Electron microscope histochemical evidence for a partial or total block of the tricarboxylic acid cycle in the mitochondria of presynaptic axon terminals.

Respiration-linked, massive accumulation of Sr(2+) is used to reveal the coupled oxidation of pyruvate, alpha-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no alpha-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of alpha-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr(2+) accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO(2) into some of the pyruvate.

Dystrophin splice variants are distinctly localized in the hippocampus.

It has previously been demonstrated that Dp71, the most abundant dystrophin protein in the brain, is mainly localized in the postsynaptic densities. Here we show the localization of Dp71f, one of the splice variants of this protein, within the CA3 region of the hippocampus. Immunopositivity occurs in the postsynaptic density of small asymmetrical axospinous and axodendritic synapses, while it is absent in the postsynaptic densities of the axospinous synapses of the large mossy fiber terminals. Dp71f immunoreactivity was found to be attached to the membranes of the mossy fibers in the stratum lucidum of the CA3 area. In a certain population of thin myelinated axons the protein seems to be present within the axon proper. These data support the notion of a physiological role of Dp71f distinct from other dystrophin isoforms present in the central nervous system.

Seasonal fluctuations of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat suprachiasmatic nucleus.

The pacemaker of the "biological clock", the suprachiasmatic nucleus (SCN) of the hypothalamus was studied in intact male rats for glial fibrillary acidic protein (GFAP) a specific marker for astrocytes. Immunohistochemical reactions were carried out in winter (January-February) and in summer (June-July). In winter the GFAP-immunoreactivity of the SCN was found low whereas in summer it was high. Gonadectomy reduced differences. Since photic stimuli that apparently trigger the observed differences reach the SCN through identified neuronal pathways we conluded that the reaction of astrocytes is an indicator of seasonally altered neuronal function in the SCN.

Mapping of glial fibrillary acidic protein-immunoreactivity in the rat forebrain and mesencephalon by computerized image analysis.

Computer-assisted image analysis was used to map the regional distribution of glial fibrillary acidic protein-immunoreactive (GFAP-IR) astrocytes in the rat forebrain and mesencephalon. A complete survey of packing densities of GFAP-IR structures was performed. Computer maps revealed high values in the outer and inner layers of the cortex, some hippocampal and olfactory bulb layers, prepiriform cortex, dorsal part of the caudate-putamen, globus pallidus, lateral septum, reticular thalamic nucleus, lateral habenular nucleus, circumventricular organs, nuclei of the medial hypothalamus, substantia nigra, interpeduncular nucleus, and mamillary body. These correspond to regions of the embryonic pial and ventricular brain surfaces, which undergo developmental alterations including growth and various forms of internalization. From this we conclude that in the adult brain, astrocytes of high GFAP-IR are derivatives of surface-contact glia, whereas those located in areas having developed by the local thickening of the neural tube wall show reduced or no GFAP-IR.

Reversible and irreversible neuronal damage caused by excitatory amino acid analogues in rat cerebellar slices.

Slice preparations of the developing rat cerebellum were used to investigate the light and electron microscopic correlates of reversible and irreversible neuronal injury caused by the neurotoxic excitatory amino acid receptor agonists, kainate and N-methyl-D-aspartate. The slices were examined after various periods of exposure to the agonists (up to 30 min) with or without a 90 min recovery period in agonist-free medium. N-Methyl-D-aspartate (100 microM) caused necrosis of deep nuclear neurons and differentiating granule cells, the exposure times necessary to induce non-recoverable damage (leading to necrosis), being, respectively, 10 min and 20-30 min. Exposure periods of only 2-4 min with kainate (100 microM) were needed for Golgi cells to subsequently undergo necrosis. Other cell types (Purkinje, granule and deep nuclear neurons) were altered histologically by kainate but most recovered fully from 30 min exposures. Before the recovery period, the worst affected of these cells (deep nuclear neurons) displayed increased cytoplasmic and nuclear electron density and microvacuolation due to swelling of Golgi cisterns but little or no chromatin clumping or mitochondrial expansion. The neurons which were injured irreversibly by the agonists within 30 min displayed, near the time of lethal injury, increased cytoplasmic and nuclear electron lucency, marked focal aggregation of chromatin and swelling of Golgi apparatus. Mitochondrial swelling did not appear to precede lethal injury and even after exposure times sufficient, or more than sufficient, to lead to necrosis, large numbers of mitochondria remained in a condensed configuration. The significance of the histological changes is discussed and they are compared with those occurring in other pathological conditions. The time scales required for the receptor agonists to induce irreversible cellular lesions would be consistent with this being a process which is responsible for acute neuronal necrosis in the brain.

Ionic requirements for neurotoxic effects of excitatory amino acid analogues in rat cerebellar slices.

The ionic requirements for the neurotoxic effects of N-methyl-D-aspartate and kainate in incubated slices of developing rat cerebellum were studied using light and electron microscopy. Under normal conditions, 30 min exposure to 100 microM N-methyl-D-aspartate followed by a 90 min recovery period in agonist-free medium resulted in the necrosis of differentiating granule cells and deep nuclear neurons, while the corresponding effect of 100 microM kainate was the death of Golgi cells. Substitution of 96% of the Cl- in the medium with isethionate did not prevent the toxicity of either agonist. However, all the ordinarily vulnerable cells survived and exhibited normal ultrastructure if the slices were exposed to the excitants in a Ca2+-free medium and were subsequently allowed to recover in a Ca2+-containing solution. Prior to this recovery period, granule, Golgi and deep nuclear neurons exposed to N-methyl-D-aspartate were markedly swollen but their mitochondria were hypercontracted and there was no clumping of chromatin or obvious swelling of the rough endoplasmic reticulum or Golgi apparatus, in contrast to observations made on slices exposed to this agonist in normal medium. Substitution of all the Na+ in the medium with a mixture of choline (118 mM) and Tris (25 mM) itself caused necrosis of granule cells and deep nuclear neurons and an intense microvacuolation of Purkinje cells, due, in large part, to high amplitude mitochondrial swelling. A low (25 mM) Na+ medium was well tolerated under control conditions. This medium protected granule cells but not deep nuclear neurons from the toxicity of N-methyl-D-aspartate and failed to prevent kainate-induced death of Golgi cells. It is concluded that the acute neurotoxic effects of the two excitatory amino acid receptor agonists in the slices are dependent on extracellular Ca2+ and are independent of extracellular Cl-. Where apparent, the protective effect of reducing extracellular Na+ on the toxicity of N-methyl-D-aspartate is likely to reflect the involvement of this ion in the primary depolarizing mechanism.

Amino acid neurotoxicity: relationship to neuronal depolarization in rat cerebellar slices.

It has long been proposed that the excitatory and toxic properties of acidic amino acid receptor agonists are linked. To test this hypothesis, the depolarizing effects of quisqualate, kainate and N-methyl-D-aspartate in adult and immature rat cerebellar slices have been studied in relation to their neurotoxic effects in the same tissues (reported separately). A "grease-gap" method was used to measure the depolarizing responses of Purkinje cells and granule cells in lobule VI to the agonists. The depolarizing potencies of kainate and quisqualate were apparently similar on both cell types and at both ages studied although maximal responses to kainate were always larger. N-Methyl-D-aspartate was a very weak agonist in the adult slices but was much more effective in the immature tissues, apparently on both Purkinje cells and granule cells. Comparison of the depolarizing effects of the agonists with their neurotoxic effects on Purkinje cells and granule cells suggested that: (a) the ability to depolarize is a required condition for an agonist to be neurotoxic, (b) the magnitude of depolarization, rather than depolarizing potency, is the more pertinent determinant of neurotoxic potency and (c) resistance to the neurotoxicity of an agonist is not necessarily associated with resistance to its depolarizing actions. Histological studies indicated that the neurotoxicity of N-methyl-D-aspartate and kainate in immature cerebellar slices could largely not be replicated by veratridine (50 microM) or high extracellular K+ (124 mM) indicating that receptor-mediated ionic fluxes may be needed in addition to those caused by depolarization. Exposure of the slices to anoxia in the absence of glucose partially reproduced the toxicity of the receptor agonists. Application of ouabain for 30 min caused necrosis of all the cells which are vulnerable to the agonists but spared the cells which are not vulnerable. Profound ionic imbalance thus appears to be a sufficient explanation for amino acid neurotoxicity.

Morphological response of endoplasmic reticulum in cerebellar Purkinje cells to calcium deprivation.

Mobilisable intracellular Ca2+ stores are highly enriched in the cerebellum, particularly in Purkinje cells. We have detected, by light and electron microscopy, striking morphological changes in the presumed Ca2+ stores of Purkinje cells when slices of eight-day-old rat cerebellum were incubated in Ca(2+)-deficient media. After 30 min under these conditions, the endoplasmic reticulum became thinned and elongated. By 2 h, it was transformed into multilamellar, whorl-like inclusions with electron-dense cores. These changes were reversed on reintroduction of Ca2+. Analogous changes in other neurons were not observed. The results suggest that Ca2+ storage sites within Purkinje cells are capable of dramatic morphological change depending on the availability of Ca2+. The transformations may reflect, initially, depletion of Ca2+ from the stores and then homeostatic alterations in their capacity.

Modular distribution of vasoactive intestinal polypeptide in the rat barrel cortex: changes induced by neonatal removal of vibrissae.

The distribution of vasoactive intestinal polypeptide-immunoreactive neuronal structures in the barrel cortex (posteromedial barrel subfield) of adult rats was analysed after unilateral removal of the vibrissal follicles of row C in neonatal rats. The hypothesis was tested whether the distribution of vasoactive intestinal polypeptide-immunoreactive structures depends on the normal anatomical organization of the specific sensory input. After three months survival the distribution of the vasoactive intestinal polypeptide-immunoreactive structures was morphometrically evaluated. This approach revealed alterations in the contralateral posteromedial barrel subfield, where the disappearance of barrel row C and a substantial increase in size mainly of barrel row D, but also of other rows could be detected. Increase in row D included both barrels and the interspace (septal segments between barrels in one row). As vasoactive intestinal polypeptide immunoreactivity of the barrel field was found previously to be localized in synaptic boutons involved in symmetric synapses, our present findings suggest that (i) the interspace is enriched in inhibitory vasoactive intestinal polypeptide-immunoreactive synapses as opposed to the excitatory thalamocortical input reaching the barrel hollow, (ii) the spatial distribution of the vasoactive intestinal polypeptide system in the barrel cortex is closely associated with the neuronal organization of the sensory input and reacts with a considerable plasticity to lesion-induced changes of the input, and (iii) the compensatory barrel hypertrophy in a row neighbouring the deafferented row involves an increasing number of vasoactive intestinal polypeptide-immunoreactive synapses per barrel.

Enantioselectivity at the physiologically active GABAA receptor.

A subfraction of cortical tissue from rat brain, containing membrane vesicles was prepared freshly with added protease inhibitors and antioxidant. The preparation was used to measure stimulation of transmembrane 36C1- flux and inhibition of bicuculline-sensitive [3H] muscimol binding by (+)-(S) and (-)-(R) enantiomers of dihydromuscimol at 30 degrees in physiological salt solution. Displacement of bound [3H]muscimol and stimulation of 36Cl- flux appeared in the 0.1-10 microM concentration range of the enantiomers, channel gating, however, required rather high concentrations. Degrees of enantioselectivity for channel gating, desensitization of and binding to GABAA receptors were estimated by the concentration ratios of dihydromuscimol enantiomers, [(-)-(R)]/[(+)-(S)], at the same level of response or displacement. Different enantioselectives were observed for channel gating (6 +/- 3), receptor binding (3 +/- 2) and desensitization (no selectivity). The low and concentration-dependent enantioselectives found for channel gating and receptor binding can be explained by desensitization and heterogeneity of GABAA receptors.

Differential distribution of dystrophin in postsynaptic densities of spine synapses.

Dystrophin immunocytochemistry was carried out using monoclonal antibody against the C-terminal part of dystrophin (Dys2) in rat cerebral cortex, cerebellum and hippocampus containing a high number of spine synapses of similar morphological character. Dys2 immunoreactivity was found in the spine component of spine synapses. Particularly heavy label was observed on the postsynaptic densities. In the cerebral and cerebellar cortices all spines were immunopositive. In the hippocampus some postsynaptic densities were intensely immunostained, whereas those of adjacent synapses remained unstained. The findings suggest that dystrophin is an integral protein of the postsynaptic component of spine synapses but is lacking in a subpopulation of hippocampal spine synapses. The heterogeneity of input to the hippocampal spiny sites by contrast to the homogeneous population of fibres synapsing with cerebral and cerebellar cortical spines is suggested to account for differences.

Glial fibrillary acidic protein (GFAP)-immunoreactivity is reduced by castration in the interpeduncular nucleus of male rats.

The interpeduncular nucleus of adult male rats was investigated for glial fibrillary acidic protein immunoreactivity. In intact animals the nucleus had an outstandingly intense immunostaining, particularly at its periphery, including the rostral, lateral, dorsomedial and dorsolateral subnuclei where, in addition to neuropil astrocytes, a substantial amount of perivascualr glia was found. Four weeks after castration, immunostaining decreased markedly in the core region of the nucleus corresponding to the caudal and medial subnuclei, and to a much lesser extent at the periphery. The immunoreactivity in pericapillary astrocytes proved to be insensitive to castration. Testosterone, if administered after castration prevented or restituted the loss of immunoreactivity. Beyond 4 months after castration, the effect of testosterone gradually declined. It is concluded that testosterone stimulates the expression of glial fibrillary acidic protein immunoreactivity in the interpeduncular nucleus. Our findings support the argument that gonadal steroids can influence astrocytes also in non-endocrine areas of the brain.

Pre- or postembedding immunocytochemistry: which to choose for the localization of glial fibrillary acidic protein (GFAP)?

Immunostaining for glial fibrillary acidic protein was performed in the hippocampus and cerebellum of adult rats in order to compare the distributions of immunolabelling after pre- and postembedding procedures. The reactions of protoplasmic astrocytes and pericapillary astrocyte processes were investigated at the electron microscopic level. After the pre-embedding reaction, visualized with 3,3'-diaminobenzidine tetrahydrochloride, a granular precipitate was observed to decorate the rough endoplasmic reticulum of the astrocyte cell bodies and a precipitate filled the cytoplasm in the astrocyte processes. In studies with the postembedding procedure, immunogold particles were observed to be attached exclusively to the intermediate filaments of the astrocytic cytoskeleton both in the cell body and in the processes. It is concluded that the diaminobenzidine precipitate diffuses in the cytosol, pre-embedding immunocytochemistry is therefore, suitable for the overall labelling of astrocytes, whereas the postembedding procedure reveals the exact intracytoplasmic localization of glial fibrillary acidic protein. This explains the similar pre-embedding immunostaining patterns of astrocytes with markedly different amounts of glial filaments (e.g. protoplasmic, fibrous and reactive) and stresses the importance of the use of the postembedding method when an exact intracellular localization is required. The results also suggest that, in spite of claims of soluble cytoplasmic pools of this protein, the glial filaments are the exclusive domains of immunoreactive glial fibrillary acidic protein.

Slices from the rat olfactory bulb maintained in vitro. Morphological aspects.

Transverse, 400-microns-thick slices of 8-day-old rat olfactory bulb were incubated in Krebs-Henseleit medium with and without oxygenation. Following incubation, slices were fixed in aldehyde-osmium and embedded in resin for light and electron microscopy. After 2 h of incubation oxygenated preparations showed a structural preservation comparable to that of the freshly fixed olfactory bulb. Under hypoxic conditions mitral cells located on the medial side of the bulb were the most sensitive to the interruption of gassing, while ventricular cells and glomeruli were remarkably resistant as judged by morphological standards. The effects of short-term (up to 30 min) interruptions of gassing proved to be reversible. Our findings suggest that the incubated olfactory bulb slice may be a useful preparation for functional morphological studies.

Immunocytochemical demonstration of radial glia in the developing rat olfactory bulb with antibodies to glial fibrillary acidic protein.

Olfactory bulbs of 8-, 12-, 16- and 30-day-old rats were studied by means of immunocytochemistry using antibodies to glial fibrillary acidic protein (GFAP) and with the Toluidine blue-staining of semithin sections. Until day 12 the GFAP-reaction revealed a radial glia system, the fibres of which extended from the axial ventricular cleft to the surface. From day 16 onwards radial fibres were gradually replaced by typical astrocytes. The lack of proliferative activity within the bulb during the early postnatal period suggests that its cells are generated at and migrating from an external site. An intensely proliferating area was detected in the frontal lobe subventricular layer from where a bundle of migratory cells extends into the bulb. Radial glia may thus be of importance in guiding the migration of cells from this axial bundle to more peripheral regions of the olfactory bulb.

The demonstration of immunoreactive dystrophin and its developmental expression in perivascular astrocytes.

Immunoreactivity of dystrophin family proteins was observed in the astrocytes of the adult and immature rat hippocampus and cerebral cortex by using Dys2, a monoclonal antibody recognizing both 427 kDa and short dystrophin isoforms. As revealed by light and electron microscopy, immunostaining of the ribosomal apparatus and of pericapillary endfeet was particularly pronounced in the adult. In the pericapillary astrocyte processes immunostaining appeared between postnatal days 10 and 20, and reached the intensity seen in the adult by postnatal day 30. In the pericapillary astrocyte process, the membrane facing the endothelial basal lamina was the earliest structure to show the immunoreaction. At later stages, the pericapillary astrocyte process was gradually filled up with immunoprecipitate. Findings suggest that dystrophins are expressed coinciding with the development of the blood-brain barrier, and it is assumed that they contribute to the formation of this system.

Immunohisto- and cytochemical localization of cortical nicotinic cholinoceptors in rat and man.

A monoclonal antibody (WF 6) raised against purified Torpedo nicotinic acetylcholine receptor was applied to study the cellular and subcellular receptor distribution in human and rat neocortex. In both species, immunostaining was most prominent in perikarya and dendrites of the projection neurons in layers III and V. In layer VI fusiform cells displayed immunoreactivity while in layers I, II and IV some round-shaped cells were immunostained. Subcellularly, immunoprecipitate was found in neuronal perikarya, dendrites and in the postsynaptic thickenings, indicating intracellular sites of synthesis, transport and membrane incorporation of receptor protein. The results suggest that WF 6-immunocytochemistry is a useful tool to label nicotinic cholinergic receptors rendering new information about the specific cell-type and subcellular receptor distribution hardly obtainable by using conventional receptor autoradiography.

Ovarian cycle-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The interpeduncular nucleus (IPN) of female rats was studied across the estrous cycle to observe whether the expression of the astroglial marker, glial fibrillary acidic protein (GFAP) reacts to hormonal changes in an area not belonging to the 'endocrine brain'. A marked reduction of immunoreactive GFAP was observed in estrus as compared to the immunoreactivities in met- and proestrus. This finding is consistent with earlier observations in the endocrine hypothalamus, but also proves that gonadal steroids influence astroglia in brain regions not involved in neuroendocrine regulation. Since cyclic fluctuations of synaptic numbers in the female have been described only for the endocrine hypothalamus, decrease of immunoreactive GFAP in the IPN during estrus may reflect a down-regulation of GFAP synthesis.

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. III. Isolation of large fragments of the cerebellar glomeruli.

By a combination of differential and sucrose density gradient (both discontinuous and linear) centrifugation, large fragments of the cerebellar glomeruli were isolated in high purity from hand homogenised tissue. The final preparation contained only about 1% of the tissue protein, but over 90% of its volume was accounted for by the glomerulus particles. The ultrastructure of the glomerulus particles was well preserved. The enzyme profile was characteristic: the glomerulus particles were enriched in glutamate decarboxylase (GAD) activity (relative specific activity (RSA), 2.54), but the RSA of choline acetyltransferase (ChAc) was only 1.05. These findings are consistent with the view that GAD activity is very high in the inhibitory Golgi terminals, which occupy only a small fraction of the total volume of the particles, and acetylcholine may be a transmitter only in a relatively small fraction of the mossy fibre terminals. The glomerulus particles also contained a high concentration of succinate dehydrogenase (SDH) activity (RSA, 1.91), whereas the RSA of glutamate dehydrogenase (GDH) was only 1.15. The great asset of this preparation for future investigations is that it is composed almost exclusively from pre- and postsynaptic neuronal structures. Fractions containing neuropil fragments of non-glomerular origin were also obtained, but the profile of the estimated enzymes did not indicate unique characteristics.

Separation of cell types from the developing cerebellum.

The heterogenous population of perikarya (cells) obtained by dissociating cerebellar tissue of developing postnatal rats was separated by sedimentation at unit gravity. Peak fractions, defined by monitoring the distribution of different size classes with a particle analyzer, were enriched in ultrastructurally well-preserved and metabolically competent specific cell types. These fractions included the peak of rapidly sedimenting cells comprising large neurones, such as Purkinje cells, which accounted for about 50% of the cells (vs. 2% in the initial cell suspension) and for a much greater proportion of the total cell mass (over 80%). More slowly sedimenting peak fractions contained 2-5-fold enrichments in replicating cells (assessed in terms of [3H]thymidine incorporation into DNA or by the frequency of mitotic cells), astroglia-like cells and external and differentiating granule cells, respectively. Separated, replicating cells continued synthesizing DNA in vitro; the [3H]thymidine incorporation rate was about 5-fold greater in the fraction enriched in proliferating cells than in the total cell suspension. Besides their structural integrity, the viability of the cells was also indicated by the finding that the proportion of trypan blue-excluding cells in all fractions exceeded 80%. Moreover, protein synthesis, in terms of incorporation of labelled amino acids, continued in the separated cells at a linear rate for a relatively long time. The rate per cell was highest in the large neuronal fraction, and lowest in the astroglia-like fraction.