Distribution of class I, III and IV alcohol dehydrogenase mRNAs in the adult rat, mouse and human brain.

The localization of different classes of alcohol dehydrogenases (ADH) in the brain is of great interest because of their role in both ethanol and retinoic acid metabolism. Conflicting data have been reported in the literature. By Northern blot and enzyme activity analyses only class III ADH has been detected in adult brain specimens, while results from riboprobe in situ hybridization indicate class I as well as class IV ADH expression in different regions of the rat brain. Here we have studied the expression patterns of three ADH classes in adult rat, mouse and human tissues using radioactive oligonucleotide in situ hybridization. Specificity of probes was tested on liver and stomach control tissue, as well as tissue from class IV ADH knock-out mice. Only class III ADH mRNA was found to be expressed in brain tissue of all three investigated species. Particularly high expression levels were found in neurons of the red nucleus in human tissue, while cortical neurons, pyramidal and granule cells of the hippocampus and dopamine neurons of substantia nigra showed moderate expression levels. Purkinje cells of cerebellum were positive for class III ADH mRNA in all species investigated, whereas granular layer neurons were positive only in rodents. The choroid plexus was highly positive for class III ADH, while no specific signal for class I or class IV ADH was detected. Our results thus support the notion that the only ADH expressed in adult mouse, rat and human brain is class III ADH.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.     

Immunocytochemical localisation of transferrin in the human brain.

Immunocytochemical studies on the adult human brain have shown that transferrin is localized within three main compartments in the adult human brain. Oligodendrocytes and some astrocytes together with cells of the choroid plexus showed the highest intensity of staining. Neuronal staining occurred mainly within pyramidal or large polygonal cells, but this showed considerable regional variation being most marked in areas such as the cerebral cortex, amygdala, hippocampus, brainstem and cerebellar Purkinje cells. Small neurones such as caudate interneurones and granule cells showed relatively low activity. Diffuse immunostaining of the neuropil was evident, particularly where heavy neuronal or glial staining occurred. Immunostaining was also observed in white-matter fibre tracts. This pattern of distribution helps to provide a model for the mechanisms responsible for iron homeostasis in the normal brain.     

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II–III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.     

Differential neuronal loss following early postnatal alcohol exposure

Neonatal rats were exposed to 6.6 g/kg of alcohol each day between postnatal days 4 and 10 while artificial-rearing procedures were used, in a manner which produced high peak and low trough blood alcohol concentrations each day. Gastrostomy controls were reared artificially with maltose/dextrin isocalorically substituted for alcohol in the milk formula, and suckle controls were reared normally by dams. The pups were sacrificed on day 10 and tissue sections (2 microns thick) were obtained in the sagittal plane through the cerebellum and in the horizontal plane through the hippocampal formation. Overall area measures were obtained for the hippocampus proper, area dentata, and cerebellum, along with areas of the cell layers of these regions. In the hippocampal formation, cell counts were made of the pyramidal cells of the hippocampus proper, the multiple cell types of the hilus, and the granule cells of the area dentata. In the cerebellum, cell counts of Purkinje cells, granule cells of the granular layer, granule cells of the external granular layer, and mitotic cells of the external granular layer were obtained from lobules I, V, VII, VIII, and IX. Alcohol selectively reduced areas and neuronal numbers in the cerebellum but had no significant effects on neuronal numbers in the hippocampal formation. Purkinje cells exhibited the greatest percent reductions, and cerebellar granule cells were significantly reduced in the granular layer but not in the external granular layer. All lobules showed these effects, but lobule I was significantly more affected than the other four lobules that were analyzed. The results demonstrate the differential vulnerability of selected neuronal populations to the developmental toxicity of alcohol exposure during the brain growth spurt.     

Permanent neuronal deficits in rats exposed to alcohol during the brain growth spurt

The purpose of this study was to determine whether developmental alcohol exposure could induce permanent neuronal deficits, whether the peak blood alcohol concentration (BAC) influences the severity of the effects, and whether the effects are gender related. Rat pups were reared artificially over postnatal days (PD) 4 through 11 (a period of rapid brain growth, comparable to part of the human third trimester). Alcohol treatments were administered on PD 4 through 9. Patterns of alcohol exposure that produce different peak BACs have been shown to affect differentially the amount of brain weight deficits and neuron loss shortly after the exposure period, so this study investigated whether the pattern of alcohol exposure was also effective in producing permanent deficits. Two groups received a daily alcohol dose of 4.5 g/kg, condensed into either four or two feedings. A third group received a higher daily alcohol dose of 6.6 g/kg administered in 12 uniformly spaced daily feedings. Pups were fostered back to dams on PD 11 and perfused on PD 90. Brain weights were measured, and Purkinje cells and granule cells were counted in each of the 10 lobules of the cerebellar vermis. In the hippocampal formation, cell counts were made of the pyramidal cells of fields CA1 and CA2/3, the multiple cell types of CA4 and the granule cells of the dentate gyrus. The groups receiving the lower daily dose (4.5 g/kg) condensed into either four or two feedings were exposed to higher peak BACs and suffered significant permanent brain weight deficits and neuronal losses, relative to controls. The group receiving the higher daily dose (6.6 g/kg) in continuous fractions had no significant brain weight reductions or neuronal loss. Vulnerability to alcohol-induced neuronal loss varied among regions and cell populations and as a function of peak BAC. In the hippocampus, only the CA1 pyramidal cells were significantly reduced in number and only in group receiving the most condensed alcohol treatment. In the cerebellum, the severity of Purkinje cell and granule cell losses varied among lobules, and Purkinje cell vulnerability appeared to depend on the maturational state of the neuron at the time of the alcohol exposure, with the more mature Purkinje cells being the more vulnerable.     

Expression, localization and potential physiological significance of alcohol dehydrogenase in the gastrointestinal tract.

ADH1 and ADH4 are the major alcohol dehydrogenases (ADH) in ethanol and retinol oxidation. ADH activity and protein expression were investigated in rat gastrointestinal tissue homogenates by enzymatic and Western blot analyses. In addition, sections of adult rat gastrointestinal tract were examined by in situ hybridization and immunohistochemistry. ADH1 and ADH4 were detected along the whole tract, changing their localization and relative content as a function of the area studied. While ADH4 was more abundant in the upper (esophagus and stomach) and lower (colorectal) regions, ADH1 was predominant in the intestine but also present in stomach. Both enzymes were detected in mucosa but, in general, ADH4 was found in outer cell layers, lining the lumen, while ADH1 was detected in the inner cell layers. Of interest were the sharp discontinuities in the expression found in the pyloric region (ADH1) and the gastroduodenal junction (ADH4), reflecting functional changes. The precise localization of ADH in the gut reveals the cell types where active alcohol oxidation occurs during ethanol ingestion, providing a molecular basis for the gastrointestinal alcohol pathology. Localization of ADH, acting as retinol dehydrogenase/retinal reductase, also indicates sites of active retinoid metabolism in the gut, essential for mucosa function and vitamin A absorption.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Chronic prenatal ethanol exposure-induced decrease of guinea pig hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density

The brain is a key target of ethanol teratogenicity, in which ethanol can produce neurodegeneration in selected areas, including the hippocampus and cerebellum. The research objective was to test the hypothesis that chronic prenatal ethanol exposure, via maternal ethanol administration, produces differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells. Timed pregnant guinea pigs received chronic oral administration of ethanol, isocaloric-sucrose/pair-feeding, or water throughout gestation (term, about gestational day (GD) 68), and the offspring were studied at GD 62 (near-term fetus), postnatal day (PD) 1 (neonate), PD 5, and PD 12 (early postnatal life). Ethanol treatment, compared with isocaloric-sucrose/pair-feeding and water treatments, decreased brain, hippocampal, and cerebellar weights at GD 62, PD 1, PD 5, and PD 12. Hippocampal CA1 pyramidal cell linear density and cerebellar Purkinje cell linear density were unaffected at GD 62. Ethanol treatment produced 25, 30, and 30% decreases in linear density of hippocampal CA1 pyramidal cells at PD 1, PD 5, and PD 12, respectively, and a 30% decrease in linear density of cerebellar Purkinje cells at PD 12 only. At PD 5, Purkinje cell profile linear density remained unaffected; however, ethanol treatment appeared to increase linear density of apoptotic Purkinje cell nuclei, as determined by a modified TUNEL method. The data demonstrate that chronic prenatal ethanol exposure produces apparent differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells in the developing guinea pig.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Abstract

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.     

Do neurons in the vertebrate CNS migrate on laminin?

In adult rat brain the extracellular matrix glycoprotein, laminin, is found only in basement membranes, but is transiently expressed by astrocytes after brain injury. Here, I show that laminin also appears in immature brain cells during CNS development, and that its presence coincides with phases of neuronal migration. In early embryos, laminin is seen throughout the whole thickness of the forming brain, and is apparently synthesized by the cells, as judged by its intracytoplasmic localization. As development proceeds, intracellular laminin becomes restricted to the periventricular regions while punctate deposits of laminin follow the course of vimentin-positive radial glial fibers. In most brain regions, the adult pattern of laminin expression is achieved by birth. In the post-natal rat cerebellum, however, laminin is detected in external granule cells, in Purkinje cells, and in punctate deposits along the radial Bergmann glial fibers. By day 24 after birth, when the migration of external granule cells is complete, all laminin immunoreactivity disappears from these structures. The transient expression of laminin in regions where neurons are migrating raises the possibility that laminin plays a role in neuronal migration during CNS development.     

Mammalian DSCAMs: roles in the development of the spinal cord,cortex, and cerebellum?

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral-dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal-Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal-ventral patterning and cell-fate specification in the developing CNS.     

Region- and neuronal phenotype-specific expression of NELL2 in the adult rat brain

NELL2, a neural tissue-enriched protein, is produced in the embryo, and postembryonically in the mammalian brain, with a broad distribution. Although its synthesis is required for neuronal differentiation in chicks, not much is known about its function in the adult mammalian brain. We investigated the distribution of NELL2 in various regions of the adult rat brain to study its potential functions in brain physiology. Consistent with previous reports, NELL2-immunoreactivity (ir) was found in the cytoplasm of neurons, but not in glial fibrillary acidic protein (GFAP)-positive glial cells. The highest levels of NELL2 were detected in the hippocampus and the cerebellum. Interestingly, in the cerebellar cortex NELL2 was observed only in the GABAergic Purkinje cells not in the excitatory granular cells. In contrast, it was found mainly in the hippocampal dentate gyrus and pyramidal cell layer that contains mainly glutamatergic neurons. In the dentate gyrus, NELL2 was not detected in the GFAP-positive neural precursor cells, but was generally present in mature neurons of the subgranular zone, suggesting a role in this region restricted to mature neurons.     

Developmental and Age-Dependent Changes of 28-kDa Calbindin-D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS)