Amino acid neurotransmitter candidates in rat cerebellum: Selective effects of kainic acid lesions

Kainic acid injections directly into the cerebellum destroy Purkinje, stellate, basket and Golgi II cells selectively with much less damage to granule cells. We have utilized such kainic acid lesions to evaluate the disposition of amino acid transmitter candidates in different neuronal populations of the cerebellum. Kainic acid lesions produce a 65-70% decrease in high affinity [3H]GABA uptake into synaptosomal fractions and a similar decrease in glutamic acid decarboxylase with a 50% reduction in endogenous GABA. Synaptosomal accumulation of [3H]glutamate and [3H]-aspartate is reduced 25-30% following such lesions while no decline in uptake of numerous other amino acids is observed. No significant changes are found in endogenous levels of glycine and serine are elevated following such lesions. These findings are consistent with the possibility that glutamate is the transmitter of granule cells and that GABA is the transmitter of the other cell types in the cerebellum.     

Substrate specificity and developmental aspects of a presynaptic GABA receptor regulating glutamate release in the rat cerebellum

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.     

Impact of STZ‐induced hyperglycemia and insulin‐induced hypoglycemia in plasma amino acids and cortical synaptosomal neurotransmitters

In this work, we evaluated the effects of streptozotocin (STZ)‐induced hyperglycemia and an acute episode of insulin‐induced hypoglycemia in plasma amino acids and cortical neurotransmitters. For that purpose, we used citrate (vehicle)‐treated Wistar rats, STZ‐treated rats [i.p., 50 mg/kg body weight], and STZ‐treated rats injected with insulin [s.c., dose adjusted with blood glucose levels] 1 h prior to sacrifice to induce an acute episode of hypoglycemia. Plasma was collected for determination of amino acids levels. In addition, cortical synaptosomal preparations were obtained and the total levels of neurotransmitters, levels of aspartate, glutamate, taurine, and GABA released by the action of KCl, iodoacetic acid (IAA), ouabain, and veratridine, membrane potential and ATP levels were evaluated. Compared with control rats, plasma from hypoglycemic rats presented increased levels of aspartate, glutamate, glutamine, and taurine whereas GABA levels were decreased in STZ and hypoglycemic rats. Similarly, glutamate and taurine levels were increased in hypoglycemic synaptosomes while GABA decreased in hypoglycemic and STZ‐diabetic synaptosomes. The depolarizing agent KCl promoted an increase in aspartate, glutamate, and taurine release from hypoglycemic synaptosomes. The highest release of neurotransmitters occurred in the presence of veratridine and ouabain, two other depolarizing agents, in all groups of experimental animals. However, a higher release of glutamate was observed in the diabetic and hypoglycemic synaptosomes. No alterations were observed in synaptosomal membrane potential and ATP levels. These results show that in the presence of a metabolic insult a higher release of excitatory amino acids occurs, which may underlay the neuronal injury observed in type 1 diabetic patients under insulin therapy. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Uptake of [3H]L-serine in rat brain synaptosomal fractions

Accumulation of [3H]L-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na(+)-dependent and Na(+)-independent components. The accumulation of [3H]L-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [3H]L-proline, [3H]L-glutamate and [3H]GABA. It was fully inhibited by L-glutamine, L-asparagine, L-cysteine, L-alanine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine, L-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by beta-alanine, taurine, glycine nor was it inhibited by the marker for the A system, L-2-methylamino isobutyric acid. D-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [3H]L-serine. We conclude that L-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na(+)-dependent one of lower affinity (K(m) in mM range) and a Na(+)-independent system of higher affinity (K(m) approximately 20-100 micro M). Characteristics of [3H]L-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical 'A', nor by the 'large neutral', amino acid transport systems but predominantly by transporters belonging to the recently identified LAT (L-amino acid transporter) family.     

Decrease of uptake and exchange of neurotransmitter amino acids after depletion of their synaptosomal pools.

Synaptosomes prelabeled at 37 degrees C with radioactive amino acids (GABA, glutamate, glycine, taurine, alpha-aminoisobutyric acid, phenylalanine, leucine) and then washed at 0 degrees C on Millipore filters (DAWP 02500) lost 60-70% of the accumulated radioactivity. The loss was similar with exogenous tritiated GABA and glutamate, and with [14C]GABA and [14C]glutamate metabolically derived from [14C]glucose. In contrast, radioactive norepinephrine, dopamine and 5-hydroxytryptamine were almost totally retained by cold shocked synaptosomes. After pretreatment with reserpine and nialamide the loss of norepinephrine became significantly greater (about 25%). The uptake of radioactive GABA, glutamate and clycine after cold shock was about 50% reduced, whereas that of radioactive biogenic amines was less affected (reduction of 22% for norepinephrine, 29% for 5-hydroxytryptamine and 35% for dopamine). The loss of amino acids and the reduction of uptake could be minimized by performing the cold shock in hypertonic conditions. In synaptosomes prelabeled with [3H]GABA, a good correlation was observed among magnitude of amino acid pool depletion induced by cold shock or by 56 mM KCl, decrease of subsequent accumulation of [14C]GABA, and decrease of [14C]-GABA-stimulated [3H]GABA release (homoexchange).     

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Glutamate receptor agonists release [3H]GABA preferentially from horizontal cells

A total of 5-6 different cell types in vertebrate retinas accumulate [3H]gamma-aminobutyric acid (GABA). In frog retina, specific populations of cells in the horizontal, amacrine and ganglion cell layers are labeled autoradiographically after a 15-min in vitro incubation with [3H]GABA. Cells which may be bipolar or interplexiform cells are also labeled. Similar autoradiographic patterns are observed in chick retina except for the absence of labeled bipolar or interplexiform cells. In rat retinas, [3H]GABA uptake is limited primarily to Muller and amacrine cells. Depolarizing glutamate receptor agonists (glutamate, aspartate and kainic acid) applied in an in vitro perfusion system, stimulated massive release of [3H]GABA from frog and chick retina but not from rat retina. Under these conditions, autoradiographic labeling of horizontal cells was virtually depleted, while labeling of other cell types remained robust. In contrast, potassium caused release of the label from all 3 types of retina, and loss of autoradiographic labeling occurred uniformly in all cell types. We conclude that [3H]GABA-accumulating horizontal cells possess depolarizing glutamate receptors and that activation of these receptors leads to a release of GABA stores. On the other hand, Muller cells and the various subclasses of [3H]GABA-accumulating amacrine, bipolar and/or interplexiform cells, do not release GABA in response to glutamate receptor stimulation and thus appear to be relatively insensitive to excitatory amino acids.     

Glutamate release induced by activation of glycine and GABA transporters in spinal cord is enhanced in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a progressive and fatal neurodegenerative disease, involving both upper and lower motor neurons, the cause of which is obscure, although glutamate (GLU)-induced excitotoxicity has been suggested to play a major role. We studied the release of [3H]d-aspartate ([3H]d-ASP) and endogenous glutamate evoked by glycine (GLY) or GABA from spinal cord synaptosomes in mice expressing a mutant form of human SOD1 with a Gly93Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, in mice expressing the non-mutated form of human SOD1 [SOD1+], and in non-transgenic littermates [SOD1(-)/G93A(-)]. In parallel experiments, we also studied the release of [3H]GABA evoked by GLY and that of [3H]GLY evoked by GABA. Mutant mice were killed at advanced phase of pathology or during the pre-symptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice GLY evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]GLY, release. The GLY-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]GLY, were more pronounced in SOD1-G93A(+) than in SOD1(+) or SOD1(-)/G93A(-) mice. Furthermore, the excessive potentiation of [3H]d-ASP by GLY or GABA was already present in asymptomatic 30-40 day-old SOD1-G93A(+) mice. The releases of endogenous glutamate and GABA also were enhanced by GLY and the GLY-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in control animals. Potentiation of the spontaneous amino acid release is likely to be mediated by activation of a GLY or a GABA transporter, since the effect of GLY was counteracted by the GLY transporter blocker glycyldodecylamide but not by the GLY receptor antagonists strychnine and 5,7-dichlorokynurenate while the effect of GABA was diminished by the GABA transporter blocker SKF89976-A but not by the GABA receptor antagonists SR9531 and CGP52432. It is concluded that the glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

The expression of GABA in mossy fiber synaptosomes coincides with the seizure-induced expression of GABAergic transmission in the mossy fiber synapse

Although the granule cells of the dentate gyrus are glutamatergic, they contain the machinery for the synthesis and vesiculation of GABA. Furthermore, glutamic acid decarboxylase and the vesicular GABA transporter mRNA are expressed in the granule cells and mossy fibers in an activity-dependent manner, suggesting that these cells release GABA in addition to glutamate. Supporting this hypothesis, we found that seizures induce simultaneous glutamatergic and GABAergic transmission in the mossy fiber projection. To further explore this expression of inhibition, we looked for the presence and expression of endogenous GABA in a synaptosomal preparation enriched with mossy fiber nerve endings of kindled rats. We also studied the capacity of this preparation to capture and release [(3)H]GABA under control conditions and after kindling epilepsy. In accordance with our hypothesis we show that the mossy fiber synaptosomal preparation of the kindled rats has a significantly higher content of endogenous GABA than controls. We also found that the protein content in the mossy fiber synaptosomal preparation of kindled rats was significantly augmented, which is consistent with mossy fiber sprouting. Due to this, the total [(3)H]GABA incorporated in the synaptosomal preparation was also augmented. However, [(3)H]GABA uptake (expressed in % radioactivity/mg protein) and its evoked release were similar in both groups. With the present results, we provide further support for the hypothesis of the emergence of GABAergic transmission in the mossy fiber synapse that can constitute a protective mechanism in response to seizures.     

Lipid peroxidation and aluminium effects on the cholinergic system in nerve terminals

In the present study, we analyzed how aluminium and oxidative stress induced by ascorbate/Fe²⁺ affect the mechanisms related with the cholinergic system in a crude synaptosomal fraction isolated from rat brain. [³H]Choline uptake, [³H]acetylcholine release, membrane potential and Na⁺/K⁺-ATPase activity were determined in the presence or in the absence of aluminium in control conditions and in the presence of ascorbate (0.8 mM)/Fe²⁺ (2.5 μM). The extent of lipid peroxidation was measured by quantifying thiobarbituric acid reactive substances (TBARS). Under oxidizing conditions aluminium increased the formation of TBARS by about 30 %, but was without effect when the synaptosomal preparation was incubated in the absence of oxidants. Additionally, aluminium potentiated the inhibition of the high-affinity [³H]choline uptake observed following lipid peroxidation and had the same effect on the Na⁺/K⁺-ATPase activity. [³H]Acetylcholine release induced by 4-aminopyridine, and membrane potential were not significantly affected under oxidizing conditions, either in the absence or in the presence of aluminium. We can conclude that aluminium, by potentiating lipid peroxidation, affects the uptake of choline in nerve endings. This effect, occurring during brain oxidative injury, might contribute to the cholinergic dysfunction and neuronal cell degeneration known to occur in Alzheimer’s disease.     

Effect of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA

The effects of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA were examined in attempts to understand the mechanism of ascorbic acid attenuation of MPTP neurotoxicity. [3H]Dopamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of ascorbic acid. Ascorbic acid inhibited [3H]MPP+ uptake (IC50 = 0.1 mM) and [14C]GABA uptake (IC50 = 10 mM). Washout of ascorbic acid restored uptake of [3H]dopamine and [3H]MPP+, suggesting that ascorbic-acid-induced lipid peroxidation was not involved in the effect on uptake. In addition to the possible involvement of antioxidant mechanisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct effect of ascorbic acid in inhibiting the uptake of MPP+ into dopaminergic nerve terminals.     

Evidence for a hippocampal-septal glutamatergic pathway in the rat.

Kainic acid lesions of the hippocampal formation resulted in degeneration of axons and terminals in the lateral septal nuclei and in the nucleus accumbens. The degeneration of the hippocampal-septal projection was associated with a selective reduction in the synaptosomal sodium-dependent uptake of l-[³H]glutamate (?30 to 40%) and in the concentration of l-glutamate in the P₂ fraction (?20%). Presynaptic neurochemical markers for cholinergic, GABAergic, and noradrenergic neurons in the septal nuclei were not reduced by the hippocampal lesion. Transection of the hippocampal-septal projection at the fornix reduced the synaptosomal uptake of l-[³H]glutamate 40% but did not significantly reduce [³H]GABA uptake in the septal nuclei. The hippocampal-septal pathway, an excitatory projection, may thus be glutamatergic.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Heterogeneity of sodium-dependent excitatory amino acid uptake mechanisms in rat brain

The pharmacologic and kinetic characteristics of sodium-dependent uptake of [3H]L-glutamate, [3H]D-aspartate, and [3H]L-aspartate into crude synaptosomal preparations of rat corpus striatum and cerebellum have been examined in vitro. In cerebellum the apparent Kts and Vmax for the three excitatory amino acids were identical whereas in striatal synaptosomes, the Vmax for [3H]L-glutamate was 30% greater (P less than or equal to .001) than for [3H]D-aspartate and 50% greater (P less than or equal to .001) than for [3H]L-aspartate. L-Amino adipic acid inhibited the uptake of the three amino acids in both regions of brain was 15- to 20-fold more potent in cerebellum than in striatum. In contrast, dihydrokainic acid inhibited transport processes in the corpus striatum but was without activity in cerebellar preparations. The neurotoxin kainic acid blocked only a portion (60%) of [3H]L-glutamate and [3H]D-aspartate uptake in cerebellum while completely inhibiting amino acid transport in corpus striatum. Three days post kainic acid lesion, [3H]D-aspartate uptake was attenuated more than [3H]L-glutamate uptake in the corpus striatum; destruction of corticostriatal afferents reduced [3H]L-glutamate to a greater extent than [3H]D-aspartate. Various lesions of the cerebellum affected excitatory amino acid transport processes to a similar extent. These results suggest that excitatory amino acid transport systems are pharmacologically distinct in different brain regions and may be heterogeneous within a single region.     

Effects of zinc on markers of glutamate and aspartate neurotransmission in rat hippocampus

Receptor binding and synaptosomal uptake of L-[3H]glutamate and L-[3H]aspartate were measured in hippocampus derived from rats maintained on zinc restricted diets from weaning. Despite near lethal zinc deficiency, these markers of excitatory amino acid neurotransmission were unaffected compared to zinc-supplemented controls. However, addition of zinc in vitro markedly inhibited binding of glutamate and aspartate to hippocampal membranes. These data suggest that zinc can modulate the receptor affinities for glutamate and aspartate and may function as a tonic inhibitor of excitatory synapses in vivo.     

Histological and neurochemical effects of fetal treatment with methylazoxymethanol on rat neocortex in adulthood

Forebrain microencephaly results when developing rats are exposed to methylazoxymethanol acetate (MAM) at 15 days of gestation (DG). This potent alkylating agent is selectively cytotoxic for dividing cells. Since distinct neuronal populations in neocortex vary greatly with respect to timing of mitotic activity during gestation, it was predicted that some groups would be differentially reduced by treatment. Histological examination of neocortex from treated rats grown to adulthood revealed major losses of laminae II--IV with relative preservation of deeper layers. The atrophic adult neocortex was further characterized by assay of several defined pre- and postsynaptic neurochemical markers. Total markers for GABAergic neurons were greatly reduced (glutamate decarboxylase -71%, [3H]GABA synaptosomal uptake -63% and endogenous GABA -59%). Total [3H]GABA binding to cortical membranes was reduced 67%. Total [3H]glutamate synaptosomal uptake and endogenous glutamate were reduced 71% and 65% respectively. In contrast, total presynaptic markers for noradrenergic innervation were minimally altered but concentration of tyrosine hydroxylase, [3H]norepinephrine synaptosomal uptake and endogenous norepinephrine were increased by 275%, 130% and 133%, respectively. Concentration of cholinergic presynaptic markers was also increased (choline acetyltransferase +97%, endogenous acetylcholine +64%) in atrophic cortex, but to a lesser degree than for noradrenergic innervation. Specific binding of muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate and the beta-adrenergic receptor antagonist [3H]dihydroalprenolol was reduced 25% and 29% respectively in treated cortex. Thus, MAM treatment at 15 DG severely reduces intrinsic neuronal populations including GABAergic and glutamatergic neurons, and produces a shrunken cortex relatively hyperinnervated by noradrenergic and cholinergic neurons. MAM-induced microencephaly is a useful model system for producing relatively selective lesions of telencephalic neurons and for study of altered neurochemical relationships following developmentally incurred brain damage.     

An excitatory amino acid projection from rat prefrontal cortex to periaqueductal gray

High affinity D-[3H]aspartate and [3H]GABA uptake, and amino acid concentrations were examined in synaptosome-enriched preparations of rat periaqueductal gray matter 6-7 days following N-methyl-D-aspartate lesions confined to medial prefrontal cortex. Specific reductions were observed in the high affinity uptake of D-[3H]aspartate (78% of control, p less than 0.025), but not of [3H]GABA. Concentrations of glutamate, aspartate, GABA, glycine and alanine were not significantly reduced in lesioned animals. These results suggest the presence of glutamatergic and/or aspartatergic projections from medial prefrontal cortex to periaqueductal grey matter.     

Selective uptake of neuroactive amino acids by both oligodendrocytes and astrocytes in primary dissociated culture: a possible role for oligodendrocytes in neurotransmitter metabolism

CNS glia may be involved in the modulation of neuronal excitability through their capacity to accumulate and metabolize neuroactive amino acids. To investigate the possible role of oligodendrocytes in amino acid neurotransmitter metabolism, we have used light microscopic autoradiography, following the uptake of 3H-labelled amino acids by dissociated cultures of neonatal mouse brain, characterized immunocytochemically using cell-type specific markers. Oligodendrocytes, recognized by their characteristic galactocerebroside membrane staining, rapidly accumulated [3H] gamma-aminobutyric acid (GABA), becoming intensely labelled over cell body and processes after short incubations. In contrast, oligodendrocytes became only lightly labelled with [3H]L-glutamate and aspartate, which preferentially labelled astrocytes. [3H]D-aspartate, a non-metabolized analogue of L-glutamate, was avidly accumulated by oligodendrocytes, labelling cell bodies and processes after short incubations, to a similar extent as GABA. Thus, oligodendrocytes possess a transport mechanism for these excitatory amino acids, but rapidly metabolize them and release the metabolites. Not only the GC-positive cells but also the GC-negative undifferentiated oligodendrocyte precursors accumulated both GABA and D-aspartate, suggesting that this may be a function expressed early in the differentiation of oligodendrocytes. Net uptake of [3H] beta-alanine and [3H]glycine by oligodendrocytes was not observed under any conditions tested. A small number of oligodendrocytes were labelled with [3H]taurine after longer incubations. The uptake of certain neuroactive amino acids is thus a property shared by astrocytes and oligodendrocytes, the latter acting in a protective fashion around neuronal perikarya and axons.     

Biochemical development of the human brain. III. Benzodiazepine receptors, free gamma-aminobutyrate (GABA) and other amino acids

Benzodiazepine receptor binding and the concentration of the free tissue GABA, glutamate, glutamine, aspartate, and 13 other amino acids were estimated in specimens of cerebral cortex and cerebellum taken from normal foetuses and from perinatal and postnatal infants and adults postmortem. Compared with the ontogenetic increase, previously reported, in [3H]muscimol binding, that of [3H]flunitrazepam binding developed more rapidly in the cerebellum and less rapidly in the cerebral cortex. The concentration of GABA in both brain parts increased with age more rapidly than did that of glutamate decarboxylase activity. The overall pattern of developmental changes in the amino acid concentrations in the human brain showed many similarities with those reported in the brains of experimental animals. The concentration of the amino acids associated with the tricarboxylic acid cycle increased with age. Although the general downward trend in the levels of essential amino acids (with the exception of Met) was also evident in the human cerebral cortex, this was not the case in the cerebellum where the concentrations of most amino acids were relatively low in the foetus whereas the concentrations exceeded the cerebral cortical levels in the adult.