Histochemical demonstration of copper in normal rat brain and spinal cord

Summary The high sensitivity of the magnesium-dithizonate silver-dithizonate (MDSD) staining procedure makes this method very suitable for the histochemical localization of copper in different regions of the central nervous system of adult rats. In the telencephalon (bulbus olfactorius, nucleus caudatus-putamen, septum pellucidum and are dentata), diencephalon (nucleus habenulae medialis, nuclei of the hypothalamus in the vicinity of the third ventricle, and corpus mamillare), mesencephalon (substantia nigra), cerebellum (mainly in the nodulus), pons (locus coeruleus, nucleus vestibularis), medulla oblongata (nucleus tractus solitarii) and spinal cord, the glial cells exhibit specific copper staining. The glial cells of some circumventricular organs (e.g. the subfornical organ) are also stained using the MDSD method. The significant staining observed in whitematter glial cells (e.g. in the corpus callosum, cerebellum and spinal cord) further indicates the very high sensitivity of this method. In glial cells of the same regions, the presence of copper can likewise be demonstrated using the modified sulphide silver method. On the basis of the present histochemical results, it is suggested that copper may play an important role in the normal physiological functioning of glial cells and also, via glia-neuron interactions, in neuronal processes.     

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Histochemical localization of FMRFamide-like immunoreactivity in the rat brain

Molluscan cardioexcitatory neuropeptide or FMRFamide is present in the invertebrate central nervous system (CNS) and FMRFamide like peptide has been demonstrated in the mammalian CNS. In this study, the distribution of FMRFamide immunoreactivity was studied in rat brain using the indirect immunofluorescent method. The highest number of FMRFamide staining cell bodies was found in the nucleus (n) arcuatus. N. paraventricularis, n. hypothalamus, n. ventromedialis, n. dorsomedialis and n. tractus solitarii also contained high numbers. FMRFamide positive nerve fibers and terminals were widely distributed. The septal complex contained high densities, especially in n. interstitialis striae terminalis. N. paraventricularis hypothalami, n. paraventricularis, n. hypothalamicus, n. ventromedialis and n. dorsomedialis showed a high to very high degree of immunoreactivity. In myelencephalon, n. tractus solitarii had the densest innervation. Spinal cord had a dense band of FMRFamide positive fibers in lamina I and II of the dorsal horn. The present findings support a neurotransmitter role for a FMRFamide like peptide in the mammalian brain, possibly related to endocrine and autonomic regulation as well as pain modulation.     

The renin-angiotensin system in the rat brain. Immunocytochemical localization of angiotensinogen in glial cells and neurons

The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem. Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

Histochemical and immunohistochemical localization of hexokinase isoenzymes in normal rat liver

Summary Histochemical and immunohistochemical procedures have been used to examine the localization of three of the four hexokinase isoenzymes present in the liver of fed female Wistar rats. Distinctive distribution patterns were found for hexokinase type I and glucokinase but hexokinase type II was not detectable. Hexokinase type I was identified in sinusoidal cells and in bile duct epithelia, nerves and arteries in the portal triad. Glucokinase, the major isoenzyme, was confined to parenchymal cells where it was present in much higher amounts in perivenous compared with periportal hepatocytes. Staining within these two zones was not homogeneous and each had a mosaic appearance caused by the presence of a few hepatocytes containing little or no glucokinase amongst the majority of darkly stained cells in perivenous areas and a few darkly stained cells amongst the majority of unstained cells in periportal areas. Hence, hepatocytesin situ are a strikingly heterogeneous population of cells. Their metabolic status cannot be controlled simply by the differential supply of oxygen, substrates and hormones to different regions of the liver acini as proposed in the metabolic zonation model. Phenotypic differences may exist between cells within a given metabolic zone which influence their ability to respond to different environmental conditions.     

Histochemical localization of the soluble arylsulphatase activities in rat brain

Summary The modified method of Goldfischer has been used for the localization of soluble arylsulphatase activities in the rat brain. The highest activity of these enzymes was found in some parts of the drive system and in nervous tracts. The bulk of activity in neurons and glial cells is localized in the lysosomes. Some arylsulphatase activity has been found to be bound to myelin sheaths. We have not been able to ascribe this activity to any defined subcellular structures, by light microscopy.The method of Goldfischer does not permit differentiation of the two soluble arylsulphatases (enzymes: A and B). We suggest however, that these enzymes may have different functions in the brain. Arylsulphatase A (cerebrosidesulphatase) may be primarily connected with the nervous tracts, a hypothesis supported by the character of disorders caused by lack of this enzyme in metachromatic leucodystrophy. Arylsulphatase B, hydrolysing sulphuric esters of catecholamines, may be involved in the function of the drive system.Arylsulphate sulphohydrolase, EC 3. 1.6. 1.     

Glutamate-, kainate- and NMDA-evoked membrane currents in identified glial cells in rat spinal cord slice

The effect of L-glutamate, kainate and N-methyl-D-aspartate (NMDA) on membrane currents of astrocytes, oligodendrocytes and their respective precursors was studied in acute spinal cord slices of rats between the ages of postnatal days 5 and 13 using the whole-cell patch-clamp technique. L-glutamate (10(-3) M), kainate (10(-3) M), and NMDA (2x10(-3) M) evoked inward currents in all glial cells. Kainate evoked larger currents in precursors than in astrocytes and oligodendrocytes, while NMDA induced larger currents in astrocytes and oligodendrocytes than in precursors. Kainate-evoked currents were blocked by the AMPA/kainate receptor antagonist CNQX (10(-4) M) and were, with the exception of the precursors, larger in dorsal than in ventral horns, as were NMDA-evoked currents. Currents evoked by NMDA were unaffected by CNQX and, in contrast to those seen in neurones, were not sensitive to Mg2+. In addition, they significantly decreased during development and were present when synaptic transmission was blocked in a Ca2+-free solution. NMDA-evoked currents were not abolished during the block of K+ inward currents in glial cells by Ba2+; thus they are unlikely to be mediated by an increase in extracellular K+ during neuronal activity. We provide evidence that spinal cord glial cells are sensitive to the application of L-glutamate, kainate and transiently, during postnatal development, to NMDA.     

Acetylcholine estimate in glial cells of the cat spinal cord

Structures containing acetylcholinesterase were found in the motor nuclei of the cervical enlargement of the cat spinal cord by light and electron microscopy in material stained by the Karnovsky-Roots method. The specific response was observed not only in neurons of the motor nuclei, but also in some satellite cells, astrocytic glial cells, and Schwann cells. A positive reaction for acetylcholinesterase was found in some of the satellite cells located close to both cholinergic and noncholinergic neurons. As a result of electron microscopy, an electron-dense deposit of copper ferrocyanide was found on the structures of the nucleolus, on the surface of the inner and outer layers of the nuclear membrane, in the pores of the nuclear membrane, in the perinuclear space, and in the endoplasmic reticulum of the perikaryon of some satellite cells, as well as on the outer and inner surfaces of the cytoplasmic membrane of the Schwann cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev, Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 48–51, January–February, 1977.     

Modulation of NADPH-diaphorase and glial fibrillary acidic protein by progesterone in astrocytes from normal and injured rat spinal cord

Progesterone (P4) can be synthesized in both central and peripheral nervous system (PNS) and exerts trophic effects in the PNS. To study its potential effects in the spinal cord, we investigated P4 modulation (4 mg/kg/day for 3 days) of two proteins responding to injury: NADPH-diaphorase, an enzyme with nitric oxide synthase activity, and glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. The proteins were studied at three levels of the spinal cord from rats with total transection (TRX) at T10: above (T5 level), below (L1 level) and caudal to the lesion (L3 level). Equivalent regions were dissected in controls. The number and area of NADPH-diaphorase active or GFAP immunoreactive astrocytes/0.1 mm(2) in white matter (lateral funiculus) or gray matter (Lamina IX) was measured by computerized image analysis. In controls, P4 increased the number of GFAP-immunoreactive astrocytes in gray and white matter at all levels of the spinal cord, while astrocyte area also increased in white matter throughout and in gray matter at the T5 region. In control rats P4 did not change NADPH-diaphorase activity. In rats with TRX and not receiving hormone, a general up-regulation of the number and area of GFAP-positive astrocytes was found at all levels of the spinal cord. In rats with TRX, P4 did not change the already high GFAP-expression. In the TRX group, instead, P4 increased the number and area of NADPH-diaphorase active astrocytes in white and gray matter immediately above and below, but not caudal to the lesion. Thus, the response of the two proteins to P4 was conditioned by environmental factors, in that NADPH-diaphorase activity was hormonally modulated in astrocytes reacting to trauma, whereas up-regulation of GFAP by P4 was produced in resting astrocytes from non-injured animals.     

Histochemical demonstration of purine nucleoside phosphorylase (PNPase) in microglial and astroglial cells of adult rat brain

The histochemical localization of enzymes associated with purine nucleoside metabolism indicates that glial cells might participate in the regulation of these compounds in the central nervous system. In the present study we examined the histochemical localization of purine nucleoside phosphorylase (PNPase) in sections from adult rat brain. Some sections were also sequentially stained immunocytochemically for astroglial or microglial cells utilizing glial fibrillary acidic protein (GFAP) or OX-42 antibodies, respectively. Our observations showed that PNPase was restricted to glial cells, whereas neurons always remained negative. Brain sections stained for both PNPase and GFAP showed that the GFAP-positive astroglial cells were always PNPase positive. Other PNPase-positive but GFAP-negative cells were also observed. These cells resembled microglial cells, and brain sections reacted for both PNPase and OX-42 confirmed this by showing that the major part of OX-42-positive microglial cells were PNPase positive. In these sections, the PNPase-positive but OX-42-negative cells present resembled astroglial cells. From our double staining experiments, we conclude that PNPase is present in both astroglial and microglial cells in normal adult brain.     

Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Histochemical demonstration of mercury induced changes in rat neurons.

A histochemical method modified for ultrastructural studies of mercury induced changes is described. Rat neurons from areas known to be influenced by mercury are used as examples. The histochemical reaction, suggested to be caused by polymercury sulphide complexes, is localized to "dense bodies" where it is visible 14 days after initiation of peroral mercury treatment (20 mg HgCl2/l drinking water).