METABOLITE LEVELS IN BRAIN FOLLOWING EXPERIMENTAL SEIZURES: THE EFFECTS OF ISONIAZID AND SODIUM VALPROATE IN CEREBELLAR AND CEREBRAL CORTICAL LAYERS

Abstract— Levels of glucose, lactate, GABA and cyclic nucleotides were examined in discrete layers of the cerebellum and cerebral cortex of mice following treatment with the anticonvulsant, sodium valproate, and/or the convulsant, isoniazid. The concentrations of the metabolites were essentially uniform among the layers of each region, whether from control or from drug-treated mice. Metabolite concentrations in the isoniazid-treated mice were determined either 30 min after administration (preconvulsive state), or immediatley after the onset of seizures. Glucose and lactate, two markers of energy status in the brain, were only minimally affected by drug treatment. However, the levels of GABA and cyclic nucleotides were markedly different from control values in the drug-treated animals. In the preconvulsive state, GABA levels in cerebellar layers were depressed and the cyclic nucleotides were elevated in most layers of both regions. At the onset of seizures, the reduction of GABA and the elevation of cyclic AMP in both regions was more pronounced than during the preconvulsive state. While the concentration of cyclic GMP remained elevated in the cerebellar layers at the onset of seizures, the level in the cerebral cortex returned to control values. Valproate elevated GABA in all the layers of both regions and decreased the cyclic GMP in the cerebellar layers. Generally, when valproate was administered in combination with isoniazid, it dampened the isoniazid induced changes in the metabolites. The events leading up to a seizure as well as those that sustain it may be reflected by the disparate responses of the metabolites in the cerebellum and cerebral cortex.     

THE EFFECTS OF ELECTROSHOCK SEIZURES ON POTASSIUM TRANSPORT WITHIN SYNAPTOSOMES FROM RAT BRAIN

—Potassium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one or two maximal electroshock (ES) convulsions daily. No significant change in endogenous K content was present after 2 consecutive days of ES-seizures. However, K decreased 22 % and 41 % after 4 and 6 days of convulsions respectively. After 6 days, synaptosomes from ES rats were able to accumulate K to the same extent as controls and were inhibited by ouabain to an equivalent extent (50%). When these synaptosomes from ES rats were preloaded with high concentrations of K, K leaked out at an increased rate. When diphenylhydantoin (DPH) was administered intraperitoneally from the second to the sixth day of ES-convulsions, the decrease in endogenous K induced by chronic convulsions was corrected. In ion-free media or with 50 mM Na, DPH had no effects on the enhanced efflux of K observed in vitro after ES-seizures. However, with high Na (50–100 mm ) and low K (0.2–1 mm ) DPH stimulated K uptake but did not affect the ouabain inhibition. Under conditions optimal for K uptake (50 mm Na and 10 mm K), DPH did not affect K accumulation but it prevented in vitro ouabain inhibition. Our results indicate that repeated ES-convulsions decreased K content within isolated nerve terminals by enhancing its passive leakage. In vivo DPH prevented the effects of ES-seizures by stimulating the Na-K pump and not by directly blocking the K leak.     

EFFECT OF OLIGEMIA ON REGIONAL METABOLITE LEVELS IN CAT BRAIN

Abstract— Incomplete cerebral ischemia (oligemia) was produced in cat by carotid occlusion combined with arterial hypotension. Lowering arterial pressure to 50–60 Torr for 20 min caused marked alterations of the ATP, phosphocreatine, and lactate content of subcortical white matter. In contrast, metabolite levels in cerebral cortex and caudate nucleus were only moderately perturbed from control values. More severe oligemia resulted when arterial pressure was lowered to 30 Torr for 20 min following carotid occlusion. Metabolite levels in cortex, caudate nucleus, and white matter were greatly altered from control. In the gray matter there was regional heterogeneity of metabolic alteration, as evidenced from the pattern of NADH tissue fluorescence. The cortex contained micro-patches (0.1mm) of increased NADH, which frequently exhibited a columnar orientation.
These findings demonstrate two distinct types of cerebral inhomogeneity of metabolic failure with reduced blood flow; white matter fails before gray matter, and there is micro-heterogeneity of metabolic failure in the gray matter.     

ELECTROSHOCK-INDUCED SEIZURES AND THE TURNOVER OF BRAIN PROTEIN IN THE RAT

Abstract— A total of ten electroshock seizures (two seizures per day) were induced in rats beginning 3 days after an injection of [U-¹⁴C]glucose. Despite the intense stimulation, the labelling of the protein and nucleic acid fractions in the brains of convulsed animals decreased only slightly and not significantly. During the first 2 days after administration of [¹⁴C]glucose to untreated animals, there was a slight decrease in the specific activity of protein-bound glutamic acid relative to that of aspartic acid and the total protein fraction, suggesting the presence of a protein with a high content of glutamic acid and a rapid turnover.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: THE TEMPORAL RELATIONSHIP OF CHANGES INDUCED IN ADENOSINE 3',5'-MONOPHOSPHATE AND GUANOSINE 3',5'-MONOPHOSPHATE FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

–Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) levels increase about 5-fold in the cerebral cortex and 2-fold in the cerebellum following electroconvulsive shock (ECS). The peak levels of cyclic AMP occur at 45 s after ECS in the cerebral cortex, and at 15 s in the cerebellum. In the cerebral cortex, ECS produces twice the cyclic AMP accumulation as does decapitation in a comparable time period; however, the relative effect of a number of neurotropic agents on the cyclic AMP accumulation is essentially the same, whether stimulated by decapitation or by ECS. In the cerebellum, the levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) also increase following ECS. The cyclic GMP levels are greatest at 60 s after ECS during the postictal depression. An association between elevated cerebellar cyclic GMP and depression seems unlikely, since CNS depressants either lowered or had no effect on cyclic GMP levels. From these results, cyclic nucleotide profiles following treatments such as ECS or decapitation may be useful in elucidating the molecular events involved in seizures, brain injury and ischemia.     

INTERACTIVE EFFECTS OF CYCLOHEXIMIDE AND PUROMYCIN IN ALTERING BRAIN POLYRIBOSOMES AND NEURAL AND BEHAVIOURAL RESPONSES TO ELECTROSHOCK IN MICE

Abstract— Interactive effects of puromycin and cycloheximide on brain polyribosomes and cortical electrical activity were investigated. The time courses of action of the drugs on these parameters, in comparison to their inhibitory actions on protein synthesis, were also observed. The results indicate that the disruption of brain polyribosomes by cycloheximide was independent of its inhibition of protein synthesis, whereas the two processes were closely linked in the case of puromycin. For both the disruption of polyribosomes and the alteration of electrical activity, the order in which the drugs were administered was critical, with preadministration of cycloheximide having a protective effect. In contrast to the massive effect of cycloheximide on brain polyribosomes, the drug had no such effect on polyribosomes from liver.     

EFFECTS OF PRE-WEANING AND POST-WEANING UNDERNUTRITION ON ACETYLCHOLINE LEVELS IN RAT BRAIN

Abstract— Studies were made of the effects of undernutrition during the neonatal period and also protein deficiency and undernutrition during the post-weaning period on brain acetylcholine. Rats undernourished from birth to 4 weeks so as to result in a body wt deficit of 43 per cent had an associated deficit in brain wt of 14 per cent, but the concentration of acetylcholine in the brain was not affected. In the case of post-weaning undernutrition, acetylcholine concn was found to be affected in protein deficiency as well as in severe calorie restriction.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: EFFECT OF HYPOTHERMIA ON ADENOSINE 3'',5'' MONOPHOSPHATE, GLYCOGEN PHOSPHORYLASE, GLYCOGEN SYNTHASE AND METABOLITES FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

Abstract —The accumulation of adenosine-3',5'-cyclic monophosphate (cyclic AMP) has been investigated in murine brain following electroconvulsive shock and decapitation. Animals were made hypothermic (20°C) to minimize the freezing time of the brain and to delay metabolic events. Cyclic AMP concentrations were decreased in the cerebral cortex of hypothermic rats and mice. Furthermore, the changes in cyclic AMP elicited by electroconvulsive shock and decapitation were delayed. In hypothermic animals, the metabolic rate as determined by high energy phosphate use was decreased to 65% of control values. The interconversions of the active and inactive forms of glycogen phosphorylase and glycogen synthase were sufficiently retarded in hypothermic animals to correlate with changes in cyclic AMP concentrations. The conversion of phosphorylase b to a and synthase a to b occurred when cyclic AMP concentrations had increased from 2 to 5 μmol/kg, following either electroconvulsive shock or decapitation. The results indicate that cyclic AMP plays a role in regulation of glycogen metabolism in cerebral cortex.     

LONG-TERM EFFECTS OF TEFLUTIXOL ON THE SYNTHESIS AND ENDOGENOUS LEVELS OF MOUSE BRAIN CATECHOLAMINES

—The long term effects on accumulation of ¹⁴C-labelled dopamine and noradrenaline after [¹⁴C]tyrosine administration and on the endogenous levels of catecholamines in mouse brain were studied after treatment with a new potent thioxanthene neuroleptic, teflutixol. The drug was given as a single dose (5 mg/kg i.p.), as repeated daily doses (1·25 mg/kg p.o.), or as a single dose of the palmitic ester in Viscoleo® (20 mg/kg s.c). After a single dose, teflutixol increased catecholamine synthesis (100%). Noradrenaline synthesis rapidly returned to normal, whereas decreased dopamine synthesis was seen from the third to sixth day, after which it was normal. When the receptors were continuously exposed to teflutixol, either by daily dosage or by the depot preparation, catecholamine synthesis was increased for the first few days but then returned to normal, indicating development of tolerance. Endogenous concentrations of catecholamines were only decreased during the first few days, when the increase in synthesis was greatest. The findings are in accordance with results obtained by Møller Nielsen & Christensen (1975), who found that receptor blockade was followed by receptor supersensitivity after treatment with a neuroleptic compound. The receptor blockade may activate a feedback mechanism that induces increased nervous firing with increased amine synthesis as a consequence. The resulting supersensitivity, if sufficiently great, may lead to reduced nervous firing, followed by slowing of dopamine synthesis.     

褪黑素对谷氨酸致痫大鼠海马内谷氨酸、GABA及其受体水平的影响

目的探讨褪黑素(melatonin,MT)对谷氨酸(glutamate,Glu)致痫大鼠海马内Glu及GluR2、γ-氨基丁酸(γ-aminobutyric-acid,GABA)及其受体GABRA1水平的影响,进而研究褪黑素的抑痫作用机制。方法随机将健康SD雄性大鼠40只分为A、B、C、D组,每组10只。A组:生理盐水组;B组:MT Glu组;C组:Glu致痫组;D组:Luzidole MT Glu组。观察并记录行为学变化,采用免疫组化法进行Glu、GluR2、GABA和GABRA1免疫组化染色和图像分析。结果行为学观察结果显示,C组和D组大鼠均有不同程度的癫痫发作,B组大鼠癫痫发作不明显,A组无发作;免疫组化结果显示,C组和D组海马内CA1-CA3区和齿状回Glu阳性反应较A组增强(P<0.05),GluR2、GABA和GABRA1均较A组减弱(P<0.05),B组Glu较C组和D组阳性反应有显著性减弱(P<0.05),GluR2、GABA和GABRA1阳性反应均较C组和D组有显著性增强(P<0.05),而B组与A组无明显差异性。结论MT通过增加GABA及其受体GABRA1和GluR2的作用和抑制Glu作用对Glu致痫大鼠癫痫发作发挥抑制作用。     

银杏类黄酮对大鼠脑损伤后机能改变的影响

观测了银杏类黄酮（flavonoid of Ginkgo biloba,FGb)对损伤大鼠运动皮层后机能改变过程的影响。结果表明：（1）FGb明显促进运动平均能力的恢复;（2）损伤后脑水肿明显,FGb促进脑水肿症状的恢复;（3）挫伤3h后脑内的游离氨基酸递质水平明显下降,FGb促进脑内氨基酸水平的恢复。这可能是银杏叶提取物促进脑损伤修复的机制之一。     

DOPAMINE IN THE MESOLIMBIC SYSTEM OF THE RAT BRAIN: ENDOGENOUS LEVELS AND THE EFFECTS OF DRUGS ON THE UPTAKE MECHANISM AND STIMULATION OF ADENYLATE CYCLASE ACTIVITY

Abstract— High concentrations of dopamine were found in the nucleus accumbens and olfactory tubercle of the rat brain using a radiochemical enzymatic assay technique. An active uptake system for [³H]dopamine that is temperature sensitive and dependent on external sodium ions is present in synaptosome-rich homogenates of these two brain areas. This uptake process is potently inhibited by benztropine (IC₅₀= 2.0 × 10^-7m ). Dextroamphetamine d was 4.5 times more potent than 1-amphetamine in inhibiting dopamine uptake in the nucleus accumbens and six times more potent in the olfactory tubercle and corpus striatum. Low concentrations of dopamine caused an increase in adenosine 3′5′-monophosphate (cyclic AMP) formation in homogenates of both the nucleus accembens and olfactory tubercle. This effect was potently blocked by chlorpromazine. The α-adrenoceptor antagonist phentolamine weakly antagonized the stimulation of this adenylate cyclase by dopamine, but the β-adrenoceptor antagonist propranolol did not.     

银杏类黄酮对大鼠脑损伤后机能改变的影响

观测了银杏类黄酮(flavonoid of Ginkgo biloba, FGb)对损伤大鼠运动皮层后机能改变过程的影响.结果表明:(1) FGb明显促进运动平衡能力的恢复;(2) 损伤后脑室水肿明显,FGb促进脑水肿症状的恢复;(3)挫伤3 h后脑内的游离氨基酸递质水平明显下降,FGb促进脑内氨基酸水平的恢复.这可能是银杏叶提取物促进脑损伤修复的机制之一.     

EFFECTS OF EPILEPTIC SEIZURES ON BRAIN RIBOSOMES: MECHANISM AND RELATIONSHIP TO CEREBRAL ENERGY METABOLISM

Epileptic seizures were induced electrically in rats paralyzed and ventilated with oxygen. Dissociation of brain polysomes and increase in ribosomal dimers were observed after multiple (25 or more) seizures, but were not seen after shorter treatments (up to 10 seizures). Polysomal dissociation and dimer formation occurred in vivo and were accompanied by decreased amino acid incorporation into brain proteins in cell-free systems in vitro. These events paralleled changes in brain energy reserves rather than changes in behavior.     

THE EFFECTS OF REPEATED-LYSERGIC ACID DIETHYLAMIDE INJECTIONS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN REGIONS

Daily injections of 100 μg/kg of d -lysergic acid diethylamide (LSD) for 14 days produced a significant decrease in the dopamine level in rat brain corpus striatum which was still apparent 15 days after the last LSD treatment. Further LSD injections did not change the amount of dopamine depletion. In cerebral cortex, 14 days of LSD injections produced a significant decrease in the norepinephrine level and a significant increase in tyrosine hydroxylase activity. The elevated tyrosine hydroxylase activity was still present 15 days after the final LSD injection but only in those animals receiving daily vehicle injections during this period. Pre-treatment of rats with daily saline injections for 2 weeks before the 2 week period of LSD treatment prevented both the reduced norepinephrine content and elevated tyrosine hydroxylase activity usually found 24 h after the last LSD injection.     

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION-RAPID POST-MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum. Activities of the enzymes tyrosine hydroxylase, DO PA decarboxylase, monoamine oxidase, and catechol-o-methyltransferase in the striatum were also examined. These enzymes were totally inactivated by the microwave heating, except catechol-o-methyltransferase which was decreased approx 80%. These results support either (1) the existence of a substantial pool of dopamine in the striatum with a very rapid turnover rate or (2) a decapitation-related release and destruction of striatal dopamine. Measurements of 3-methoxytyramine in the striatum exhibit post-mortem increases corresponding to the decreases of dopamine. Use of the rapid tissue enzyme inactivation technique suggests that in vivo levels of this O-methylated dopamine metabolite are an order of magnitude lower than the results normally obtained after killing by decapitation.     

BRAIN TYROSINE HYDROXYLATION: ALTERATION OF OXYGEN AFFINITY IN VIVO BY IMMOBILIZATION OR ELECTROSHOCK IN THE RAT

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5-hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare-immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.     

THE EFFECTS OF SOME NEWER ANAESTHETICS ON THE IN VITRO ACTIVITY OF GLUTAMATE DECARBOXYLASE AND GABA TRANSAMINASE IN CRUDE BRAIN EXTRACTS AND ON THE LEVELS OF AMINO ACIDS IN VIVO

—The effects of several anaesthetic and hypnotic compounds with well-defined excitatory side-effects on glutamate decarboxylase and γ-aminobutyric acid transaminase activity have been examined. The dissociative anaesthetics ketamine and γ-hydroxybutyric acid produced competitive inhibition of glutamate decarboxylase with respect to glutamate at concentrations which had no effect on GABA transaminase activity. The inhibitor constant (K_i) values were, ketamine: 13.3 mm , γ-hydroxybutyric acid; 8.8 mm . The steroid anaesthetic alphaxalone was also a potent competitive inhibitor of glutamate decarboxylase K_i= 4.1 mm ). Pentobarbitone, thiopentone and methohexitone non-competitively inhibited both glutamate decarboxylase and GABA-transaminase but only at high concentration (> 20 mm ). None of the drugs tested produced any significant change in brain GABA or glutamate levels following the injection of an hypnotic or anaesthetic dose. It is proposed that an alteration in the rate of GABA synthesis as a result of the inhibition of glutamate decarboxylase could explain the convulsive properties of the dissociative anaesthetics when given at high doses.     

THE LEVELS OF LABILE INTERMEDIARY METABOLITES IN MOUSE BRAIN FOLLOWING RAPID TISSUE FIXATION WITH MICROWAVE IRRADIATION1

The levels of several labile glycolytic and organic phosphate metabolites in mouse brain were determined following rapid inactivation with 2450 MHz microwave irradiation. The levels of ATP in mouse brain following a 0·25 s exposure in a 6 kW microwave oven was found to be 2·416 ± 0·061. Whole brain levels of 8 labile intermediary metabolites in 0·4 s irradiated samples were comparable to those reported using the previously-described methods of freeze-blowing or whole-animal immersion. Analysis of these same metabolites in 4 gross areas of brain did not reveal any anoxic changes betwen superficial and deeper brain areas. The advantages of the mcrowave irradiation inactivation technique for regional brain studies of labile intermediary metabolites is discussed.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.