nerve fibers associated with the epithelium and in nerve cell bodies in local ganglia of the tracheal wall, galanin was found to coexist with several other neuropeptides (neuropeptide Y, vasoactive intestinal peptide and substance P) suggesting co-expression of multiple neuropeptide genes in the same population of neurons.

Adrenergic nerves and 5-hydroxytryptamine-containing cells in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus.

The adrenergic innervation of the pulmonary vasculature of the file snake Acrochordus granulatus was examined by use of glyoxylic acid-induced fluorescence. Perivascular plexuses of blue-green fluorescent nerves are observed around the common pulmonary artery, the anterior and posterior pulmonary arteries, the arterioles leading to the gas exchange capillaries of the lung, the venules draining the lung, and the anterior and posterior pulmonary veins. Adrenergic nerves are also associated with the visceral smooth muscle of the lung septa and other tissues. Thus, adrenergic control of pulmonary blood flow may occur either at the common pulmonary artery or more regionally within the lung. Regional control of blood flow in the elongate lung of this snake may be important in matching pulmonary perfusion with the distribution of respiratory gas. Glyoxylic acid-histochemistry and immunohistochemistry revealed that populations of cells located in the common pulmonary artery contain the indoleamine 5-hydroxytryptamine. Many of the cells are intimately associated with varicose blue-green fluorescent nerves. It is proposed that the 5-hydroxytryptamine-containing cells may be involved in intravascular chemoreception.

Age-dependent changes in the capacity of rat sympathetic neurons to form dendrites in tissue culture.

We compared the ability of prenatal and postnatal rat sympathetic neurons to form dendrites in tissue culture. Dendrites were distinguished from axons by light microscopic criteria after intracellular dye injection and by differential immunostaining with antibodies to microtubule-associated protein-2 and to both non-phosphorylated and phosphorylated forms of the M and H neurofilament subunits. When maintained in the absence of serum and non-neuronal cells, most (72%) prenatal neurons were unipolar and had only an axon. In contrast, most (89%) neurons derived from postnatal ganglia were multipolar and extended both axons and dendrites. The dendritic morphology of postnatal neurons was usually simple with cells commonly having 2-5 short (50-200 microns), relatively unbranched dendrites. Thus, as the development of the dendritic arbor progresses in situ, sympathetic neurons acquire an enhanced ability to extend dendrites in tissue culture. To determine whether changes in the capacity to develop dendrites might occur with aging in vitro, ganglia were removed from prenatal rats and grown as explants for 3 weeks in the presence of non-neuronal cells; under these conditions, prenatal neurons within the explant became multipolar. When neurons derived from aged explants were subsequently maintained in dissociated cell culture, most formed dendrites. In cultures treated with an antimitotic agent, neurons typically had 1-4 unbranched dendrites; greater amounts of dendritic growth occurred in cultures in which ganglionic non-neuronal cells were allowed to proliferate. We conclude that: (1) the acquisition of the capacity to form dendrites in dissociated cell culture does not require either normal afferent input or physical contact with the target tissue; and (2) even after aging in vitro, sympathetic neurons remain responsive to the dendrite-promoting activity of ganglionic non-neuronal cells.

Light and electron microscopic immunocytochemical analysis of the noradrenaline innervation of the rat visual cortex.

Immunocytochemistry with an antiserum against noradrenaline was used to examine the organization and morphology of noradrenergic axons in the rat visual cortex. Observations with the light microscope confirmed earlier reports concerning the distribution pattern of noradrenergic fibres, and provided some further clues about their intracortical organization. Particularly striking was the finding of fibres which followed an oscillating course within the boundaries of layers II-IV as they ran in the mediolateral direction. Examination of the morphological characteristics of noradrenaline-containing axon terminals in serial ultrathin sections has provided further evidence that the vast majority (87.6%) form conventional synapses in the visual and frontoparietal cortex, and has given clues about the postsynaptic elements involved in these synaptic contacts; they are, in decreasing frequency, spines, dendritic shafts of various diameters, and pyramidal and non-pyramidal somata. In addition, a few labelled terminals were visualized in close association with intracerebral capillaries.

The amygdala directly innervates adrenergic (C1) neurons in the ventrolateral medulla in the rat.

The innervation of adrenergic (C1) neurons in the ventrolateral medulla by the central amygdaloid nucleus (Ce) was investigated using immunohistochemical detection of phenylethanolamine N-methyltransferase (PNMT) combined with anterograde tracing through Phaseolus vulgaris leucoagglutinin (PHA-L) transport and lesion-induced axonal degeneration. Injections of PHA-L into the medial Ce labelled axons in close proximity to PNMT-immunoreactive dendrites and somata in the ventrolateral medulla. The PNMT-immunoreactive neurons within the rostral part of the nucleus reticularis rostroventrolateralis were preferentially innervated by the amygdaloid terminals. Degenerating terminals formed synaptic contacts on PNMT-immunoreactive cells of the ventrolateral medulla in animals with lesions of the Ce. The synaptic contacts were mainly found on dendrites and were usually of the symmetrical type. The present findings provide evidence that cells within the amygdala directly innervate presumed adrenergic cells in the ventrolateral medulla. This pathway may be part of the anatomical substrates that are activated during amygdaloid-mediated sympathetic activity.

Destruction of sympathetic and sensory neurons in the developing rat by a monoclonal antibody against the nerve growth factor (NGF) receptor.

The ability of the monoclonal antibody, 192-IgG, directed against the rat nerve growth factor (NGF) receptor to mimic or inhibit the actions of NGF was examined in vitro and in vivo. 192-IgG had no effect on morphology, survival, or protein synthesis rates of sympathetic neuronal cultures. When injected into newborn rats, destruction of sympathetic, but not sensory, neurons was produced. Injection prenatally produced more dramatic destruction of sympathetic neurons and, in addition, destruction of neural crest-derived sensory neurons. Therefore, although 192-IgG had no discernible effects in vitro, it produced a pattern of neuronal destruction in vivo qualitatively similar to that produced by antibodies to NGF itself.

Age-related alterations in noradrenergic input to the hippocampal formation: structural and functional studies in intraocular transplants.

Intrinsic versus extrinsic determinants of age-related alterations in hippocampal noradrenergic transmission were investigated using intraocular allografts in rats. Three groups of animals were examined: young hippocampal transplants in young hosts, old transplants in old hosts and young transplants in old hosts. Postsynaptic sensitivity to noradrenaline (NA) was measured by extracellular recordings of spontaneous activity and superfusion with known concentrations of catecholamines in the anterior chamber of the eye. Hill plots demonstrated that the dose-response relationships of NA-induced depressions were linear and parallel in the 3 groups. Aged hippocampal grafts displayed a highly significant subsensitivity to NA of one order of magnitude. The EC50 for this group was 203.1 microM as compared to 29.2 in young grafts. Young intraocular grafts in old hosts responded similarly to transplants in young hosts, with an EC50 of 32.4 microM for the depressant actions of NA. Collaterals of the host iris sympathetic ground plexus invaded the hippocampal grafts. The density of this noradrenergic innervation was estimated by immunohistochemistry for tyrosine hydroxylase. A slightly increased density and fluorescence intensity of the noradrenergic fibers were observed in the old transplants as compared to the young transplants in young and old hosts. This was correlated with a significantly (P less than 0.01) increased content of NA in old transplants, as measured with high performance liquid chromatography. The old transplants also contained a large number of autofluorescent lipofuchsin granules, which were absent in the young transplants, regardless of the recipient age. Taken together, these results suggest the existence of alterations in pre- as well as postsynaptic noradrenergic mechanisms in the aging hippocampus. These changes were dependent on transplant age rather than host age, thus suggesting an involvement of intrinsic rather than extrinsic determinants in this model system.

Electrophysiological characterization of putative C1 adrenergic neurons in the rat.

Recent studies in the rat have demonstrated that at least two populations of sympathoexcitatory reticulospinal neurons reside in the nucleus reticularis rostroventrolateralis. It appears that only one of these populations consists of C1 adrenergic neurons. The present study used both double-labeling (one retrograde tracer and immunohistochemistry) and triple-labeling (two retrograde tracers and immunohistochemistry) to determine if C1 adrenergic neurons, which are immunoreactive for phenylethanolamine N-methyltransferase, exhibit a projection pattern that is sufficiently unique to permit the electrophysiological discrimination between C1 adrenergic and non-adrenergic neurons in the nucleus reticularis rostroventrolateralis. Double-labeling experiments indicated that 71% (range: 53-80) of phenylethanolamine-N-methyltransferase-immunoreactive neurons in the nucleus reticularis rostroventrolateralis could be retrogradely labeled from the thoracic cord, as were 76% (range: 67-94) following tracer injection in the central tegmental tract at pontine levels. Triple-labeling experiments indicated that 88% (range: 82-93) of nucleus reticularis rostroventrolateralis neurons with projections to both spinal cord and central tegmental tract were phenylethanolamine-N-methyltransferase-immunoreactive. Single-unit recording, in nucleus reticularis rostroventrolateralis, was used to identify antidromic potentials elicted from stimulation sites in the spinal cord and/or central tegmental tract. Since clonidine is known to reduce central adrenaline turnover, sensitivity to this drug was used to identify putative adrenergic neurons. Twenty-six nucleus reticularis rostroventrolateralis neurons with axonal projections to both the ipsilateral spinal cord and the central tegmental tract were recorded in halothane-anesthetized rats. All these cells were barosensitive, pulse-modulated, and 16 of the 16 cells tested exhibited a 66 +/- 8% reduction in activity upon the intravenous administration of clonidine (20 micrograms/kg). Most (13 out of 16) exhibited a strong respiratory modulation. The conduction velocity of their spinal collateral was generally low (0.9 +/- 0.1 m/s) and their firing rate moderate (7.4 +/- 1.2 spikes/s). Forty-three nucleus reticularis rostroventrolateralis cells with axonal projections exclusively to the thoracic cord were studied for comparison. These cells were strongly barosensitive and pulse-synchronous, had a high discharge rate (25 +/- 3 spikes/s) and a moderate conduction velocity (3.4 +/- 0.3 m/s). Only one of the 15 cells tested was inhibited by clonidine and only two to these 15 cells exhibited a detectable respiratory modulation. Thus barosensitive nucleus reticularis rostroventrolateralis neurons with axonal projections to both the spinal cord and the central tegmental tract likely belong to the C1 adrenergic cell group. It is concluded that this subgroup of adrenergic neurons probably subserves a vasomotor function.

5-Hydroxytryptamine demonstrated immunohistochemically in rat cerebrovascular nerves largely represents 5-hydroxytryptamine uptake into sympathetic nerve fibres.

This study has re-examined, by immunohistochemistry, a proposed serotonergic innervation of major cerebral vessels in the rat. Previous studies had demonstrated a dense perivascular plexus of 5-hydroxytryptamine immunoreactive nerve fibres upon major cerebral vessels in this and many other species. The present work has shown, however, that 5-hydroxytryptamine immunoreactive nerve fibres are rarely observed in cerebral vessels prepared by perfusion-fixation in situ, and only form a well-developed plexus in vessels prepared, as in previous studies, by immersion-fixation. Prior treatment with a predominantly noradrenergic uptake inhibitor desmethylimipramine but not the serotonergic uptake inhibitor fluoxetine produced a major diminution in the 5-hydroxytryptamine immunoreactive plexus visualized in these immersion-fixed vessels. In addition, 5-hydroxytryptamine immunoreactive nerves were only occasionally observed in immersion-fixed vessels from animals that had been pretreated with 6-hydroxydopamine to produce adrenergic denervation. The removal, firstly, of vessel-contained blood, by left ventricular perfusion with Krebs' solution, prior to vessel dissection and immersion-fixation, resulted in an absence of 5-hydroxytryptamine immunoreactivity in perivascular nerves. Immunoreactivity could then be restored by briefly incubating vessels in Krebs' solution containing either blood or 5-hydroxytryptamine before fixation. It would appear therefore that 5-hydroxytryptamine is rarely present under normal circumstances in the perivascular nerves of major cerebral vessels, and that previous descriptions of a dense serotonergic nerve plexus represent 5-hydroxytryptamine in blood released during vessel dissection being taken up via the noradrenaline-uptake system into perivascular sympathetic nerves. The possibility is thus raised that 5-hydroxytryptamine uptake and interaction within perivascular adrenergic nerves could occur in those cerebrovascular disorders where blood is released.

Adrenergic development of neural crest cells grown in a defined medium under a reconstituted basement-membrane-like matrix.

The embryonic neural crest of vertebrates is the source of a wide variety of adult cell types. We have demonstrated previously that the presence of a reconstituted basement-membrane-like (RBM) gel overlay can dramatically stimulate the development of adrenergic cells in neural crest cultures grown in a complex medium containing horse serum and chick embryo extract. In the present experiments we have analyzed the differentiation of neural crest cells grown in a defined medium with an RBM gel overlay. We found that the presence of the RBM gel promoted the development of catecholamine-containing (CA+) cells in neural crest cultures grown in defined medium compared to cultures grown in this same medium in the absence of the gel. The number of CA+ cells which developed in cultures grown in defined medium in the presence of the RBM gel overlay was similar to that seen in cultures grown in complex medium in the absence of the RBM gel.

Comparison of transmitter release properties of embryonic sympathetic neurons growing in vivo and in vitro.

The functional behavior of embryonic chick sympathetic neurons was determined by inducing release of [3H]norepinephrine by electrical stimulation of sympathetic neurons growing in the chick heart and in culture, with and without heart cells. A very close correspondence between the functional behavior of neurons developing with the heart cells, either in vivo or in vitro, was demonstrated. For example, the outflow of tritium from [3H]norepinephrine loaded sympathetic neurons of 15-day-old chick heart was about three times more at 10 Hz than at 1 Hz. In contrast, the outflow of tritium from 12-day-old [3H]norepinephrine loaded cultured sympathetic neurons was inversely related to the frequency of stimulation (outflow at 1 Hz was about three time more than at 10 Hz). When neurons were co-cultured with the heart cells, the frequency-outflow relationship reverted to that seen in the intact heart. Electrically-evoked outflow of tritium from the heart was reduced in a concentration-dependent manner by 3-30 nM tetrodotoxin, abolished in 0.25 mM Ca medium, and potentiated by 3 mM tetraethylammonium. In sharp contrast, the outflow evoked by stimulation of cultured neurons was neither blocked by 30-300 nM tetrodotoxin, low Ca, nor potentiated by tetraethylammonium. However, when neurons were co-cultured with heart cells, the evoked outflow was blocked by 30 nM tetrodotoxin and low Ca, and potentiated by tetraethylammonium. Veratrine (10 microM) had very little effect on the outflow from cultured neurons but induced a massive outflow from co-cultures as well as hearts. Neurons grown in a medium conditioned by the heart cells were not sensitive to tetrodotoxin and veratrine. It is implied that cultured sympathetic neurons are endowed mostly with Ca channels, and that the Na channels become functional only when neurons are grown with the target cells. This dramatic alteration in the functional behavior of neurons co-cultured with heart cells indicates that the effector organ has an important role in the development of ionic conductances of sympathetic neurons growing in the body and in culture.

Demonstration of adrenergic and dopaminergic receptors in cultured sympathetic neurons--their coupling to cAMP but not to the transmitter release process.

Experiments were carried out on cultured sympathetic neurons of the chick embryo; first, to demonstrate the presence of adrenergic and dopaminergic receptors, and then to see if these receptors are involved in regulation of transmitter release. We show that alpha 2-agonists, norepinephrine, epinephrine and clonidine, had no effect on neuronal cyclic 3',5'-adenosine monophosphate content. Forskolin enhanced neuronal cyclic 3',5'-adenosine monophosphate from a control value of about 20 pmoles/mg protein to 150 pmoles/mg protein. In the presence of alpha 2-agonists and forskolin the cyclic 3,5'-adenosine monophosphate content increased between 340 and 430 pmoles/mg protein. The alpha 1-agonist, phenylephrine, had no such effect. The facilitatory effect of alpha 2-agonist on forskolin-stimulated cyclic 3',5'-adenosine monophosphate production was blocked by the alpha 2-antagonist, yohimbine, but not the alpha 1-agonist, prazosin. Dopamine did not affect neuronal cyclic 3',5'-adenosine monophosphate content, but forskolin-stimulated increase in cyclic 3',5'-adenosine monophosphate was further facilitated by dopamine, and this effect was blocked by haloperidol. Activation of neuronal alpha 2-receptors by norepinephrine, epinephrine and clonidine did not interfere with electrically induced release of tritium from [3H]-norepinephrine-loaded sympathetic neurons. However, if sympathetic neurons were co-cultured with heart cells, clonidine, norepinephrine and epinephrine markedly inhibited the stimulation-induced release. Yohimbine or phentolamine partially reversed the inhibitory effects of alpha 2-agonists. alpha 2-Agonists and -antagonists also modified stimulation-induced release of tritium from [3H]norepinephrine-loaded hearts of the chick embryo.(ABSTRACT TRUNCATED AT 250 WORDS)

Sympathetic and afferent somata projecting in hindlimb nerves and the anatomical organization of the lumbar sympathetic nervous system of the rat.

The anatomy of the sympathetic pathways from the spinal cord to the lumbar sympathetic trunk and the inferior mesenteric ganglion was studied systematically in the rat. Details of the arrangements of white and gray rami communicantes, sympathetic trunk ganglia, the intermesenteric nerve, and the lumbar splanchnic nerves are summarized. A modified nomenclature for the segmental ganglia of the paravertebral sympathetic chain is proposed. Cell bodies of sensory and sympathetic axons projecting to the skin and skeletal muscle of the rat hindlimb were labeled retrogradely with horseradish peroxidase (HRP) in order to study numbers, segmental distribution, and location of the somata of these neurons quantitatively. HRP was applied to the nerves supplying skeletal muscle (gastrocnemius-soleus, GS), hairy skin (sural, SU; saphenous, SA) and to a mixed nerve (tibial, TI). All sensory somata and 96.4% of the sympathetic cell bodies were located ipsilaterally. Sensory somata were commonly restricted to two adjacent dorsal root ganglia (usually L3-4 for SA; L4-5 for GS, TI; L5-6 for SU). Although the sympathetic somata were more widely distributed rostrocaudally (four to six segments), their maximum was always located one or two segments more cranially than the sensory outflow, i.e., corresponding to the rami communicantes grisei. From the data, it is estimated that 420 sympathetic and 530 afferent neurons project into GS, 590 and 3,610 into SU, 920 and 3,750 into SA, and 1,070 and 5,760 into TI. These absolute neuron numbers are compared with electron microscopic fiber counts from the literature.