Target specificity of neuropeptide Y-immunoreactive cranial parasympathetic neurons

We recently showed that neuropeptide Y (NPY)-like immunoreactivity occurs in subpopulations of neurons in 3 cranial parasympathetic ganglia: the otic, sphenopalatine, and ciliary. The present work identifies the target tissues innervated by cranial parasympathetic NPY-immunoreactive neurons. Plexuses of NPY-immunoreactive fibers were observed in the parotid gland, the target of the otic ganglion, and in the intraorbital lacrimal gland and palate, targets of the sphenopalatine ganglion. NPY-immunoreactive fibers of apparent parasympathetic origin innervated glandular acini in all 3 structures and were also present around small blood vessels in the parotid and intraorbital lacrimal glands. These fibers were presumed to be parasympathetic because they were not affected by removal of the superior cervical ganglion and because their distribution was coextensive with that of vasoactive intestinal polypeptide (VIP) immunoreactivity, which we have previously shown to be colocalized with NPY in the cell bodies of otic and sphenopalatine ganglion neurons. In contrast, no NPY-immunoreactive fibers were observed in the iris or ciliary body of acutely sympathectomized rats, suggesting that NPY-immunoreactive neurons in the ciliary ganglion do not normally transport detectable levels of NPY to their terminals. The target specificities of cranial parasympathetic NPY-immunoreactive neurons are different from those of sympathetic NPY-immunoreactive neurons. Sympathetic NPY-immunoreactive fibers innervated the iris and ciliary body, and the blood vessels but not the parenchymal cells of all the glands examined. In contrast, parasympathetic NPY-immunoreactive fibers primarily innervated glandular acini. NPY-immunoreactive neurons in the sphenopalatine ganglion displayed an additional level of specificity in their projection pattern in that they innervated only a subset of the ganglion's array of target glands: they innervated the intraorbital lacrimal gland and the seromucous glands of the palate but not the exorbital lacrimal gland or the glands of the nasal mucosa. The finding that NPY immunoreactivity is present in the parasympathetic innervation of secretory acini in several craniofacial glands raises the possibility that NPY plays a role in the parasympathetic control of glandular secretion. The observed overlap in the distributions of NPY- and VIP-immunoreactive fibers in these glands further suggests that NPY may interact with VIP to stimulate secretion.     

Chemical coding of sympathetic neurons controlling the tarsal muscle of the rat

Sympathetic axons in the upper eyelid and in tissues in the superior retro-orbital space were examined for NPY immunoreactivity. Sympathetic nerve terminals containing co-localised NPY were associated with blood vessels, the conjunctiva and the Meibomian glands. The acini of the Harderian gland completely lacked sympathetic innervation. Sympathetic axons lacking NPY were only found in the tarsal muscle. In addition, a minority of terminals, located in the more proximal part of the tarsal muscle, contained weak immunoreactivity to NPY. Injections of the retrograde tracer, Fast Blue, into the eyelid or retro-orbital space labelled postganglionic somata in the superior cervical ganglion. While many retrogradely labelled somata were immunoreactive for NPY, around half lacked NPY immunoreactivity and so are likely to project to the tarsal muscle. Most of the retrogradely labelled postganglionic somata lacking NPY were surrounded by terminals immunoreactive for met-enkephalin, leu-enkephalin and met-enkephalin arg-gly-leu which were all found to be present in the same nerve terminals. Sectioning the cervico-sympathetic trunk eliminated all enkephalin-immunoreactive pericellular baskets. Many enkephalin-immunoreactive pericellular terminals contained co-localised VAChT, calretinin and calbindin immunoreactivity, but completely lacked nitric oxide synthase immunoreactivity. A second population of nerve terminals that were immunoreactive for nitric oxide synthase also surrounded tarsal muscle-projecting neurons, but these terminals lacked immunoreactivity to enkephalin. Thus, postganglionic neurons projecting to the tarsal muscle are of at least two chemical phenotypes (with or without NPY) and they receive convergent input from at least two populations of preganglionic neurons with distinctive chemical phenotypes.     

gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.     

GABAergic innervation of the ciliary ganglion in macaque monkeys – A light and electron microscopic study

The vertebrate ciliary ganglion (CG) is a relay station in the parasympathetic pathway activating the iris sphincter and ciliary muscle to mediate pupillary constriction and lens accommodation, respectively. While the postganglionic motoneurons in the CG are cholinergic, as are their inputs, there is evidence from avian studies that GABA may also be involved. Here, we used light and electron microscopic methods to examine the GABAergic innervation of the CG in Macaca fascicularis monkeys. Immunohistochemistry for the gamma aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) revealed that all CG neurons are contacted by ChAT‐positive terminals. A subpopulation of 17.5% of CG neurons was associated with terminal boutons expressing GAD‐immunoreactivity in addition. Double‐labeling for GAD and synaptophysin confirmed that these were synaptic terminals. Electron microscopic analysis in conjunction with GABA‐immunogold staining showed that (1) GAD‐positive terminals mainly target dendrites and spines in the perisomatic neuropil of CG neurons; (2) GABA is restricted to a specific terminal type, which displays intermediate features lying between classically excitatory and inhibitory endings; and (3) if a CG neuron is contacted by GABA‐positive terminals, virtually all perisomatic terminals supplying it show GABA immunoreactivity. The source of this GABAergic input and whether GABA contributes to a specific CG function remains to be investigated. Nevertheless, our data indicate that the innervation of the ciliary ganglion is more complex than previously thought, and that GABA may play a neuromodulatory role in the control of lens or pupil function. J. Comp. Neurol. 525:1517–1531, 2017. © 2016 Wiley Periodicals, Inc.     

Location and peptide content of pelvic neurons supplying the muscle and lamina propria of the rat vas deferens.

Retrograde tracing and immunohistochemistry have identified the location within the rat pelvic plexus of neurons which project to the vas deferens, and their neurochemical properties. The fluorescent tracers, Fast Blue and FluoroGold, were injected into the wall of the vas deferens and labelled neurons located within the ventral part of the major pelvic ganglion (MPG) and the adjacent accessory ganglia (AG). Most neurons were located in ganglia ipsilateral to the injection site. Noradrenergic neurons were defined as those containing immunoreactivity for tyrosine hydroxylase (TH). Five groups of dye-labelled neurons could be identified immunohistochemically, noradrenergic neurons containing neuropeptide Y (NPY) (60-70%), and four types of non-noradrenergic neurons, NPY-only neurons (5-10%), NPY neurons containing vasoactive intestinal peptide (VIP) (3-5%), neurons containing only VIP (15-25%) and neurons containing galanin (GAL) (2-5%). Noradrenergic axons, and axons containing NPY or GAL were primarily located within the muscle, whereas most VIP axons were found as a dense plexus within the lamina propria. Very few peptide-containing varicose nerve terminals surrounded dye-labelled (vas deferens-projecting) pelvic neurons. Thus, no peptide marker was found for most of the preganglionic inputs supplying postganglionic neurons which project to the vas deferens. These studies have shown that pelvic neurons supplying the vas deferens have a discrete location within the rat pelvic ganglia and that they comprise at least five neurochemical groups, providing innervation to the muscle and lamina propria. The preganglionic connections with these noradrenergic and non-noradrenergic (possible cholinergic) pathways, and further examination of the role of mucosal innervation remain to be determined.     

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.     

Co-existence of neuropeptides in sympathetic, cranial autonomic and sensory neurons innervating the iris of the guinea-pig

We have used double-labelling immunofluorescence to identify the peptide content of autonomic and sensory neurons innervating the iris of albino guinea-pigs. Four major classes of neurons were identified on the basis of their distributions, origins and immunohistochemical characteristics. A dense plexus of noradrenergic axons in the constrictor and dilator muscles of the iris originated from the superior cervical ganglion, and contained immunoreactivity (IR) to both neuropeptide Y (NPY) and dynorphin (DYN). The constrictor and dilator muscles were also supplied with a dense plexus of axons with IR to substance P (SP). These axons probably originated from SP-IR nerve cell bodies located along the ciliary nerves, and are almost certainly the same axons as those producing cholinergic pupilloconstriction. The iris was also innervated by unmyelinated, capsaicin-sensitive axons with IR to both SP and calcitonin gene-related peptide. Most of these axons also contained IR to DYN and some were also IR for cholecystokinin. These axons are almost certainly sensory. Axons containing IR to both NPY and vasoactive intestinal peptide (VIP) were common in the ciliary processes, and also formed a sparse plexus near the ciliary margin of the dilator muscle. Following surgical sympathetic denervation these axons showed IR for dopamine-beta-hydroxylase; they seemed to originate from the sphenopalatine ganglion. These results demonstrate that there are well-defined patterns of coexistence of neuropeptides in the autonomic and sensory neurons supplying the iris of guinea-pigs. To understand the physiological roles of these peptides, it will be necessary to consider the possibility of complex interactions between them.     

Regional innervation of rabbit ciliary ganglion cells by the terminals of preganglionic axons

In the rabbit, ciliary ganglion neurons with dendrites maintain inputs from several different axons during the period of synaptic rearrangement that occurs in early postnatal life. Neurons without dendrites, on the other hand, lose the majority of their initial inputs and are innervated in maturity by the terminals of only one or two axons (Purves, D., and R.I. Hume (1981) J. Neurosci. 1: 441-452; Hume, R.I., and D. Purves (1981) Nature 293: 469-471). We have explored the basis of this phenomenon by individually marking preganglionic axons and the neurons they innervate with horseradish peroxidase. In general, the innervation of geometrically complex (multiply innervated) neurons by individual preganglionic axons is regional. That is, the synaptic contacts made by an axon on these neurons are limited to a portion of the postsynaptic surface that includes some, but not all, of the dendrites. This regional innervation of target neurons is consistent with the view that dendrites allow multiple innervation to persist by providing relatively separate postsynaptic domains for individual preganglionic axons. Such regional innervation may mitigate competitive interactions between the several axons which initially innervate the same neuron.     

CRF-like immunoreactivity selectively labels preganglionic sudomotor neurons in cat.

Immunohistochemical and neuronal tracing methods were used in cats to determine which type of postganglionic sympathetic neuron is innervated by preganglionic neurons which contain corticotrophin releasing factor-like immunoreactivity (CRF-LI). Preganglionic neurons with CRF-LI have their cell bodies at two restricted levels of the spinal cord and terminate in the stellate and lower lumbar ganglia. CRF-LI terminal baskets in stellate and lumbar ganglia surrounded cell bodies, 96-99% of which showed no tyrosine hydroxylase (TH)-LI (presumptive cholinergic neurons). Calcitonin gene-related peptide (CGRP)-LI was used to label the cholinergic ganglion cells which innervate sweat glands: 96-99% of those were confirmed as lacking TH-LI, while the remainder showed weak staining. Every one of over 6000 CRF-LI terminal baskets counted in 4 stellate and 6 lumbar ganglia was found to surround a cell body with CGRP-LI; conversely, 81-86% of the cell bodies showing CGRP-LI were surrounded by CRF-LI terminal baskets. In 3 cats, the retrograde tracer fluorogold was used to label postganglionic neurons projecting to the paw pads (a population which includes both cholinergic sudomotor neurons and noradrenergic vasoconstrictor neurons). Between 26 and 38% of the retrogradely labelled ganglion cells were surrounded by CRF-LI terminal baskets. We conclude that in cats, preganglionic sympathetic neurons with CRF-LI are sudomotor in function.     

Ultrastructural analysis of the distribution of synaptic boutons from labeled preganglionic axons on rabbit ciliary neurons

The number of preganglionic inputs that innervate rabbit ciliary ganglion cells is directly correlated with the number of dendrites arising from each ganglion cell (Purves and Hume, 1981). In general, the innervation of multiply innervated ciliary neurons by individual preganglionic axons is regionally restricted to a portion of the postsynaptic surface that usually includes the cell body and some, but not all, of the dendrites (Forehand and Purves, 1984). These observations suggest that dendrites modulate convergence to each cell by providing relatively separate postsynaptic domains for individual inputs. To examine this possibility further, I have assessed the distribution of synaptic boutons from individually labeled preganglionic axons on ciliary ganglion cells at the ultrastructural level. The results show that at least a third of the dendrites of these neurons are contacted exclusively by synaptic boutons from a single preganglionic axon. However, at least half of the dendrites (and nearly all of the cell bodies) of multiply innervated ganglion cells are innervated by at least 2 different preganglionic axons. Moreover, synapses from 2 different inputs often coexist in close proximity on the postsynaptic surface. Thus, individual preganglionic axons do not require exclusive dominion over a particular part of a postsynaptic cell in order to maintain their connection with the cell. These results suggest that competitive interactions between the inputs to these cells occur between the sets of boutons arising from different inputs, rather than at the level of individual boutons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.     

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.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Origin of neuronal nitric oxide synthase (NOS)-immunoreactive fibers in guinea pig parasympathetic cardiac ganglia

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.     

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the mudpuppy cardiac septum

An immunohistochemical study was undertaken to determine the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the cardiac septum of the mudpuppy, Necturus maculosus. Numerous long, CGRP-immunoreactive nerve fibers course across the septum, run in the nerve trunks connecting clusters of postganglionic parasympathetic cells, form complexes over groups of ganglion cells and make pericellular networks around individual ganglion cells. The postganglionic parasympathetic neurons and small intensely fluorescent (SIF)-like cells did not exhibit CGRP immunoreactivity. Most of the CGRP-immunoreactive nerve fibers also are labeled for substance P. In freshly dissected preparations, the staining pattern for CGRP was not similar to that obtained using an antiserum against synaptic vesicle membrane, which appears to preferentially label cholinergic preganglionic terminals on all postganglionic parasympathetic cells in the mudpuppy preparation. Further, in explanted ganglia (maintained 10 days in culture) almost no reactivity was obtained with the antivesicle antiserum whereas numerous nerve fibers still exhibited CGRP-immunoreactivity. These observations demonstrate that the CGRP-immunoreactive nerve fibers are not parasympathetic preganglionic axons. Rather we suggest that the CGRP-immunoreactive nerve fibers are processes of primary sensory fibers.     

Double labeling of the paravertebral sympathetic C system in the bullfrog with antisera to LHRH and NPY

The specificity of synaptic contacts between pre- and postganglionic cells in the sympathetic C system has been examined by immunocytochemical localization of two neuropeptides. Sections of bullfrog paravertebral sympathetic ganglia were stained with antibodies to luteinizing hormone releasing hormone (LHRH) and neuropeptide Y (NPY). Preganglionic synaptic boutons containing LHRH immunoreactivity were found to make contact with a subpopulation of postganglionic cell bodies and with some clusters of small intensely fluorescent (SIF) cells. In ganglia 9 and 10, 95.8% of the neurons contacted by LHRH-containing boutons were also positive for NPY-like immunoreactivity and conversely, 99.3% of the neurons that contained NPY-like immunoreactivity were contacted by LHRH-containing boutons. Qualitatively similar results were found in most other paravertebral ganglia. These observations support the conclusions that preganglionic C axons selectively innervate C-type ganglion cells and that virtually all C-type ganglion cells and some SIF cells receive a direct LHRH input. Moreover, they suggest that a pattern of specific connections between two sets of peptidergic neurons is expressed throughout most of the paravertebral sympathetic chain of the bullfrog.     

Preganglionic and sensory origins of calcitonin gene-related peptide-like and substance P-like immunoreactivities in bullfrog sympathetic ganglia

These experiments further define the organization of peptidergic pathways in the paravertebral sympathetic system of the bullfrog. Populations of axons and synaptic boutons in sympathetic ganglia 9 and 10 were found to express calcitonin gene-related peptide-like immunoreactivity (CGRP-IR) and substance P-like immunoreactivity (SP-IR). CGRP-IR is present in fibers that run through the ganglia and in boutons that make contact with almost half of the principal neurons. SP-IR is also present in fibers within the ganglia and in a rare class of synaptic boutons that are found on less than 1% of the principal neurons. Both forms of immunoreactivity are coexpressed in some nerve fibers and in the rare synaptic boutons that contain SP-IR. Neuropeptide Y-like immunoreactivity (NPY-IR), a marker for C-type postganglionic neurons, was used to identify the postsynaptic targets of boutons containing CGRP-IR and SP-IR. Ninety-five percent of the ganglion cells contacted by CGRP-IR boutons were negative for NPY-IR and are therefore likely to be B-type postganglionic neurons. Similarly, 100% of the ganglion cells contacted by boutons containing SP-IR were negative for NPY-IR. Lesions of the sympathetic chain demonstrated that synaptic boutons containing CGRP-IR arise from neurons whose axons enter the chain rostral to ganglion 7. Cutting the chain between ganglia 8 and 9 eliminates all preganglionic B and C inputs to ganglia 9 and 10. The destruction of the preganglionic C pathway by this lesion was verified by staining ganglia 9 and 10 for luteinizing hormone releasing hormone (LHRH). This lesion also eliminated boutons containing CGRP-IR and drastically reduced the number of ganglionic fibers that stained for CGRP-IR and SP-IR. By contrast, cutting the sympathetic chain between ganglia 6 and 7 spared LHRH-IR in the preganglionic C pathway but still eliminated the boutons that normally express CGRP-IR and reduced the amount of staining for SP-IR. CGRP-IR in the sympathetic ganglia arises from preganglionic and sensory neurons whereas ganglionic SP-IR is purely sensory in origin. In the spinal cord, the preganglionic B and C neurons that innervate ganglia 9 and 10 are located in different segments. In segments that contain preganglionic B cells, but not those that contain C cells, there were 243 +/- 37 (mean +/- SD) neurons in the intermediolateral cell column that express CGRP-IR. However, no cell bodies containing SP-IR were found in this region of the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development and aging of cholinergic synapses. I. Endogenous levels of acetylcholine and choline in developing autonomic ganglia and iris of the chick

Acetylcholine (ACh) and choline (Ch) levels were measured by means of a sensitive micromethod in developing irises, ciliary and sympathetic ganglia of the chick, starting at 5--7 days of incubation (DI) up to 1 year of age. The neurotransmitter is present in all three organs at relatively low levels (1--10 pmol) from 5 DI (iris) or 7 DI (ciliary and sympathetic ganglion). This is followed by a rapid and sustained 9-fold increase in all three tissues up to 14 DI. Thus, it appears that low levels of ACh may be sufficient for neurotransmission to occur in the primitive ciliary ganglion and iris. After hatching, total ACh levels continue to increase up to 1 year of age in the iris and ciliary ganglion and up to 3 months in the sympathetic ganglion. The increase may depend on either an increased functional demand for the neurotransmitter or an increased number of preganglionic terminals. In general Ch levels parallel closely the levels of ACh in each organ throughout development. It is concluded that ACh and Ch are present since the earliest detectable stages of development in the ganglia and iris, and their first increase seems to be correlated to the phase of innervation of the organs. The subsequent increase probably correlates to synaptogenesis. The close relationship between Ch and ACh levels indicates a regulatory role of Ch for ACh synthesis during neuronal development.     

Ultrastructural analysis of enkephalinergic terminals in parasympathetic ganglia innervating the urinary bladder of the cat

Leucine enkephalin immunoreactivity was identified in axons and varicosities in parasympathetic ganglia located in the pelvic plexus and on the surface of the urinary bladder of the cat. Electron microscopic immunohistochemical studies revealed that varicosities containing leucine enkephalin exhibited large dense core vesicles and small, clear, spherical vesicles, which were similar to those found in cholinergic terminals. Leucine enkephalin immunoreactivity was primarily associated with large dense core vesicles. The varicosities formed axodendritic and axosomatic synapses with principal ganglion cells. Axoaxonic synapses were not detected. Some axosomatic enkephalinergic synapses were detected embedded within or invaginating the principal ganglion cells. Varicosities containing flattened and/or small dense core vesicles did not exhibit enkephalin immunoreactivity. Bladder ganglion cells identified by retrograde HRP tracing from the urinary bladder exhibited similar leucine enkephalinergic synapses. These observations, coupled with previous reports that leucine enkephalin is present in sacral preganglionic neurons and released by preganglionic nerve stimulation, suggest that leucine enkephalin and acetylcholine are cotransmitters stored and released from the same nerve terminals in bladder parasympathetic ganglia.