Brain Cortex Slices Research Articles

Studies on the relationship between GABA synthesis and protein synthesis in brain.

Abstract— The synthesis of γ‐aminobutyric acid (GABA) in mouse brain was decreased by treatment of the animals with pyridoxal phosphate‐ γ‐glutamylhydrazone, an inhibitor of glutamate decarboxylase in vivo. Under these experimental conditions the following parameters were studied: (1) the incorporation of labeled leucine in vivo, into protein of brain subcellular fractions; (2) the brain polysome profile; (3) the incorporation of labeled leucine into protein in vitro, in ribosomal preparations isolated from brain tissue. In other experiments, GABA synthesis was also decreased in brain cortex slices by preincubation with aminooxyacetic acid. The incorporation of [3H]leucine or [14C]leucine into protein in these slices was studied, and samples from the proteins were subjected to acrylamide‐sodium dodecylsulfate gel electrophoresis. Radioactivity was counted in slices of the gel. The results of the experiments in vivo and in vitro indicate that the previously reported decrease of protein synthesis induced by an inhibition of GABA synthesis affects proteins of all subcellular fractions and all populations of protein as separated by gel electrophoresis. The polysome profile from brains of mice with decreased GABA synthesis was similar to that of control mice. This result differs from that found when brain protein synthesis is inhibited by dopamine and serotonin.

UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+‐INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3‐O‐methyl‐D‐glucose and α‐methyl‐D‐glucoside were not metabolized in brain tissue.The uptake of these two sugars into the intracellular compartment of brain cortex slices was investigated using media with normal and low Na+ concentration (replacement of all NaCl with choline Cl). The cellular transport was not Na+‐dependent. The transport mechanism clearly distinguished between the two sugars in both normal and low Na+ media.

Factors Influencing the Insulin Space in Cerebral Cortex Slices from Adult and 7‐Day‐Old Rats

The inulin spaces and swelling of brain cortex slices from adult and 7-day-old rats were measured under differing experimental conditions. At 37 degrees C inulin penetrated into larger compartments than at 0 degrees C in both age groups. At 37 degrees C the inulin space increased with decreasing concentration of inulin in the medium. At 0 degrees C the inulin space did not depend on the inulin concentration. The swelling of the slices at 37 degrees C diminished with increasing concentrations of inulin. Anaerobic conditions, 0.2, and 1.0 mmol/l sodium cyanide and 0.1 mmol/l dinitrophenol reduced the inulin space and caused increased swelling of the slices. 1.0 mmol/l sodium iodoacetate was ineffective. In anaerobic conditions the inulin space did not change significantly as a function of the inulin concentration. A serious disadvantage of the inulin space as an indicator of extracellular space is that its size depends on the inulin concentration used. The inulin itself may influence the size of the space to be measured. Inulin also in osmotically inactive concentrations considerably reduces the swelling of brain cortex slices.

Postnatal alterations in effects of potassium on uptake and release of glutamate and GABA in rat brain cortex slices.

Abstract—The uptake and release of glutamate and of GABA, as well as the effect of high potassium concentrations (35 or 80 mM) hereupon, were studied by aid of 14C‐labelled amino acids in brain cortex slices from rats of different ages between birth and adulthood. Both the extent of the uptake (i.e. the tissue/medium ratio of 14C at, or close to, equilibrium) and the rate of uptake (i.e. the tissue/ medium ratio of 14C after short (5 min) incubation periods) increased with age. Differences were, however, found between glutamate and GABA, and the extent of the GABA uptake had a distinct maximum during the second postnatal week. At all ages, high concentrations of potassium caused a decrease in the rate of GABA uptake but were without effect on the rate with which glutamate was taken up. The release of the two amino acids occurred with approximately the same half‐time (50 min) in slices from animals of at least 14 days of age. Before that time the release of glutamate was somewhat faster, whereas that of GABA was much slower, especially during the first postnatal week (half‐time 90 min). The ontogenetic alterations in the effect of excess potassium were complex and varied both between the two potassium concentrations used and between the two amino acids. The results are thus compatible with the existence of different transport systems for the two amino acids, They also suggest that glutamate may exert other functions in addition to its role as a putative transmitter.

The action of triethyltin on the respiration of rat brain cortex slices.

Abstract—Glucose oxidation by rat brain cortex slices is inhibited by low concentrations of triethyltin and this sensitive inhibition is shown to be chloride‐dependent. Pyruvate oxidation is only inhibited at high concentrations of triethyltin whether chloride is present or not. In contrast, the stimulation of both glucose and pyruvate oxidation observed with low concentrations of triethyltin prior to inhibition is chloride‐dependent. The results are discussed in relation to the chloride‐hydroxide exchange reaction known to be mediated across the inner mitochondrial membrane by triethyltin and other organo‐metals.

Cerebral uptakes and exchange diffusion in vitro of L- and D-glutamates.

Abstract— 1. Whereas exogenous l‐glutamate enters rat brain cortex slices incubated in a glucose‐physiological saline medium by both low affinity (Km= 0.7 mm) and high affinity (Km= 27−30 μM) processes, the uptake of d‐glutamate occurs only by a low affinity (Km= 2mm) system. 2. d‐glutamate appears to release l‐glutamate from incubated rat brain cortex slices only to a very small extent, whether the tissue l‐glutamate is of endogenous or exogenous origin. 3. Competitive inhibition takes place between l‐ and d‐glutamates at the low affinity carrier. This indicates that a common carrier exists for l‐ and d‐glutamates for the low affinity uptake process. 4. Apparently non‐competitive inhibition by d‐glutamate of l‐glutamate uptake occurs at the high affinity carrier, but the affinity of d‐glutamate for this carrier is about 0.4% of that of l‐glutamate. 5. Both d‐, and l‐glutamate exchange freely with labelled d‐glutamate taken up by preliminary incubation of the brain slices with this amino acid. Whereas l‐glutamate exchanges freely with labelled l‐glutamate taken up by preliminary incubation, d‐glutamate shows little or no exchange. 6. The uptake of labelled d‐glutamate by exchange diffusion into brain slices previously loaded with unlabelled d‐glutamate proceeds by a low affinity system. Therefore, the process of exchange diffusion does not necessarily involve a high affinity uptake component. 7. Whereas ouabain suppresses both high and low affinity concentrative uptakes of l‐ and d‐glutamate it has little apparent effect on the exchange diffusion process. 8. Sensitivity to tetrodotoxin of evoked release of l‐ and d‐glutamates, taken up by brain slices by preliminary incubation with these amino acids, indicates that the major proportion of the uptake of exogenous l‐ or d‐glutamate proceeds into non‐neuronal structures (presumably the glia). 9. At 0°C non‐carrier mediated (passive) diffusion of labelled d‐ and l‐glutamate takes place in brain slices.

Distribution of protein-bound radioactivity in brain slices of the adult rat incubated with labelled leucine

The distribution of protein-bound labelled leucine in brain cortex slices, prepared from adult rats by various methods and incubated with [ 14C]- or [ 3H]leucine, was investigated by autoradiography. In the first and second slices a marked gradient of incorporated radioactivity from the cut surface to the slice interior was observed. Very high labelling of leptomeningeal cells and vessels enhanced further the inhomogeneity of radioactivity distribution. Light microscopic examination of incubated slices revealed morphological alterations of neurones, especially in the vicinity of the cut surface. The comparison of grain density over neurones and their satellite glia indicated markedly higher incorporation into the latter. The ATP level in slices at the end of incubation reflected the method of slice preparation and morphological integrity. Inhomogeneity of incorporated radioactivity distribution in brain slices contrasted with the uniform labelling of cortical cells in vivo, and may represent at least one reason for the low estimates of protein synthesis rate in brain cortex slices.

EFFECTS OF Na+ AND OTHER MONOVALENT CATIONS ON Ca-EFFLUX FROM SYNAPTOSOMES

Effects of monovalent cations on Ca-efflux were examined in rat brain cortex slices, synaptosomes and synaptic plasma membranes. Effluxes of 45Ca from brain slices and synaptosomes were stimulated by Na+ in medium and the effects of Na+ on the 45Ca-effluxes disappeared at low temperature. Furthermore, we observed that Rb+ had more effect than Na+ on 45Ca-efflux from the synaptosomes, while Li+ had less effect. On the other hand, release of 45Ca from the synaptic plasma membranes which had been preincubated with ATP, Mg++ and 45Ca was stimulated by Na+ and Li+. But Cs+ and Rb+ were less effective on the release. These results indicate occurrence of Na-dependent Ca-efflux at nerve endings. However, the assumption that ATP-dependent Ca-binding with synaptic plasma membranes may be a partial reaction of Ca-efflux was not supported by these experiments.

Effects of pharmacological agents on [ 14C]-nicotine distribution and movements in slices from different rat brain areas

The effects of various pharmacological agents on [ 14C]-nicotine accumulation, distribution and movements in rat brain cortex, striatum, hypothalamus and cerebellum slices were studied. When present during incubation, mecamylamine (2.0 m m) decreased the [ 14-nicotine tissue space and both mecamylamine and nonradioactive nicotine (0.62 m m) acted primarily to decrease the amount of [ 14C]-nicotine present in the slower component of a two-component washout of [ 14C]-nicotine. Procaine (3.67 m m) also decreased [ 14C]-nicotine uptake; this decrease occurred almost exclusively within the slower component. Lobeline (0.62 m m), physostigmine (1.0 m m) and, to a lesser extent, metanephrine (1.0 mm) also decreased the [ 14C]-nicotine tissue space in a competitive manner. No direct interaction with various cholinergic agents was demonstrated. When added during washout of [ 14C]-nicotine, both mecamylamine and procaine increased [ 14C]-nicotine efflux. It appears that the agents employed compete with [ 14C]-nicotine for cellular binding sites or, in the case of procaine, might possibly also inhibit access of [ 14C-nicotine to the intracellular sites.

GABA metabolism and cerebral protein synthesis

Glutamate decar☐ylase (GAD) activity and [ 14C]leucine incorporation into brain protein in vivo were measured in mice injected with l-glutamic acid -γ-hydrazide and 60 min later with pyridoxal-5′-phosphate; this combined treatment produces a decrease in GAD activity and an increase in GABA levels. Under such conditions, protein synthesis was inhibited to the same extent as GAD activity. A parallelism between the decrease in GAD activity and that in protein synthesis was also observed in brain cortex slices treated with GAD inhibitors. This treatment did not affect leucine incorporation into protein in liver slices. The results support the previously suggested hypothesis that a certain pool of GABA may have a role in the regulation of protein synthesis in brain, and further suggest that the synthesis of GABA may be more important in this respect than its total concentration.

Metabolism of amino acids and ammonia in rat brain cortex slices in vitro: a possible role of ammonia in brain function.

Abstract— (1) The sum of the values of total (tissue + medium) amino acid‐N of glutamate, glutamine, γ‐aminobutyrate, and aspartate (referred to as the glutamate system) and of ammonia‐N of incubated rat brain cortex slices is approximately constant under a variety of metabolic conditions (presence or absence of glucose or of oxygen or in the presence of metabolic inhibitors such as aminooxyacetate, malonate, methionine sulfoximine, fluoroacetate, ouabain, 2:4 dinitrophenol, or Amytal). Fluctuations in the value of one constituent are compensated by fluctuations in the values of other constituents. The same applies to infant rat brain cortex slices and to rat brain synaptosome preparations. It is suggested that the constancy of the glutamate‐ammonia system implies a coupling of neurons and glia in such a manner that glutamate released from the neurons during excitation is taken up by the glia and there converted to glutamine. The glutamine is returned to the neurons where it is hydrolysed to glutamate and ammonia. The glia, on this view, exercise an important buffering effect on the extracellular content of the excitatory amino acid, glutamate, and possibly on that of other functionally active amino acids emanating from the neurons. (2) The magnitude of the glutamate‐ammonia system in the infant rat brain cortex is about 43% of that in the adult. It is suggested that, with maturity, the development of the glutamate‐ammonia system is linked with the development of the citric acid cycle of operations. (3) The ammonia in the system is tightly linked to the activity of the ATP‐controlled glutamine synthetase. (4) Proteolytic ammonia and amino acids are formed, during the incubation, to values that seem to be independent of a wide variety of metabolic conditions. The total value is approximately 10 μmol/g in the first h of incubation. (5) As the ammonium ion is necessary for the return of glutamate to the neuron in the form of glutamine, it is inferred that the ion plays a functional role in the nervous system by helping to maintain the steady state of glutamate in the neuron.

Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats.

Abstract—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7‐day‐old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7‐day‐old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7‐day‐old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7‐day‐old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.

In vivo incorporation of 14-C-leucine into brain protein of methylmercury treated rats.

In an attempt to explain the sensitivity of the brain to methylmercury, investigators have monitored various biochemical parameters in systems poisoned with this compound. YOSHINO et al. (1966) measured changes in brain cortex slices from rats severely intoxicated with methylmercury. Although several alterations were observed in animals showing neurological symptoms, only protein synthesis decreased during both the latent period and after the onset of symptoms. It has recently been observed that in vivo brain protein synthesis tended to decrease in rats mildly intoxicated with methylmercury. No investigations have been reported concerning the effects of low or extremely high doses of methylmercury on in vivo protein synthesis. We, therefore, measured in vivo /sup 14/C-leucine incorporation into brain protein in 48 hour old rats which received low levels of the mercurial during the fetal stage, and also in severely poisoned adult female rats showing neurological symptoms.

THIAMINE PHOSPHATE METABOLISM AND POSSIBLE COENZYME‐INDEPENDENT FUNCTIONS OF THIAMINE IN BRAIN1

Abstract—The effect of depolarization of rat brain cortex slices on the relative distribution of thiamine among its various phosphate esters and on the efflux of thiamine was studied as a probe of possible coenzyme‐independent neurophysiological functions of thiamine. Electrical pulses for 30 min increased lactate production but did not affect the levels of thiamine esters. Depolarization with 41 mM‐potassium decreased thiamine diphosphate by only 3 percent (P= 0.05). Thiamine triphosphate levels (TTP) were unaffected by depolarization but doubled during incubation for 1 h in which time efflux of 40 percent of the total thiamine from the slices as unesterified thiamine occurred. Depolarization by potassium released a small but highly variable portion of the thiamine content of superfused cortex slices above the basal rate of efflux. The basal efflux was partially sodium dependent. Thiamine efflux was unaffected by acetylcholine, ouabain, or tetrodotoxin, compounds previously reported to increase thiamine efflux. The incorporation of 32P1 into the endogenous thiamine phosphates of cortex slices was studied. Incorporation into thiamine diphosphate reached only 20 percent of the specific activity of its precursor, ATP, after 2h of incubation while the incorporation into TTP approached equilibrium with ATP in 15‐30 min indicating that the TTP pool was the most rapidly turning over of the thiamine phosphates. The data suggest that only a small portion of the TDP pool undergoes rapid turnover and serves as a precursor for TTP. The rapid turnover of TTP phosphoryl groups is consistent with specific functions for this compound related to its potential for phosphorylation reactions. An analog of TTP with the β, γ oxygen bridge replaced by a methylene group decreased TDP levels and increased thiamine when incubated with cortex slices, but did not effect thiamine monophosphate or triphosphate levels indicating inhibition of thiamine pyrophosphokinase.

Further studies on ammonia formation in brain slices: the effect of hadacidin.

Hadacidin, a specific inhibitor of adenylosuccinate synthetase, inhibited the ammonia formation of respiring brain cortex slices in a substrate-free medium. Compared with the inhibitory effect of 5-bromofuroate, a specific inhibitor of glutamate dehydrogenase, the effect of hadacidin was considerably weaker at all concentrations. For both inhibitors, the concentration which produced half-maximum inhibition was approximately 10 mM. With combinations of the two inhibitors, additivity or even potentiation of the separate effects were observed at concentrations up to 10mM. At higher concentrations, the effects of the more potent inhibitor, 5-bromofuroate, were still enhanced by the addition of hadacidin, but in decreasing degrees. The effects of hadacidin or 5-bromofuroate, at concentrations up to 10 mM, were also enhanced by the addition of cycloheximide and the combined effect was almost additive. It is concluded that the glutamate dehydrogenase system and the purine nucleotide cycle are both involved in the ammonia formation of brain slices and that the inhibitory effect of cycloheximide is due to its inhibition of electron transport.

The utilization of glucose in the brain slices during uraemia.

Rat brain cortex slices were incubated in diluted sera obtained from patients with renal failure and from healthy persons. It was found that uraemic sera inhibited glucose uptake and the incorporation of14C-D-glucose into aspartate, glutamate and gamma-aminobutyrate (GABA). The concentration of these amino acids in the brain slices did not change.

Mescaline-induced changes of brain cortex ribosomes: Effect of mescaline on the binding of aminoacyl-transfer ribonucleic acid to ribosomes of brain tissue

The effect of mescaline sulfate on the binding of aminoacyl-tRNA by brain cortex ribosomes was studied. The poly U-directed binding of 14C-phenylalanyl-tRNA in vitro was moderately inhibited by mescaline. The pretreatment of normal ribosomes with mescaline caused a decrease of their phenylalanyl-tRNA binding capacity. The pretreatment of brain cortex slices with mescaline also decreased the phenylalanyl-tRNA binding capacity of the ribosomes isolated from the slices so treated. This was true in assays with phenylalanyl-tRNAs from both Escherichia coli and yeast. This inhibitory effect was dependent on the concentration and length of treatment of brain cortex slices with mesaline.

Effects of oxygen deprivation on incorporation of [U- 14C]-glucose into macromolecules of guinea pig cerebral cortex slices

Incorporation of [U- 14C]-glucose into lipid, protein, RNA and DNA of guinea pig brain cortex slices was studied in vitro for varying time intervals under conditions of anoxia and recovery from anoxia. In addition, pH values ranging from 6.8 to 8.0 were also studied to examine their effect on postanoxic cellular metabolism. The following observations were made: (i) Maximal incorporation of [U- 14C]-glucose into macromolecules of slices was observed during 15 min of anaerobic incubation; longer incubation produced no further uptake. (ii) Following incubation under anaerobic conditions for 15 min, slices regained the capacity to incorporate glucose radiocarbon into macromolecules when transferred to an oxygenated incubation medium, but exhibited lower rates than those of controls which were not exposed to anoxia. While protein synthesis showed significant recovery immediately after anoxia, no further incorporation occurred following an initial period of uptake. The same was true for protein synthesis after increased intervals of anaerobic preincubation. However, the rates of incorporation into DNA and RNA were not significantly affected. Synthesis of lipid was maintained at the highest level of all molecular classes studied. (iii) Studies at different pH values showed that 7.4 was the optimum pH for incorporation. (iv) High concentration of unlabelled glucose in the incubation medium greatly improved postanoxic recovery. In conclusion, exposure to anoxic conditions irreversibly inhibited further protein synthesizing activity in brain slices after an initial constant increment of residual synthesis, while synthesis of nucleic acids and lipid were reversible, albeit depressed.

The relative significance of CO2-fixing enzymes in the metabolism of rat brain.

AbstractTo evaluate the relative significance of CO2‐fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP‐isocitrate dehydrogenase and NADP‐malate dehydrogenase, as well as the fixation of H14CO3− by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol‐pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP‐isocitrate dehydrogenase and NADP‐malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H14CO3− at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H14CO3− in the presence of α‐ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H14CO3− at a rate of about 7 nmol/g of tissue/min and it was increased by two‐fold when pyruvate was added to the incubation medium. The carboxylation of α‐ketoglutarate and pyruvate by the reversal of the cytosolic NADP‐isocitrate dehydrogenase and NADP‐malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP‐isocitrate dehydrogenase and NADP‐malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO2‐fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α‐ketoglutarate by a sequential action of aconitate hydratase and NADP‐isocitrate dehydrogenase. The operation of the citrate‐cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO2‐fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO2‐fixation in the brain.

Effects of ethanol, tert. butanol, and clomethiazole on net movements of sodium and potassium in electrically stimulated cerebral tissue.

AbstractThe sodium and potassium contents of rat brain cortex slices incubated in bicarbonate medium were measured and the intracellular quantities estimated on the basis of the inulin distribution. Ethanol (109 mM) did not affect the ion content of unstimulated tissue during passive leakage in cold medium or aerobic incubation at 37o C.The effects of ethanol (109 mM), tert. butanol (34 mM) and clomethiazole (0.39 mM) were tested on the response to electrical stimulation (entry of sodium and loss of potassium in the non‐inulin space) as well as on the restoration of the ionic gradients occurring after a period of stimulation. All agents selectively inhibited the entry of sodium on stimulation with very little effect on potassium loss. The result is considered compatible with preferential action of the drugs on the activation step of the sodium channel controlling movement of sodium during the excitation cycle, and presumably located on the outside of the membranes. During recovery, no inhibition of sodium expulsion was observed, whereas both alcohols‐ but not clomethiazole‐slightly decreased the accumulation of potassium. Considered together with other observations, this finding suggests a special affinity of ethanol for the potassium binding site in the ion transport system.