Cytoplasm Of Cell Bodies Research Articles

Reactions of Cysteine and Cysteinyl Derivatives with Dopamine-o-quinone and Further Insights into the Oxidation Chemistry of 5-S-Cysteinyldopamine: Potential Relevance to Idiopathic Parkinson′s Disease

Recent studies suggest that elevated rates of autoxidation of dopamine (DA) in the cytoplasm of neuromelanin-pigmented dopaminergic cell bodies in the substantia nigra (SN) and/or reactions of its proximate oxidation product, DA-o-quinone, with glutathione (GSH) or L-cysteine (CySH) might yield endotoxins that play roles in the pathogenesis of idiopathic Parkinson′s Disease (PD). In this study the reactions between DA-o-quinone and CySH and some cysteine derivatives have been studied. The reactions between DA-o-quinone and CySH or cysteine methyl ester are rapid and give the corresponding 5-S-cysteinyl conjugates of DA as the predominant products. By contrast, the reaction between the sterically hindered D-penicillamine methyl ester (PME) and DA-o-quinone is much slower to give, initially, a mixture of the 2-S-, 5-S-, and 6-S-PME conjugates of DA. These conjugates are then further oxidized by unreacted DA-o-quinone to give a complex mixture of products which include 7-(2-aminoethyl)-5-hydroxy-2,2-dimethyl-1,4-benzothiazine-3-carboxylic acid methyl ester (13). Large increases in the 5-S-cysteinyldopamine (5-S-CyS-DA)/DA concentration ratio have been measured in the Parkinsonian SN and have been interpreted to reflect elevated rates of DA autoxidation in this structure that degenerates in PD. However, the present study reveals that at physiological pH 5-S-CyS-DA is not only more easily oxidized than DA but a bicyclic o-quinone imine (5) intermediate is formed that can chemically oxidize the former conjugate in a reaction that leads to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (6). Compound 6 is lethal when administered into the brains of mice. However, this putative dihydrobenzothiazine endotoxin is even more easily oxidized than 5-S-CyS-DA in a rather complex reaction that ultimately forms 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (15). Although this benzothiazine could not be isolated it was identified by comparison of its electrochemical and spectroscopic properties in solution with those of 13. The results of this study suggest that benzothiazine 15 might serve as a better analytical marker molecule than 5-S-CyS-DA for either elevated rates of DA autoxidation and/or for roles of GSH and CySH in the neurodegenerative mechanisms in the SN that contribute to PD.

Localization of a nicotinic acetylcholine receptor-like antigen in the thoracic nervous system of embryonic locusts, Schistocerca gregaria.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent immunoblotting of neuronal membrane proteins derived from thoracic ganglia of adult Locusta and Schistocerca reveal that a polyclonal antiserum raised against the Locusta nicotinic acetylcholine receptor (nAChR), binds strongly to an identical polypeptide band corresponding to 65 kDa in both locust species. This polyclonal antiserum was used to analyze the distribution of antigenic sites within the developing thoracic central nervous system of Schistocerca embryos. Axonal outgrowths from the earliest differentiated neurons are first labeled between 30% and 35% development. By 40% to 45% development, labeled granules appear in the cytoplasm of neuronal cell bodies. When the developing neuropil is first enclosed at approximately 45% to 50% development, it appears uniformly labeled, but by 55% development, unlabeled areas appear that represent the sites of future tracts and commissures. By 75%, an adult pattern of neuropil immunogenicity is established in which synaptic regions are stained but tracts and commissures are not. This suggests that during the early development of the thoracic nervous system nAChR-like antigenic sites are evenly distributed, but later become concentrated in the developing synaptic areas.

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique.

The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2-3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures -30 degrees C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intracellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions.

Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation

The mRNAs for two forms of glutamic acid decarboxylase (GAD65 and GAD67) were localized in the rat hippocampal formation by nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. Some neurons in all layers of the hippocampus and dentate gyrus were readily labeled for each GAD mRNA, and the patterns of labeling for GAD65 and GAD67 mRNAs were very similar. All major groups of previously described GAD- and GABA-containing neurons appeared to be labeled for each GAD mRNA. Such findings suggest that most GABA neurons in the hippocampal formation contain both GAD mRNAs. When the labeling of neurons in the hippocampal formation and cerebral cortex was compared in the same sections, the intensity of neuronal labeling for GAD67 mRNA was generally similar in the two regions. However, the intensity of labeling for GAD65 mRNA was generally stronger for many neurons in the hippocampal formation than for most neurons in the cerebral cortex. Neurons in the hilus of the dentate gyrus were particularly well labeled for GAD65. The nonradioactive labeling for the GAD mRNAs was confined to the cytoplasm of neuronal cell bodies, and this allowed a clear visualization of the relative number and location of labeled neurons. Several distinct patterns of GAD mRNA-containing neurons were observed among different regions of the hippocampal formation. In the hilus of the dentate gyrus, GAD mRNA-containing neurons were numerous in the regions deep to the granule cell layer as well as in more central parts of the hilus. Within CA3, the densities (quantities) of labeled neurons varied among the regions. In the inner or hilar segment of CA3, the density of labeled neurons was often lower than that in the outer part of CA3 where numerous labeled neurons were distributed throughout all layers. In CA1, GAD mRNA-labeled neurons were distributed in a relatively laminar pattern with the highest density in stratum pyramidale and moderate densities in stratum oriens and at the interface between strata radiatum and lacunosum-moleculare. Lower densities were found within the latter two layers. The prominent localization of the two GAD mRNAs in the hippocampal formation suggests that a dual system for GABA synthesis is necessary for normal GABAergic function in this brain region. Most putative GABA neurons contain relatively high levels of GAD67 mRNA as might be expected if this GAD form is responsible for the synthesis of GABA for metabolic and baseline synaptic function.(ABSTRACT TRUNCATED AT 400 WORDS)

Polarized distribution of the trans-Golgi network marker TGN38 during the in vitro development of neocortical neurons: effects of nocodazole and brefeldin A.

Neurons are polarized secretory cells whose cytoplasm and plasma membrane are polarized to form two compartments: dendrites and axons. In mature, fully polarized neurons, the microtubule-associated protein Map2 is targeted to dendrites, while tau is mainly restricted to axons. However, the intraneuronal distribution of secretory pathway organelles, such as the endoplasmic reticulum and the Golgi complex, which give rise to all constitutive, regulated and lysosome vesicles, is poorly understood. Thus, to investigate the distribution of the trans-Golgi network during the development and maturation of rat neocortical neurons in vitro, we have utilized an antibody recognizing a 38 kDa trans-Golgi network-specific protein, TGN38, and immunofluorescence microscopy. Before neurons have established polarity. TGN38 immunoreactivity outlines several vesicles dispersed throughout the cell body cytoplasm; these converge close to a major Map2-immunopositive process during the establishment of neuronal polarity, and later merge into a single structure located at the base of a thick Map2-immunopositive process, approximately 18 h after plating. At this stage TGN38 immunoreactivity is located within 45 degrees of the major Map2-immunoreactive process in 54% of neurons, while in only 6% of cells it is located at the opposite pole. After 3 days in vitro, during the segregation of microtubule-associated proteins to either dendrites or axons. TGN38 immunoreactivity clusters continue to be located close to a major dendrite, and in some neurons these clusters begin to enter a major Map2-immunoreactive process. At 10 days in vitro TGN38 immunoreactivity extends into a major dendrite for 5-30 microns in many neurons. Thus, the distribution of TGN38 immunoreactivity becomes polarized, being localized within a single, usually the major, neocortical dendrite. Our results also show that the morphological appearance of TGN38-immunoreactive structures is microtubule-dependent, since nocodazole treatment of polarized neurons induces scattering of TGN38-immunoreactive vesicles throughout the cell body's cytoplasm. Treatment with brefeldin A induces scattering of small TGN38-immunoreactive vesicles throughout the neuronal cytoplasm and processes, a different response to that observed in non-neuronal cells.

Light and electron microscopic immunocytochemical localization of AMPA‐selective glutamate receptors in the rat spinal cord

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors are probably the most widespread excitatory neurotransmitter receptors of the central nervous system, and they play a role in most normal and pathological neural activities. However, previous detailed studies of AMPA subunit distribution have been limited mainly to the brain. Thus, a comprehensive study of AMPA receptor subunit distribution was carried out on sections of rat spinal cord and dorsal root ganglia, which were immunolabeled with antibodies made against peptides corresponding to C-terminal portions of the AMPA receptor subunits: GluR1, GluR2/3, and GluR4. In the spinal cord, labeling was most prominent in the superficial dorsal horn, motoneurons, and nuclei containing preganglionic autonomic neurons. Immunostaining also was observed in neurons in other regions including those known to contain Renshaw cells and Ia inhibitory cells. Although overall immunostaining was lighter with antibody to GluR1 than with GluR2/3 and 4, there were neurons that preferentially stained with antibody to GluR1. These "GluR1 intense" neurons were usually fusiform and most concentrated in lamina X. In dorsal root ganglia, immunostaining of ganglion cell bodies was moderate to dense with antibody to GluR2/3 and light to moderate with antibody to GluR4. Possible neuroglia in the spinal cord (mainly GluR2/3 and 4) and satellite cells in dorsal root ganglia (GluR4) were immunostained. Electron microscopic studies of the superficial dorsal horn and lateral motor column showed staining that was restricted mainly to postsynaptic densities and associated dendritic and cell body cytoplasm. In dorsal horn, colocalization of dense-cored vesicles with clear, round synaptic vesicles was observed in unstained presynaptic terminals apposed to stained postsynaptic densities. Subsynaptic dense bodies (Taxi-bodies) were associated with some stained postsynaptic densities in both the superficial dorsal horn and lateral motor column. Based on several morphological features including vesicle structure and presence of Taxi-bodies, it is likely that at least some of the postsynaptic staining seen in this study is apposed to glutamatergic input from primary sensory afferent terminals.

Glutamate transporter mRNA and glutamate-like immunoreactivity in spinal motoneurones.

Glutamate is the major excitatory neurotransmitter in the central nervous system. The release of glutamate is terminated by rapid uptake of glutamate into the presynaptic nerve terminals and into surrounding glial cells. Recently, a neuronal glutamate transporter was cloned from rabbit small intestine, thereby providing the possibility to study the distribution of cells that express glutamate transporter mRNA. Using oligonucleotide probes and in situ hybridization, glutamate transporter mRNA was demonstrated in large cell bodies, presumably motoneurones, in the thoracic spinal cord of the rabbit. Immunohistochemical analysis with rabbit polyclonal antibodies to glutamate showed immunoreactivity in the cytoplasm of large cell bodies in the ventral horn, presumably motoneurones, of the rat spinal cord. Glutamate-LI was in addition demonstrated in the motor end plate in hindlimb muscle of the rat, as visualized by double-labelling with mouse monoclonal antibodies to synaptophysin. Taken together, these data raise the possibility that glutamate has a function at the vertebrate neuromuscular junction.

Immunohistochemical localization of peripheral nitric oxide synthase‐containing nerves using antibodies raised against synthesized C‐ and N‐terminal fragments of a cloned enzyme from rat brain

Antibodies were raised in rabbits against C- or N-terminal fragments of a cloned nitric oxide synthase (NOS) enzyme from rat cerebellum, and used for demonstration of NOS-immunoreactive (NOS-IR) nerves in different tissues from the rat (colon, duodenum, adrenal gland, aorta, caval vein, penis and urethra). Both antisera demonstrated the same neuronal elements, although with differences in intensity in the immunoreaction in some tissues. Sections incubated with antisera preabsorbed with excess of the antigens showed no NOS immunoreactivity. In duodenum and colon, NOS-immunoreactivity was found in the cytoplasm of numerous cell bodies in myenteric ganglia and in some nerve cell bodies in the submucosa. NOS-IR nerve fibres were numerous in the circular muscle layer, while few were found in the longitudinal layer or the mucosa and submucosa. In the penis, strong NOS immunoreactivity was found in nerves surrounding the deep penile and dorsal arteries, and in nerves in the stroma of the cavernous tissue. In the urethra, NOS immunoreactivity was found in nerves in the mucosa. No NOS immunoreactivity was found in the urothelium. The adrenal medulla, and occasionally the cortex, contained nerve cell bodies with strong cytoplasmic NOS immunoreactivity as well as scattered nerve fibres. No NOS immunoreactivity was found in the abdominal aorta or inferior caval vein. Combined NOS immunostaining and NADPH diaphorase staining showed that virtually all NOS-IR nerve structures were also NADPH diaphorase-positive. However, thin nerve fibres and cell linings were sometimes better visualized by NOS-immunohistochemistry. Furthermore, the adrenal cortex, which only occasionally showed NOS immunoreactivity, was strongly NADPH diaphorase-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of hippocalcin mRNA and immunoreactivity in rat brain

Distribution of hippocalcin in rat brain was analysed by in situ hybridization and immunohistochemical methods. Hippocalcin mRNA and immunoreactivity were expressed most intensely in the pyramidal cells of the hippocampus, intensely in the Purkinje cells of the cerebellum, moderately in the dentate granule cells and pyramidal cells of cerebral cortex layers II–VI and weakly in the large neuronal cells of the caudate-putamen. Some discrepancies in the localization of hippocalcin mRNA and immunoreactivity were noted in the mamillary nuclei, anterior part of the thalamus and the septal nuclei. In most cell types, hippocalcin immunoreactivity was localized in the cytoplasm and plasma membrane of cell bodies and dendrites.

The anterograde neuroanatomical tracer biotinylated dextran-amine: comparison with the tracer Phaseolus vulgaris-leucoagglutinin in preparations for electron microscopy

We investigated the properties of biotinylated dextran-amine (BDA) as a neuroanatomical tracer at the electron microscopic level and we compared the results with those obtained previously with another tracer, the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). BDA was injected into various brain areas of rats. Following survival and fixation, vibratome sections were cut, subjected to a freeze-thaw treatment, and incubated overnight with avidin-biotin complex (ABC). Following reaction with diaminobenzidine (DAB)-hydrogen peroxide, the sections were processed for electron microscopy. In the electron microscope we observed that the reaction product occurred in the cytoplasm of cell bodies and in the matrices of dendrites, axons and axon terminals following ABC histochemistry of BDA-containing brain sections. The ultrastructural details of BDA-labelled neurones were generally better preserved than in PHA-L-labelled material, whereas at the same time penetration of the reagent into the sections was complete (incomplete in sections of PHA-L material). We conclude that the use of BDA as a neuroanatomical tracer in electron microscopy is a good substitute for PHA-L. The detection method for transported BDA is much faster and less complicated than that for PHA-L, while the results are better; that is, there is improved penetration coinciding with good preservation of ultrastructure. Keeping the limitations of BDA as a neuroanatomical tracer in mind, e.g., retrograde transport into local collaterals of axons that intermingle with anterogradely labelled axons, BDA seems well suited as a neuroanatomical tracer for electron microscopy.

Light and Electron Microscopic Localization of the Neutral Metalloendopeptidase EC 3.4.24.16 in the Mesencephalon of the Rat.

The topographic and cellular distribution of the neurotensin-hydrolysing neutral metalloendopeptidase 24.16 (EC 3.4.24.16) was examined by light and electron microscopic immunohistochemistry in adult rat mesencephalon. Light microscopic immunoradioautography revealed a ubiquitous distribution of the enzyme throughout the midbrain with a relative enrichment of grey matter areas including the substantia nigra, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, rostral and caudal linear raphe nuclei, central grey and superficial grey of the superior colliculus. Peroxidase - antiperoxidase immunocytochemistry revealed two distinct cellular patterns of immunostaining: (1) weakly labelled neuronal perikarya more or less uniformly distributed throughout the grey matter, and (2) intensely immunoreactive glial cells heterogeneously distributed across the mesencephalon. Areas exhibiting dense concentrations of endopeptidase 24.16-containing glial cells corresponded to those displaying enhanced immunoreactivity in immunoradioautographs, suggesting that a major proportion of brain endopeptidase 24.16 is associated with glia. Electron microscopic examination of the substantia nigra and ventral tegmental area confirmed the association of the enzyme with a subpopulation of neurons and allowed identification of labelled glial cells as protoplasmic astrocytes. In neurons, endopeptidase 24.16 immunoreactivity was distributed heterogeneously within the cytoplasm of perikarya, dendrites and axons. Reaction product was also characteristically associated with restricted zones of the plasma membrane and underlying neuroplasm. In astrocytes, endopeptidase 24.16 immunostaining was densely and uniformly distributed throughout the cytoplasm of cell bodies and processes. Many of these processes were in direct contact with endopeptidase 24.16-immunopositive neuronal elements. The present results demonstrate that within the midbrain, endopeptidase 24.16 is both intracytoplasmic and membrane-associated in neurons and predominantly intracytoplasmic in glia. The presence of a large number of immunostained elements within areas of the midbrain known to display high levels of neurotensin and/or neurotensin receptors, together with the demonstrated catabolic activity of the enzyme on neurotensin in vitro, is consistent with a role of endopeptidase 24.16 in the functional inactivation of endogenous neurotensin in this region of the brain.

Regional distribution of low-Km mitochondrial aldehyde dehydrogenase in the rat central nervous system.

To clarify the regional capacity of the brain to oxidize biogenic aldehydes and ethanol-derived acetaldehyde, a quantitative immunohistochemical study of the microregional and cellular expression of low Km mitochondrial aldehyde dehydrogenase (mALDH; EC 1.2.1.3) in the rat central nervous system was undertaken, using antiserum raised in rabbit against low-Km aldehyde dehydrogenase purified from rat liver mitochondria. mALDH-specific immunoreactivity (IR) was observed to various extent in the majority of structures in all brain and spinal cord areas. Staining was strong in the extranuclear cytoplasm of neuronal and glial cell bodies but less pronounced in their processes and terminals, the conducting tracts, white matter and neuropile and in blood vessels. Immunostaining density was 2 to 3 times higher in neuronal perikarya as compared with neuropile. mALDH-positive neurons were found in all brain regions, being strongest in the inferior olive and hippocampus stratum pyramidale and weakest in substantia nigra. The percentage of morphologically identifiable ALDH-positive neurons ranged from 40% in the arcuate hypothalamic nucleus to 88% in the cerebellar Purkinje cells. A comparison of the heterogeneous expression of mALDH in various rat CNS regions and cells, as observed in the present study, with the corresponding previously published distributions of the potential acetaldehyde-producing enzymes ADH and cytochrome P450 2E1 indicates major differences, which may help in understanding potential acetaldehyde-mediated CNS effects of ethanol. Knowledge of the regional distribution of high-affinity aldehyde dehydrogenase should also throw light on the neurophysiological role of local regulation of the metabolism of biogenic aldehydes in the brain.

Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostriatal dopaminergic neurons

We previously demonstrated the existence of a retrograde axonal transport of radioactivity to the substantia nigra pars compacta following injection of mono-iodinated neurotensin in rat neostriatum. In the present study, the topographical and cellular distribution of this retrogradely transported material was examined by light and electron microscopic autoradiography. Four and a half hours after unilateral injection of [ 125I]neurotensin in the caudoputamen, retrogradely labelled neuronal cell bodies were detected by light microscopic autoradiography throughout the ipsilateral substantia nigra pars compacta as well as within the ventral tegmental area and retrorubral field. In semithin sections, silver grains were prevalent over the perinuclear cytoplasm of neuronal cell bodies but were also detected over neuronal nuclei. Analysis of electron microscopic autoradiographs revealed that the vast majority (> 85%) were associated with neuronal perikarya, unmyelinated and myelinated axons, dendrites and terminals. Within the soma, a significant proportion of silver grains (16% of somatic grains) was detected over the nucleus. However, the majority were identified over the cytoplasm where they often encompassed cytoplasmic organelles, including rough endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes and multi-vesicular bodies. In dendrites and axons, a substantial percentage of silver grains (63–89%) was localized over the plasma membrane. A minor proportion (13% of total) of the autoradiographic labelling was detected over myelin sheaths, astrocytes and oligodendrocytes. The present results are consistent with previous light-microscopic evidence for internalization and retrograde transport of intrastriatal neurotensin within nigrostriatal dopaminergic neurons. They further suggest that retrogradely transported neurotensin may be processed along a variety of intracellular pathways including those mediating degradation in lysosomes and recycling in rough endoplasmic reticulum. The detection of specific autoradiographic labelling in the nucleus supports the concept that neurotensin alone, or complexed to its receptor, might be involved in the regulation of gene expression through direct or indirect interactions with nuclear DNA. Consequently, the retrograde transport of neurotensin in nigrostriatal dopaminergic neurons might provide a vehicle through which events occurring at the level of the axon terminal may initiate long-term biological responses.

Biochemical and immunohistochemical demonstration of a tightly bound form of prostaglandin E 2 in the rat brain

Basal levels of prostaglandin E 2 in the rat brain were determined by radioimmunoassay to be 0.68–0.79 pmol/g brain. About one-third of the prostaglandin E 2 (0.23–0.28 pmol/g) was resistant to extraction with ethanol, but could be recovered with a mixture of ethanol and 1 N HC1 (9:1, v/v), indicating that a tightly bound form of prostaglandin E 2 exists in the brain. The amount of the bound form of prostaglandin E 2 was almost unchanged by pentylenetetrazole-induced convulsion or by transcardial perfusion with a formaldehyde solution, although these treatments resulted in 40- to 80-fold increases in prostaglandin E 2 content extracted with ethanol at neutral pH. A polyclonal antibody against prostaglandin E 2-albumin conjugates recognized the bound form of prostaglandin E 2, giving a punctate appearance in many neuronal cell bodies in the brain. Although almost all of the neuronal perikarya were immunoreactive for prostaglandin E 2, intense immunoreactivity was observed in the mitral cell layer of the olfactory bulb, layer V of the cerebral neocortex, anterodorsal and reticular nuclei of the thalamus, supraoptic, paraventricular, accessory neurosecretory and lateral mammillary nuclei of the hypothalamus, mesencephalic trigeminal nucleus, nucleus of the trapezoid body and deep cerebellar nuclei. When the cerebral neocortical regions were observed electron microscopically, immunoreaction products were seen as fine granules which were clustered into small patches in the cytoplasm of neuronal cell bodies and proximal dendrites. No immunoreaction products were seen in glial cells or endothelial cells. These results suggest that prostaglandin E 2 is involved in fundamental processes of neurons.

Acrylamide disrupts elemental composition and water content of rat tibial nerve: II. Schwann cells and myelin

The effects of subchronic and subacute acrylamide (ACR) intoxication on elemental composition (Na, P, S, Cl, K, Ca, Mg) and water content of Schwann cell body cytoplasm and myelin were assessed in rat tibial nerve. Electron probe X-ray micro-analysis demonstrated that, in control rats, peripheral nerve glia and myelin exhibited highly characteristic distributions of elements and water and that ACR intoxication was associated with disruption of this normal subcellular distribution. When rats were intoxicated with ACR by either the oral (2.8 m m in drinking water for 15, 22, 30, and 60 days) or the intraperitoneal (50 mg/kg/day ×5 and 10 days) route, an exposure-dependent loss of cytoplasmic Na, K, P, Cl, Mg, and water regulation was detected in Schwann cell cytoplasm. Maximum development of elemental deregulation occurred after 30 days of oral ACR exposure and 10 days of ip treatment. The cytoplasmic elements involved and their corresponding quantitative changes were similar regardless of the route of ACR intoxication. Analysis of myelin revealed that both oral and parenteral ACR exposure caused early, persistent increases in dry weight Na, P, and water content. However, Cl dry weight concentrations were increased by oral exposure and decreased by ip ACR injection. Results of this study indicate that ACR intoxication is associated with a significant disturbance of subcellular element and water distribution in tibial nerve Schwann cells and myelin. The pattern of elemental disruption is typical of reversible cell damage and, therefore, Schwann cell injury might play a role in the expression of ACR neurotoxicity.

Light and electron immunocytochemical localization of AMPA‐selective glutamate receptors in the rat brain

Since four AMPA-type excitatory amino acid receptor subunits have been cloned recently, it is now possible to localize these important molecules in the nervous system. A comprehensive study of AMPA receptor immunocytochemistry was carried out on vibratome sections of rat brain, which were immunolabeled with antibodies made against peptides corresponding to the C-terminal portions of AMPA-receptor subunits: GluR1, GluR2/3, and GluR4. Labeling was most prominent in forebrain structures such as the olfactory bulb and tubercle, septal nuclei, amygdaloid complex, hippocampus, induseum griseum, habenula, and interpeduncular nucleus, and in the cerebellum. Different patterns of immunolabeling were evident with the antibodies to the four subunits, with marked contrast between densely and lightly stained structures with antibody to GluR1, widespread dense staining with antibody to GluR2/3, and moderate staining with antibody to GluR4. In the parietal cortex, some non-pyramidal neurons were more densely stained than pyramidal cells with antibodies to GluR1. Neurons of the main olfactory bulb, other than granule cells, were most densely stained with antibody to GluR1. In the cerebellum, Bergmann glia were densely stained with antibodies to GluR1 and 4, while neurons, other than granule cells, were most densely stained with antibody to GluR2/3. Immunolabeling patterns of all antibodies were consistent with that of previous in situ hybridization histochemistry studies and with the overall pattern of 3H-AMPA binding. Electron microscopy of thin sections taken from immunolabeled vibratome sections of hippocampus and cerebral cortex showed staining which was restricted mainly to postsynaptic densities and adjacent dendritoplasm, and to neuron cell body cytoplasm. We saw no convincing examples of stained presynaptic terminals, and only limited evidence of glial staining, excepting Bergmann glia.

Ultrastructural Localization of Neuropeptide Y in the Hypothalamus

Immunoreactive NPY neurons are widely distributed in the hypothalamus of several mammalian species. In the rat, dense NPY fiber networks are found in the paraventricular, suprachiasmatic and arcuate nuclei. NPY-containing cell bodies are mostly found in the arcuate nucleus. Studies performed at the electron microscope level clearly indicate that NPY is concentrated in dense core vesicles in the cytoplasm of cell bodies as well as in terminals. Only a small percentage (about 20%) of the NPY endings are making synaptic contacts with nerve processes, especially dendrites. These ultrastructural data suggest that NPY might play a neurotransmitter/neuromodulator role. NPY has been shown, when injected into hypothalamic areas, to exert a variety of effects, including modifications in food intake, energy balance and pituitary secretion. In an attempt to define the exact role of NPY in hypothalamic functions, we have designed experiments to study the interactions of NPY with other neurotransmitter systems. In the suprachiasmatic nucleus, both NPY and 5-HT terminals have been shown to establish synaptic junctions sometimes with the same neurons. Occasionally, axoaxonic junctions between these two types of endings have been observed. These results suggested that both 5-HT and NPY might be involved in the complex regulation of circadian rythms. In the arcuate nucleus, nonsynaptic appositions between 5-HT nerve endings and NPY-containing neurons were demonstrated. In this nucleus, direct appositions between TH- and NPY-containing neurons were also detected. These appositions were of axosomatic, axodendritic or axoaxonic types. Since it has been demonstrated that arcuate NPY neurons are projected to other hypothalamic areas, such as the paraventricular and dorsomedial nuclei, it might be speculated that arcuate 5-HT/NPY and catecholamines/NPY interactions might be involved in regulation of behavior and neuroendocrine functions.

Presence of neuropeptide messenger RNAs in neuronal processes

The messenger RNAs coding for vasopressin, oxytocin, luteinizing hormone releasing-hormone and somatostatin have been detected in tissue sections of the rat brain, especially in the hypothalamus with radioactive and biotinylated oligonucleotide probes. The results demonstrate that neuropeptide mRNAs are present in the cytoplasm of cell bodies, in processes and in punctate structures in the vicinity of the cell bodies. These results demonstrate that neuropeptide mRNAs can be transported outside the cell body most probably in proximal dendrites but also in some of their branching, and possibly at synaptic contacts. These data suggest that neuropeptide mRNA could undergo a specific compartmentation that could contribute to the targetting of the corresponding peptide inside neurons.

Antibody specific to the alpha subunit of the guanine nucleotide-binding regulatory protein Go: developmental appearance and immunocytochemical localization in brain.

A polyclonal rabbit antibody (9120) against the alpha subunit of the "other" guanine nucleotide-binding protein Go (alpha o) was raised against a synthetic alpha o peptide fragment (Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val) attached to a branched core system. Antiserum 9120, at a final dilution of 1:400, can detect alpha o in as little as 0.2 microgram of Go on immunoblots, and, at a final dilution of 1:20,000, can detect alpha o in 1 microgram of Go on immunoblots. Antiserum and affinity-purified antibody are specific to alpha o. No cross-reactivity was detected to the alpha subunits of the stimulatory or inhibitory guanine nucleotide-binding regulatory proteins or of transducin (alpha s, alpha i, or alpha T) or to the beta or gamma subunits. Immunoblots revealed a high density of alpha o in rat brain and lung membrane preparations, but other tissues (such as adipose tissue, heart, erythrocytes, and liver) have no detectable alpha o. Developmental studies showed that alpha o in rat brain was low before birth, increased after birth, and reached the full adult level at 4 weeks of age. In contrast, ADP-ribosylation of 40-kDa proteins increased for up to 1 week and then decreased. Immunocytochemistry revealed that alpha o was localized to somatic and synaptic membranes in rat brain, whereas little or no alpha o was detected in the cytoplasm of neuronal cell bodies. Our observations suggest that Go in brain might have a role in membrane signal transduction at synaptic and extrasynaptic sites.

Light and electron microscope localization of the microtubule- associated tau protein in rat brain

We have studied the distribution of microtubule-associated tau proteins in rat brain using monoclonal and affinity-purified polyclonal antibodies. Tau staining is prominent in axons in white matter areas of brain, as reported by Binder et al. (1985). In addition, we also find tau protein in neuron cell bodies, especially in the brain stem and basal ganglia and in the cell bodies of interfascicular oligodendroglia. Using electron microscopy, tau antibodies and colloidal gold-labeled second antibodies, gold particles are found associated with microtubules in axons and in the cytoplasm of cell bodies, while the nuclei, mitochondria, and myelin remain unlabeled. In double-staining experiments, tau staining co-localizes with that of tubulin. Our studies indicate that tau proteins are more widely distributed in brain than previously reported and cannot be used as an exclusive marker for axons.