Brain Cortex Slices Research Articles

Extracellular 5-hydroxytryptamine inhibits 5-hydroxytryptamine release from rat brain cortex slices.

Rat brain cortex slices preincubated with 3H-5-hydroxytryptamine were superfused with physiological salt solution and stimulated electrically, or they were superfused with Ca2+-free solution containing 25 mM K+ and stimulated by introduction of 1.3 mM CaCl2 for 2 min. After blockade of neuronal 5-hydroxy-tryptamine (5-HT) uptake with clomipramine or paroxetine, the 3H overflow evoked by both methods of stimulation was decreased by unlabelled 5-HT and increased by methiothepin. The inhibition caused by 5-HT was antagonized by simultaneous administration of methiothepin. The inhibition by 5-HT of Ca2+-induced overflow was also observed in the presence of tetrodotoxin. These results suggest that 5-HT regulates its own release from central serotoninergic neurones by activating presynaptic 5-HT autoreceptors, thus decreasing the availability of Ca2+ for stimulus-release coupling.

Effects of anoxia and depolarization on the movement of carbon atoms derived from glucose into macromolecular fractions in rat brain slices.

Incorporation of U-14C-glucose into macromolecules (lipid, protein and nucleic acid fractions) of rat brain cortex slices was studied in vitro under conditions of anoxia and reoxygenation. Additionally, the influence of depolarization on control and postanoxic U-14C-glucose metabolism was investigated. Postassium-induced depolarization of the slices lowered their capacity to incorporate 14 from U-14C glucose into proteins and nucleic acids without any changes in the labeling the lipids. Fifteen and 30 minutes of anoxia depressed the rate of 14C incorporation into each of the above macromolecules was partly restored compared to the control. Excess of potassium in the medium during the reoxygenation period inhibited restoration of the synthetic capacity of the slices execpt lipids, into which incorporation of 14C was even stimulated under depolarizing conditions. The influence of anoxia and depolarization were investigated also in different classes of lipids and proteins. 14C incorporation into SDS-extractable and residual proteins and phospholipid fraction containing phosphoinositol was closest to the control during reoxygenation which suggests the relatively highest resistance of these fractions of anoxia.

Glutamine and glutamate as precursors of the releasable pool of gaba in brain cortex slices

The release of labeled GABA, synthesized from [ 14C]glutamine and from [ 14C]glutamate, was studied in mouse brain cortex slices. The spontaneous release of radioactive GABA derived from glutamate was higher than that of GABA synthesized from glutamine. In contrast, depolarization by 48 mM K + notably stimulated the release of GABA synthesized from glutamine but did not affect significantly the release of GABA formed from glutamate. It is concluded that glutamate is a better precursor than glutamine of the GABA pool spontaneously released, whereas the reverse is true for the pool releasable by K +-depolarization. The release of [ 14C]glutamate was stimulated by K + depolarization both when it was exogenously supplied and when it was derived from [ 14C]glutamine.

Citrate as the precursor of the acetyl moiety of acetylcholine.

Abstract— Rat brain cortex slices were incubated with glucose labeled with either 3H or 14C in the 6‐position. The 3H/14C ratios and the incorporation of radioactivity into lactate, citrate, malate and acetylcholine were determined. While the 3H/14C ratio of lactate was close to that of glucose, the ratios in the acetyl moiety of acetylcholine and the acetyl (C‐4,5) portion of citrate decreased in a similar proportion. This was interpreted as indirect evidence for the participation of citrate as a precursor to the acetyl moiety of acetylcholine. Two inhibitors of the citrate cleavage pathway: n‐butylmalonate, an inhibitor of citrate transport and (‐)‐hydroxycitrate, an inhibitor of ATP‐citrate lyase were studied for their effect on acetylcholine synthesis. N‐butylmalonate (10 mM) and (‐)‐hydroxycitrate (7.5 mM) led to a decrease in the per cent of 14C recovered as acetylcholine. In each instance the 3H/14C ratio in acetylcholine was higher in the presence of inhibitor while the corresponding ratios in lactate and citrate (C‐4.5) remained unchanged. From the results, it is suggested that citrate is involved in the transport mechanism of acetyl units from its site of synthesis in mitochondria to the site of acetylcholine synthesis in the cytosol.

Brain edema: induction in cortical slices by polyunsaturated fatty acids.

The presence of polyunsaturated and saturated fatty acids in leukocytic membranes prompted study of their possible role in the induction of brain edema. Polyunsaturated fatty acids including sodium arachidonate, sodium linoleate, sodium linolenate, and docasahexaenoic acids induced edma in slices of rat brain cortex. This cellular edema was specific, since neither saturated fatty acids nor a fatty acid containing a single double bond had such effect.

Effects of histidine on the transport of L-dopa by slices of brain cortex and striatum of the rat.

Abstract— L‐Histidine inhibits the uptake of L‐DOPA by brain cortex or striatum slices in short‐term experiments, but increases it in long‐term experiments. The potentiation of the steady‐state accumulation of L‐DOPA by histidine is a result of the cis‐ and trans‐effects of histidine on the transport of L‐DOPA at both sides of the cell membrane. The results demonstrate the importance of hetero‐exchange phenomena in determining the cellular level attained for a given amino acid at the steady‐state.

Electrically induced, calcium-dependent release of endogenous GABA from rat brain cortex slices

Electrically induced, calcium-dependent release of endogenous GABA from rat brain cortex slices

Iso-renin secretion from rat brain cortex slices in vitro under the influence of ions and electrical stimulation.

The aim of this study was to obtain additional data concerning the influence of changes in osmolarity and ionic composition on iso-renin secretion from rat brain cortex slices. The slices were isolated and superfused in vitro according to the technique of Pull and McIlwain (1972). The secretion of iso-renin was strongly stimulated by an increase of Na+ concentration. Iso-renin output almost doubled upon raising NaCl from 120 to 240 mM. Decrease of the NaCl concentration to 60 mM resulted in a reduced iso renin secretion while addition of tetrodotoxin (TTX) (60 micron) did not significantly alter the iso-renin content of brain slices. Changes in osmotic pressure were without significant influence. Electrical stimulation or elevation of extracellular K+ concentrations enhanced iso-renin secretion. Furthermore, Ca2+ ions seem to increase both the content and the release of cerebral iso-renin.

Effects of ammonium ions on spontaneous action potentials and on contents of sodium, potassium, ammonium and chloride ions in brain in vitro.

Abstract—The effects of ammonium ions on the frequency of spontaneous action potentials in guinea‐pig cerebellar slices, recorded with an extracellular microelectrode, and on the contents of sodium, potassium and chloride ions in incubated guinea‐pig cerebellar, and rat brain cortex, slices have been investigated. The frequencies of the spontaneous action potentials are partially suppressed by concentrations of NH4Cl less than 2 mm and completely abolished by concentrations exceeding 2 mm. The amplitudes of the spike discharges are unaffected. A lag period of at least 15 s precedes the inhibition. The suppressing action of NH on the spike frequency is reversible, as shown by complete recovery on removal of NH after short time intervals. Deficiency of Cl− in the superfusion medium causes conversion of inhibition by NH to excitation. Reduction of [K+], or of [Na+], causes increase of inhibition by NH in a normal [Cl1], and reduction of excitation in a low [Cl1], medium. The inhibitory effects of NH on spike frequency are unaffected by picrotoxin or strychnine. NH4Cl, even at 1 or 2 mm, causes a significant increase of aerobic glycolysis. It is suggested that the lag period preceding the suppression of the frequency of spike discharges by NH is partly due to a metabolic change induced by NH, perhaps a transient lowering of pH in the responsible neurons, causing changed permeability to Cl− and possibly to K+ and Na+, NH promotes, in guinea‐pig cerebellar slices, an inward flow of Na+ and an outward flow of K+, the latter being greater than that due to exchange of K+ for NH. NH4Cl at 1 or 2 mm causes an outward flow of K+ and an inward flow of Cl− in rat brain cortex slices. The movement of Cl− is biphasic, the first phase, seen with low [NH], consisting of an increase of tissue content of Cl− with little or no fluid uptake and a second phase, seen with high (> 5 mm) concentrations of NH, in which the uptake of Cl− is directly proportional to the fluid uptake. It is suggested that the first phase is largely neuronal in location whilst the second is largely glial. In infant rat brain cortex slices, there seems to be predominantly an equal exchange of NH for K+. There is little evidence of energy assisted concentrative uptake of NH by brain slices and this is thought to be due largely to the rapid diffusion of undissociated NH3 across cell membranes. It is suggested that some NH (amounting to about 2 mequiv/1) may be bound in the brain. It is concluded that changes in ionic permeabilities, particularly that of Cl−, partly due to a metabolic action, may be responsible for some of the acute cerebral effects of NH administration.

Effects of tetrodotoxin, elevated calcium and calcium antagonists on electrically induced 3H-noradrenaline release from brain slices

Electrically induced release of 3H-noradrenaline from superfused rat brain cortex slices was completely inhibited by tetrodotoxin (1 μg/ml) if 0.5 or 1 V stimuli were used, while inhibition was 97% with 2 V, and nil with 12 V stimuli. 20 mM calcium depressed noradrenaline release at all applied potentials, and shifted the release versus voltage curve to higher potentials. Manganese (5 mM) and D-600 (10–100 μg/ml) also considerably inhibited release. This suggests that stimuli of up to 2 V induce transmitter release by exciting intracortical noradrenergic axons in their non-terminal regions.

Uptake of glucose analogues by rat brain cortex slices: membrane transport versus metabolism of 2-deoxy-D-glucose.

Abstract— —The uptake of the glucose analogue 2‐deoxy‐d‐glucose by rat brain cortex slices was studied in order to compare the rate of membrane transport with the rate of phosphorylation in the concentration range 5–12 mM‐glucose plus 0.5–15 mM‐2‐deoxy‐glucose. The comparison was carried out by fitting a model of the brain slice to uptake data and by determination of 2‐deoxy‐glucose and 2‐deoxy‐glucose‐6‐phosphate by ion exchange chromatography.The rate of membrane transport exceeded the rate of phosphorylation by at least one order of magnitude. The membrane transport was so rapid that the extracellular diffusion became rate limiting for the uptake. The membrane transport could therefore only be determined as a minimum value and it was not possible to determine unidirectional flux across the cell membranes (initial rate). Accordingly, characterization of the membrane tranport with respect to maximal transport rate and affinity was not possible. The phosphorylation reaction, however, was so slow that it was accessible for exact determination and only the phosphorylation reaction was responsible for the fact that the cellular uptake of 2‐deoxy‐glucose was of the Michaelis‐Menten type, thus emphasizing the importance of dissociation between membrane transport and metabolism when transport is studied of a substance which can undergo metabolism.The data indicate that glucose transport across glial and neuronal membranes is not rate limiting for glucose metabolism of brain tissue in vitro.

Effect of elevated extracellular potassium on the release of labelled noradrenaline, glutamate, glycine, β-alanine and other amino acids from rat brain cortex slices

The effect of an elevated concentration (46 m m) of extracellular potassium on the release, from superfused rat brain cortex slices, of labelled γ-aminobutyrate, glutamate, glycine, α- and β-alanine, aspartate, taurine and α-aminoisobutyrate has been studied; and a comparison made with the release of soluble (non-vesicular) noradrenaline (i.e. that transported into slices taken from the brains of animals pretreated with reserpine; the slices were incubated with nialamide). In all cases, with the possible exception of l-α-alanine, elevated potassium induced an increased efflux of these substances. Omission of calcium in the superfusing fluid markedly diminished the release of all the test substances, except, in part, that of β-alanine. It was also found that the time course of the induced efflux varied considerably for the different substances: substances such as noradrenaline and γ-aminobutyrate that are transported predominantly into axons and axon terminals, showed a ‘peak’ time course, with a maximal release within 2–4 min following potassium elevation, and the rate of release diminished rapidly in spite of the continued presence of high potassium. Such a decreased release was not due to exhaustion of the tissue stores of these substances. On the other hand, glutamate and glycine, substances that are thought to be transported predominantly into glial cells, attained their maximal release rates only 10 min after potassium was elevated, and such release was maintained without decrement, β-alanine showed a mixed type of release, with a small initial peak resembling that of the axonally located substances, and a delayed release similar to that of glycine and glutamate. The release of the rest of the amino acids also resembled that of glycine and glutamate. A correlation was found ( r = 0.99, P < 0.01) between the area of the efflux curve that had a ‘peak’ shape, and the percentage of the substance that was transported into an axonal compartment. It is concluded that although elevated potassium concentrations can release substances from both axons and glia, and that a calcium dependency may exist in both cases, the time course of the induced efflux is markedly different when the substances are located in axons or in glial cells. Such a procedure may prove valuable for establishing grossly into which compartment a given substance is transported.

Effects of acetylcholine on potassium-induced changes of some amino acid uptakes and release in cerebral cortex slices from the rat.

High concentrations (105 μequiv./ml) of potassium ions in the incubation medium bring about reduced uptakes of L-glutamate, L-aspartate, and glycine but not of L-glutamine into rat brain cortex slices incubated aerobically in a physiological saline – glucose medium. The reductions are suppressed by acetylcholine (20 μM – 2 mM) in presence of eserine (0.1 mM) and not by tetrodotoxin (3 μM). The effect of acetylcholine is calcium dependent. It is diminished by atropine but not by d-tubocurarine (1 mM). Protoveratrine (5 μM) inhibition of amino acid uptake is not affected by acetylcholine but it is suppressed by tetrodotoxin. Acetylcholine and tetrodotoxin act independently of each other. Acetylcholine suppresses the potassium-evoked release of endogenous glutamate, aspartate, or glycine from incubated rat brain cortex slices. Its action on release is calcium dependent. Acetylcholine also suppresses the potassium-induced release of amino acids from rat brain cortex slices that have been previously incubated with 2 mM sodium L-glutamate or 2 mM sodium L-aspartate.It is suggested that increased cell concentrations of calcium ions, owing to high concentrations of potassium ions in the incubation medium, cause an increased glial permeability to sodium ions, with a resultant diminution of the sodium gradient. This diminution is considered to be responsible for the diminished concentrative uptake of L-glutamate, L-aspartate, or glycine, and the increased release of these amino acids. Acetylcholine suppresses the permeability change due to high concentrations of potassium ions and reverses the changed sodium gradient and the consequent change in amino acid uptake and release. It would seem that accumulation of acetylcholine in the intracellular spaces may affect glia, as well as neurons, modifying permeability to sodium ions and to various amino acids now assuming importance as possible transmitters.

Effects of acetylcholine on potassium-induced changes of water and sodium uptakes in cerebral cortex slices from the rat.

The increases in uptakes of water and of sodium ions that occur in rat brain cortex slices when they are incubated in a physiological saline-glucose medium in presence of a high concentration of potassium ions (105 muequiv./ml) are abolished by acetylcholine in presence of eserine but not by choline. Acetylcholine is effective at 20 micron but its optimal effect occurs at about 0.7 micron. Its action is suppressed by atropine and not by d-tubocurarine. The potassium-induced change of permeability of brain cell membranes to sodium ions occurs at a site different from the tetrodotoxin-sensitive channel of sodium entry, because the suppressive effects of acetylcholine and tetrodotoxin are apparently independent of each other. The acetylcholine effect does not occur in the absence of calcium ions from the incubation medium. It is suggested that the increase of cell calcium ions, brought about by high concentrations of potassium ions in the incubation medium, induces an increase of glial permeability to sodium ions, with a resultant change in the sodium gradient, and that this increase is suppressed by acetylcholine.

Effects of acetylcholine on potassium-induced changes of GABA and taurine uptakes and release in cerebral cortex slices from the rat

Acetylcholine, in presence of eserine, has little or no effect on the potassium-ion-suppressed concentrative uptakes of GABA and taurine by rat brain cortex slices in contrast with its effect on those of L-glutamate, L-aspartate, and glycine. Potassium ions at a concentration of 30 muequiv./ml in the incubation medium has a marked suppressive effect on the uptakes of GABA and taurine when there is no apparent change in the sodium ion content of the brain tissue. It is concluded that some factor, besides the change in sodium gradient, operates in the mechanism of potassium suppression of GABA and taurine uptakes. Acetylcholine diminishes the potassium-evoked release of endogenous GABA and taurine from brain slices. Its action is Ca2+ dependent and is diminished by atropine. Acetylcholine does not affect the potassium-accelerated release of GABA from brain slices previously loaded with this amino acid. The differences in uptake and release phenomena exhibited by GABA and taurine from those of L-glutamine and L-aspartate may be due to differences between the mechanisms, as well as the sites, of cerebral uptake and release of these two groups of amino acids.

Mescaline-induced changes of brain-cortex ribosomes. Mescaline demethylase activity of brain-cortex soluble supernatant.

Brain-cortex slices demethylate mescaline and p-methoxyacetanilide, a reference O-demethylating substrate, though the rate of demethylation of mescaline is about one third that of the reference substrate. The demethylase activity is localized mostly in the soluble supernatant (105 000 x g). It is purified 47-fold with respect to the demethylation of mescaline by ammonium sulfate precipitation and DEAE cellulose chromatography. The partially purified demethylase, which is stable for 3-5 days at -5 degrees C in the presence of dithiothreitol and glutathione and is inhibited by p-chloromercuribenzoate, has maximal activity at pH between 7.2 and 8.0. It demethylates mescaline into 3,4-dimethoxy-5-hydroxyphenethylamine and 3,5-dimethoxy-4-hydroxyphenethylamine and some unidentified derivatives.

Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

Mechanism of intestinal absorption and brain uptake of L-5-hydroxytryptophan in rats, as compared to those of L-3,4-dihydroxyphenylalanine.

L-5-Hydroxytryptophan (5-HTP) and L-3, 4-dihydroxyphenylalanine (DOPA) were comparatively investigated of their mechanisms of intestinal absorption, metabolism in the intestine and liver tissues and mechanisms of passage through the blood-brain barriers, by means of in vitro and/or in situ techniques using rat tissues. The corresponding D-isomers were also investigated for comparison. L-5-HTP, but not the D-isomer, was found to be absorbed from the intestine by an active transport mechanism in the same way as L-DOPA. In vitro studies using rat brain cortex slices proved that both L-5-HTP and L-DOPA, but not their D-isomers, are taken up by the brain tissues by an active transport mechanism which is saturable, energy-dependent and competitive. There was no substantial difference between L-5-HTP and L-DOPA in the rate of intestinal absorption and that of brain uptake. It was indicated, however, that L-5-HTP was decarboxylated in the intestine to a much less extent than L-DOPA during the absorption and the decarboxylation activity of rat intestine and liver homogenates was found to be about four and seven times higher for L-DOPA than for L-5-HTP, respectively. The latter results suggest that the oral dose of L-5-HTP can be much more easily transferred into the circulating blood without being suffered from decarboxylation in the peripheral organs than L-DOPA, resulting in a more efficient penetration of L-5-HTP into the brain as a serotonine precursor.

Potassium-induced release of [3H] GABA and of [3H] noradrenaline from normal and reserpinized rat brain cortex slices, Differences in calcium-dependency, and in sensitivity to potassium ions.

Abstract—The release of [3H]GABA induced by elevated extracellular potassium (K)o, from thin rat brain cortex slices, has been compared with that of [3H]noradrenaline ([3H]NA), released by the same procedures, both from normal slices, and from slices pre‐treated with reserpine and nialamide, [3H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM‐(K)o released considerably more [3H]NA from normal than from drug‐treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [3H]NA from drug‐treated slices increased in proportion to (K)o, up to 36‐46 mM and then declined considerably with higher (K)o. The dependency of potassium‐induced release on the concentration of calcium in the medium, indicated that release of [3H]NA from normal slices was proportional to calcium up to 1.5‐2 mM, while that of [3H]NA from drug‐treated slices increased up to 0.5 mM‐Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM‐calcium, but no further changes were observed at higher concentrations. The calcium antagonist D‐600 inhibited high (K)o‐induced release of [3H]NA from normal slices to a greater extent than that of [3H]GABA or of [3H]NA from drug‐treated slices. These results, in which elevated (K)o‐induced release of [3H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [3H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)o as a stimulus for inducing transmitter secretions.

Increase in ACh Release from the Rat Brain Cortex Slices by 3’,5’-Cyclic Adenosine Monophosphate

Increase in ACh Release from the Rat Brain Cortex Slices by 3’,5’-Cyclic Adenosine Monophosphate