Ambrish J. Patel

Exposure toN-methyl-d-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [3H]ARA release. The half maximal effect was obtained at about 15 microM NMDA, whereas the maximum concentration (50 microM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg2+ which blocks NMDA receptor-linked Ca2+ ion channels. The removal of external Ca2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca2+ ionophore) greatly increased the [3H]ARA release. The inhibitors of phospholipase A2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca2+ brought about by opening the voltage-sensitive channels by high K+ had no effect on the release of [3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca2+ entry through this route is more efficiently coupled to the activation of phospholipase A2. Treatment with NMDA had no significant effect on [3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.

Topographical localization of neurons containing parvalbumin and choline acetyltransferase in the medial septum-diagonal band region of the rat

The normal morphology and distribution of parvalbumin-containing neurons (shown in a previous study to be GABAergic nerve cells) of the medial septal-diagonal band region of the adult rat brain have been studied, and the findings compared with observations on choline acetyltransferase-immunoreactive neurons. The two antigens were visualized either in the same sections using a double-label immunohistochemical procedure for the simultaneous localization of parvalbumin and choline acetyltransferase, or in immediately adjacent sections. In double-stained sections of the whole medial septal-diagonal band complex, about 34% of the total neurons showed immunoreactivity to parvalbumin; the proportion of parvalbumin-labelled neurons was slightly higher in the medial septal-vertical limb of the diagonal band region, and much lower in the horizontal limb of the diagonal band region. The distribution of parvalbumin- and choline acetyltransferase-containing neurons also varied markedly between different mediolateral subdivisions of the medial septum: about 30, 65 and 2% of the parvalbumin-immunoreactive neurons were present in the midline, medial and lateral part of the medial septum, respectively. At different rostrocaudal levels, the proportion of parvalbumin- and choline acetyltransferase-positive neurons varied in a consistent manner, and the largest number of parvalbumin-containing neurons was found at the level 1.9 mm anterior to the bregma. In the absence of reliable immunocytochemical methods for the localization of glutamate decarboxylase and GABA, parvalbumin may serve as a good marker for studying the distribution of GABAergic neurons in the medial septum-diagonal band region. Moreover, the precise maps reported in the present study of the topographic localization of parvalbumin-containing GABAergic and choline acetyltransferase-immunoreactive cholinergic nerve cells in the medial septal-diagonal band complex will serve as a useful guide in future morphological and electrophysiological studies on the septum and its efferents.

Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-D-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 microM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 microM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca2+ ion channel blockers, MK 801 and Mg2+. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K+ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca2+ mediated through the NMDA induced increase in Ca2+ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.

Regulation of β-amyloid precursor protein isoform mRNAs by transforming growth factor-β1 and interleukin-1β in astrocytes

Abstract In cultured astrocytes, all three major transcripts of β-amyloid precursor protein (APP) were expressed with the ratio for APP695, APP751 and APP770 isoform mRNAs being 1:4:2. In comparison with controls, treatment of astrocytes with transforming growth factor-β1 (TGF-β) produced about 6 fold increase in total APP mRNA, while elevation in the interleukin-1β (IL-1β) treated group was small and may relate to the mitogenic effect of IL-1β on astrocytes. Treatment of astrocytes with cytokines also produced marked changes in the upregulation in expression of different APP isoforms. The net increase in mRNAs of KPI-containing isoforms APP751 and APP770 was relatively more than for the APP695 isoform. This phenomenon was mainly related to the differences in the expression of KPI-containing APP isoforms and APP695 isoform in the controls. The present findings provide further evidence for the involvement of astrocytes in a cascade of events leading to the development of senile plaques in Alzheimers disease and Downs syndrome.

Cell-specific expression of β-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes

Abstract The abnormal accumulation of β -amyloid (A β ) in senile plaques appears to be a central pathological process in Alzheimers disease. A β is formed by proteolysis of β -amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A β region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was ≈90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A β is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.

Concentration of free amino acids in primary cultures of neurones and astrocytes

The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and γ‐aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was

Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development

65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8‐day‐old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the “small” compartment of glutamate to glial cells and the “large” compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.

Developmental changes in the amount of glial fibrillary acidic protein in three regions of the rat brain

The cerebellar granule cells are believed to be glutamatergic neurons. During the normal development of granule cells grown in a chemically defined medium, the specific activity of phosphate-activated glutaminase increased from 60 at 3 days to 150 (nmol/h/mg protein) at 15 days in vitro. Treatment with 25 mM K+ for the last 2 days elevated glutaminase activity in an age-dependent manner: about 100% at 3 and 6 days, 75% at 10 days, and 40% at 15 days in vitro. The enhancement of glutaminase in granule cells was dose-dependent. The half-maximal effect was obtained at about 20 mM K+, whereas the maximum concentration, which produced about a 2.5-fold increase in 3-day-old cultures was about 40 mM K+. The voltage-sensitive Na+ channel inhibitor tetrodotoxin had no effect on the depolarization-induced activity in granule cells. However, the increase in glutaminase by 25 mM K+ was significantly blocked by both organic (nifedipine) and inorganic (Ni2+ and Mg2+) calcium antagonists, indicating that elevation in activity may be mediated through transmembrane Ca2+ entry into granule cells. In contrast to neurons, in cultured cerebellar astrocytes, the activity of glutaminase slightly decreased during development, and treatment with 25 mM K+ had no significant effect on this enzyme activity. The present findings, together with previous observations, would indicate that depolarization with K+, which is believed to mimic in vivo presynaptic stimulation, could be one of the mechanisms that selectively controls the development and function of neurons, when measured in terms of the activity of the enzymes involved in the synthesis of cell-specific neurotransmitters.

Development of the cholinergic fibres innervating the cerebral cortex of the rat

Glial fibrillary acidic (GFA) protein, extractable in 50 mM phosphate buffer, pH 8, was measured in the olfactory bulbs, forebrain and cerebellum of the rat during development using a double antibody radioimmunoassay. Each brain region showed a different pattern of development for GFA protein. At birth GFA protein per mg protein was highest in olfactory bulbs followed by forebrain and cerebellum, and these amounted to 15, 10 and 8% of the adult values, respectively. The relative increase in GFA protein was more marked during the first 2 postnatal weeks than in the following 7 weeks after birth. When values were expressed per brain region, the developmental increase in the amount of GFA protein from birth to adulthood was about 100-fold in olfactory bulbs, 85-fold in forebrain and 485-fold in cerebellum. The patterns of developmental increases in GFA protein and in glutamine synthetase activity, another protein enriched in astrocytes, were similar in the forebrain and olfactory bulbs, but differed markedly in the cerebellum. The major increase in content of the GFA protein during development was found to correspond with the maturation of astrocytes rather than with their proliferation; however, a small but significant amount of GFA protein acquired at an early age may be related to increase in astroglial cell numbers in the cerebellum.

Induction of β-amyloid precursor protein isoform mRNAs by bFGF in astrocytes

The ontogeny of innervation of the cholinergic fibres from the basal forebrain into the cingulate, frontal, parietal and piriform cortices of the rat has been examined using a modified histochemical method of acetylcholinesterase (AChE). The method produced crisp fibre staining with enhanced visibility and a clear back‐ground, and a pattern of the distribution of these fibres was comparable to that achieved by choline acetyltransferase (ChAT) immunocytochemistry. In the rat, the AChE‐stained fibres developed progressively from the deep cortical white matter towards the cortex itself. In general, a few AChE‐positive fibres were seen in the subcortical white matter and the cingulum bundle, entering into the cerebral cortex by about 5 postnatal days. The number of these AChE‐positive processes increased dramatically during the following two weeks. Thereafter, the general appearance of the overall pattern of distribution of the AChE fibres changed little, but the staining density became gradually more intense and by about 28 days after birth it was virtually indistinguishable from that in the adult. The onset and the development of the AChE‐positive fibre network varied considerably between individual cortical regions, and indicated, in general, an anterior to posterior gradient. Within the dispersed AChE fibre network in the cerebral cortex, three bands of relatively enriched cholinergic processes, namely the deep cortical, mid‐cortical and superficial layers, developed in an ‘inside‐out’ fashion. The exact position of some of these AChE‐rich bands varied from one cortical region to another and during development. A striking correlation during ontogeny was observed in the cerebral cortex between the changing patterns of AChE fibre network and the activity of ChAT, the enzyme synthesizing acetylcholine. The present findings can also provide an important anatomical baseline for future studies related to the factors controlling the expression of ChAT activity and the development of cholinergic neurotransmitter system in the rat.