Cholinergic innervation of the mouse superior cervical ganglion: light-and electron-microscopic immunocytochemistry for choline acetyltransferase

Summary The cholinergic innervation of the mouse superior cervical ganglion was investigated by means of immunocytochemistry using a well-characterized monoclonal antibody against choline acetyltransferase (ChAT). Immunopositive nerve fibers entered the superior cervical ganglion from the cervical sympathetic trunk. Light-microscopically, these fibers appeared to be heterogeneously distributed among the principal ganglion cells. The rostral part of the ganglion contained more ChAT-positive fibers then the middle or the caudal one. The axons branched several times before forming numerous varicosities. Most of the ChAT-stained fibers and varicosities aggregated in glomerula-like neuropil structures that were surrounded by principal ganglion cell bodies, whereas others were isolated or formed little bundles among principal neurons. None of the neurons or other cell types in the ganglion exhibited ChAT-positivity. ChAT-immunoreactive fibers disappeared from the ganglion 5 or 13 days after transection of the cervical sympathetic trunk. At the ultrastructural level, most axon terminals and synapses showed ChAT-immunoreactivity. An ultrastructural analysis indicated that immunostained synapses occurred directly on the surface of neuronal soma (1.8%) and dendritic shafts (17.6%). Synapses were often seen on soma spines (18.4%) and on dendritic spines (62.2%). All immunoreactive synapses were of the asymmetric type. The results provide immunocytochemical evidence for a heterogeneous cholinergic innervation of the ganglion and the principal neurons.     

The ultrastructure of the cardiac ganglion of the desert scorpion,Paruroctonus mesaensis (Scorpionida: Vaejovidae)

Light and electron microscopy of the pacemaker ganglion of the scorpion heart indicate that it is about 15 mm long and 50 μm in diameter and extends along the dorsal midline of the heart. The largest cell bodies (30–45 μm in diameter) occur in clusters along the length of the ganglion. The ganglion appears to be innervated with fibers from the subesophageal and first three abdominal ganglia. The cardiac ganglion is surrounded by a neurilemma and a membranous sheath. The latter is apparently derived from connective tissue cells seen outside the ganglion. Nerve fibers other than those in the neuropil areas are usually surrounded by membrane and cytoplasm of glial cells. Often there are several layers of glial membrane, forming a loose myelin. The cardiac nerves to the heart muscle are also surrounded by a neurilemma, and the axons are surrounded by glia. The motor nerves contain lucent vesicles 60–100 nm and opaque granules 120–180 nm in diameter. In the cardiac ganglion, some nerve cell bodies have complex invaginations of glial processes forming a peripheral trophospongium. In the neuropil areas, nerve cell processes are often in close apposition. The septilaminar configuration typical of gap junctions is common, with gap distances of 1–4 nm. In tissues stained with lanthanum phosphate during fixation, we found gaps with unstained connections (1–2 nm diameter) between nerve-nerve and glial-nerve cell processes. Annular or double-membrane vesicles in various stages of formation were also seen in some nerve fibers in ganglia stained with lanthanum phosphate. Nerve endings with electron-lucent vesicles 40–60 nm in diameter are abundant in the cardiac ganglion, suggesting that these contain the excitatory transmitter of intrinsic neurons of the ganglion. Less abundant are fibers with membrane-limited opaque granules, circular or oblong in shape and as much as 330 nm in their longest dimension. Also seen were some nerve endings with both vesicles and granules.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Neurobiology of the scallop. II. structure of the parietovisceral ganglion lateral lobes in relation to afferent projections from the mantle eyes

The lateral lobes of the scallop parietovisceral ganglion have been examined morphologically with respect to their functional role as optic lobes. The gross morphology of the lateral lobe and projections of optic nerve fibers within it were investigated by 1) supravital methylene blue staining, and 2) autoradiography using tritiated proline injected intraocularly for incorporation and transport by the optic fibers. Ultrastruc‐turally, the lateral lobe was examined using standard electron microscopic techniques. The lateral lobe is composed of a cortical rind of cells, 8–15 μm in diameter at the ventral surface and 15–20 μm in diameter at the ventral surface, surrounding a central neuropil. The neuropil contains three distinct regions: 1) the glomerular neuropil, a series of densely staining spherical subunits associated with the eyes and pallial nerves, 2) the subcellular neuropil, a synaptic region adjacent to the ventral cell layer also having a visual function, and 3) the subglomerular neuropil, the remaining, rather unspecialized neuropil of the lateral lobe. Synaptic profiles with symmetrical membrane thickenings, a 32 nm synaptic cleft, and three types of vesicles are seen throughout the neuropil, although the density of synapses is greater in the glomerular region. Clear, dense core and neurosecretory vesicles are seen individually or as mixed populations in the presynaptic terminals. Autoradiographic experiments have revealed that optic fibers enter the lateral lobe and project directly to the subcellular neuropil where they synapse with cells located on the ventral surface of the lateral lobe cells. These cells in turn form the dense glomerular structures previously identified as visual association centers and send efferent fibers into the pallial nerves. The projection of optic fibers to the ventral surface of the lobe is consistent with previous electrophysiological recordings of visual activity at this site.     

Metamorphosis of wing motor system in the silk moth,Bombyx mori L. (Lepidoptera: Bombycidae): Anatomy of the sensory and motor neurons that innervate larval mesothoracic dorsal musculature,stretch receptors,and epidermis

Anatomy of dorsal mesothoracic structures, such as muscles, sensory organs, and innervation, was studied in the silkworm, Bombyx mori L. (Lepidoptera : Bombycidae), and compared with the adult wing motor system. Musculature and nerve innervation were investigated by dissection and electron micrograph; and central projection of sensory fibers and morphology of somata and dendrites of motor neurons by cobalt back-filling, followed by silver intensification. There are 23 muscle bundles (DLM) and 2 stretch receptors (SR). The DLMs, SRs, and epidermis are innervated by a branch of the dorsal nerve trunk emerging from the mesothoracic ganglion (MSG). The branch bifurcates into a dorsal sensory branch of about 300 sensory fibers and a dorsal motor branch of 14 fibers. The sensory fibers project mainly to a longitudinal portion near the mid line in the ventral neuropil of MSG and the metathoracic ganglion. Several fibers extend into the prothoracic ganglion (PG) and a few into the subesophageal and 1st abdominal ganglia. At least 13 (probably 14) motor neurons send axons to DLMs: 9 (probably 10) in PG, and 4 in MSG. Their dendrites are located mostly on the dorsoipsilateral side of the neuropil, but several branches cross the mid line and give rise to many fine branches on the contralateral side. Comparison between the larval (present study) and adult motor system shows a significant similarity in the musculature, peripheral nerve pattern, and motor neurons with some peculiarities.     

Glutamic acid decarboxylase (GAD)-like immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary A substance immunologically related to vertebrate glutamic acid decarboxylase (GAD) has been visualized in the pedal ganglion of Mytilus with the pre-embedding peroxidase-antiperoxidase method, by use of an antiserum raised in sheep against rat brain GAD. The results show that GAD-like immunoreactivity is present both in neuronal perikarya and in nerve fibers. Positive neurons are located mainly among the fibers of the ganglion neuropil at the commissural level, and more rarely close to unreactive cortical cell bodies. Immunoreactive nerve fibers are observed throughout the neuropil and also in cerebropedal and pedal nerves.Supported by Ministero Pubblica Istruzione (40%)     

Evidence for GABAergic fibers entering the superior cervical ganglion of rat from the preganglionic nerve trunk

The origin of gamma-aminobutyric acid immunoreactive (GABA-IR) nerve fibers present in the superior cervical ganglion (SCG) of rat was investigated. With immunocytochemical techniques many nerve fibers showed GABA-like positivity in the cervical sympathetic trunk, whereas similar staining could not be revealed in the internal carotid nerve or in the external carotid nerve. Ligation of the cervical sympathetic trunk for 24 h resulted a dramatic reduction in the staining density in the ganglion and in the cervical sympathetic trunk distal to the ligature. After transection of the preganglionic nerve fibers for eleven days or more, very few fibers staining for GABA were seen in the ganglion. The immunohistochemical results suggest that a major source of GABA within the SCG is a population of GABAergic axons entering from the preganglionic trunk.     

Evidence for GABAergic fibers entering the superior cervical ganglion of rat from the preganglionic nerve trunk

Summary The origin of gamma-aminobutyric acid immunoreactive (GABA-IR) nerve fibers present in the superior cervical ganglion (SCG) of rat was investigated. With immunocytochemical techniques many nerve fibers showed GABA-like positivity in the cervical sympathetic trunk, whereas similar staining could not be revealed in the internal carotid nerve or in the external carotid nerve. Ligation of the cervical sympathetic trunk for 24 h resulted a dramatic reduction in the staining density in the ganglion and in the cervical sympathetic trunk distal to the ligature. After transection of the preganglionic nerve fibers for eleven days or more, very few fibers staining for GABA were seen in the ganglion. The immunohistochemical results suggest that a major source of GABA within the SCG is a population of GABAergic axons entering from the preganglionic trunk.     

Granule-containing cells in the human superior cervical ganglion.

The fine structure of granule-containing cells in the human superior cervical ganglion is described. These cells are larger than the typical SIF cells in mammals and exhibit green-yellow fluorescence. They are characterized by numerous granular vesicles (80-140 nm in diameter) in the cytoplasm, but have many features in common with ordinary ganglion cells. They emit several long processes which form bundles together with ordinary nerve fibers. No synapses are found where the cells are presynaptic, although a few synapses are observed there where nerves are prosynaptic on the perikarya and processes of the cells. No close topographical relations are seen between the cells and blood vessels. It is suggested that the granule-containing cells are a special type of postganglionic aminergic neurons.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

Axonal sprouts of the hypoglossal nerve implanted in the superior cervical ganglion of adult rats establish synaptic contacts under long-lasting GABA effect. An experimental degeneration study

Experimental degeneration was used in this study to determine if the hypoglossal nerve implanted already in the superior cervical ganglion of adult rat under GABA treatment has established morphologically-identifiable synapses with the dendrites of principal ganglion cells. The implanted hypoglossal nerve trunk was cut in a re-operation, and the ganglionic samples were studied by electron microscopy after 0, 6, 12, 24 and 48 h survival times. First signs of degenerative changes were found in the myelinated and non-myelinated axons alike, 6 h after axotomy. The fine-structural signs of degeneration resembled those of the preganglionic nerve fibres. Degenerating nerve terminals establishing synaptic contacts with the dendrites of the principal ganglion cells were also seen, indicating that the axonal sprouts of the implanted hypoglossal nerve established synaptic contacts with the ganglion cells. It remained, however, to be elucidated whether or not these synapses of the hypoglossal nerve are functionally active contacts while the preganglionic innervation is also present within the ganglion.     

Acetylcholine content in superior cervical ganglion following reinnervation of vagal afferent fibers in cat

The superior cervical ganglion (SCG) was reinnervated by vagal afferent fibers by cross anastomosis between the cranial end of nodose ganglion and the caudal end of SCG in cats. Formation of functional synapses was evidenced by unilateral mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or to electrical stimulation of the afferent vagus. The acetylcholine (ACh) content in the cross-anastomosed SCG (reinnervated by vagal afferent fibers) was measured. In anastomosed SCG, the ACh content was about half of normal SCG, but significantly higher than chronically decentralized SCG. Also the ACh content in nodose ganglion (NDG) was investigated in situations in which there was anastomosis, chronic supra, infra, or supra-/infranodose vagotomy. The ACh content of anastomosed NDG was near that of supranosdose vagotomized ganglion. The ACh content of supra-/infranodose vagotomized NDG, which can be considered the NDG itself, was as much as that of normal intact NDG. It was found that the ACh content of infranodose vagotomized NDG was increased, possibly the result of vagal efferent axonal flow or transport. The ACh content of vagal trunk with or without infranodose vagotomy was also measured. The ACh content of vagal trunk with infranodose vagotomy was smaller than that of the normal trunk, but there was still a considerable quantity of ACh. There was no significant change in wet weight of the SCG and NDG before or after the operations. From these results we have concluded that the transmission of the cross-anastomosed SCG (reinnervated with vagal afferent nerve) was cholinergic; and that the vagal afferent nerve have afferent cell bodies not only in NDG but also in peripheral vagal trunks (infranodose portion). These results strongly suggest that vagal afferent fibers are in part cholinergic.     

Some observations on the fine structure of the corpus striatum of the rat brain

Summary The rat corpus striatum was perfused vitally with glutaraldehyde, immersed in OsO₄ and then observed under an electron microscope.Numerous small cells in the neostriatum show a simple cytoplasmic structure, while the large cells possess a complicated fine structure. These are differentiated under the elctron microscope into two kinds, which seem to have functional differences. The large pallidal cells containing much pale cytoplasm are covered with many varied axonal boutons from the cell body to the dendritic terminal making numerous axo-somatic or axo-dendritic trunk synapses. Numerous axo-dendritic, or spine synapses are recognized in the neostriatal neuropil.These numerous axon terminals, which belong to striatal nerve cells or other nuclei of the brain, are classified morphologically into several types. At least five types of synaptic vesicles are distinguished by their size or by the presence of fine dense granules on their membranes, and seem to be specific to the neostriatum.Many myelin interruptions and several kinds of glial cells in the corpus striatum are observed. Moreover, the ventricular wall of the caudate nucleus, namely, the ependyma, and two kinds of subependymal cells are described and discussed with reference to the subependymal layer.     

Electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion

An electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion shows that there are, on the everage, 603 more nerve fibers in the portion of the nerve distal to the geniculate ganglion than there are in the part proximal to the ganglion. The average distal increase in the number of unmyelinated fibers is 444 and that in the myelinated fibers is 165. The somatic motor nerve fibers and the parasympathetic fibers in the mouse facial nerve may not contribute to the distal excess. It is possible that the increase in the number of unmyelinated fibers distal to the geniculate ganglion is mainly due to the presence of postganglionic sympathetic fibers in the facial trunk distal to the geniculate ganglion and the greater petrosal nerve. The distal increase in the number of myelinated fibers may be mainly contributed by the sensory fibers.     

Observations on the ultrastructure of ganglion cells in the circumvallate papilla of rat and mouse

Ganglion cells in the circumvallate papilla of adult rodents are described as typical autonomic neurons. Some neurons are aggregated to form a discrete structure in the base of the papilla; others are scattered through the core, along the nerve bundles, and particularly near the dome. The term "circumvallate ganglion" is applied to the entire population. Satellite cells completely ensheathe each neuron. Preganglionic fibers, containing clear vesicles, synapse on the soma and stumpy dendrites of the neurons. Axons, containing dense-cored vesicles, are observed in close proximity to the neurons. However, these fibers do not establish true morphological synaptic contacts with the neurons. We have not observed serial or reciprocal synapses on or in the vicinity of the ganglion cells. The hypothesis that the axons of the circumvallate ganglion neurons act as parasympathetic vasodilators is indicated by the proximity of the two structures and by nerve terminations on the arteriole muscle cells. Direct modulation of taste transduction by these neurons is ruled out.     

Fine structure of the area postrema of the rabbit brain

Summary The area postrema of the rabbit, which was perfused with glutaraldehyde and postfixed in osmium tetroxide, was observed under the electron microscope. This area showed neuronal and neuroglial structures similar to those of ordinary cerebral tissue, except for rich blood capillaries, which were surrounded by conspicuous perivascular spaces. Parenchymal cells included a moderate number of small neurons and large numbers of specific astrocyte-like cells. The neuropil consisted of a small number of thin myelinated and many non-myelinated nerve fibers of varying calibers, axo-dendritic synapses, and neuroglial cell processes, leaving no spaces between them. The axons and synaptic terminals contained moderate amounts of granular vesicles, which were similar in size to those found in the hypothalamus and were supposed to contain catecholamine. Glycogen paticles were demonstrated mainly in the cytoplasm of the astrocyte-like cells.     

Morphological characteristics of chemosensory,visual, and statocyst pathways in the snailHelix lucorum

The following structural characteristics of the chemosensory, visual, and vestibular pathways of the snail (Helix lucorum) were demonstrated by using a variety of histological techniques. Large and small neurons of the tentacle ganglion, the bipolar cells of the olfactory nerve, and a proportion of optic tentacle bulb chemoreceptors within the olfactory nerve all send their processes to the CNS of the mollusk. Here they are divided up into numerous bundles of fibers in the neuropil of the ipsilateral cerebral ganglion. They are joined by processes from the central nervous system put out by all neurons of the protocerebrum and the cluster of cells of the commissural section of the metacerebrum. Ocular receptors do not send processes down below the enlargement of the upper optic nerve. This enlargement is also the site where processes from cells within the CNS and the nerve itself terminate. An area of arborization of processes from the visual pathway cells is located in the neuropil of the pleural portion of the metacerebrum. Hair cells of statocysts put out processes to the cerebral ganglion, whence axons of small metacerebral neurons extend towards the organ of balance. Some processes from vestibular pathway cells form an arborization zone at the ipsilateral cerebral ganglion, while others pass through the cerebral commissure to form their area of arborization in the contralateral ganglion. Processes from vestibular and visual pathway cells arborize in exactly the same area.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 7–16, January–February, 1986.     

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation

Summary The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of preand postganglionic NF-positive nerves in the ganglion. Cuts in both preand postganglionic nerves result in a marked increase in GFAP immunoreactivity in SCG, whereas NF immunoreactivity increases in nerve cell bodies after preganglionic cuts. Only a few ganglion cells show NF immunoreactivity in the normal SCG of guinea pig. All intraganglionic NF-positive nerves are of preganglionic origin; decentralization abolishes NF immunoreactivity in these nerve fibers. The inferior mesenteric ganglion, the hypogastric nerves and colonic nerves in guinea pigs contain large numbers of strongly NF-immunoreactive nerve fibers.When the SCG of adult rat is grafted to the anterior eye chamber of adult rat recipients, both ganglionic cell bodies and nerve fibers, forming on the host iris from the grafted ganglion, are NF-positive. As only the perikarya of these neurons normally exhibit NF immunoreactivity, and the terminal iris arborizations are NF-negative, it appears that the grafting procedure causes NF immunoreactivity to become more widespread in growing SCG neurons.     

Innervation of the parathyroid in the european starling (Sturnus vulgaris)

Anatomical studies were conducted to characterize the source, type, and distribution of parathyroid gland innervation in European starlings. Denervation experiments demonstrated that the parathyroid glands and adjacent carotid bodies are innervated by nerve fibers originating in the nodose ganglion of the vagus nerve. In the parathyroid parenchyma, these fibers terminate adjacent to chief cells or near vascular smooth muscle. Vagal fibers also form synapses with catecholamine-containing glomus cells of the carotid body. Blood that first perfuses the carotid body subsequently perfuses the parathyroid parenchyma. These observations suggest that vagal innervation may influence parathyroid function in starlings either through direct chief cell innervation or through alteration of vascular perfusion. A neurohemal relationship also may exist between the carotid body and parathyroids.     

Substance P-like immunoreactivity in the trigeminal sensory nuclei of an infrared-sensitive snake,Agkistrodon blomhoffi

Summary With the peroxidase-antiperoxidase immunohistochemical method we ascertained the presence of substance P-like immunoreactivity (SPLI) in fibers and cell bodies of the trigeminal sensory system of the pit viper, Agkistrodon blomhoffi. There are a few SPLI fibers each in the principal sensory nucleus and the main neuropil of the lateral descending nucleus (i.e., the infrared sensory nucleus); a moderate number in the descending nucleus; and a large number in the caudal subnucleus, the medial edges of the interpolar subnucleus, and the marginal neuropil of the lateral descending nucleus. About 30% of the cell bodies in the ophthalmic and maxillo-mandibular ganglia show SPLI, and of the two craniocervical ganglia, the proximal ganglion has many more cells with SPLI than the distal ganglion. The SPLI distribution in the common trigeminal sensory system is similar to that of mammals, and suggests that the function of this system is also similar. In the infrared sensory system, the differing distribution in the main and marginal neuropils suggests separate functions for these two structures in the system.