NUCLEOSIDE PHOSPHATASE AND CHOLINESTERASE ACTIVITIES IN DORSAL ROOT GANGLIA AND PERIPHERAL NERVE

In dorsal root ganglia and peripheral nerve of the rat and other species, nucleoside phosphatase and unspecific cholinesterase reaction products are found in the plasma membranes and spaces between them at two sites: (1) Schwann cell-axon interfaces and mesaxons of unmyelinated fibers, and (2) sheath cell-perikaryon interfaces and interfaces between adjacent sheath cells. Acetylcholinesterase reaction product is found in the perikaryon (within the endoplasmic reticulum) and the axon (axoplasmic surface). Nucleoside phosphatase reaction product is also found in the numerous vacuoles at the surface of perineurium cells, ganglion sheath cells, and cells surrounding some ganglion blood vessels. Nucleoside phosphatase activities in the sections fail to respond, in the manner described for "transport ATPase," to diisopropylphosphofluoridate, sodium and potassium ions, and ouabain. Nucleoside diphosphates are hydrolyzed more slowly than triphosphates in unmyelinated fibers, and are not hydrolyzed at the perikaryon surface. Nucleoside monophosphates are either not hydrolyzed or hydrolyzed very slowly. In contrast to these localizations, which are believed to demonstrate sites of enzyme activity, it is considered likely that diffusion artifacts account for the nucleoside phosphatase reaction product frequently found along the outer surfaces of myelinated fibers and within vacuoles at the Schwann cell surfaces of these fibers. The diffuse reaction product seen in basement membranes of ganglion and nerve may also be artifact.     

Localization of neuron-specific (gamma gamma) enolase in proliferating (supportive and neoplastic) Schwann cells. An immunohisto- and electron-immunocyto-chemical study of ganglioneuroblastoma and schwannomas

Neuron-specific (gamma gamma) enolase, a glycolytic enzyme used as a relatively specific marker for normal neurons and neuroendocrine cells, has recently been found in a variety of neoplastic cells and in reactive astrocytes. Its localization was investigated by immunohisto- and electron-immunocyto-chemistry, in the proliferating supportive Schwann cells of a peripheral ganglioneuroblastoma and in the neoplastic Schwann cells of four acoustic tumours. By light microscopy, the neoplastic Schwann cells showed moderate uneven diffuse immunopositivity for enolase. By electron-immunocytochemistry, both types of Schwann cells demonstrated immunopositivity discretely limited to their cell surface membranes. The neoplastic ganglion cells and axons of the ganglioneuroblastoma and the normal neurons and axons included in the schwannomas were, as expected, intensely immunopositive. The visualization of gamma gamma enolase on the cell surface membranes of both neoplastic and non-neoplastic proliferating Schwann cells suggests that increased glycolytic activity may occur on the surface of these proliferating cells irrespective of the nature of the proliferation.     

Cytochemical localization of ouabain-sensitive, K+ -dependent p-nitrophenylphosphatase and Ca++-stimulated adenosine triphosphatase activities in human parotid and submandibular glands

K+ -dependent p-nitrophenylphosphatase (pNPPase) and Ca++ -stimulated adenosine triphosphatase (ATPase) activities were studied in human parotid and submandibular glands using cytochemical methods at the ultrastructural level. In both glands, only the striated-duct epithelium showed K+ -pNPPase reaction product, thereby indicating the localization of Na+, K+ -ATPase. The precipitate was concentrated on the deep invaginations of the basolateral plasma membranes, in close association with their cytoplasmic surface. Ca++ -ATPase activity was also found on the basolateral plasma membranes, but two striking differences from the K+ -pNPPase distribution were observed: firstly, Ca++ -ATPase appeared in both acinar and ductal cells, and secondly, it was localized on the outer side of the plasma membranes.     

Structural Repair and Functional Recovery Following Cerebral Ganglion Removal in the Pulmonate Snail Melampus

SYNOPSIS. Regeneration of the nervous system of Melampus followingcerebral ganglion removal proceeds through tract, bud, and ganglionstages Each stage can represent a terminal condition in someanimals Early events of regeneration appear to include rolesfor chemotactic and growth-promoting agents and axonal guidanceby preferential adhesion to a connective tissue sheath Thislatter proposed mechanism accounts for the observed sequencein which the neural elements unite in the tract stage and forthe pattern of failures that result when the sheath is disruptedIn the tract stage of regeneration, communication through thesite of the missing ganglion is restored within the centralnervous system, and between neurons of non-excised ganglia andthe denervated periphery Some behavioral recovery results Thebud stage of regeneration is characterized by neuropil developmentand associated swelling at the site of confluence of the tractsSerotonin immunohistochemistry of bud stage preparations andretrograde dye transport via bud nerves show tracts and numeroussynaptic vancosities, but the neuron somata that are labeledare located in other ganglia Ultrastructural examination oflate bud/early ganglion stage tissue reveals the presence ofsmall undifferentiated cells By six to seven months postoperative,some snails have clearly reached a ganglion stage of regenerationcharacterized by the appearance of differentiated neurons withinthe bud The origin of these new neurons is currently under investigation.     

Herpes simplex virus in the vestibular ganglion and the geniculate ganglion—role of loose myelin

This study presents the first direct evidence for herpes simplex virus type 1 (HSV-1) infection in the neurons of the vestibular ganglion. Although many investigators have reported electron microscopic evidence of HSV-1 infection in sensory ganglia, HSV-1 infection in the vestibular ganglion has not been described. Vestibular ganglion neurons have a unique structure, with a loose myelin sheath instead of the satellite cell sheath that is seen in other ganglia. This loose myelin is slightly different from compact myelin which is known as too tight for HSV-1 to penetrate. The role of loose myelin in terms of HSV-1 infection is completely unknown. Therefore, in an attempt to evaluate the role of loose myelin in HSV-1 infection, we looked for HSV-1 particles, or any effects mediated by HSV-1, in the vestibular ganglion as compared with the geniculate ganglion. At the light microscopic level, some neurons with vacuolar changes were observed, mainly in the distal portion of the vestibular ganglion where the communicating branch from the geniculate ganglion enters. At the electron microscopic level, vacuoles, dilated rough endoplasmic reticulum and Golgi vesicles occupied by virus were observed in both ganglia neurons. In contrast, viral infections in Schwann and satellite cells were observed only in the geniculate ganglion, but not in the vestibular ganglion. These results suggest that loose myelin is an important barrier to HSV-1 infection, and it must play an important role in the prevention of viral spread from infected neurons to other cells.     

Peripheral nerve connections influence the appearance of neurosecretary material in neural sheath of ventral ganglion of the fly Sarcophaga bullata: an immunocytochemical study

This study examined the role of the brain and peripheral connections with the target organs in the appearance of neurosecretary material within the dorsal neural sheath of the ventral ganglion of the fly S. bullata. Specifically, the accumulation of the neuropeptide FMRFamide (the neurosecretary material) was examined by immunocytochemistry. Immunoreactions were performed on: (1) a normal intact ventral ganglion, (2) an isolated ventral ganglion that was cultured in vivo, and (3) a ventral ganglion that was isolated by transection from the brain, but retained its peripheral nerve connections. The results demonstrate that (a) the neurons of the ganglia survive and exhibit FMRFamide immune reaction independent of their peripheral connections, and (b) the accumulation of neuropeptide in the dorsal neural sheath is controlled by intact peripheral nerve connections with the ganglion. It is suggested that in the absence of their peripheral connections, the axons of FMRFamide immunoreactive neurons fail to invade the neural sheath resulting in the accumulation of neurosecretary material.     

FMRFamide-like immunoreactivity in the ventral ganglion of the fly Sarcophaga bullata: metamorphic changes.

1. Localization of FMRFamide-like immunoreactivity was examined in the ventral ganglion of the fly Sarcophaga bullata using the indirect immunofluorescent method. 2. There are six large cells in the thoracic ganglion which are highly immunoreactive at all stages of development. 3. During metamorphosis the thoracic FLI neurons shift their position from ventrolateral to mid-ventral position and their axons terminate and elaborate a highly immunoreactive dorsal neural sheath. 4. It is suggested that the dorsal neural sheath may function as a neurohaemal organ from which FMRFamide-like substances may be released into the haemolymph to act as neurohormones.     

Adducin: Ca++-dependent association with sites of cell-cell contact

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly

Summary Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, ut overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the lightmicroscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leuenkephalin and Met-enk-Arg⁶-Phe⁷ (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.     

Intracellular localization of the P21rho proteins

We have surveyed the proteins expressed at the surface of different primary neurons as a first step in elucidating how axons regulate their ensheathment by glial cells. We characterized the surface proteins of dorsal root ganglion neurons, superior cervical ganglion neurons, and cerebellar granule cells which are myelinated, ensheathed but unmyelinated, and unensheathed, respectively. We found that the most abundant proteins are common to all three types of neurons. Reproducible differences in the composition of the integral membrane proteins (enriched by partitioning into a Triton X-114 detergent phase) were detected. These differences were most striking when the expression of glycosylphosphatidyl-inositol (GPI)-anchored membrane proteins by these different neurons was compared. Variations in the relative abundance and degree of glycosylation of several well known GPI-anchored proteins, including Thy-1, F3/F11, and the 120-kD form of the neural cell adhesion molecule (N-CAM), and an abundant 60-kD GPI-linked protein were observed. In addition, we have identified several potentially novel GPI-anchored glycoproteins on each class of neurons. These include a protein that is present only on superior cervical ganglion neurons and is 90 kD; an abundant protein of 69 kD that is essentially restricted in its expression to dorsal root ganglion neurons; and proteins of 38 and 31 kD that are expressed only on granule cell neurons. Finally, the relative abundance of the three major isoforms of N-CAM was found to vary significantly between these different primary neurons. These results are the first demonstration that nerve fibers with diverse ensheathment fates differ significantly in the composition of their surface proteins and suggest an important role for GPI-anchored proteins in generating diversity of the neuronal cell surface.     

Membrane properties of cultured rat sympathetic neurons: Morphological studies of adrenergic and cholinergic differentiation

Dissociated neurons from the newborn rat superior cervical ganglion were grown under conditions which lead to either adrenergic or cholinergic differentiation. Lectins and toxins were used to detect differences in the cell membrane associated with transmitter status, age of the neurons, or location on the neurons. These ligands were made visible in the light or electron microscope by coupling to rhodamine or colloidal gold. The density of binding sites for concanavalin A (Con A), ricin (RCA₆₀), and wheat germ agglutinin (WGA) increased with age in culture on both adrenergic and cholinergic cells. Soybean agglutinin (SBA) binding increased about threefold on adrenergic axons, but failed to increase on neurons induced to become cholinergic by medium conditioned by rat heart cells (CM). The effect of CM on SBA binding paralleled previously described effects of CM on transmitter production; the CM binding pattern developed slowly and was not readily reversible. Mature adrenergic neurons also appeared to bind more WGA than neurons in CM cultures. Tetanus toxin gold binding was uniform, but low, on axons of adrenergic and cholinergic neurons at all ages. In contrast, cholera toxin binding decreased with age on adrenergic axons. Binding sites for SBA and tetanus toxin were found to be less numerous on the cell body surface than on the axonal surface. Thus growth in CM induces fundamental changes in the phenotype of developing sympathetic neurons involving the cell membrane as well as transmitter choice. Differences also appear with maturation and between axonal and somatic cell surface membranes.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Summary Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetycholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immnoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholingergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.