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.     

Tracing of sensory neurones and spinal motoneurones of the pigeon by injection of fluorescent dyes into peripheral nerves

The projection of peripheral sensory and motor nerves was investigated in the pigeon (Columba livia) by means of retrogradely transported fluorescent dyes. Two combinations of fluorescent tracers were used that could be identified within the same cell when excited by light of 405 nm: 1) Propidium iodide and Bisbenzimide, which label the cytoplasm orange and the nucleus blue, respectively; 2) Fast Blue, which labels the cytoplasm blue, and Nuclear Yellow, which labels the nucleus (especially the nucleolar ring) yellow. The presence of the tracers in a given cell was confirmed microspectrophotometrically. Following injection of the tracers into peripheral nerves, labelled sensory neurones were seen in the dorsal root ganglia and motoneurones of the spinal cord. The peroneal and tibial nerves projected to L2-L5 and L2-L7, respectively, whereas the median and ulnar nerves projected to C12-Th2 and C13-Th1. Double-labelled sensory neurones were observed when both peroneal and tibial, or median and ulnar nerves were injected with different tracers. This indicates that some sensory neurones possess peripheral processes that dichotomize to pass down two different peripheral nerves. Double labelling was never seen in motoneurones, or in sensory neurones after tracer injection into the sciatic and femoral nerves.     

PROTEIN COMPOSITION OF AXONS and MYELIN FROM RAT and HUMAN PERIPHERAL NERVES

Abstract— Proteins of rat and human peripheral nerves were studied in whole nerve homogenates and in purified myelin and axonal preparations of peripheral nerve. Both myelin and axonal fractions were obtained from desheathed and minced nerve segments by flotation and sedimentation, respectively, in 0.85 m -sucrose following hypotonic treatment. The purity of myelin and axonal preparations was confirmed by electron microscopic examination of pelleted material. Nerve proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3 and 7.4. Major protein bands of fresh whole nerve homogenates corresponded to polypeptide bands of either the purified myelin or axon preparations. The most prominent electrophoretic band in peripheral nerve was identified as a myelin glycoprotein with molecular weight of 27,000. The major polypeptides of axon preparations had molecular weights of 200,000, 150,000, 69,000, 55,000 and 27,000. The latter two proteins were believed to represent tubulin and residual major myelin protein, respectively. The three largest axonal polypeptides were believed to be derived from neurofilaments, which represented the predominant organelle of the purified axons. Collagen was also seen in whole nerve homogenates and in purified axons but could be distinguished by its metachromatic staining with Coomassie blue.     

Egg laying inAplysia

1. Central pathways for bag cell activation were identified by examining the frequency of spontaneous egg laying episodes in animals with central connective lesions. Bilateral lesions of the cerebropleural (but not the cerebropedal) connectives abolished spontaneous egg laying. In contrast, bilateral lesions of all cerebral ganglion peripheral nerves did not abolish spontaneous egg laying, suggesting that sensory input to the cerebral ganglion is not necessary for activating the bag cells. 2. Backfilling either pleuroabdominal connective labelled cell bodies in the cerebral ganglia (via the ipsilateral cerebropleural connective) that could project to the bag cells. Focal extracellular stimulation of these stained clusters activated the bag cells in isolated brains. 3. Central pathways for initiating egg laying behaviors were identified by selectively eliciting bag cell discharges in animals with central connective lesions. Bilateral lesions of the cerebropedal (but not the cerebropleural) connectives completely abolished elicited egg laying behaviors. 4. Pathways for motor output during rhythmic head and neck movements were identified by eliciting bag cell discharges in animals with peripheral nerve lesions. Bilateral lesions of the four tegumentary nerves in combination with the anterior pedal nerve completely abolished elicited egg laying behaviors, indicating that these nerves are necessary for normal motor output. A normal pattern of egg laying behaviors occurred when the four tegumentary and the anterior pedal nerves were left intact and all other pedal ganglion nerves were lesioned bilaterally, indicating that these nerves are also sufficient for normal motor output.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endopeptidase-24.11, a Cell-Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.     

Organization of motoneurones in the prothoracic ganglion of the cockroach Periplaneta americana (L.).

The location within the prothoracic ganglion of neurone somata with axons in identified peripheral nerves is examined by the cobalt iontophoresis technique. Axons are filled with cobalt by diffusion through their cut ends and the cobalt is then precipitated as the black sulphide inside the neurone. It is assumed that neurones with axons in peripheral nerves and somata in central ganglia are either motor or neuro-secretory. Fifteen nerves are examined and maps of the location of somata with axons in each nerve are presented. The axon distribution in peripheral nerves of three common inhibitory neurones is described. Dendritic morphology of one common inhibitory neurone and two coxal depressor motoneurones is illustrated. It is proposed that some individual neurones can be reliably identified from their soma dimensions and location within the ganglion. The number of motoneurones with somata in the prothoracic ganglion and their homology with cells in the other thoracic ganglia are discussed.     

Immunohistochemical identification of pancreatic hormones,neuropeptides and cytoskeletal proteins in pancreas of the camel (Camelus dromedarius)

The patterns of distribution of insulin (INS), glucagon (GLU), atrial natriuretic peptide (ANP), neuropeptide-Y (NPY), cholecystokinin-octapeptide (CCK-8), neurofilament-200 protein (NF), S-100 protein (S-100), and vimentin (VIM) in the pancreas of the one-humped camel (Camelus dromedarius) were investigated using immunohistochemical techniques. INS-immunoreactive cells were observed in the central and peripheral parts of the islets of Langerhans, but some solitary INS-positive cells were found outside the islets. INS-positive cells constituted 44.26–90.91% [mean ± standard deviation (std): 67.34 ± 14.20] of the total number of islet cells. GLU-immunopositive cells were located mainly in the peripheral region of the islets, and they constituted 11.43–44.44% [mean ± std: 23.54 ± 8.27] of the total number of islet cells. ANP and CCK-8 immunoreactivity was observed in neurons and perivascular nerves fibers. NPY was identified in pancreatic neurons and in some peripheral and central cells of the islets of Langerhans. VIM immunoreactivity was observed in the endothelial cells of blood vessels and the nerves located in the perivascular, interlobular and periacinar regions. VIM was also detected immunohistochemically in the periductal nerves of the pancreas. NF occurred only in nerves. S-100 was discerned mainly in the nerves of the interlobular connective tissue and in nerves lying close to blood vessels and acinar tissue. It is concluded that INS, GLU, ANP, NPY, CCK-8, NF, S-100, and VIM are well distributed in the pancreas of the camel. J. Morphol. 231:185–193, 1997. © 1997 Wiley-Liss, Inc.     

Neutrophils and B-cells in Atlantic cod (Gadus morhua L.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.     

Desert hedgehog-patched 2 expression in peripheral nerves during Wallerian degeneration and regeneration

Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh-receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT-PCR, however, ptc1 was undetectable under the same experimental condition. Using RT-PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild-type (WT) and dhh-null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT-PCR in WT mice. In dhh-null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh-null mice, minifascicular formation was even more extensive than in dhh-null intact nerves. Both dhh and ptc2 mRNA levels were down-regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh-Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury.     

Determination of the topical organization of outputs of identified molluscan neurons

In experiments on mollusks (Planorbis corneus) the topical organization of outputs of neurons RPal and RPa2 of the right parietal ganglion was investigated. Outputs were identified by coherence analysis of accumulated electrical activity in dissected nerves during activation of the above neurons. The analysis was based on a number of features described in this paper. Axons of the test neurons were found to be present in the left and right pallial nerves; polysynaptic pathways activated by these neurons also were discovered. The topical organization of outputs of the test neurons was shown to be invariant for different preparations.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 458–463, July–August, 1984.     

Demonstration of catecholamines in peripheral adrenergic nerves in stretch preparations with fluorescence induced by aqueous solution of glyoxylic acid.

Fluorescence induced by aqueous solution of glyoxylic acid and formaldehyde-induced fluorescence of catecholamines were compared for the demonstration of peripheral adrenergic nerves in stretch preparations. Glyoxylic acid was better than formaldehyde for the demonstration of the adrenergic nerves. On the other hand, the formaldehyde was better than glyoxylic acid for the demonstration of biogenic amines in cell bodies.     

Axonal regrowth is impaired during digit tip regeneration in mice

Mice are intrinsically capable of regenerating the tips of their digits after amputation. Mouse digit tip regeneration is reported to be a peripheral nerve-dependent event. However, it is presently unknown what types of nerves and Schwann cells innervate the digit tip, and to what extent these cells regenerate in association with the regenerative response. Given the necessity of peripheral nerves for mammalian regeneration, we investigated the neuroanatomy of the unamputated, regenerating, and regenerated mouse digit tip. Using immunohistochemistry for β-III-tubulin (β3T) or neurofilament H (NFH), substance P (SP), tyrosine hydroxylase (TH), myelin protein zero (P0), and glial fibrillary acidic protein (GFAP), we identified peripheral nerve axons (sensory and sympathetic), and myelinating- and non-myelinating-Schwann cells. Our findings show that the digit tip is innervated by two digital nerves that each bifurcate into a bone marrow (BM) and connective tissue (CT) branch. The BM branches are composed of sympathetic axons that are ensheathed by non-myelinating-Schwann cells whereas the CT branches are composed of sensory and sympathetic axons and are ensheathed by myelinating- and non-myelinating-Schwann cells. The regenerated digit neuroanatomy differs from unamputated digit in several key ways. First, there is 7.5 fold decrease in CT branch axons in the regenerated digit compared to the unampuated digit. Second, there is a 5.6 fold decrease in myelinating-Schwann cells in the regenerated digit compared to the unamputated digit that is consistent with the decrease in CT branch axons. Importantly, we also find that the central portion of the regenerating digit blastema is aneural, with axons and Schwann cells restricted to peripheral and distal blastema regions. Finally, we show that even with impaired innervation, digits maintain the ability to regenerate after re-amputation. Taken together, these data indicate that nerve regeneration is impaired in the context of mouse digit tip regeneration.     

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue,cryostat sections and whole-mounts

Summary This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.     

Responses to magnetic stimuli recorded in peripheral nerves in the marine nudibranch mollusk <Emphasis Type="Italic">Tritonia diomedea</Emphasis>

Prior behavioral and neurophysiological studies provide evidence that the nudibranch mollusk Tritonia orients to the earth’s magnetic field. Earlier studies of electrophysiological responses in certain neurons of the brain to changing ambient magnetic fields suggest that although certain identified brain cells fire impulses when the ambient field is changed, these neuron somata and their central dentritic and axonal processes are themselves not primary magnetic receptors. Here, using semi-intact animal preparations from which the brain was removed, we recorded from peripheral nerve trunks. Using techniques to count spikes in individual nerves and separately also to identify, then count individual axonal spikes in extracellular records, we found both excitatory and inhibitory axonal responses elicited by changes in the direction of ambient earth strength magnetic fields. We found responses in nerves from many locations throughout the body and in axons innervating the body wall and rhinophores. Our results indicate that primary receptors for geomagnetism in Tritonia are not focally concentrated in any particular organ, but appear to be widely dispersed in the peripheral body tissues.     

Vimentin in the Central Nervous System

Intermediate filament proteins were identified by two-dimensional gel electrophoresis in urea extracts of rat optic nerves undergoing Wallerian degeneration and in cytoskeletal preparations of rat brain and spinal cord during postnatal development. The glial fibrillary acidic (GFA) protein and vimentin were the major optic nerve proteins following Wallerian degeneration. Vimentin was a major cytoskeletal component of newborn central nervous system (CNS) and then progressively decreased until it became barely identifiable in mature brain and spinal cord. The decrease of vimentin occurred concomitantly with an increase in GFA protein. A protein with the apparent molecular weight of 61,000 and isoelectric point of 5.6 was identified in both cytoskeletal preparations of brain and spinal cord, and in urea extracts of normal optic nerves. The protein disappeared together with the polypeptides forming the neurofilament triplet in degenerated optic nerves.     

The distribution of putative nitric oxide releasing neurones in the locust abdominal nervous system: a comparison of NADPHd histochemistry and NOS-immunocytochemistry

Nitric oxide is well established as a signalling molecule in the nervous system of vertebrates and invertebrates. In this study we evaluate the usefulness of NADPHdiaphorase histochemistry and immunocytochemistry for detecting the presence of nitric oxide synthase in locusts. We describe the distribution of putative nitric oxide releasing neurones and stained neuropiles in the locust ventral nerve cord, in particular the abdominal ganglia and abdominal neuromeres. NADPHdiaphorase histochemistry revealed prominent staining in all neuropilar regions and a specific distribution pattern of stained cell bodies in all examined ganglia. Nitric oxide synthase immunocytochemistry, using a commercially available universal antibody, labelled cells in corresponding positions within the ganglia. This was confirmed by double labelling of alternate sections. Western blot analysis demonstrated that in locusts this universal NOS-antibody binds to a protein of similar size to nitric oxide synthase identified in other insect species. The antibody also labelled axons in most peripheral nerves of all examined ganglia, whereas NADPHdiaphorase histochemistry only revealed such stained fibres within peripheral nerves in some preparations, because they may have been masked by intense background staining. We therefore conclude that nitric oxide synthase-immunocytochemistry and NADPHd histochemistry are both good markers for the presence of nitric oxide synthase in the locust ventral nerve cord, and that nitric oxide may be used as a signalling molecule by efferent neurones in locusts.     

Neural pathways to cardioaccelerator neurons in the isopod crustacean Bathynomus doederleini: Cholinergic activation by somatic movements

We investigated the excitatory and inhibitory input to cardioaccelerator (CA) and cardioinhibitor (CI) neurons located in the thoracic ganglia of the isopod crustacean Bathynomus doederleini by extracellular and intracellular recording. Electrical stimuli applied to the anterior and posterior connectives of single-ganglion preparations, containing either the 2nd or 3rd thoracic ganglion alone, and each of three paired ganglionic nerve roots produced excitatory postsynaptic potentials (EPSPs) in the cell body of a CA neuron. Artificial movements of appendages, such as the thoracic limbs and the swimmerets, also evoked EPSPs in the CA neuron. Electrical stimuli applied to the peripheral nerves running to appendages induced inhibitory postsynaptic potentials (IPSPs) in a CI neuron. Since artificial movements of the appendages caused decrease of CI impulse rate, these IPSPs in the CI neuron may be caused by mechanoproprioceptors in the appendages. Since tachycardia was accompanied by excitation of CA neurons and inhibition of CI neurons, activation of the mechanoproprioceptors may be responsible for tachycardia. EPSPs in CA neurons produced by stimulation of peripheral nerves were augumented by eserinization and blocked by curarization. The activation of CA neurons by ganglionic roots may be mediated by cholinergic processes ascending from mechanoproprioceptors.     

Schwann-spheres derived from injured peripheral nerves in adult mice--their in vitro characterization and therapeutic potential

Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call "Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these "Schwann-spheres" would provide a more potential autologous cell source for such transplantation.     

Galanin-like immunoreactivity in extrinsic and intrinsic nerves to the gut of the Atlantic cod,Gadus morhua,and the effect of galanin on the smooth muscle of the gut

The presence of galanin-like immunoreactivity in nerves to the stomach of the Atlantic cod has been investigated by immunohistochemistry. The distribution of ganglion cells showing galanin-like immunoreactivity was compared with the total distribution in nerves and ganglia. Projection studies were made to determine the origin of the galanin neurons. The effect of galanin was studied in smooth muscle strip preparations of the gut wall and arteries. Galanin-like immunoreactive ganglion cells frequently occurred along the vagal branches to the stomach. Most of them projected cranially. Immunoreactive nerve fibres were present in all layers of the gut and around arterial branches on the surface of the stomach. Ligations of the vagus and splanchnic nerves produced accumulations of immunoreactive material on both sides of the ligature. Galanin produced weak contractile effects unaffected by tetrodotoxin on the gut wall and on gut arteries. It is concluded that a population of the ganglion cells along the vagus nerve in the Atlantic cod contains a galanin-like peptide. Some of these cells may be parts of autonomic parasympathetic pathways innervating the gut of the Atlantic cod, having direct excitatory effects on the smooth muscles of the gut wall and gut arteries.     

Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder

The morphological characteristics of smooth muscle cells (SMCs) and their innervation of the suburothelial microvasculature of the mouse bladder were investigated by immunohistochemistry. Whole mount bladder mucosal preparations were immune-stained for α-smooth muscle actin (α-SMA) and/or neuronal markers and examined using confocal laser scanning microscopy. Suburothelial arterioles consisted of α-SMA-immunopositive circular smooth muscle cells, while the venular wall composed of α-SMA-positive SMCs that displayed several processes which extended from their cell bodies to form an extensive meshwork. In larger venules, a complex meshwork of stellate-shaped SMCs were observed. NG2 chondroitin sulphate proteoglycan-immunoreactive cell bodies of capillary pericytes were not immunoreactive for α-SMA. In the rat bladder suburothelial venules, circular SMCs were the dominant cell type expressing α-SMA-immunoreactivity. Since α-SMA-positive SMCs in suburothelial arterioles and venules in the mouse bladder had quite distinct morphologies, the innervation of both vessels could be examined by double labelling for α-SMA and various neuronal markers. Varicose nerve bundles immunoreactive for tyrosine hydroxylase (sympathetic nerves), choline acetyltransferase (cholinergic nerves) or substance P (primary afferent nerves) were all detected along side suburothelial arterioles. Single varicose nerve fibres positive for these three neuronal markers were also detected around the venules. Thus, whole mount preparations are useful when examining the morphology of α-SMA-positive SMCs of the microvasculature in the suburothelium of mouse bladder as well as their relationship with their innervations. In conclusion, arterioles and venules of the bladder suburothelium are the target of sympathetic, cholinergic and primary afferent nerve fibres.