Serotonergic terminals in the neural sheath of the blowfly nervous system: electron microscopical immunocytochemistry and 5,7-dihydroxytryptamine labelling

With serotonin immunocytochemistry we have demonstrated an extensive plexus of immunoreactive varicose fibres in the neural sheath of the nervous system of the blowfly, Calliphora. These fibres are located in the neural sheath of the following regions: the maxillary-labial and labrofrontal nerves of the cerebral ganglia, the cervical connective, the dorsal surface of the thoracicoabdominal ganglia, two pairs of prothoracic nerves and the median abdominal nerve. We identified the serotonin-immunoreactive neural processes in the electron microscope by means of the peroxidase-antiperoxidase method. Immunoreactivity was seen in large granular vesicles (ca 100 nm), on membranes of smaller (ca 60 nm) and larger (ca 100 nm) agranular vesicles, along the inner surface of the axolemma, along neurotubules and outer membranes of mitochondria. By conventional electron microscopy we found numerous varicose neural processes in the neural sheath of some of the above regions. These varicosities are of at least two types. One type corresponds to the serotonin-immunoreactive profiles. A second type contains large granular vesicles (ca 200 nm) of variable electron density. 5,7-Dihydroxytryptamine injected into the head capsule labelled varicosities in the neural sheath, corresponding to the ones identified with serotonin immunocytochemistry. The electron-dense labelling was seen in flattened vesicles within these varicosities. We propose that the serotonin-immunoreactive fibers in the neural sheath constitute neurohemal regions for the release of serotonin into the circulation. The finding of another morphological type of varicose fibers in the neural sheath suggests the presence of further putative neurohormones in these regions.     

An immunogold electron microscopic analysis of FMRFamide-like immunoreactive neurons in the CNS ofHelix pomatia: ultrastructure and synaptic connections

Summary The ultrastructure and synaptic connections of FMRFamide-like immunoreactive neurons were investigated in the CNS of the snailHelix pomatia, following the application of a post-embedding immunogold method. For comparison, first, we analyzed the ultrastructure and granule content of the identified FMRFamide-containing C3 neuron in the cerebral ganglion. Three types of unidentified immunoreactive neuronal perikarya, and five types of varicosities could be distinguished on the basis of granule content. The different granule types revealed a highly selective accumulation of gold particles. One granule type contained by one FMRFamide-like immunoreactive neuron type (N1) and by one varicosity type (T₂) showed similar ultrastructure to that of the granules seen in the C3 cell. In the neuropil, the majority of FMRFamide-like immunoreactive varicosities (four of the five varicosity types) established specialized synaptic contacts with unidentified postsynaptic profiles. In the connective tissue sheath around the ganglia, three types of FMRFamide-like immunoreactive varicosities were found to establish unspecialized contacts with smooth muscle fibres or to be free in the mass of collagen fibres. On the basis of these observations, we suggest (1) an extensive diversity of the localization of FMRFamide (and related substances) at the ultrastructural level; (2) the involvement of FMRFamide-like immunoreactive varicosities in synaptic, modulatory and neurohormonal regulatory processes in theHelix nervous system.     

Ultrastructure of the nerve plexuses of the mammalian intestine: The enteric glial cells

The ultrastructure of the glial cells in the enteric plexuses of the rat, guinea-pig, rabbit, cat and sheep has been investigated by freeze-fracture and by thin-section electron microscopy. In all the ganglia studied, glial cells outnumber neurons. They are readily identified by their shape, position and ultrastructure (particularly the abundant amount of gliofilaments) but could not be subdivided into separate types. They provide a partial sheath to the ganglion neurons (but large areas of neuronal membrane lie directly beneath the basal lamina and collagen fibrils) and have long laminar processes extending between nerve processes. Most nerve processes are in direct membrane-to-membrane contact with each other; the glial cells only separate groups of them and rarely form a sheath around an individual neurite.The gliofilaments are anchored to conspicuous dense bodies beneath the cell membrane at the surface of ganglia. The possible significance of these systems of gliofilaments (and the high number of intermediate junctions) is discussed in the light of the severe mechanical stresses imposed on the ganglia by the contractile activity of the gut wall.Numerous specialized contacts, of unknown significance, are found between vesicle-containing nerve varicosities and glial cell bodies or glial processes. In freeze-fracture preparations (cat and guinea-pig), a specific pattern of intramembrane particles allows the cell membrane of the enteric glial cells to be readily identified.     

Serotonin immunoreactivity in the circumesophageal nervous system of Hermissenda crassicornis

Immunohistochemical techniques were used to examine the distribution of serotonin-immunoreactive (5-HT-IR) neurons and processes in the circumesophageal nervous system of Hermissenda. Both the pedal and the cerebropleural ganglia contained immunoreactive neuronal somata, with the majority occurring in the pedal ganglia. Immunoreactive fibers and varicosities were identified in portions of the central neuropil, where we noted a consistent and specific relationship between 5-HT-IR axons, the optic nerve and the synaptic region in the neuropil near the photoreceptor terminals.     

Ultrastructure of synaptic connections of a bimodal pacemaker giant neuron in the central nervous system of Helix pomatia L.

Following intracellular labelling with horseradish peroxidase, the arborization and synaptic connections of the bimodal pacemaker giant neuron (RPal) of Helix pomatia were investigated in the right parietal and visceral ganglia. The RPal neuron possesses extensive axonal branching, the elements of which could be observed and traced within the entire neuropil region of both ganglia. The main axonal branches showed further arborization. The thin axon processes enter the synaptic neuropil, where they receive numerous synapses. At least six ultra-structurally different terminals form synaptic contacts on peroxidase-labelled axon processes of the cell. On the basis of their vesicle and granule content, they are likely to contain different neurotransmitters. Some intraganglionic efferent contacts of the RPal neuron were also observed.It is suggested that, besides its peripheral efferent connections, this cell might also serve as an interneuron.     

Peripheral connections of FMRFamide-like immunoreactive neurons in the snail,Helix pomatia: an immunogold electron microscopic study

Summary Using a postembedding immunogold electron microscopic method, the ultrastructure and synaptic connections of FMRFamide-like immunoreactive varicosities were investigated in different peripheral organs of the snailHelix pomatia, including the heart (auricle), intestine, hepatopancreas, upper tentacle and salivary gland. The FMRFamide-like immunoreactive varicosities contained granules and vesicles as described in a previous study of the CNS of this species, and additionally, based on their granule content, two novel types of varicosities were found in the auricle. A selective accumulation of gold particles over the granules could be demonstrated. The FMRFamide-like immunoreactive varicosities formed unspecialized contacts with postsynaptic target cells in all peripheral organs investigated, with the exception of the tentacle retractor muscle. Both the neuro-muscular and the neuro-glandular contacts were characterized by either unspecialized close apposition of the pre- and postsynaptic membranes or the immunoreactive elements faced the target cell(s) across a relatively wide extracellular space. In the tentacle retractor muscle some of the neuromuscular contacts showed appositions of electron dense material along the presynaptic membrane, clustering of agranular synaptic vesicles and intersynaptic cleft material. The present observations support previous electrophysiological findings and suggest a versatile modulatory role of FMRFamide and related substances in theHelix PNS.Dedicated to Professor Rolf Elofsson on the occasion of his 65th birthday.     

Differentiation/maturation of neuropeptide Y neurons in the corpus callosum is promoted by brain-derived neurotrophic factor in mouse brain slice cultures

Neuropeptide Y (NPY) is widely distributed throughout both the central and peripheral nervous systems in mammals, and plays a role in various functions such as neural modifications affecting feeding, cardiovascular dynamics, or neural diseases. Many NPY neurons exist not only in gray matter in the central nervous system or ganglia in the peripheral system, but also in white matter such as the corpus callosum (cc) especially during development. The functions and regulation of callosal NPY neurons are not well understood, though NPY neurons in the cerebral cortex or hypothalamus are known to be regulated by neurotrophic factors such as brain-derived neurotrophic factor (BDNF). We examined the effect of BDNF on NPY neurons in the cc using organotypic slice cultures to clarify the regulation of callosal NPY neurons. A 3-week administration of BDNF significantly increased the number of NPY-immunopositive neuronal cell bodies and fibers in the cc rather than in the cerebral cortex as assessed with immunohistochemistry. Electron microscopy demonstrated that the NPY immunoreactivity mainly occurred in the regions associated with accumulating synaptic or cored vesicles. NPY-positive fibers had some contacts with several other neuronal fibers and glial processes. BDNF affected these fine structures of NPY neuronal fibers in the cc. These results suggest that BDNF takes part in the development, maturation, and maintenance of NPY neurons in the cc.     

Light- and electron-microscopic characterization of electrophysiologically-identified,horseradish peroxidase-injected magnocellular neuroendocrine cells in goldfish preoptic nucleus

We recorded intracellularly from neurons in the goldfish preoptic nucleus which were anti dromically identified by electrical stimulation of the pituitary gland and marked by intracellular injection of horseradish peroxidase for subsequent localization. At the light-microscopic level, labeled neurons resembled profiles of Golgi-impregnated neurons and lay in the magnocellular portion of the preoptic nucleus. Densely labeled axons and dendrites projected to the lateral forebrain bundle, the medial forebrain bundle, fiber tracts in the preoptico-hypophysial tract, small blood vessels and capillaries, the ependymal lining of the third ventricle and toward other preoptic neurons. Occasionally, a lightly-labeled, large perikaryon lay adjacent to a large, heavily-labeled magnocellular neuron. Ultra structural examination of these identified cells revealed dense reaction product in neuronal perikarya and processes. Heavily labeled perikarya had elaborate networks of endoplasmic reticulum, extensive Golgi apparatus, occasional somatic spines and infrequent axo-somatic contacts from unlabeled neurons. These labeled perikarya which were frequently in close somatic apposition with unlabeled profiles were sometimes adjacent to a large, lightly-labeled perikaryon. A thin glial sheath separated most labeled neurons and processes from brain capillary endothelium. Labeled dendrites had heavily labeled spines and axo-dendritic contacts from unlabeled neurons. Labeled axons abutted unlabeled axons and -dendrites. Synaptic boutons innervating labeled structures always contained small clear synaptic vesicles and some boutons also contained large dense-core vesicles.These results demonstrate the complex connections of goldfish preoptic magnocellular neuroendocrine cells with other neurons, fiber systems, brain capillaries, ventricular ependyma and the pituitary and provide further support for non-endocrine as well as endocrine functions of magnocellular neurons.     

Direct contacts between Auerbach's ganglia and smooth muscle cells in the small intestine of the rat

The present electron microscopic study demonstrated direct contacts between Auerbach's ganglia and longitudinal smooth muscle cells in the rat small intestine. The muscle cells were often observed to extend small, foot-like processes to the Auerbach's ganglia. These processes were in contact with glial cells in the ganglia without an intervening basal lamina, or were in contact with intraganglionic axon varicosities containing many synaptic vesicles. The processes in contact with glial cells may anchor the muscle cells to the ganglia during muscle contraction and those in contact with axon varicosities may function as synaptic sites between ganglion and longitudinal muscle cells.     

Distribution of serotonin-immunoreactivity in juvenile Aplysia

Serotonin-immunocytochemistry has been applied to whole mounts of the central nervous system and of several peripheral tissues from stage 12 juvenile Aplysia californica. The small size of animals at this stage permits visualization of the three-dimensional distribution of structures containing serotonin-immunoreactivity in unsectioned tissues. Many neuronal cell bodies are stained in addition to the giant cerebral neuron of the cerebral ganglion and cells in the RB cluster of the abdominal ganglia which previously had been characterized biochemically and pharmacologically as being serotoninergic. Neuronal cell bodies, both in central ganglia and in the wall of the gut, are encircled by plexuses of serotoninergic varicosities. The neuropil of ganglia and the eye also contain fine, immunoreactive axons bearing varicosities. Intraganglionic connectives and nerves contain many stout fluorescent axons. Serotoninergic varicosities are also observed in the connective tissue sheath surrounding central ganglia and nerves, as well as in heart and body muscle, blood vessels and gut.     

Immunoelectronhistochemical evidence for innervation of brain microvessels by vasopressin-immunoreactive neurons in the rat

Peroxidase-antiperoxidase (PAP) immunocytochemistry at the electron microscopic level was used to describe the fine structural characteristics of vascular connections between vasopressin (VP)-immunoreactive neuronal elements and cerebral microvessels in the rat. In the majority of connections, somata or neural processes (mainly dendrites) showing VP-like immunoreactivity were separated from the vessel wall by thin glial processes. In addition, some VP-positive elements could establish direct contacts with the basal lamina of the endothelium or of a pericyte associated with the capillary bed. The findings provide immunocytochemical evidence that the vasopressinergic neuronal elements can directly innervate microvessels in the brain and thereby participate in regulating the local permeability of and the flow through the cerebral microvessels.     

Scanning electron microscopic studies of bullfrog sympathetic neurons exposed by enzymatic removal of connective tissue elements and satellite cells

Summary The ninth and tenth abdominal sympathetic ganglia of bullfrogs were studied by light microscopy and transmission and scanning electron microscopy after the removal of the connective tissue elements overlying the neurons. Digestion of tissues with trypsin and subsequent acid hydrolysis exposed the unipolar neurons, which remained covered by their satellite cells. The preganglionic innervation was visible on the proximal segment and axon hillock region of the postganglionic neurite. Clusters of small cells seen at the periphery of ganglia probably corresponded to groups of cells with abundant catecholamine-containing granules (SIF cells). Digestion with collagenase and protease removed some or all of the satellite cells in addition to the connective tissue. The true neuronal surfaces had short finger-like processes, whereas the external surfaces of satellite cells were smooth. Preganglionic nerve varicosities were clearly visible on the proximal segment of the postganglionic neurite, on the axon hillock and on the cell body of neurons. A few axonal varicosities were fractured to reveal the synaptic vesicles within. The possible effects of the distribution and glial ensheathment of nerve varicosities on their function are discussed.     

Routes of excretion of neuronal lysosomal dense bodies after ventricular infusion of leupeptin in the rat: a study using ubiquitin and PGP 9.5 immunocytochemistry

Summary To determine the rate and routes of removal of lysosomal, lipofuscin-like dense bodies from neurons, the protease inhibitor, leupeptin, was infused into the lateral ventricle of rats for up to nine days. After seven days a number of animals were then allowed to recover. The formation and later disappearance of dense bodies was followed by morphology and immunocytochemistry. After 48 h of infusion lysosomal dense bodies in large numbers appeared in cortical, hippocampal and cerebellar neurons, which also showed increased ubiquitin immunoreactivity, as well as in other cell types. By 3–4 days ubiquitin-immunoreactive dense bodies were equally distributed between neurons and astroglia. After seven to nine days of infusion ubiquitin immunoreactive dense bodies filled neuronal perikarya, dendrites and expanded initial segments of many axons and were abundant in glial processes. All dense bodies studied by electron microscopy were ubiquitin immunoreactive. After four days of recovery dense bodies were markedly fewer in neuronal perikarya, and virtually all were now within glial processes. From 7 to 28 days of recovery, when most neurons appeared normal, lipofuscin bodies remained in axon initial segments and in reduced numbers in glial processes, particularly around blood vessels and beneath the pia of hippocampus and of cerebellar cortex. Thus, neurons probably have a steady passage of short lived proteins through the lysosomal excretory pathway. The observed temporal sequence of events on recovery suggests that secondary lysosomes probably pass rapidly from neuronal perikarya and dendrites to astrocytes and thus to the vascular bed or pia-arachnoid. The mechanism of cell-to-cell transfer is not clear from this study.     

Peptidergic innervation of the mammalian sinus nodes: Vasoactive intestinal polypeptide,neurotensin, substance P

By the use of the peroxidase-antiperoxidase (PAP) technique in the sinus node of several mammalian species, vasoactive intestinal polypeptide (VIP), neurotensin (NT) and substance P (SP) immunoreactive structures were detected. VIP and NT as well as SP immunoreactive fibers were found in close association to the vasculature. While the innervation by SP immunoreactive fibers was restricted to blood vessels, VIP and NT immunoreactive fibers and varicosities were also in contact to nodal cells. Juxtanodal intracardiac ganglia and single intranodal ganglionic cells were supplied by VIP, NT and SP immunoreactive varicosities. In addition, VIP immunoreactive perikarya were present. The results suggest an involvement of VIP, NT and SP in the regulation of sinus node blood flow, in impulse generation as well as in extrinsic and intrinsic cardiac reflex mechanisms.     

Immunocytochemical localization of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase, in the hypothalamus of cattle

Immunocytochemical staining for the presence of catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine β-hydroxylase, was used to characterize the regional distribution of catecholaminergic neurons in the hypothalamus and adjacent areas of domestic cattle, Bos taurus. In steers, heifers and cows, tyrosine hydroxylase-immunoreactive perikarya was located throughout periventricular regions of the third cerebral ventricle, in both anterior and retrochiasmatic divisions of the supraoptic nucleus, suprachiasmatic nucleus, and ventral and dorsolateral regions of the paraventricular nucleus, dorsal hypothalamus, ventrolateral aspects of the arcuate nucleus, along the ventral hypothalamic surface between the median eminence and optic tract, and in the posterior hypothalamus. Immunostained perikarya ranged from small (10–20 μm, parvicellular) to large (30–50 μm, magnocellular) and were of multiple shapes: round, triangular, fusiform or multipolar, often with 2–5 processes of branched arborization. There were no dopamine-β-hydroxylase immunoreactive perikarya observed within the hypothalamus and adjacent structures. However, both tyrosine hydroxylase and dopamine-β-hydroxylase immunoreactive fibers and punctate varicosities were observed throughout regions of tyrosine hydroxylase immunoreactivity perikarya. Generally, the location and pattern of hypothalamic tyrosine hydroxylase immunoreactivity and dopamine-β-hydroxylase immunoreactive were similar to those reported for most other large brain mammalian species, however, there were several differences with commonly used small laboratory animals. These included intense tyrosine hydroxylase immunoreactivity of perikarya within the retrochiasmatic division of the supraoptic nucleus (ventral A15 region), the absence of tyrosine hydroxylase immunoreactive perikarya below the anterior commissure or within the bed nucleus of stria terminalis (absence of the dorsal A15 region), an abundance of tyrosine hydroxylase immunoreactive perikarya within the ependymal layer of the median eminence, heavy innervation of the arcuate nucleus with dopamine-β-hydroxylase immunoreactive fibers and varicosities, and the paucity of dopamine-β-hydroxylase immunoreactive throughout the median eminence.     

Embryogenesis of the central nervous system of the pond snail Lymnaea stagnalis L. An ultrastructural study

The ultrastructural characteristics of the developing CNS of the pond snail, Lymnaea stagnalis, were investigated, with special attention paid to three specific stages of embryonic development, representing distinctly different phases of both the body morphogenesis and gangliogenesis. These were the 35% (veliger), the 50–55% (metamorphic), and the 75% (post-metamorphic, adult-like) stages of embryonic development. Also, a brief comparison was done with the CNS of hatchlings (100% of embryonic development). During embryogenesis specialized axo-axonic synapses and elements of the glial system, including the ganglionic (neural) sheath, were rarely observed, whereas the frequent occurrence of unspecialized axo-somatic contacts could be demonstrated. Synapse-like axo-axonic connections could be found first in 75% embryos, showing asymmetric vesicle clustering on the presynaptic side and increased electron density of the apposed membranes. These phenomena may reflect the dominance of modulatory processes in the CNS during embryogenesis, and the absence of the neural sheath may facilitate trophic and/or hormonal influences within the developing ganglia. The gradual increase in the size of ganglia and the diameter of their neuropils was not accompanied by any widening of the cell body layer or increasing diameter of the nerve cell bodies until the very end of embryogenesis. With respect to the ultrastructural organization of the neuropil, and possibly the entire CNS, a determining stage seems to be that of metamorphosis. Two types of neuropil could be observed at this time; the metamorphosing neuropil with an irregular organization of wavy axon profiles, and well-structured neuropil with a regular organization of axon profiles. Ganglia with irregular or regular neuropil occurred simultaneously in the developing CNS.     

Vagal afferents innervating the gastrointestinal tract and CCKA-receptor immunoreactivity.

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA-receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA-receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C-terminal (Ab-1) nor an N-terminal (Ab-2) specific antibody produced any specific staining; in the myenteric plexus, non-vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR-immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR-immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab-1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR-immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA-receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals.     

Distribution of protein tracers in the nervous system of the crayfish (Astacus astacus L.) following systemic and local application

Summary A study was made on the penetration and cellular uptake of two protein tracers, albumin labelled with Evans blue (EBA) and horseradish peroxidase (HP), in the nervous system of the crayfish following systemic and local administration. Followingsystemic injection, EBA did not diffuse freely from the cerebral vessels into the brain parenchyma. When the tracers werelocally applied on the surface of the ventral nerve cord their penetration into the nervous parenchyma was to some extent restricted by the nerve sheath. However, unlike the perineurium of vertebrate peripheral nerves, which acts as an efficient diffusion barrier, the crayfish nerve sheath allowed the diffusion of small amounts of tracers into the ganglia. The tracers could more readily penetrate into peripheral nerves in the vicinity of ganglia. Inside the ganglion the tracers spread in extracellular spaces, between glial cell membranes and reached the neuronal surfaces. The proteins were taken up by pinocytosis in glial cells, and also in axons.     

Tunnel crossing fibers and their synaptic connections within the inner hair cell region in the organ of corti in the maturing mouse

 Combined ultrastructural and immunocytochemical studies reveal that in the adolescent 12- to 17-day-old mouse the afferent tunnel crossing fibers that innervate outer hair cells receive synaptic contacts from three distinct sources: the GABAergic fibers (GABA= gamma-aminobutyric acid) of the lateral olivocochlear bundle, the non-GABAergic efferent tunnel crossing fibers, and the inner hair cells themselves. The GABAergic fibers give off collaterals that synapse with the afferent tunnel fibers as they cross the inner hair cell region. These collaterals also form synapses with afferent radial dendrites that are synaptically engaged with the inner hair cells. Vesiculated varicosities of nonGABAergic efferent tunnel fibers also synapse upon the outer spiral afferents. Most of this synaptic activity occurs within the inner pillar bundle. Distinctive for this region are synaptic aggregations in which several neuronal elements and inner hair cells are sequentially interconnected. Finally, most unexpected were the afferent ribbon synapses that inner hair cells formed en passant on the shafts of the apparent afferent tunnel fibers. The findings indicate that: (1) the afferent tunnel (i.e., outer spiral) fibers may be postsynaptic to both the inner and the outer hair cells; (2) the non-GABAergic efferent and the afferent tunnel fibers form extensive synaptic connections before exiting the inner pillar bundle; (3) the GABAergic component of the lateral olivocochlear system modulates synaptically both radial and outer spiral afferents. Accepted: 7 May 1998     

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.