Characterization of the N-methyl-D-aspartate (NMDA) receptor in the embryonic chick brain.

Relatively little is known about the properties of the NMDA receptor in avian species. In the present study, NMDA receptor pharmacology and function has been studied in the embryonic chick brain. The competitive antagonist ligand [3H]CGP 39653 bound to telencephali membranes from 17-day-old chick embryos with KD and Bmax values of 6.6 nM and 3.3 pmol/mg protein, respectively. The binding was inhibited by CGS 19755, L-glutamate, R-CPP and D-AP5 with pI50 values of 7.57, 7.49, 7.28 and 7.08, respectively. L-glycine, S-AMPA, kainate and MK-801 only weakly affected the binding. The inhibitory potency of NMDA (pI50 value 6.98), however, was greater than seen in other species, suggesting a species difference in receptor structure. Such a possibility was investigated by use of two NMDA-R1 antibodies, AB 1516 and 69921 A, which recognize the C-terminal region and an intracellular domain of the rat receptor, respectively. Immunoreactive signals (approximately 116 kDa) were found for a rat brain homogenate, whereas only 69921A gave an immunoreactive signal in the chick. The functional status of the NMDA receptors was investigated in serum-free cultures of neurons from 8-day-old chick embryos. Glutamate and NMDA were found concentration-dependently, and in a manner that could be antagonized by CGS 19755 and MK-801, to produce cell death. Thus, the present study indicates that chick embryonic brain expresses functional NMDA receptors that are pharmacologically and possibly structurally different from their rodent counterparts.

Correlation between cortical EEG and striatal microdialysis in soman-intoxicated rats.

In vivo microdialysis and EEG recording have been used in order to study the combined neurochemical and electrophysiological events during intoxication with soman (o-1,2,2-trimethylpropyl methylphosphono-fluoridate), a potent inhibitor of acetylcholinesterase (AChE), in the freely moving rat. All rats exposed to soman exhibited signs of AChE inhibition. The duration of EEG recorded seizures after soman intoxication averaged 43 +/- 24 min. The extracellular striatal levels of dopamine and GABA, increased significantly during the EEG seizure periods. Using an EEG based differentiation between seizure and non-seizure conditions, we found that intrastriatal release of dopamine, but not glutamate, during soman intoxication is highly correlated with seizures. Our results suggest that excitatory amino acids (EAA) involvement in soman-induced seizures, as demonstrated in hippocampus, may not be relevant in the striatum. Our data, instead, may indicate the importance of dopamine as a neurotoxic agent.

Expression pattern of glutamate decarboxylase (GAD) in the developing cortex of the embryonic chick brain.

The development of the GABAergic system in the chick embryo telencephalon has been studied. Special emphasis was placed on the development of glutamate decarboxylase (GAD) between embryonic day 8 (E8) and E17. The GABA immunoreactivity and neuron-specific enolase expression was detected simultaneously in glutardialdehyde fixed sections, which confirmed that GABAergic cells exhibit neuronal phenotype. The GAD expression was studied by means of immunohistochemistry on cryo-sectioned material both at the light and electron microscopic levels. Furthermore, the presence and localization of GAD65 and GAD67 mRNAs were studied with an in situ hybridization technique with digoxigenin-labeled RNA probes. Protein expression as well as mRNA appearance mostly coincided both temporally and spatially. In the parahippocampal area, as well as in other regions of the developing cortex, GAD staining was seen from E8 onwards. The number of positive cells increased as did the intensity of staining up to E14. As observed in the electron microscope, the GAD protein was co-localized with GABA in most cases, although some GAD-positive cells devoid of GABA-staining also were observed. The pattern of GAD mRNA expression was in general similar to that of GAD immunostaining. Both GAD65 and GAD67 mRNA were detected during the entire period. Furthermore, GAD67 mRNA localization spatially was more correlated with GAD protein expression. The study provides evidence for the notion that development of the GABAergic system occurs rapidly during embryogenesis and, as suggested from mRNA data, that two forms of GAD with slight difference in distribution can contribute to this.

Release of dopamine, GABA and EAA in rats during intrastriatal perfusion with kainic acid, NMDA and soman: a comparative microdialysis study.

There is an increasing amount of experimental evidence that excitatory amino acids (EAAs) are involved in the brain lesions observed after severe intoxication with the highly toxic organophosphorus compound soman. This study was undertaken to compare the acute actions of soman, and the glutamatergic receptor agonists kainic acid and N-methyl-D-aspartate (NMDA) on striatal release of dopamine and amino acids. The neurotoxic compounds were administered in high (10 mM) concentrations by unilateral intrastriatal microdialysis perfusion in freely moving rats. During the microdialysis the animals were observed for toxic signs related to convulsion. The glial fibrillary acidic protein (GFAP) was monitored as a marker of neurotoxicity in parts of prefrontal cortex, hippocampus, striatum and cerebellum. Acetylcholinesterase (AChE) inhibition in six brain regions was measured after soman perfusion in order to assess its cerebral distribution. We found that soman perfusion induced a major release of dopamine, GABA and aspartate in the striatum. Kainic acid also induced a release of dopamine and aspartate. NMDA was not as potent an inducer of striatal neurotransmitter release as soman and kainic acid. Soman and kainic acid perfusion produced convulsive behaviour in the rats. The main neurochemical event in the striatum during soman- and kainate-induced convulsions is the release of dopamine. We suggest that this major dopamine release might be as important as an increase in EAA in the cascade of pathological events leading to the brain damage in the striatum observed after soman intoxication.

Differences in the release of L-glutamate and D-aspartate from primary neuronal chick cultures.

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release of D-[3H]aspartate and L-[3H]glutamate. The D[3H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependent L-[3H]glutamate release was calcium dependent, and furthermore L-[3H]glutamate release was optimal at potassium concentrations < 30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1 -aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux of D-[3H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulated L-glutamate release. We believe that L-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture. D-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.

Morphological and GABA-immunoreactive development of the embryonic chick telencephalon.

The development of neurons utilizing gamma-aminobutyric acid (GABAergic neurons) in prosencephalon and telencephalon from chicken embryonic days 4-14 (E4-E14) was studied by means of immunohistochemistry. Furthermore, routine histology and transmission electron microscopy. respectively, were performed in order to study the morphological development in the designated area. The main finding is that development of GABAergic neurons in the chick telencephalon is rapid; the GABA neurons are appearing in bulk at day 8, being "overexpressed" at days 10-11, decreasing in numbers thereafter and achieving mature morphology on day 14, which is considerably faster than in the rodent. Morphological analysis revealed that the prosencephalon mainly consisted of a thin layer of undifferentiated neuroblasts in the E4 embryo. By E6, the prosencephalon had increased in thickness and occasional cells outside the neuroepithelium showed a more mature morphology with a few cells weakly staining positive for GABA. At E8, the prospective granular and subventricular layers had developed. At E14, the appearance of the telencephalon is approximating that of the adult since both ependymal cells and morphologically mature neurons can be seen.

Inhibition of SKF 89976-A of the gamma-aminobutyric acid release from primary neuronal chick cultures.

Neuronal cultures were made from the 8-d-old embryonic chick telencephalon. The primary culture model was further improved, the medium composition was modified, and the cells grown for 10 d, which allowed the development of relatively differentiated neurones. A superfusion protocol was developed and applied to study the release of [3H]-gamma-aminobutyric acid ([3H]GABA). High endogenous activity levels of glutamate decarboxylase (GAD) and of a Ca-dependent potassium stimulated [3H]GABA release were used as criteria for GABAergic differentiation. The influence of the non-substrate inhibitor of GABA transport, SKF 89976-A, on the GABA release, was studied using the primary neuronal culture. The release was found to be inhibited by SKF 89976-A at higher concentrations (= 400 microM).

Net taurine transport and its inhibition by a taurine antagonist.

P2-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [3H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using 86Rb+ as a marker for intracellular K+. Taurine was synthesized in the P2-fraction when incubated in taurine free medium. At external taurine concentrations below 370 microM a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P2-fraction was achieved at external taurine concentrations exceeding 370 microM. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [3H]taurine transport into the P2-fraction. As the external concentration of taurine was increased, the accumulation of 86Rb+ into the P2-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.

The protective effect of nimodipine, a calcium antagonist, and its influence on soman clearance in the anaesthetized rabbit.

The effect of pretreatment with a vasoactive compound, nimodipine, on soman intoxication in peripheral organs of rabbits was studied by measuring changes in the cholinesterase and acetylcholinesterase activity and by measuring clearance of soman in blood using gas chromatography/high resolution mass spectrometry. In animals receiving soman only, initial blood concentrations were approximately 100 ng mL-1 and were still detectable after 5 min. The clearance rate of soman in blood markedly increased following nimodipine pretreatment such that soman was below the detection limit (0.002-0.003 ng mL-1) in all samples. Soman injection caused a significant inhibition of the acetylcholinesterase activity in serum, and in brain. In rabbits pretreated with nimodipine, no significant inhibition of acetylcholinesterase activity occurred after soman injection. In view of the effects of nimodipine on soman clearance and on the acetylcholinesterase and cholinesterase inhibition during soman intoxication, we suggest that nimodipine has profound circulatory effects, which during soman intoxication, increase the vascular perfusion through the body and thereby increase the detoxifying capacity.

Inhibition of transporter mediated gamma-aminobutyric acid (GABA) release by SKF 89976-A, a GABA uptake inhibitor, studied in a primary neuronal culture from chicken.

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the gamma-aminobutyric acid (GABA) transporter, on the release of radioactive GABA and D-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [3H] D-aspartate and [14C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [3H] D-aspartate and [14C] GABA was increased by [K+] and time, and a minimum value was obtained at [Ca2+] 1.05 mM. The release of both [3H] D-aspartate and [14C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.

On the activity of gamma-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes.

The uptake of radioactive gamma-aminobutyric acid (GABA) and D-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P2-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [3H]GABA and for D-[3H]aspartate with an increasing Vmax during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P2-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake of D-aspartate. We found no non-saturable uptake of D-aspartate.

On the inhibition of glutamic acid decarboxylase and gamma-aminobutyric acid transaminase by sodium cyanide.

The effects of sodium cyanide (NaCN) on the gamma-aminobutyric acid metabolizing enzymes glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid transaminase (GABA-T) were studied in vitro. With no pyridoxal-5-phosphate added, GAD was non-competitively inhibited by NaCN, with an IC50 of 280 microM. GAD was also inhibited when exposed to an equimolar amount of NaCN and pyridoxal-5-phosphate. NaCN inhibited GABA-T. The inhibition kinetics suggests that NaCN may react with more than one of the substrates and products present during the reaction, i.e. pyridoxal-5-phosphate, alpha-ketoglutarate and/or succinic semialdehyde. The presence of pyridoxal-5-phosphate in the reaction mixture completely protected GABA-T from inhibition by NaCN. The gamma-aminobutyric acid synthesizing enzyme, GAD may thus be inhibited in vivo by NaCN or by a reaction product of NaCN and pyridoxal-5-phosphate. The gamma-aminobutyric acid catabolizing enzyme, GABA-T is not as vulnerable to inhibition by NaCN, since the cyanide-pyridoxal-5-phosphate complex is ineffective as inhibitor.

On the inhibition of GABA release from a preparation of rat brain nerve-endings by SKF 89976-A, a GABA uptake inhibitor.

Fractions enriched in nerve-terminals were made in order to investigate the release of gamma-aminobutyric acid (GABA). A P2-preparation made from whole rat brain was pre-labeled with [3H]GABA and thereafter incubated in high concentrations (1 mM) of the GABA uptake inhibitor SKF 89976-A. The nerve-terminals were thereafter incubated in depolarizing concentrations of KCl and finally the medium and nerve-terminals separated by a filtration procedure. The amount of [3H]GABA present on the filter and in the supernatant was measured by liquid scintillation counting and used to assess the release rate. SKF 89976-A reduced the KCl-induced efflux of [3H]GABA from the nerve-terminals. The inhibition was as effective against KCl-induced [3H]GABA efflux in the presence as well as in the absence of Ca2+. Our finding may demonstrate an inhibition of the inside-out activity of the GABA transporter.

Development of cholinergic markers in the neuroblastoma, N1E-115.

The neuroblastoma, N1E-115, was grown for 9 days after subcultivation. The development of acetylcholinestrase (AChE), choline acetyltransferase (CAT), QNB-binding, choline uptake, and acetylcholine release was measured on days 1, 3, 6, and 9. In parallel experiments the irreversible AChE inhibitor soman was added to neuroblastoma on day 1 and the development of the above parameters except for release, was followed. AChE activity in the normal cells was found to develop also after confluency, whereas CAT activity and QNB-binding followed the development of most of the cellular proteins, i.e., ceased to develop at confluency. Both choline uptake and acetylcholine release appeared independent of cellular development. In the soman-treated cells the development of AChE was inhibited for up to 6 days and thereafter developed with the same rate as in the normal cells. CAT and QNB-binding developed as in the normal cells, but at a significantly reduced level. The development of choline uptake was not significantly different in soman-treated and normal neuroblastoma. It is concluded that the development of the cholinergic marker QNB-binding is intimately associated with that of the "presynaptic" marker CAT, whereas the development of AChE seems to be unrelated to these cholinergic parameters. The choline transport and the acetylcholine release seem to be equally well expressed on all the days studied.

On the use of diazepam and pro-diazepam (2-benzoyl-4-chloro-N-methyl-N-lysylglycin anilide), as adjunct antidotes in the treatment of organophosphorus intoxication in the guinea-pig.

Diazepam and pro-diazepam (2-benzoyl-4-chloro-N-methyl-N-lysylglycin anilide) have been used as adjunct antidotes to pyridostigmine and atropine against the organophosphate, soman, in the guinea-pig. Both added significant protection to the pyridostigmine/atropine treatment. Animals pretreated with diazepam, 60 min before soman, were "better" protected than animals given an equimolar dose of pro-diazepam therapeutically 1 min after soman. A pretreatment with diazepam for three days further increased the protection. A therapeutic dose of pro-diazepam, 1 min after soman, gave no further protection, to the three day diazepam pretreatment. The serum concentrations of diazepam (given i.p.) and desmethyldiazepam (given i.m.) were determined by GLC after diazepam (i.p.) and pro-diazepam (i.m.) were given. The protection, relative to the control, provided by the diazepam pretreatment (60 min before and for three days before soman) correlated linearly, r = 0.9898, with the serum values of diazepam achieved at these times. Our data suggest that diazepam as adjunct to pyridostigmine and atropine administered as pretreatment gives a "safer" protection, than an equimolar dose of pro-diazepam given therapeutically.

Depolarization of the neuronal membrane caused by cotransport of taurine and sodium.

C 1300 neuroblastoma cells were cultured and used to study the effect of sodium dependent taurine transport on the membrane potential. Measuring net accumulation of taurine and the depolarization caused by externally applied taurine, we found both processes become active at an external concentration of taurine of 1 mM or more. Net accumulation had Km of 13 mM and a Vmax of 126 nmol x mg of protein-1 x min-1. The taurine induced depolarization of the neuroblastoma cell was parallelled by a 25 per cent decrease in its membrane impedance. The transport of taurine, the depolarization caused by taurine and the effect of taurine on the membrane impedance, all, had a similar dependence on the external sodium concentration. Our results on the depolarizing cotransport between taurine and sodium at the neuronal membrane, may illustrate an additional mechanism for the control of the electrical activity of neuronal cells.

Is the concentration of gamma-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of gamma-aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [3H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K+ and Ca2+ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [3H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca2+, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.

On the composition and characteristics of microvessels isolated from the rabbit and bovine brain.

Brain capillaries (microvessels) were isolated from the rabbit and bovine brain. Extensive morphological examinations were performed at the light and electron microscopical levels. The relative contribution of endothelium (52%), basal membrane (32%) and pericytes (16%) to the composition of the microvessel was assessed. The ability of the endothelium from bovine brains to maintain a membrane potential, i.e. to accumulate the lipophilic cation [3H]TPMP, was shown. The transmitter catabolizing enzymes MAO and AchE were shown to be, and COMT and GABA-T not to be associated with the microvessel fraction isolated from rabbits.

Calcium-independent release of gamma-aminobutyrate from nerve processes in the developing rabbit retina.

Retinas from 3- and 10-day-old rabbits, and from young (29 days), or adult animals were used to study in parallel the development of synaptic vesicles in amacrine cells and the Ca2+ dependence of the K+-stimulated [3H]gamma-aminobutyrate release from them. Few synaptic vesicles were observed in the amacrine cell processes in retinas from the 3-day-old rabbits. The number of vesicles significantly increased between 3 and 10 days and increased further between day 10 and the adult animal. The Ca2+ dependence of the K+-stimulated release decreased with increasing age. There is thus a poor correlation between the Ca2+ dependent transmitter release and the number of synaptic vesicles in the nerve terminal, favouring the existence of a Ca2+ dependent nonvesicular process for the [3H]gamma-aminobutyrate release in the rabbit retina.