HPLC-荧光检测法测定大鼠脑组织中的单胺类神经递质及相关物质

目的 采用HPLC-荧光检测方法测定大鼠脑组织中的单胺类递质及相关物质.方法 脑组织匀浆用提取液(含0.1 mol · L-1 HClO4和0.2 mmol · L-1 EDTA)提取后进样.色谱柱用Waters Nova-Pak C18(150 mm×3.9 mm,4 μm);流动相为甲醇-缓冲液(14:86);流速1.0 ml · min-1;荧光检测波长λEX=280 nm,λEM=330 nm.结果 7种单胺类神经递质及相关物质的标准曲线在一定浓度内线性关系良好,方法的加样回收完全,精密度好.结论 所建方法简便、快速、准确,可用于脑组织中单胺类神经递质及相关物质的测定.     

The activities of monoamine oxidase-A and-B,succinate dehydrogenase and acid phosphatase in the rat brain after hemitransection

Summary The activities of monoamine oxidase-A and-B were determined in four brain regions (limbic system, occipitotemporal cortex, hemispheres and striatum) of the rat 0, 3, 6, 9 and 14 days after hemitransection of the left side. No larger or consistent change in the activity of monoamine oxidase-A towards 5-hydroxytryptamine was found for the left (hemitransected) side with respect to the right side for any of the rats. The monoamine oxidase-B activity towards -phenethylamine increased in the left side striatum to a significant level by 3 days, and in the hemispheres and occipito-temporal cortex on the left side, with respect to the right side by 9 days, but no significant changes were found for the limbic system. A small decrease in the activity of succinate dehydrogenase was found in the striatum on the left side by 9 days after hemitransection, but no change in the activity of acid phosphatase was found in this brain region.     

Interactions of tricyclic antidepressant drugs with human and rat monoamine oxidase type B

Summary The effect of tricyclic antidepressant drugs on the deamination of phenylethylamine and benzylamine by monoamine oxidase (MAO) type B was investigated in vitro in human brain cortex, human platelet, and rat brain preparations. These drugs inhibited MAO activity as expected; however, an atypical biphasic response was observed with the tertiary amine tricyclic, clomipramine, and, to a somewhat lesser extent, with two other tertiary amine tricyclics, imipramine and amitriptyline, when benzylamine was used as the substrate in human tissue preparations. This atypical biphasic pattern was not found when we used the secondary amine antidepressant drugs, desipramine, desmethylclomipramine, or fluoxetine, or used phenylethylamine as the substrate, or used rat rather than human brain tissue. For the tricyclics exhibiting normal inhibition patterns, the same rank order of inhibition was observed with benzylamine as a substrate in all three types of tissue; however with phenylethylamine, differences in inhibition were found between rat and human tissues. These tricyclic-MAO interactional data suggest that secondary and tertiary amine tricyclics interact differently with human MAO type B, that rat and human MAO type B are not functionally identical, and also support other data that phenylethylamine and benzylamine are deaminated by different mechanisms. Send offprint requests to A. A. Reid at the above address     

无机汞对大鼠脑单胺类神经递质代谢的影响

用大鼠脑组织匀浆、游离脑细胞和脑细胞亚组分作实验材料，较全面地观察了氯化汞对单胺类神经递质含量及其合成、释放、摄取、降解等重要代谢环节的影响。结果表明，汞通过抑制脑线粒体外膜单胺氧化酶使得脑组织中５－羟色胺、多巴胺、去甲肾上腺素含量增高，５－羟吲哚乙酸含量下降，呈良好的剂量－效应关系；汞还明显改变了游离脑细胞对单胺类神经递质的释放和摄取能力。     

Effect of stress on norepinephrine-stimulated cyclic AMP formation in brain slices.

Experiments were conducted to determine if stressful procedures, which increase brain norepinephrine (NE) release in rats, lower the responsiveness of central noradrenergic receptors as measured by the catecholamine (CA)-induced cAMP accumulation in hypothalamic and cerebral cortical slices. No conclusive evidence of subsensitivity was found after either acute or chronic electric footshock or continuous restraint. Failure to find a significant reduction after stress may have resulted from several methodological problems. These include (a) the inhibition of phosphodiesterase activity with isobutylmethylxanthine, which may have obscured possible adaptive changes in cAMP degradation and/or adenosine-dependent adrenergic receptors; (b) a low initial responsiveness to NE in these animals as suggested by the greater case in inducing supersensitivity with reserpine than subsensitivity with amphetamine; and (c) the use as a test agent of exogenous NE which may stimulate a far broader population of receptive sites in brain slices than are activated during stress by the local release of endogenous NE.     

Effect of (+)amphetamine on monoamine synthesis and metabolism after axotomy in rat forebrain

Summary (+)Amphetamine is known to produce feedback inhibition of catecholamine-containing neurons in the central nervous system due to its indirect receptor stimulating properties. This feedback control may involve postsynaptic receptors and neuronal loops or presynaptic receptors and may be restricted to the catecholaminergic terminal system.In order to study the effect of (+)amphetamine on catecholamine synthesis and metabolism in the terminal system changes in impulse flow were eliminated by cutting the ascending monoaminergic axons.Axotomy of the ascending monoaminergic fibers by means of a complete transverse cerebral hemisection resulted in a 3-fold increase in Dopa formation in the lesioned forebrain during 30 min after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine HCl, 100 mg/kg i.p. (+)-Amphetamine sulfate, 10 mg/kg i.p. antagonized the hemisection-induced increase in Dopa formation and reduced the formation of 5-hydroxytryptophan. Pre-treatment with haloperidol, 5 mg/kg i.p. failed to counteract the effect of (+)amphetamine. In the intact forebrain the stimulation of Dopa accumulation was more than additive after combined treatment with haloperidol and (+)amphetamine.Hemitransection retarded the disappearance of dopamine and noradrenaline after administration of -methyl-p-tyrosine methylester HCl, 300 mg/kg. (+)-Amphetamine, 10 mg/kg accelerated the utilization of dopamine on the lesioned side.Hemitransection reduced the formation of 3-methoxytyramine during 1 h after pargyline, 75 mg/kg i.p. After (+)amphetamine 3-methoxytyramine formation in the intact forebrain was 3 times higher than in the lesioned forebrain.The action of (+)amphetamine on dopamine synthesis and release appears to be dependent on the firing rate in dopamine neurons.A preliminary report has been given recently (Kehr and Speckenbach, 1975)Data of this communication are part W.S.'s thesis for an M.D.     

The role of brain norepinephrine in the anorexic effects of dextroamphetamine and monoamine oxidase inhibitors in the rat

The effects of d-amphetamine on food and water intake and brain monoamine concentrations in rats that had been deprived of food and water for 24 h were compared with those of two MAO inhibitors: tranylcypromine which has prominent amphetamine-like activity; and, pargyline which does not. All drugs produced dose-related depressions of food and water intake. The anorexic effects of the MAO inhibitors were correlated, over a 16-fold dose range, with elevated levels of norepinephrine, dopamine and serotonin. The anorexic effect of d-amphetamine was blocked by -methyltyrosine, an inhibitor of catecholamine synthesis. -Methyltyrosine failed to block the depression of food and water intake caused by the MAO inhibitors, although the rise in catecholamine levels was prevented. It was concluded that the mechanisms by which d-amphetamine produces anorexia may differ from those of the MAO inhibitors. Central adrenergic mediation appears to play a role in the anorexic activity of d-amphetamine, but may not be essential for the anorexic effect of tranylcypromine and pargyline.Publication No. 1023 of the Division of Basic Health Sciences of Emory University. This investigation was suported by NIMH Grant MH 12870-03.Postdoctoral trainee of NIH Graduate Pharmacology Training Grant GM-179 during part of this work.     

Effects of methylenedioxymethamphetamine on the release of monoamines from rat brain slices

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamine release were investigated in superfused slices of rat striatum and hippocampus. MDMA (10 microM) increased the resting release of radioactivity from slices incubated in [3H]dopamine, [3H]5-hydroxytryptamine or [3H]noradrenaline. These effects of MDMA (10 microM) were blocked by the neuronal uptake inhibitors, cocaine (10 microM), fluoxetine (1 microM) and desmethylimipramine (1 microM), respectively. MDMA (10 microM) enhanced the stimulation-induced efflux of radioactivity from slices incubated in [3H]noradrenaline but not from slices incubated in [3H]5-hydroxytryptamine or [3H]dopamine. These results demonstrate for the first time a direct noradrenaline-releasing action of MDMA and differential effects of MDMA on the stimulation-induced release of noradrenaline, dopamine and 5-hydroxytryptamine from rat superfused brain slices.     

Effect of lisuride and LSD on monoamine synthesis after axotomy or reserpine treatment in rat brain

Summary The effect of d-lysergic acid diethylamide (LSD) and lisuride on the synthesis of monoamines was evaluated in rat brain regions using an in vivo method in which the accumulation of dopa and 5-hydroxytryptophan (5-HTP) is measured after inhibition of the aromatic amino acid decarboxylase by means of 3-hydroxybenzylhydrazine.LSD (50–1000 g/kg i.p.) caused a dose-dependent increase in dopa formation and a slight elevation of tyrosine and tryptophan concentrations in the intact rat forebrain; moreover, it reduced the accumulation of 5-HTP.The increase in dopa formation in the terminal system of the rat forebrain following an axotomy of the ascending monoaminergic fiber system was antagonized by LSD at a dose of 0.5 mg/kg i.p. Haloperidol (2 mg/kg i.p.) counteracted the effect of LSD.The increase in dopa formation in c. striatum and the dopamine-rich part of the limbic system following treatment with reserpine was antagonized by lisuride as well as by LSD. However, lisuride was at least 10 times as potent as LSD. In reserpinized animals LSD counteracted the inhibitory effect on dopa accumulation of the direct dopamine receptor stimulant apomorphine while lisuride did not. The data suggest that LSD is a weak agonist at dopamine receptors in vivo with partial receptor blocking properties at higher doses, while the action of lisuride on dopamine receptors is a potent, purely agonistic one.with technical assistance of Ruth NeumeisterPart of this paper has been presented at the Spring Meeting 1977 of the German Pharmacological Society at Mainz (Kehr, 1977b)     

Electrically induced release of endogenous noradrenaline and dopamine from brain slices: pseudo-one-pulse stimulation utilized to study presynaptic autoinhibition

Summary Slices of rat hypothalamus (noradrenaline experiments) or rabbit caudate nucleus (dopamine experiments) were prepared, superfused, and field-stimulated using series of monophasic rectangular pulses. Noradrenaline, dopamine and the main dopamine metabolite, dihydroxyphenylacetic acetic acid (DOPAC), were determined using HPLC with electrochemical detection. Electrical stimulation was performed using the following protocols: 1) 4 pulses delivered at 100 Hz; this type of stimulation is referred to as pseudo-one-pulse stimulation (POP); its short duration of only 32 ms does not allow the development of autoinhibition; 2) 2 bursts of 4 pulses at 100 Hz, delivered 1 s apart (2-POP-stimulation); 3) 8 pulses at 1 Hz (dopamine experiments only); 4) 36 pulses at 3 Hz. Noradrenaline experiments. The ₂-adrenoceptor antagonist yohimbine (1 mol/l) did not enhance noradrenaline overflow following POP stimulation, but enhanced the overflow following 2-POP-stimulation by about 50% and that following 36-pulse-stimulation by almost 100%. Dopamine experiments. The D₂-dopamine receptor antagonist sulpiride (3 mol/l) facilitated the overflow of dopamine elicited with 2-POP-stimulation (66%), 8 pulses/1 Hz (92%), and 36 pulses/3 Hz (140%). It did not significantly facilitate the overflow of dopamine following POP-stimulation (19%). The overflow of DOPAC was not, or only slightly, increased by electrical stimulation, and its spontaneous outflow was more than three times higher than that of dopamine. Furthermore, the electrically induced overflow of dopamine did not exceed the outflow of DOPAC at any of the stimulation conditions employed.The results of the present study bear out important claims of the autoreceptor theory and confirm the data obtained in previous experiments using labelled transmitters. Correspondence to E. A. Singer at the above address     

Influence of monoamine oxidase inhibition on the release of 3H-dopamine elicited by potassium and by amphetamine from the rat substantia nigra and corpus striatum

Summary The effects of monoamine oxidase inhibition on the release of ³H-dopamine induced by exposure to amphetamine or to potassium were studied in the corpus striatum and in the substantia nigra of the rat using identical in vitro experimental conditions. Marked differences between the two areas of the dopaminergic neurone were found. In the corpus striatum, release of ³H-dopamine was observed during exposure to low concentrations of amphetamine, while considerably higher concentrations of amphetamine were required to elicit ³H-dopamine release from the substantia nigra. The difference in the effects of amphetamine on the release of ³H-dopamine from the substantia nigra and the rat corpus striatum was observed independently of whether monoamine oxidase activity was intact or inhibited by pargyline. Exposure to potassium was also more effective in inducing ³H-dopamine release from the striatum than from the substantia nigra, but the difference was less pronounced than that observed for amphetamine.Since inhibition of monoamine oxidase by pargyline results in changes in the proportions of vesicular and axoplasmic dopamine, conclusions about the in vivo effects of amphetamine on dopaminergic neurotrans-mission should not be based on in vitro studies in which the enzyme is inhibited. In addition, in vitro studies on dopamine release by nerve depolarization should be carried out under the physiological conditions in which monoamine oxidase is not inhibited by drugs.     

3H-Catecholamine binding to alpha-receptors in rat brain: enhancement by reserpine.

(+/-)-3H-Epinephrine and (-)-3H-norepinephrine bind to rat cortex membranes in a saturable manner with dissociation constants of 16.7 and 27 nM respectively. The maximum number of 3H-catecholamine binding sites, 10--12 pmoles/g tissue, and the pharmacological characteristics of (+/-)-3H-epinephrine binding, indicate that the catecholamines label the same alpha-noradrenergic receptor in the rat as does 3H-clonidine. At 25 degrees, (+/-)-3H-epinephrine binding associates rapidly to equilibrium, and dissociates in a biphasic manner. The affinities of alpha-agonists at the 3H-catecholamine binding site are 2--4 fold weaker in the rat than in the calf cortex under the same experimental conditions. Ergot alkaloids and phenoxybenzamine have similar affinities in the two tissues, whereas phentolamine and WB-4101 are 8--10 times weaker in the rat. Reserpine (0.25 mg/kg s.c. per day for 3 weeks) causes 25 and 46% increases in the numbers of (+/-)-3H-epinephrine and 3H-WB-4101 alpha-receptor binding sites respectively, and a 51% increase in the number of 3H-dihydroalprenolol beta-receptor sites, in rat forebrain. Reserpine pretreatment does not alter the affinities of either alpha- or beta-receptor 3H-ligands.     

Amphetamine induced release of endogenous dopamine in vitro is not reduced following pretreatment with reserpine

Summary The release of endogenous dopamine evoked by electrical stimulation or by exposure to (+)-amphetamine (10 M) was determined in superfused striatal slices of the rat.The spontaneous and the electrically-evoked release of dopamine were significantly increased in the presence of nomifensine (10 M). After reserpine pretreatment (5 mg/kg, s.c., 24 h), the striatal dopamine content was reduced by about 90%. Exposure to 10 M (+)-amphetamine during 2 min released similar amounts of dopamine from striatal slices of untreated or reserpine pretreated rats. Similar results were obtained when monoamine oxidase activity was inhibited in vivo with pargyline.Pretreatment with reserpine does not modify the (+)-amphetamine-induced release of dopamine, in spite of the marked reduction of the striatal dopamine content. These results provide direct evidence for the view that (+)-amphetamine releases dopamine from a special, reserpine-resistant pool of newly synthetized transmitter.Some of the results described in this publication have been presented at the British Pharmacological Society Meeting (Arbilla et al. 1984a)     

Chronic amphetamine treatment reduces NGF and BDNF in the rat brain

Amphetamines (methamphetamine and d-amphetamine) are dopaminergic and noradrenergic agonists and are highly addictive drugs with neurotoxic effect on the brain. In human subjects, it has also been observed that amphetamine causes psychosis resembling positive symptoms of schizophrenia. Neurotrophins are molecules involved in neuronal survival and plasticity and protect neurons against (BDNF) are the most abundant neurotrophins in the central nervous system (CNS) and are important survival factors for cholinergic and dopaminergic neurons. Interestingly, it has been proposed that deficits in the production or utilization of neurotrophins participate in the pathogenesis of schizophrenia. In this study in order to investigate the mechanism of amphetamine-induced neurotoxicity and further elucidate the role of neurotrophins in the pathogenesis of schizophrenia we administered intraperitoneally d-amphetamine for 8 days to rats and measured the levels of neurotrophins NGF and BDNF in selected brain regions by ELISA. Amphetamine reduced NGF levels in the hippocampus, occipital cortex and hypothalamus and of BDNF in the occipital cortex and hypothalamus. Thus the present data indicate that chronic amphetamine can reduce the levels of NGF and BDNF in selected brain regions. This reduction may account for some of the effects of amphetamine in the CNS neurons and provides evidences for the role of neurotrophins in schizophrenia.     

GABA potentiates potassium-stimulated 3H-dopamine release from slices of rat substantia nigra and corpus striatum

GABA (10-5--10-3 M) had no effect on the spontaneous outflow of previously accumulated 3H-DA or 3H-5HT from rat nigral or striatal slices. However, GABA markedly potentiated the potassium-stimulated release of 3H-DA in both brain regions, while the depolarization-induced output of 3H-5HT was only slightly increased. This action of GABA was blocked by pictotoxin but not by bicuculline. Amphetamine likewise evoked a dose-related efflux of 3H-DA and 3H-5HT from nigra and striatum, but these releases were unchanged by GABA. The data suggest that GABA acts presynaptically in these areas to regulate dopaminergic cell function.     

拟除虫菊酯对大鼠脑组织单胺类含量的影响

目的观察氯菊酯和溴氰菊酯两种不同类型拟除虫菊酯对大鼠脑组织单胺类递质含量的影响.方法大鼠经腹腔注射给予氯菊酯(100mg·kg     

Glucose deprivation increases basal and electrically evoked transmitter release from rat striatal slices. Role of NMDA and adenosine A1 receptors

We have investigated how glucose deprivation in vitro influences the basal and electrically evoked release of dopamine and acetylcholine from rat striatal slices and the role of endogenous activation of NMDA receptors and adenosine A₁ receptors in determining the magnitude of this response. Rat striatal slices, preincubated with [

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]dopamine and [

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]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with glucose-free Krebs' solution. Such glucose deprivation caused a 2 to 3-fold increase of the electrically evoked, calcium-dependent release of endogenous adenosine (but not hypoxanthine and inosine) and [

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]dopamine and a 30% increase in release of [

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]acetylcholine. Glucose deprivation also caused a delayed increase in the release of [

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]dopamine, but not of [

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]acetylcholine. The dopamine release was not calcium dependent. The addition of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM), a selective adenosine A₁ receptor antagonist, slightly enhanced the glucose deprivation-induced stimulatory effect on the evoked release of these two transmitters, whereas the NMDA receptor antagonist dizocilpine((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine; 3 μM) markedly attenuated the stimulatory effect of glucose deprivation. The change in basal dopamine release was not influenced by DPCPX, but was slightly attenuated by dizocilpine. In summary, the results suggest that lack of substrate induces release of both glutamate, which by actions on presynaptic NMDA receptors causes the release of dopamine, and of adenosine, which via adenosine A₁ receptors reduces the electrically evoked release of both dopamine and acetylcholine.     

Presynaptic noradrenergic alpha-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes.

The depolarization (15 mM K+)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of alpha-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K+ stimulation greatly enhanced the sensitivity of 3H-NA release to alpha-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the alpha-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons, Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less senstive to alpha-receptor-mediated presynaptic inhibition than those in the cortex.     

Reduction of 3,4-diaminopyridine-induced biogenic amine synthesis and release in rat brain by gabapentin

The anticonvulsant drug gabapentin has been shown recently to exhibit anxiolytic and analgesic actions in animals. Such actions have been postulated in part to reflect effects on biogenic amine neuronal activity. Therefore the effects of gabapentin on biogenic amine neuronal activity were assessed by measuring the synthesis of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in rat brain and on the release of [³H] NE from rat hippocampal slices both in the presence and absence of the depolarizing agent 3,4-diaminopyridine (DAP). Gabapentin (30 and 100 mg/kg, IP) did not alter the basal synthesis rates of NE and DA as assessed by the unchanged accumulation of L-dihydroxyphenylalanine (DOPA) in the NE-enriched hippocampus and cortex and in the DA-enriched striatum and mesolimbic areas. Gabapentin also did not alter 5-HT synthesis as determined by the unaltered accumulation of 5-hydroxytryptophan (5-HTP) in the same brain areas. DAP (2 mg/kg, IP) induced a modest but significant increase in DOPA accumulation in the hippocampal, mesolimbic and striatal regions. This DAP-induced increase in DOPA accumulation was antagonized significantly in the hippocampus and mesolimbic regions by gabapentin at 30 and 100 mg/kg and in striatum by 100 mg/kg; a 10 mg/kg dose was inactive. DAP increased selectively 5-HT synthesis in hippocampus and this effect was blocked by gabapentin. These findings indicate that the increased synthesis of biogenic amines induced by DAP is antagonized by gabapentin. In support of the in vivo studies, gabapentin was also shown to inhibit the DAP-evoked release of [³H]NE from hippocampal slices. Although the underlying mechanism for these effects is unclear, the present findings nevertheless demonstrate that gabapentin has inhibitory effects on stimulated NE, DA and 5-HT neurons that may be involved in explaining in part the CNS effects of this drug. Received: 6 August 1997/Final version: 12 November 1997     

The effect of CO2 on monoamine metabolism in rat brain

Summary White male albino rats were exposed to artificial atmosphere containing 20% of O₂ and 5, 10 and 15% of CO₂, for periods of time ranging from 30 min to 2 h. Monoamine synthesis in different dissected brain areas was measured by estimating Dopa and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of their decarboxylation with the L-aromatic aminoacid decarboxylase inhibitor NSD 1015 (3-hydroxybenzylhydrazine). Endogenous levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and NA and DA levels after inhibition of catecholamine synthesis with -methyltyrosine (-MT) were measured in dissected areas and whole brain.The synthesis of all three monoamines was found to be increased in all the cerebral regions. Endogenous dopamine levels with or without -MT treatment were increased while NA levels decreased. 5-HT decreased in at least one area and 5-HIAA increased in several brain areas. These observations indicate that the utilization of NA and 5-HT is increased and that of DA is diminished in acidosis, presumably as a consequence of a corresponding change in nerve impulse flow.The similarity between the monoamine changes induced by hypercarbia and by GABA are pointed out. It is suggested that GABA-ergic activity is elevated in acidosis and that GABA may be involved in the pH dependence of seizure thresholds, as well as in the increased tendency to respiratory depression induced by central nervous system depressant drugs in patients with chronic respiratory failure.