The corticopontine projection: axotomy-induced loss of high affinity L-glutamate and D-aspartate uptake, but not of gamma-aminobutyrate uptake, glutamate decarboxylase or choline acetyltransferase, in the pontine nuclei.

The corticopontine fibres were severed in the crus cerebri in rats and mice by a stereotaxically operated retractable wire-knife. The pontine nuclei were microscopically dissected from fresh slices of rats and synaptosome-containing homogenates were prepared. The high affinity uptake of radiolabelled L-glutamate (L-Glu) and D-aspartate (D-Asp) was heavily reduced five days after the lesions. The uptake was further reduced after bilateral (-75% for D-Asp and -65% for L-Glu) than after unilateral lesions (-55% for D-Asp and -45 to 50% for L-Glu on the lesioned side.) The molar ratio of the uptakes of D-Asp and L-Glu was consistently lower in pons after transection of the cortical afferents than normally (-28% after bilateral lesions). gamma-Aminobutyrate uptake and glutamic acid decarboxylase were not changed. Choline acetyltransferase was increased (+53%) after unilateral lesions, but not altered after bilateral lesions. Autoradiograms of slices from mice, incubated with tritium-labelled amino acids and fixed in glutaraldehyde, showed high affinity uptake sites for D-Asp to be enriched in the pontine nuclei, compared to neighbouring structures. After partial lesion of the crus cerebri the uptake was reduced in the area with degenerated corticopontine afferents. gamma-Aminobutyrate uptake sites were relatively less concentrated in the pontine nuclei than D-Asp uptake sites. The results indicate, along with the previous demonstration of Ca-dependent K-induced release of D-[3H]aspartate from the corticopontine terminals, that glutamate and/or aspartate may be transmitters in this pathway. The results also suggest that acidic amino acid uptake sites may differ in their relative transport rates for aspartate and glutamate.

Distinct pattern of PCR-SSCP analysis of p53 mutations in human astrocytomas.

Clarification of somatic mutations during the progression of human astrocytomas is important in order to understand the mechanisms underlying the development of these tumors. We analyzed surgical specimens of human astrocytomas for mutations in the p53 gene using single-strand conformation polymorphism analysis of polymerase chain reaction product (PCR-SSCP analysis) at a low pH. Klenow fragment treatment after PCR amplification was an effective means to get rid of some extra bands on the SSCP gel. Five mutations in three of 24 astrocytomas were identified by this improved SSCP method. The frequency of p53 gene mutations in astrocytomas examined was 12.5%. Further examination by direct sequencing showed that all five mutants had single-base substitutions resulting in missense mutations. The present studies revealed a loss of heterozygosity and two point mutations on the remaining allele in one of the fibrillary astrocytomas. Finally, the improvement of PCR-SSCP analysis using Klenow treatment and low pH showed a distinct electrophoresis gel pattern and could be relevant for the prognosis of human astrocytomas.

Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes.

We present data on the effect of elevated concentrations of K+ ions (25 mM) and polylysine (PLL) coating of the substratum on the in vitro survival and behaviour of cells derived from 8-day-old rat cerebellum. The cells were grown in Eagle's basal medium in the presence of 10% foetal calf serum and cytosine arabinoside (10 microM), as a mitotic inhibitor. The most conspicuous effect of the high potassium was to facilitate the relatively long survival of the nerve cells, whereas PLL influenced the nerve cell attachment and thereby the size of the aggregates formed in the cultures. When cells were grown in high [K+] on PLL-coated dishes (standard conditions) over 70% of the plated cells survived beyond 7 DIV, and about 95% of the cells were small interneurones, tentatively identified as predominantly granule cells. The most numerous non-neuronal cells were glial fibrillary acid protein (GFA) positive astrocytes. The beneficial effect of high potassium on nerve cell survival was most prominent after 7 DIV, when it is known that transmission-associated neurochemical functions are just becoming detectable under the standard conditions. Initially (at 3 DIV) under all the tested conditions, and throughout the experimental period under the standard conditions, the dominant type of GFA-positive cells was the process bearing 'differentiated' astrocyte. When the conditions resulted in a great decrease in nerve cell numbers, on the other hand, flat astroblast-like cells became the most abundant cells in this class. Neurones grown on polylysine in the presence of 25 mM potassium extended neurites as early as 6 h after plating, and with longer culture times, an extensive network of fibres of neuronal origin was generated. Neurites did not seem to follow the processes of GFA-positive astrocytes in the cultures. Although there was a limited tendency for neuronal cell bodies to be positioned around astrocytes at 1 DIV, this became less marked with time, and no preferential association between astrocytes and neurones could be detected in the cultures later than 2 DIV.

Excitatory amino acids rise to toxic levels upon impact injury to the rat spinal cord.

The release of glutamate, aspartate, glutamine and asparagine upon impact injury to the rat spinal cord was characterized by sample collection from the site of injury by microdialysis. Injury caused dramatic and long-lasting increases in the concentrations of the excitatory amino acids. Determination of the relationship between unperturbed extracellular levels and the levels of amino acids in the collected fluids indicates that the concentrations of these amino acids were probably high enough to kill neurons for longer than one hour following impact injury to the spinal cord. Increases in the concentrations of the metabolically related non-neurotransmitters asparagine and glutamine were considerably smaller. The latter observations suggest that much of the increase in levels of the excitatory amino acids resulted from neuronal activity rather than from simple damage.

Localization of calpain immunoreactivity in senile plaques and in neurones undergoing neurofibrillary degeneration in Alzheimer's disease.

An antibody raised against the calcium activated neutral protease (calpain) was used to investigate the possible involvement of this enzyme in the formation of plaques and tangles in Alzheimer-type dementia (ATD) brain. Our results revealed the presence of a number of strongly stained calpain positive neurones in the normal human cerebral cortex and a loss of calpain positive cells in ATD brain. Furthermore, double staining experiments revealed that calpain immunoreactivity was present in cells undergoing tangle formation, and was also present in senile plaques. These data suggest that activation of calpain may be an important factor in the abnormal proteolysis underlying the accumulation of plaques and tangles in ATD.

K+-evoked Caa+-dependent release of D-[3H]aspartate from terminals of the cortico-pontine pathway.

Pontine nuclei dissected from rat brain slices released previously accumulated D-[3H]aspartate (D-Asp) and [14C]gamma-aminobutyrate (GABA) Ca-dependently when exposed to 50 mM K. These efflux rates were substantially increased by including 0.5 mg/ml bovine serum albumin in the superfusion fluid. Degeneration of the cortico-pontine fibres 5 days after cutting the crus cerebri caused an 80% reduction in the fractional rate of Ca-dependent D-Asp release and a 60% reduction in uptake. The fractional rate of GABA efflux was significantly less reduced than that of D-Asp efflux, and GABA uptake was nearly unchanged.

Detection of a novel point mutation in the p53 gene in grade II astrocytomas by PCR-SSCP analysis with additional Klenow treatment.

Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with additional Klenow treatment and direct sequencing mutations in the p53 tumor suppressor gene were analyzed from 21 cases of human astrocytomas. Three cases had p53 gene mutations: two of them were glioblastomas showing a point mutation, one in exon 5 and the other in 6. The last one was a gemistocytic astrocytoma showing a point mutation in exon 5. The frequency of p53 gene mutations in the astrocytomas examined was 14.3% (3 out of 21). No SSCP alterations were observed in any of the p53 fragments amplified from WHO grade I pilocytic astrocytomas and WHO grade III anaplastic astrocytomas. Further examination by direct sequencing showed that two mutations of glioblastomas had single-base substitutions resulting in silent and missense mutations, whereas one of the gemistocytic astrocytomas had a double-base substitution resulting in a missense mutation. The present studies revealed that all mutations were located outside the hot spots and, interestingly, one of them disclosed a missense mutation in exon 5 at codon 166, which was first detected in a grade II astrocytoma (gemistocytic type). It is possible that the missense mutation at this codon may be associated with special risk factors for the development of astrocytic tumors in Thai patients.

Effects of the organophosphate insecticide, monocrotophos, on acetylcholinesterase activity in the nile tilapia fish (Oreochromis niloticus) brain.

The neurotoxic effects of monocrotophos on the brain of the nile tilapia fish (Oreochromis niloticus) were examined, using a static bioassay under laboratory conditions. By probit analysis the 96 h LC50 value of monocrotophos was 4.9 mg/l. After 96 h exposure to acute levels of monocrotophos, the brain acetylcholinesterase (AChE) activity decreased progressively as the concentration of monocrotophos increased. In addition, four weeks following transfer to toxicant-free water after exposure to 1 mg monocrotophos, nile tilapia fish brain regained 95% of control AChE activity. The results indicate that inhibition of AChE activity in fish exposed to monocrotophos may serve as an indicator of hazard due to application of this chemical in the natural environment.

Developmental changes in gangliosides in cultured cerebellar granule neurons.

The content and composition of gangliosides in cultures enriched in granule neurones and in astrocytes from rat cerebellum (P6-8) showed marked differences: astrocytes contained less than 10% of the amount of granule neurones and the profile was dominated by simple gangliosides with lactosyl ceramide backbone, while gangliosides of the 'b' series, which constitute about 40% in nerve cells, were virtually undetectable. Granule cell maturation was accompanied by a 16-fold increase in the ganglioside content during the initial 8 days in a serum-supplemented medium (S+), reaching a plateau much earlier and at a higher level than observed in the cerebellum in vivo. Developmental changes were characterized, as in vivo, by a pronounced decrease in the GD3 proportion and an increase in the 'b' series of gangliosides. Compared with S+, adhesion among cells and fibres is different in a serum-free medium (S-), in which the rise in cellular ganglioside content was less (30%), but the developmental changes in ganglioside profile were similar. However, in cultures in S- only, GM3 was not detectable, while the distribution of GM1 and GD3 indicated that maturation is retarded relative to cells in S+. Surface exposure of gangliosides (studied by the periodate/[3H]borohydride method) was similar under the two culture conditions. There was an initial delay, especially in S-, in the insertion of gangliosides into the plasma membrane, while the labelling of GD3 (the dominant ganglioside of immature granule cells) was very low compared with all the other species throughout the whole cultivation time.

Autoproteolysis in human seminal plasma under acidic condition.

The existence of both neutral and acidic proteases in human seminal plasma suggests a possible autohydrolysis of the proteins in the fluid. By means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the autoproteolysis in human seminal plasma was shown to occur at pH 3.5 but not at pH 5.0--7.5 conditions. At pH 3.5, most proteins of large molecular weight, except one of 28,000 daltons, were completely hydrolyzed into peptides of 13,000 daltons or smaller. The autoproteolysis was due to the action of the acidic protease since it can be blocked by 1 mM of p-bromophenacyl-bromide or 1,2-epoxy-3-(p-nitrophenoxy) propane, the specific inhibitors of the acidic protease.