Brain Cortex Slices Research Articles

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations

The possible excitatory effect of N-acetyl-α-aspartylglutamate (NAAG) was studied in 3 different systems. First on the increase in 45Ca 2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM l-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [ 3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of l-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 μg) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

Effects of mianserin on noradrenergic mechanisms

Mianserin increased stimulation-induced release of noradrenaline from guinea-pig atria and brain cortex slices. The increase still occured when neuronal reuptake of noradrenaline was blocked by cocaine. Mianserin blocked the neuronal uptake of 3H-noradrenaline only in high concentrations (1 μ M in atria produced 40% block; 10 μ M in cortex produced 57% block). Lower concentrations (0.01 μ M in atria; 1 μ M in cortex) significantly increased stimulation-induced release of noradrenaline. It is concluded that increases in transmitter release from noradrenergic nerves produced by mianserin can be attributed to blockade of the α 2-adrenoceptors involved in autoinhibitory feedback mechanism at nerve terminals, rather than to blockade of neuronal reuptake of noradrenaline. This effect on noradrenergic transmission in the central nervous system could explain the antidepressant actions of mainserin in accordance with the hypothesis of Schildkraut.

Effects of DU 24565 (6-nitroquipazine) on serotoninergic and noradrenergic neurones of the rat brain and comparison with the effects of quipazine

Rat brain cortex slices were prepared in order to study the influence of DU 24565 (6-nitroquipazine) and quipazine on the accumulation of tritium, and to investigate the effects of DU 24565 on the electrically evoked (3 Hz) tritium overflow from slices preincubated with 3H-serotonin (3H-5-HT) or 3H-noradrenaline (3H-NA). The influence of DU 24565 and quipazine on the activity of MAO was studied in rat brain homogenates, using labelled serotonin as a substrate. In cortex slices preincubated with either 3H-5-HT or 3H-NA, DU 24565 increased the electrically evoked 3H overflow (higher potency in slices preincubated with 3H-5-HT; the percentage of 3H accounted for by unmetabolized 3H-5-HT was also increased). The facilitatory effects of DU 24565 on evoked 3H overflow were abolished when the respective monoamine uptake mechanisms had previously been blocked by citalopram and cocaine, respectively. The inhibitory effects of unlabelled 5-HT and NA on the evoked 3H overflow from cortex slices preincubated with 3H-5-HT were not altered by DU 24565. Similarly, the drug did not affect the inhibition by NA of the evoked 3H overflow from cortex slices preincubated with 3H-NA. Quipazine was a weak and non-selective inhibitor of the accumulation of 3H-5-HT and 3H-NA. DU 24565 was about 65 times more potent and about 1100 times more selective than quipazine as an inhibitor of 3H-5-HT accumulation, and it was also superior to citalopram and paroxetine in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

O-136) - Effects of ketone body on free amino acids in rat brain cortex slices

O-136) - Effects of ketone body on free amino acids in rat brain cortex slices

Evidence for common pharmacological properties of [3H]5-hydroxytryptamine binding sites, presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves.

The affinities of 16 5-hydroxytryptamine (5-HT) receptor agonists (indole derivatives) and 7 5-HT receptor antagonists for [3H]5-hydroxytryptamine [( 3H]5-HT) binding sites in rat cerebral cortex membranes were determined. In addition, the potencies of the agonists for inhibiting the electrically induced tritium overflow from rat brain cortex slices preincubated with [3H]5-HT and from canine saphenous veins preincubated with [3H]noradrenaline were measured. Furthermore, the potencies of the indole derivatives for inducing contractile responses of canine saphenous veins were recorded. In addition, the interaction of the antagonists with unlabelled 5-HT at the 5-HT autoreceptor was studied in rat brain cortex slices. There was a good correlation between the binding affinities of the indole derivatives for the [3H]5-HT sites of rat brain cortex membranes and their potencies for inhibiting the evoked tritium overflow from both rat brain cortex slices and strips of canine saphenous vein. Comparison of the inhibition constants derived from the overflow experiments in both tissues again revealed a high correlation coefficient while there was only weak correlation between the binding affinities in rat brain cortex and the contractile potencies of the drugs in canine saphenous vein strips. When 5-HT receptor antagonists were investigated, metitepin and metergoline showed moderate affinities for the 5-HT autoreceptors in rat brain cortex slices, whereas quipazine had only weak affinity, and ketanserin, metoclopramide, cinanserin and cyproheptadine exhibited no antagonistic property. In binding experiments, the competition curves of most 5-HT receptor antagonists were biphasic, suggesting that the [3H]5-HT binding sites are heterogeneous.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between transmitter uptake inhibition and effects of alpha-adrenoceptor agonists on serotonin and noradrenaline release in the rat brain cortex.

Rat brain cortex slices preincubated with 3H-serotonin or 3H-noradrenaline were superfused, and tritium overflow was evoked electrically at a frequency of 3 Hz. The accumulation of tritium in slices incubated with 3H-serotonin or 3H-noradrenaline in the presence of pargyline was also studied. In conclusion, cocaine, DU 24565 and maprotiline attenuated the clonidine-induced (α2-adrenoreceptor-mediated) inhibition of monoamine release only in those neurones in which they inhibited the transmitter uptake mechanism. Therefore, it is improbable that the attenuation is due to the interaction of the compounds with a recognition site for imidazolines (such an effect should be independent of the selectivity of uptake inhibition); it is more probably related to the blockade of neuronal uptake itself which also occurs at hypothermia and which may lead to the deprivation of an intraneuronal metabolic link between uptake mechanism and α-adrenoceptors. The reduction of the inhibitory effect of clonidine on noradrenaline release may, in addition, be caused by the α2-adrenoreceptor blocking property of clonidine which comes into play at increased concentration of released noradrenaline in the biophase of the autoreceptors during blockade of neuronal uptake.

Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine

Rat brain cortex slices preincubated with 3H-5-hydroxytryptamine ( 3H-5-HT) were superfused with physiological salt solution containing paroxetine, an inhibitor of 5-hydroxytryptamine (5-HT) uptake. The effects of various indolethylamines on the electrically evoked tritium overflow (containing 66.3% unmetabolized 3H-5-HT) were investigated (the percentage of unmetabolized 3H-5-HT was not altered by the indolethylamines or metitepin). 6,7-Dihydroxytryptamine (6,7-DHT) did not affect the stimulation-evoked tritium overflow, whereas the latter was inhibited by the other tryptamine derivatives investigated; when the compounds were compared to each other on the basis of their inhibitory potencies the following rank order was obtained: unlabelled 5-HT > 5-methoxytryptamine > 4-HT > 6-HT > 5,6-DHT > tryptamine > 7-HT > 5,7-DHT. The inhibitory effects of these compounds were antagonized by metitepin. It is concluded that the indolethylamines inhibit the stimulation-evoked 3H-5-HT release by activating the presynaptic 5-HT autoreceptors on the 5-HT neurones of the rat brain cortex. Similarities may exist between these receptors and the postsynaptic 5-HT l binding sites of this brain area.

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro

Kainate (0.62–5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl- dl-aspartate (50–1000 μM), l-glutamate (0.62–5 mM), dl-homocysteate (0.62–2.5 mM), and ibotenate (6–170 μM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino N-acetyl- l-aspartate, and α-methy- dl-aspartate (both 1.25–5 mM), did not increase such influx. We suggest that enhanced calcium uptaje may represent the basis for the neurotoxic effects of these compounds.

Effects of alpha-adrenoceptor antagonists on the release of serotonin and noradrenaline from rat brain cortex slices. Influence of noradrenaline uptake inhibition and determination of pA2 values.

Rat brain cortex slices preincubated with3H-noradrenaline or3H-serotonin were superfused with physiological salt solution. The effects of α-adrenoceptor antagonists (phentolamine, BDF 6143, BE 2254 and rauwolscine) on the electrically evoked3H overflow and their interactions with clonidine or noradrenaline were studied. The effects of these antagonists on tritium accumulation by slices incubated with either3H-monoamine were also examined. In conclusion, when neuronal uptake was inhibited, all α-adrenoceptor antagonists investigated increased, i.e. disinhibited, the evoked3H overflow from slices labelled with3H-noradrenaline, whereas there were differences between the compounds, when neuronal noradrenaline uptake was operative; these differences were not related to their imidazoline, phenylethylamine or indolealkylamine structure or their own ability to inhibit the neuronal uptake of noradrenaline. The apparent pA2 values of the α-adrenoceptor antagonists were independent of the chemical structure of the agonists against which these values were determined (derivatives of imidazoline or phenylethylamine). The α-adrenoceptor on the serotoninergic nerve terminals of the cortex slices do not appear to be activated by endogenous noradrenaline released from neighbouring noradrenergic fibres.

A search for endogenous modulators of the GABA receptor in different regions of the rat CNS

The possible existence of endogenous substances other than γ-aminobutyric acid (GABA), that can also bind to rat brain GABA receptors, has been investigated in synaptic membranes derived from whole rat brain, or from cerebral cortex; as well as in isolated synaptic vesicles obtained from cerebral cortex, striatum, hypothalamus, cerebellum and spinal cord and in the superfusion fluid of electrically stimulated brain cortex slices, where a GABA-like substance is released by a calcium-dependent process. The detector used to study the presence of such presumed non-GABA endogenous ligands, were frozen and thawed rat brain synaptic membranes, that had been treated with 0.05% Triton X-100 and thoroughly washed. With this highly sensitive preparation, at least 5 pmol of GABA/ml could be detected. The extracts of the different preparations where these hypothetical ligands were looked for, were analyzed by means of gel filtration on Sephadez G-10, paper chromatography and high voltage electrophoresis. In a very great number of experiments performed, the only endogenous ligand detected was GABA itself. The possible influence of a number of peptides on binding of GABA to its receptor, was also looked for. No significant effect was found for substance P, neurotensin, cholecystokinin octapeptide sulfated, somatostatin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, methionine enkephalin (all 10 −5 M), angiotensin II (10 −4 M), ACTH (3 × 10 −7 M), poly- l-lysine (30 μg/ml) or poly- l-glutamate (30 μg/ml).

Glutamate uptake into cerebral cortex slices is reduced in the presence of a gamma-glutamyl transpeptidase inhibitor.

The uptake of L-glutamic acid into brain cortex slices of mice of different age was studied. The results suggest that GGT contributes to the transport of L-glutamic acid into the brain.

Effects of sodium valproate and acetazolamide on cerebral respiration

The effects of sodium valproate and acetazolamide on the oxygen uptake of guinea pig brain cortex slices were investigated. In calcium-free medium, sodium valproate inhibited the oxygen uptake appreciably in the presence of glucose and glutamic acid. Acetazolamide, on the other hand, was more effective in inhibiting oxygen uptake in the presence of glucose than in the presence of glutamic acid. Addition of 0.2 mM CaCl 2 in the medium containing 5 mM KCl could appreciably reverse the inhibition of oxygen uptake by acetazolamide in the presence of glucose. The inhibition of oxygen uptake by these drugs in the presence of glucose, however, could be completely reversed by increasing the dose of K + ions (100 mM) in the medium which had no effect on the inhibition of oxygen uptake in the presence of glutamic acid. When the concentration of Ca 2+ ions in the medium was elevated to 0.75 mM, the inhibitory effects of these drugs on the oxygen uptake in the presence of both glucose and glutamic acid could be completely abolished. Sodium valproate also inhibited the endogenous respiration of guinea pig brain cortex slices, whereas acetazolamide was almost without any effect. Increase in the concentration of Ca 2+ ions in the medium failed to counteract the inhibition of endogenous respiraton of guinea pig brain cortex slices by sodium valproate.

Alpha-Adrenoceptor-mediated inhibition of noradrenaline release in rabbit brain cortex slices. Receptor properties and role of the biophase concentration of noradrenaline.

Brain cortex slices from rabbits were preincubated with [3H]noradrenaline and then superfused and stimulated electrically at 3Hz. In the presence of cocaine 30 microM, unlabelled noradrenaline, alpha-methylnoradrenaline, clonidine, oxymetazoline, xylazine and guanabenz decreased, whereas yohimbine, corynanthine, phentolamine, tolazoline and azapetine increased the stimulation-evoked overflow of tritium. Phenylephrine and prazosin had no effect on the evoked overflow except at concentrations that greatly accelerated the basal outflow of tritium. The results indicate that the noradrenergic axons of rabbit brain cortex are endowed with presynaptic alpha-adrenoceptors which are exclusively of the alpha 2-type. Addition of various concentrations of cocaine, addition of pargyline, or stimulation at different current strengths was used to obtain either a high or low stimulation-evoked overflow of tritium. Independently of the method used, a low evoked overflow coincided with a large percentage inhibition produced by 0.1 micro M clonidine, whereas a high evoked overflow coincided with a smaller percentage inhibition produced by clonidine. The results indicate that drugs which block the re-uptake of noradrenaline diminish the presynaptic inhibitory effect of alpha-adrenergic agonists by increasing the biophase concentration of released noradrenaline.

Effects of tityustoxin (TsTX) from scorpion venom on the release and synthesis of acetylcholine in brain slices

Tityustoxin (TsTX) increased the release and synthesis of acetylcholine (ACh) in slices of rat brain hippocampus, hypothalamus, thalamus, cortex and striatum. The effect was highest in slices of hippocampus and cortex and smallest in hypothalamus. These effects of TsTX were dependent on the presence of Na + and Ca 2+ in the incubation medium. EGTA, 0.1 mM, blocked the effect of TsTX in all areas, except for the hippocampus and frontal striatum, where a concentration of 10mM was required. Tetrodotoxin blocked the increase in the release and synthesis of ACh induced by tityustoxin.

Acute or chronic changes of noradrenergic transmission do not affect the alpha-adrenoceptor-mediated inhibition of 3H-serotonin release in the cerebral cortex.

Rat brain cortex slices preloaded with either 3H-serotonin or 3H-noradrenaline were superfused with physiological salt solution, and tritium overflow was evoked by electrical field stimulation. It is concluded that guanethidine inhibits not only noradrenaline but also serotonin release from the corresponding neurones of the cerebral cortex and that the selectivity for serotonin uptake. Acute or chronic impairment of noradrenaline release by guanethidine and 6-OH-DA, respectively, or long-term inhibition of noradrenaline uptake by desipramine does not affect the α-adrenoceptor-mediated inhibition of serotonin release.

Disturbances of amino acid transport in rats with experimental hyperphenylalaninaemia.

AbstractLiver and brain cortex slices from rats made hyperphenylalaninaemic withp‐chlorophenylalanine showed decreased leucine uptake but increased uptake of tryptophan and glycine.

Antagonistic properties of quipazine at presynaptic serotonin receptors and alpha-adrenoceptors in rat brain cortex slices.

Rat brain cortex slices preincubated with 3H-serotonin or 3H-noradrenaline were superfused with physiological salt solution, and the effect of quipazine on the 3H overflow evoked by electrical field stimulation was examined. 1. The electrically evoked tritium overflow from brain slices preloaded with 3H-serotonin was increased by quipazine in a concentration-dependent manner. The concentration-response curves of unlabelled serotonin and noradrenaline for their inhibitory effects on impulse-evoked 3H overflow were shifted to the right by quipazine. 2. In brain slices preincubated with tritiated noradrenaline, quipazine caused an increase in electrically stimulated 3H overflow which was less marked than the effect on brain slices preloaded with 3H-serotonin. The concentration-response curve of unlabelled noradrenaline for its decreasing effect on stimulation-evoked 3H overflow was shifted to the right by quipazine. We conclude that quipazine blocks presynaptic inhibitory serotonin receptors and alpha-adrenoceptors on monoaminergic neurones of the rat brain cortex.

Beta-Hydroxybutyrate as a precursor to the acetyl moiety of acetylcholine.

Rat brain cortex slices were incubated with 10 mM-glucose and trace amounts of [6-3H]glucose and [3-14C]beta-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of beta-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H:14C ratios calculated. Incorporation of [14C]beta-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [3H-,14C]-glucose. (-)-Hydroxycitrate (5.0 mM) reduced incorporation of [3H]glucose and [14C]beta-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mM) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mM) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mM, acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats beta-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.

Characterization of the receptor subtype involved in alpha-adrenoceptor-mediated modulation of serotonin release from rat brain cortex slices.

Rat brain cortex slices preincubated with 3H-5-hydroxytryptamine and superfused with physiological salt solution were stimulated electrically at a frequency of 3 Hz. 1. The electrically evoked 3H-overflow was decreased by clonidine, noradrenaline and B-HT 920 in a concentration-dependent manner (negative logarithms of the IC30 values: 6.66, 6.55 and 4.40, respectively) 2. Phenylephrine 10-5 M (which increased basal 3H-efflux) and methoxamine 10-4 M decreased the impulse-evoked 3H-overflow by less than 25% whereas lower concentrations were ineffective. 3. Yohimbine produced a shift to the right of the concentration-response curves of noradrenaline (apparent pA2: 6.93) and clonidine (apparent pA2: 7.06) for their inhibitory effects on evoked 3H-overflow. Rauwolscine also shifted the concentration-response curve of noradrenaline to the right (apparent pA2: 7.29), whereas prazosin (10-6 and 3.2 x 10-6 M) was ineffective in this respect. These results suggest that the alpha-adrenoceptors on the serotoninergic nerve fibres belong to the alpha2-subtype.

Electrically induced release of ( 3H)-noradrenaline from rat brain cortex slices: A kinetic analysis of the dependence on extracellular calcium

Summary Release of ( 3 H)-noradrenaline, induced by electrical stimulation (sine-wave 60 Hz, 0.5 or 1 Volt applied for 10 seconds) of superfused rat brain cortex slices, shows a saturation (Michaelis-Menten) kinetics with respect to extracellular calcium. The maximal change attained in ( 3 H)-noradrenaline efflux fractional rate constant (min −1 ) was for 0.5 Volt stimuli: 0.016 ± 0.0007, and for those of 1 Volt: 0.0404 ± 0.005 (P