Localization of Mitochondrial Carnitine/Acylcarnitine Translocase in Sensory Neurons from Rat Dorsal Root Ganglia

The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.     

The ubiquitous localization of type I hexokinase in rat peripheral nerves, smooth muscle cells and epithelial cells

Summary An indirect immunoperoxidase technique has been used to determine the localization of type I hexokinase in a wide variety of Carnoy-fixed, paraffin-embedded rat tissues. The results suggest that the widespread tissue distribution of the isoenzyme is due to its ubiquitous localization in the nervous, smooth muscle and epithelial components of each tissue. The majority of the immunostaining was confined to cells with substantial energy requirements which are probably mainly satisfied through the breakdown of glucose. This observation is consistent with the known predominance of type I hexokinase in the central nervous system and with the regulatory role allotted to it in this tissue.     

Enhanced resolution of histochemical distribution of glucose-6-phosphate dehydrogenase activity in rat neural tissue by use of a semipermeable membrane

We examined the histochemical distribution of glucose-6-phosphate dehydrogenase (G6PD) activity in neural tissue using different diffusion barriers. Although polyvinyl alcohol and agar overlays permitted regional localization of G6PD, a semipermeable membrane revealed cellular differences in G6PD activity within populations of neurons. Distribution of G6PD activity in selected regions of the nervous system was examined using the membrane technique. White matter usually exhibited strong G6PD activity. The neuronal somata of the dorsal root ganglia (L4-L6) and anterior horns of the spinal lumbar enlargement demonstrated a variation in activity which was independent of somal size. Satellite cells showed intense activity when the membrane technique was used. Hippocampal pyramidal and granular cells of the dentate gyrus exhibited moderate, uniform G6PD activity, but only weak activity was seen in hippocampal and dentate molecular layers. High levels of activity were observed in the vascular endothelial cells of the brain, spinal cord, and choroid plexus, and in the ependymal cells of the spinal central canal and ventricles of the brain. The superior vestibular nucleus appeared to have little G6PD activity in either the neuron cell bodies or the surrounding parenchyma. The use of a semipermeable membrane for localization of G6PD activity in neural tissues permits enhanced resolution of neuron elements and may provide a more accurate assessment of G6PD activity in histological preparations.     

Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target β-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican β-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.     

Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.     

芳香化酶在文昌鱼神经系统、哈氏窝和性腺特异性定位:原位杂交和免疫细胞化学研究

芳香化酶活性发现在脊椎动物脑、脑垂体和性腺中,但在文昌鱼脑和哈氏窝的组织特异性定位尚无可利用资料。本文用免疫细胞化学和原位杂交技术,首次发现芳香化酶活性组织特异性定位在幼年和性腺发育不同时期雌、雄文昌鱼神经系统(脑和脊髓)、轮器、哈氏窝和性腺中。芳香化酶蛋白和转录物在前脑、中脑、脊髓、轮器和哈氏窝十分丰富,而后脑、早期卵巢和精巢不够丰富;没有芳香化酶表达的部位是哈氏窝另两种细胞(不规则形细胞和带纤毛粘液细胞)以及成熟卵巢和精巢;芳香化酶免疫活性物质分布在胞质,核为阴性。芳香化酶在文昌鱼神经系统、哈氏窝和性腺的分布模式与低等脊椎动物中的分布模式极为类似,尤其是芳香化酶在脑内调节哈氏窝分泌活动的神经内分泌中枢表达,并形成类似脊椎动物的文昌鱼原始的脑-芳香化酶调节系统。这些结果有力地证明,文昌鱼脑和哈氏窝高水平的芳香化酶活性像在其它脊椎动物中一样,对局部介导睾酮芳香化起着关键作用,同时还可能影响脑-芳香化酶系统参与调节哈氏窝的分泌活动[动物学报49(6)：800～806,2003]。     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Localization of NADPH-diaphorase and choline acetyltransferase in the central nervous system of the bivalve mollusk Mactra sulcatoria

The presence of NADPH-diaphorase and choline acetyltransferase (ChAT) in all ganglia of the Mactra sulcatoria was demonstrated by histochemical and electron histochemical methods. Pecularities of cholinergic and nitrergic neurons localization were revealed in nervous ganglia, and their relative content there was estimated. It was established that in reaction to ChAT only large neurons were marked. Ultrastructural localization of NADPH-diaphorase and ChAT was determined in neurons and neuropile. The data obtained testify that NADPH-diaphorase and ChAT are located in different types of nervous cells. The opportunity of functional cooperation in activity of cholinergic and nitrergic systems in mollusks is discussed.     

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase (AcP) activity was undertaken by combined radioautography and cytochemistry in the spleen of mice at different ages. The localization of radiolabelled thymidine was used to determine the site of DNA synthesis (cell proliferation), while AcP activity as a marker for cell lysis/death. For EM radioautography (EMRAG), the tissue sections were incubated in a medium containing 3H-thymidine and processed for radioautography, while the lanthanide-based method for the ultrastructural localization of AcP activity was employed. Quantitation of AcP activity was carried out by X-ray microanalysis. In all tissue sections examined, mostly of the labelled nuclei were observed in the hematopoietic cells. Few mitochondria of these cells were labelled. The labeling index was expressed as the percentage of labelled cells over the total number of counted cells. The labeling indices dropped considerably from day one after birth and progressively until the 10th month. The result of AcP activity correlated well with the result of a previous work (Olea, 1991). The localization of radiolabelled thymidine and AcP activity were not hindered by the simultaneous exposure of the same tissue section to 3H-thymidine and AcP cytochemical media. Interestingly enough, the spleen actively participates both in hematopoiesis and erythrophagocytosis. Prominently, it is most active during the early postnatal life. However, their influence declined considerably at the later stage of life (adult stage).     

Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Electron cytochemical localization of gamma-aminobutyric acid catabolism in rat cerebellar cortex

Summary An electron cytochemical technique is described for the localization of GABA-T, the enzyme which degrades the neurotransmitter GABA, in rat cerebellar cortex. The technique allows ultrastructural demonstration of GABA-T activity by the final deposition of an electron dense formazan precipitate at reaction sites, whilst maintaining adequate ultrastructural preservation for recognition of cellular and subcellular structures. Numerous electron dense precipitates are evident as discrete punctate deposits situated mainly in mitochondria of stellate cells, basket cells and astrocytic glial cells; they are also seen in axonal or dendritic profiles at some synaptic junctions. The technique enables the first cytochemical demonstration of the mitochondrial localization of GABA-T activity in nervous tissue to be presented. It establishes that GABA-T is present in supposed GABA neurones, in pre- or post-synaptic endings, or both, of presumed inhibitory synapses and in glial cells which may be associated with these synapses. From this seemingly ubiquitous distribution, functional aspects of GABA-T in these cells is considered.     

NPY样免疫反应在蝾螈发育过程中神经系统的定位研究

By immuno-cytochemical method the localization of neuropeptide Y (NPY) in the nervous system during embryonic development of Cynops orientalis was studied. The results revealed that NPY was first localized in the peripheral nervous system (late tail-bud stage), and later appeared in the central nervous system (larval stage) where it appeared with the appearance of glial cells. Very probably with the migration of neural crest cells NPY appeared first in the peripheral nervous system and then distributed to the central nervous system.     

Demonstration of dehydrogenase activities in paraffin sections.

The possibility of demonstrating the activity of respiratory enzymes in paraffin sections was studied. Unfixed pieces of nervous tissue were incubated at 4 degrees C, 20 degrees C, 37 degrees C and 56 degrees C for various periods ranging from 1 to 24 hours. After dehydration, the tissue pieces were mounted in paraffin. The paraffin sections obtained there of were then tested with respect to the range of penetration of the substrate into the incubated tissue samples (as judged from the resulting histoenzymic reaction), and for the distinctness with which the localization of the histochemic reaction could be assessed. From the results it may be concluded that it is possible, under well defined conditions, to demonstrate the activity of dehydrogenases in paraffin sections. The resulting morphological pictures permit a much better localization of the histoenzymic reaction products than those obtained from cryostat sections. Optimal results are obtained when tissue fragments, about 1 mm in diameter are incubated for 24 hours at 4 degrees C.     

Unique Expression of HRF20 (CD59) in Human Nervous Tissue

Damage to autologous tissue by complement is limited by several widely distributed membrane-associated glycoproteins which restrict the action of the complement in homologous species. These include decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20,CD59). Using immunohistochemical techniques, we examined the localization of these proteins in the centra] nervous system (CNS) and peripheral nervous system (PNS) using non-neurological human nervous tissue since some complement components have been demonstrated to be synthesized in the CNS. There was no evidence of parenchymal staining by anti-DAF or anti-MCP antibodies in either type of tissue except for the staining of the endothelium in capillaries. On the other hand, anti-HRF20 antibody clearly stained myelinated axons in the CNS as well as Schwann cells in the PNS. In addition, we detected positive staining by anti-DAF antibody in the PNS of a Paroxysmal nocturnal hemoglobinuria (PNH) patient who is genetically deficient in HRF20.     

Localization and regulation of mRNAs in the nervous tissue as revealed by in situ hybridization.

1. In situ hybridization histochemistry permits the study of specific mRNAs of neuropeptides, enzymes involved in the synthesis of neurotransmitters, receptors and proteins associated with glial cells in nervous tissue. 2. The central and peripheral nervous systems are composed of heterogeneous elements and specific regulatory mechanisms occur in specific cells. 3. This review will focus on the localization and regulation of different mRNAs in the nervous system from Drosophila to human, as revealed by in situ hybridization histochemistry.     

Localization of angiotensin-converting enzyme,prolyl endopeptidase and other peptidases in cultured neuronal or glial cells

To determine the cellular localization of nervous tissue peptidases, 7 peptidases and 2 lysosomal marker enzyme activities were measured in cultured mouse and rat cells. Neuronal cells of both species exhibited higher activities of angiotensin-converting enzyme (ACE) and prolyl endopeptidase (Pro-EP) than glial cells did. In contrast, arginyl endopeptidase and lysosomal enzymes (acid phosphatase, β-glucuronidase) in the neuronal cell lines were lower than those in the glial cell lines. Other peptidases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, dipeptidyl aminopeptidase) activities were not specifically localized in either cell lines. The effects of cellular differentiation on these peptidase activities in the PC 12h cell line and rat glioblasts were also examined using nerve growth factor (NGF) and glia maturation factor (GMF), respectively. Neuron specific peptidase (ACE and Pro-EP) activities were decreased in PC12h cells cultured with NGF, and Pro-EP activity was increased in the glioblast cells cultured with GMF. These results support the idea that some of the peptidases are differentially localized in neuronal or glial cells, and play physiological roles in central or peripheral neural tissues.     

Comparative data on acetylcholinesterase cytochemistry in various chromaffin cell types of Discoglossus pictus (Amphibia, Anura)

The cytochemical localization of acetylcholinesterase (AChE) activity was studied in the adrenal chromaffin cells of Discoglossus pictus. Reaction end-products were associated with all types of chromaffin cells, i.e. adrenaline (A), noradrenaline (N) and small granule chromaffin (SGC-A, SGC-N) cells, and nervous elements present in the gland. The SGC-A and SGC-N showed the same intensity of AChE reaction in A and N cells, respectively. On the whole, the A and SGC-A cells were more reactive than the N and SGC-N cells. The functional role of the SGC cells is discussed on the basis of the cytochemical results.     

Immunohistochemical detection of [corrected] TrkB in the enteric nervous system of the small intestine in pigeon (Columba livia)

The presence and cell localization of TrkB, the main receptor for the neurotrophins (NTs), was investigated immunohistochemically in the small intestine of adult pigeons, with special reference to the enteric nervous system (ENS). Several neuronal (neurofilament proteins and PGP 9.5) and glial cell (S100 protein) markers were studied in parallel. TrkB immunoreactivity (TrkB-IR) was found to be restricted to immunohistochemically-identified glial cells present in the enteric plexuses, and to Schwann cells forming the perivascular plexus. Also, TrkB-IR was detected in enterochromaffin cells and in unidentified dendritic cells within the gut-associated lymphoid tissue. The present results demonstrate that as for mammals, TrkB in the ENS is restricted to the glial cells. The possible function of the TrkB ligands, however, remains to be established.     

On the distribution of aldolase isoenzymes in subcellular fractions from rat brain

As an extension of previous studies on the adsorption of aldolase (EC 4.1.2.13) in nervous tissue, the main features of the subcellular localization of this enzyme in rat brain have been investigated. The major portion of the aldolase activity in homogenates of this tissue was demonstrated to be present in association with the particulate material, and a differential distribution of the AC isoenzymes was evident between the membranes and the cytosol. Some of the enzyme which was associated with the particulate fraction was shown to be occluded rather than absorbed to the membranes. This type of association was evident in the nuclear and mitochondrial fractions, in particular, with the occluded enzyme presenting an isoenzyme content high in C-type activity, and similar to that of the cytosol. The microsomal fraction contained a high proportion of enzyme in the bound form. Isoenzyme analysis of the enzyme in this microsomal fraction revealed a preferential association between the particulate material and A-type aldolase activity. A purified membrane fraction was prepared from the primary microsomal fraction, and identified as the main site of aldolase binding. The significance of the differential binding of aldolase isoenzymes and its localization amongst the subcellular fractions of rat brain have been discussed in relation to the structural and metabolic features of this tissue, and the coupling of energy producing sequences with energy requiring processes.