Filament proteins in central, cranial, and peripheral mammalian nerves.

Several classes of 10-nm filaments have been reported in mammalian cells and they can be distinguished by the size of their protein subunit. We have studied the distribution of these filaments in nerves from calves and other mammals. From the display on polyacrylamide electrophoretic gels of proteins in extracts from fibroblast and central, cranial and peripheral nerves, we cut the appropriate stained bands and prepared iodinated peptide maps. The similarities between the respective maps provide strong evidence for the presence of vimentin in cranial and peripheral nerves. The glial fibrillary acidic protein was found in axon preparations from the central nervous system, but was not identified in distal segments of some cranial nerves, nor in peripheral nerve.

An unusual glucocerebroside in the crustacean nervous system.

High concentrations of glucocerebroside (glucosylceramide) were found in the ventral nerve cord, brain, optic nerve, and antenna, but not in the nonneural tissue, of the brown shrimp Penaeus aztecus aztecus. This lipid contained unusual sphingoid bases consisting of 14-, 15-, and 16-carbon sphinganines and sphingenines. The fatty acids were mainly nonhydroxylated homologs 22 carbons long and longer, similar to those found in galactocerebroside but differing from those in glucocerebroside in mammalian nervous systems.

Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system.

Patterns of expression of transcripts encoding receptors for fibroblast growth factor and nerve growth factor (FGF-R and NGF-R) in the developing chick nervous system are compared using in situ hybridization histochemistry. FGF-R transcripts are expressed abundantly in the germinal neuroepithelial layer. Expression ceases as cells migrate into the mantle layer and returns during late maturation of neuronal populations, including cholinergic nuclei of the basal forebrain, brainstem reticular and motor nuclei, and cerebellar Purkinje and granule neurons. The pattern of NGF-R expression is generally reciprocal to that of FGF-R in the CNS and in some phases of development of the PNS. These results suggest that FGF and NGF may act sequentially rather than in concert during neuronal development.

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein.

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam.

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues. Synthesis of L67 is detectable in embryos 2h after fertilization; it reaches a maximum in 6h-old embryos and gradually declines thereafter. These results argue that the composition of the NL bears no obvious relationship to the structural and functional changes that take place during the embryonic development of this invertebrate.

Strategies in neuropeptide receptor binding research.

Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed.

Serotonin-containing neurons in Drosophila melanogaster: development and distribution.

Antibodies made against serotonin (5HT) were used to identify the serotonin neuronal system in the developing and adult nervous system of Drosophila melanogaster. The 5HT neuronal pattern is composed of a small number of neurons, 84 in larvae and 106 in adults, distributed in clusters composed of one to five neurons in the CNS; 5HT immunoreactive (5HT-IR) neurons appear to be predominantly intrasegmental interneurons; however, intersegmental 5HT-IR fibers are observed and at least some neurons send peripheral fibers. Acquisition of 5HT immunoreactivity in the CNS occurs late in embryogenesis, by 16-18 hours, and most if not all the 5HT neurons appear to persist into adulthood. During early metamorphosis, the intensity of 5HT-IR neuropil transiently decreases. Other changes in the CNS during this period are reflected in the appearance of two new 5HT clusters and 5HT-IR neuropil in the developing optic lobes. Comparison of the 5HT-IR pattern with other transmitter systems in Drosophila as well as comparison of the 5HT-IR pattern within different insect species is presented.

Catecholamine-containing neurons in Drosophila melanogaster: distribution and development.

The development of catecholamine-containing neurons (CA neurons) in the fruit fly Drosophila melanogaster was studied. Glyoxylic-acid-induced histofluorescence and antibodies against dopamine and tyrosine hydroxylase were used to describe catecholamine distribution in the larval central nervous system (CNS). The three techniques gave rise to a similar pattern of distribution of putative CA neurons. At all developmental stages CA neurons were distributed widely throughout the CNS but represented only a small fraction of all CNS neurons. Catecholamine-containing processes were confined to the CNS. The CA neurons are first discerned at about 18 hours of embryonic development. We suggest that these larval CA neurons are maintained throughout the ontogeny of the fly and that the adult CA pattern is composed of embryonic neurons and neurons that differentiate during metamorphosis.

Serotoninlike immunoreactivity in the central and peripheral nervous system of the scale worm Harmothoe imbricata (Polychaeta).

The distribution of serotoninergic neurons in the nervous system of the scale worm Harmothoe imbricata was visualized in the anterior half of the body by the peroxidase-antiperoxidase (PAP) immunohistochemical method with a specific antiserotonin antibody. Immunoreactive neuronal somata were localized in discrete ganglion cell masses of the dorsally situated cerebral ganglion and in segmental ganglia of the ventral nerve cord. They also make up the majority of neurons present in the parapodial ganglia. Large and small varicose fibers stained in the neuropile of all the above-mentioned ganglia but also in interganglionic connectives and segmental nerves. On the basis of soma size and location and of fiber distribution, the reactive neurons were identified as primarily interneuronal with a few motoneurons and presumptive afferent neurons. The presence of a motor component was substantiated by observations of several reactive varicose fibers spread over longitudinal muscle layers of the trunk. In addition, neurites of the subepidermal nerve plexus and enterochromaffinlike cells of the gut epithelium reacted with the serotonin antibody. It is concluded that serotoninergic pathways are ubiquitous elements in the organization of the central and peripheral nervous system of this polychaete. The significance of these findings in relation to other annelid groups and to the physiological role of serotonin is discussed.

Purification and characterization of FMRFamidelike immunoreactive substances from the lobster nervous system: isolation and sequence analysis of two closely related peptides.

In the preceding paper (Kobierski et al: J. Comp. Neurol. 266:1-15, '87) FMRFamidelike immunoreactivity (FLI) was localized to specific cells and processes in the nervous system of the lobster Homarus americanus. In an effort to establish a role for this material we have purified and characterized a variety of immunoreactive peptides that can be extracted from the secretory pericardial organs. By using gel-filtration chromatography and three different HPLC systems, it has been established that little or no authentic FMRFamide is present. Of the major immunoreactive components two peptides were purified in sufficient quantity for microsequence analysis and have been tentatively identified as the octapeptides Ser-Asp-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 3) and Thr-Asn-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 4). Both of these are novel neuropeptides with some sequence homology to the previously described FMRFamide family. The pericardial organs release FLI when depolarized with 100 mM K+ in the presence of calcium. Between 75 and 80% of this release is accounted for by FLI 3 and FLI 4. One of these peptides (FLI 4) has been synthesized and shown to cochromatograph with the endogenous immunoreactive material. Preliminary studies show that this peptide can act as a modulator of exoskeletal and cardiac neuromuscular junctions.

FMRFamidelike peptides of Homarus americanus: distribution, immunocytochemical mapping, and ultrastructural localization in terminal varicosities.

The distribution of FMRFamidelike peptides was studied in the nervous system of the lobster Homarus americanus by using immunocytochemical and radioimmunological techniques. By radioimmunoassay FMRFamidelike immunoreactivity (FLI) was found in low levels (ca. 1 pmol/mg protein) throughout the ventral nerve cord and in much higher amounts (60-100 pmol/mg protein) in the neurosecretory pericardial organs. Immunocytochemical studies showed FLI in approximately 300-350 cell bodies, and in distinct neuropil regions, neuronal fiber tracts, and varicose endings. Specificity of the immunostaining was tested by preabsorbing the antiserum with FMRFamide, with peptides having similar carboxyl termini to FMRFamide (Met-enkephalin-Arg-Phe, Phe-Met-Arg-Tyr-amide), with several amidated peptides (alpha-melanocyte-stimulating hormone, substance P, oxytocin), and with proctolin, a peptide found widely distributed in the lobster nervous system. Of these substances, only FMRFamide blocked the staining. In addition to the pericardial organs, significant levels of FLI were found in neurosecretory regions associated with thoracic second roots and in the connective tissue sheath that surrounds the ventral nerve cord. In all three regions, immunocytochemical studies showed the FLI to be localized to fine fibers and associated terminal varicosities lying close to the surface of the tissue, with no obvious target in their immediate vicinity. When examined at the ultrastructural level, the immunoreactive varicosities of the thoracic second roots and of the ventral nerve cord sheaths were found a few microns from the surface of the tissue and contained electron-dense granules. In the immunoreactive nerve cord sheath endings, in addition to the large, dense granules, small, clear vesicles were found. The appearance and location of these terminals suggest a neurohormonal role for FMRFamidelike peptides in lobsters. The observation that low levels of FLI are found in the hemolymph supports this suggestion. In addition, the localization of FLI to particular neuronal somata, fiber tracts, and neuropil regions suggests possible functional roles for these peptides in (1) integration of visual and olfactory information, (2) function of the anterior and posterior gut, and (3) the control of exoskeletal muscles.

Monoclonal antibody identifies a 63,000 dalton antigen found in all central neuronal cell bodies but in only a subset of axons in the leech.

The monoclonal antibody Lan3-8 binds to all the neuronal cell bodies in midbody, head, tail, and supraesophageal ganglia in the mud leech (Haemopis marmorata) and the medicinal leech (Hirudo medicinalis). In contrast to the general distribution of the antigen in cell bodies it is only found in a subset of axons, where electron microscopy suggests that it may be associated with a cytoskeletal filament system. Immunoblotting shows that the antibody binds to a 63,000 dalton band that is protease-sensitive. The same 63 kilodalton (kd)-antigen is found in all regions of the central nervous system, in proteins isolated from connectives (axons alone), and from hand-dissected identified cell types. The molecular weight and electron microscopic localization raised the possibility that this antigen is the core neurofilament protein, but the antigen does not comigrate with 67-kd intense coomassie blue band that binds another anti-intermediate filament antibody. The supraesophageal ganglia are known to have a different developmental or origin from the other structures in the leech central nervous system. Two-dimensional gel electrophoresis and silver staining show that, like the 63-kd antigen, many other proteins are very similar in these developmentally distinct neural structures.

Proctolin-like immunoreactive neurons in the blowfly central nervous system.

The pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is a well-studied bioactive substance in insects. With an antiserum against proctolin we have mapped proctolinlike-immunoreactive (PLI) neurons in the nervous system of the blowfly Calliphora erythrocephala. In the brain, including the suboesophageal ganglia, 80-90 neurons were found to be PLI. A further 200-250 PLI neurons innervate the lobula of the optic lobe. The thoracic ganglia contain 100-130, and the abdominal ca. 60 PLI neurons. In the brain and ventral ganglia the immunoreactive neurons are of different types: interneurons, efferents (possibly some motorneurons), and neurosecretory cells. Some of these neurons are individually identifiable; others can be identified collectively as clusters. Identifiable neurons innervate protocerebral neuropil associated with the pars intercerebralis and the beta-lobes of the mushroom bodies as well as tritocerebral neuropil. Some of the prominent clusters innervate the central body of the protocerebrum, tritocerebrum, and possibly leg motor neurons. One abdominal cluster is of special interest because it consist of efferent neurons with processes in the lateral abdominal nerves. Some of these processes are located in the neural sheath in neurohaemal regions, and electron microscopy demonstrates that their terminals are outside the blood-brain barrier. The PLI processes in the protocerebrum contain large granular vesicles and form chemical synapses with different kinds of nonimmunoreactive neural elements. Thus, in Calliphora the proctolinlike substance may be used as a central transmitter/modulator, a neuromuscular transmitter, and a neurohormone released into the circulation.

Distribution of immunoreactive somatostatin (ISRIF) in the nervous system of the squid, Loligo pealei.

Somatostatin (SRIF) is a neuropeptide with a widespread distribution in the mammalian CNS. In the present study we have examined the distribution of immunoreactive-like SRIF (ISRIF)-containing elements in the nervous system of the cephalopod mollusk Loligo pealei, or the Woods Hole squid. ISRIF was localized by light immunocytochemistry in sections of the squid-optic lobe, circumesophageal ganglia-and in stellate ganglion. In the optic lobe, ISRIF neurons were found in the internal granule cell layer and medulla and immunoreactive fibers were seen throughout the lobe and in the optic tract but were absent from the optic nerve, i.e., the projection between the retina and optic lobe. In the supraesophageal complex, ISRIF neurons were found in all lobes, but primarily in the vertical, subvertical, and frontal. In the subesophageal ganglion, ISRIF neurons were seen mainly following unilateral pallial nerve lesions; these neurons were primarily small-to-medium sized. ISRIF fibers were seen in many of the nerves exiting from the brain and in nerves extending between the sub- and supra-esophageal ganglia. In the stellate ganglion, ISRIF was present in many neurons as well as in a plexus of fibers within the ganglion; the peptide was absent from the second-order fibers and the giant axon. The data suggest that a molecule immunologically similar to vertebrate SRIF may be a major transmitter/modulator in this invertebrate. These results provide a foundation for further studies to evaluate the role of this molecule.

Distribution and partial characterization of FMRFamide-like peptides in the stomatogastric nervous systems of the rock crab, Cancer borealis, and the spiny lobster, Panulirus interruptus.

The distribution of FMRFamide-like peptides was studied in the complete stomatogastric nervous system [the paired commissural ganglia, single oesophageal ganglion, and the single stomatogastric ganglion (STG)] of two decapod crustacean species, the spiny lobster Panulirus interruptus and the rock crab Cancer borealis, by using immunocytochemical techniques. Antiserum 231 from the O'Donohue laboratory and antiserum 671C (described here) gave essentially the same staining patterns. In the commissural ganglia of both species there were ten to 20 stained neurons and dense neuropilar staining. The oesophageal ganglion of the crab had four stained neurons. Lucifer Yellow backfills followed by immunostaining showed that the two larger stained neurons of the oesophageal ganglion sent processes into the inferior ventricular nerve. The two smaller neurons sent processes into the inferior oesophageal nerves. The oesophageal ganglion of the lobster had two stained neurons that sent processes into the inferior ventricular nerve as well. None of the somata of the STG stained in either species, but in both species stained fibers were seen in the stomatogastric nerve that entered the STGs and ramified profusely throughout the neuropil. In some preparations of the crab, a stained fiber was visible in the dorsal ventricular nerve. The amounts of the FMRFamide-like peptides found in all regions of the nervous system of P. interruptus and C. borealis were determined by radioimmune assay (RIA). Column chromatography and high-performance liquid chromatography suggest that, in both species, much if not all of the RIA-assayable material is accountable for by peptides that are larger and more hydrophobic than FMRFamide.

An X-ray diffraction comparison of myelins from the human nervous system.

Earlier small-angle X-ray diffraction studies have indicated that central and peripheral nerve myelins may be significantly different structurally, although relatively few examples for each system and for individual species have been examined. In order to understand better the intra- and inter-system relationships, this study has developed more extensive information for a single species: six cases centrally and ten peripherally, featuring cranial nerves and a few others of the human nervous system. Peripheral myelin spacings (membrane pair thicknesses) are relatively similar, 184.4 +/- 1.40 A, and the ratios of diffraction peak height intensities of the second to fourth orders are also closely bunched: 1.85 +/- 0.216. Central myelin spacings and intensity ratios are distinctly different and more variable: spacings 160.3-165.8 A and intensity ratios 2.81-4.46. It appears that within a given species or between closely related (e.g., mammalian) species peripheral myelins possess relatively invariant structures, though significant spacing declines are encountered for both systems as phylogenetic relationships become more distant. The observed greater variability of CNS structures within a single species may correspond to known compositional differences between CNS regions or result from observational difficulties. In any case there is a marked discontinuity between the myelin structures of CNS and PNS nerves.

Soluble and membrane-bound muscarinic acetylcholine receptors.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

Immunohistochemical analysis of the immune reaction in the nervous system in paraneoplastic encephalomyelitis.

We examined frozen sections of frontal cortex, medulla, and dorsal root ganglia from a patient with small-cell lung cancer and paraneoplastic encephalomyelitis, involving the medulla and dorsal root ganglia, with a panel of antibodies reactive for IgG, IgM, C3, B cells, T cells, T cell subsets, macrophages, and class I and II (HLA-DR) major histocompatibility complex (MHC) antigens. We detected an antineuronal antibody (anti-Hu) in the serum and CSF of the patient and found deposits of IgG in the periphery of some neurons in dorsal root ganglia. The infiltrates were almost exclusively T cells with a predominance of CD8-positive cells. Neurons did not express class I or II MHC antigens. Satellite cells in the dorsal root ganglia from the patient and controls were HLA-DR-positive. These data indicate that CD8-positive T cells predominate in the inflammatory infiltrates of paraneoplastic encephalomyelitis. IgG deposits may be relevant in the damage of the sensory neurons.

Immunocytochemical localization of fibronectin in limb tissues of the adult newt, Notophthalmus viridescens.

This study describes the immunocytochemical localization of fibronectin, a defined connective tissue and plasma glycoprotein, and its relationship to collagen and reticulin in adult newt limb tissues. We have also isolated the plasma form of fibronectin in a related species, the adult mudpuppy. The insoluble form of fibronectin was detected with immunoperoxidase stain in basement membranes and loose connective tissue. The endoneurium and perineurium of nerve bundles and the connective tissue elements of striated muscle stained heavily for fibronectin. The dermis and blood vessel walls also reached positively with the immunoperoxidase stain. A similar distribution was observed for reticulin with conventional histologic techniques with the exception of the dermis where only trace amounts of the protein were observed. Fibronectin and collagen were codistributed in the tissues studies. Fibronectin appeared to be intercalated among larger collagenous fibers. Collagen and fibronectin form an extracellular connective tissue scaffold that abuts against many types of adherent cells in different tissues. This supports the possible role of fibronectin in cell-matrix interactions and normal cell and tissue organization.