THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.     

INCORPORATION OF 32P IN VITRO INTO TRIPHOSPHOINOSITIDE AND RELATED LIPIDS OF RAT SUPERIOR CERVICAL GANGLIA AND VAGUS NERVES

Abstract— Rat sympathetic ganglia, vagus nerve and sciatic nerve were each incubated with inorganic ³²P for various lengths of time and the resultant labelling of their inositol lipids was measured.
At all times up to 3 hr phosphatidylinositol was the most highly labelled lipid of ganglia, while triphosphoinositide was the most active lipid of vagus and sciatic nerves. Removal of calcium ions from the incubation media had no significant effect on the incorporation of phosphate into any of the inositol lipids of sympathetic ganglia.     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.     

RESPIRATION IN VITRO OF SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

Abstract— —(1) The respiratory properties of synaptosomes and mitochondria from mammalian cerebral cortex are compared.
(2) Synaptosome showed high and linear respiration with glucose and pyruvate as substrates in Krebs-Ringer media. Mitochondria showed such respiration only with pyruvate as substrate in media lacking Na and high in K and phosphate.
(3) Exposure of synaptosomes to hypotonic media caused loss of lactate dehydrogenase (LDH) and protein, and respiration diminished and became non-linear.
(4) Both ATP and phosphocreatine were synthesised by synaptosomes with glucose as substrate. ATP was synthesised by mitochondria in the presence of pyruvate.
(5) Synaptosome but not mitochondria showed some capacity for active accumulation of potassium.
(6) Both mitochondria and synaptosomes respired with glutamate as substrate. Glutamate caused 80 per cent loss of ATP and phosphocreatine in synaptosomes but did not diminish the level of mitochondrial ATP.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

THE EFFECTS OF OXYGEN DEPRIVATION ON ELECTRICALLY STIMULATED CEREBRAL CORTEX SLICES

Abstract— (1) Thin slices were prepared from guinea pig cerebral cortex and allowed to incubate in oxygenated bicarbonate-buffered medium for 30 min. Subsequent to that time the slices were made hypoxic by passing 95% N₂-5% CO₂ through the medium. Hypoxic exposure caused the slices to gain Na⁺ and to lose K⁺ ions from the non-inulin space. These shifts were especially pronounced when slices were electrically stimulated during the hypoxic period. Thus, after 30 min of hypoxia plus stimulation, non-inulin Na⁺ had risen from 30 to 84, μequiv./g wet wt., and non-inulin K⁺ had fallen from 50·5 to 14·3 μequiv./g wet wt.
(2) The above shifts were in part reversible, but when reoxygenated slices were subsequently electrically stimulated in oxygenated media, they failed to lose K⁺ or to gain Na⁺.
(3) The induced inexcitable state could not be attributed to inability of the slices to replenish ATP and phosphocreatine and may indicate an alteration in membrane constituents necessary for preservation of membrane excitability.     

AMINO ACID DISTRIBUTION AND INCORPORATION INTO PROTEINS IN ISOLATED, ELECTRICALLY-STIMULATED CEREBRAL TISSUES

—The uptake of radioactive amino acid by incubated cerebral cortex slices is found to be a first order process. Incorporation of the radioactive amino acid into tissue protein is from a precursor pool that has first equilibrated with the intracellular endogenous free amino acids. Ways of calculating the amino acid incorporation in molar quantities from the observed incorporation of radioactivity are discussed, and it is concluded that the specific radioactivity of the intracellular acid-soluble fraction is the best basis for such estimates. The in vitro incorporation of leucine into tissue protein is estimated to be approximately 1±2 mμnol/mg protein/h, and of valine 0±4 mμmol/mg protein/h. Addition of free amino acids to the media had little or no effect on the calculated rates of incorporation. On incubation for 1 h the total free valine in tissue and medium increased by 0±43 μmol/g and leucine increased by 0±55 μmol/g. Estimates of amino acid incorporation based on the specific radioactivity of the media amino acids can give misleading results if this considerable release of amino acids into the medium is not taken into account. Electrical stimulation of neocortical slices with a variety of types of pulses was either without effect or decreased incorporation into portein. The decrease could not be directly correlated with changes in tissue K⁺ nor with the utilization of ATP. Mild, local stimulation of the lateral olfactory tract of piriform cortex slices was without effect on tissue phosphocreatine, K⁺ or amino acid incorporation.     

EFFECTS OF ADDED SUBSTANCES ON RNA AND PROTEIN SYNTHESIS IN IMMATURE NEURONS

Abstract— A newly described method for the isolation of morphologically intact neurons from newborn rat brain was used to study the influence of inhibitors and neuroactive substances on RNA and protein synthesis in these cells in vitro . Incorporation of [¹⁴C]-uridine into RNA and [³H]leucine into protein proceeded rapidly and continued up to 3 h. When the incorporation mixture was chased at 20 min with an excess of nonradioactive uridine and leucine, hardly any degradation of labelled RNA was noted during the following 2 h 40 min. In contrast, the specific radioactivity of proteins decreased by 22 per cent indicating turnover of cellular proteins.
Incorporation of labelled leucine into protein was markedly inhibited in the presence of NaF and cycloheximide but not affected in the presence of chloramphenicol or pancreatic RNase. A mixture of ATP + GTP depressed the incorporation by 38 per cent. The responses to ATP + GTP and RNase indicated that the incorporation system was typically cellular. Acetylcholine, γ-aminobutyrate, noradrenaline and phenylalanine in the incubation medium depressed the incorporation of labelled uridine into RNA by 10–30 per cent and 5-hydroxytryptamine by 75 per cent. Acetylcholine, γ-aminobutyrate and noradrenaline had no effect on protein synthesis, while 5-hydroxytryptamine and phenylalanine inhibited the incorporation by 60–80 per cent. Testosterone and prednisolone depressed both RNA and protein synthesis while thyroxine caused slight but non-significant stimulation.     

青霉素诱发癫痫及耳穴电针后大鼠前脑生长抑素mRNA的变化

应用地高辛标记的生长抑素（ＳＳ）ＲＮＡ探针原位杂交法观察大鼠在青霉素诱发的癫痫及耳穴电针抑制癫痫发作后有关的脑区中ＳＳｍＲＮＡ含量的变化。结果显示,青霉素致痫后２４小时梨状皮质、额叶皮质、扣带回、隔外侧核、杏仁基底核海马ＣＡ１－ＣＡ４区和齿状回颗粒细胞层、多形层等脑区ＳＳｍＲＮＡ的表达显著增加,与正常对照组比较Ｐ＜００５。耳穴电针抑痫（８０１００ＨＺ,６ｍＡ）后额叶皮质、杏仁基底核、海马齿状回、前梨状皮质ＳＳｍＲＮＡ的表达明显减少,Ｐ＜００５。提示前脑结核中的生长抑素与青霉素致痫和耳针抑痫有关。     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

CHOLINERGIC STIMULATION OF PHOSPHOLIPID LABELLING FROM [32P]ORTHOPHOSPHATE IN GUINEA-PIG CORTEX SYNAPTOSOMES IN VITRO: SUBSYNAPTOSOMAL LOCALIZATION

Abstract— Subsynaptosomal localization of stimulation of phospholipid labelling by cholinergic agents was investigated. Synaptosomes prepared from guinea-pig cortex were incubated with [³²P]orthophosphate in the presence or absence of 10⁻³ m carbamylcholine. Following incubation and osmotic shock, lysed synaptosomes were subjected to density gradient fractionation. Subsynaptosomal fractions were examined by electron microscopy and analysed for enzyme activities and ³²P-labelled lipids.
In the absence of carbamylcholine, labelled phosphatidate and phosphatidylinositol were recovered in layers and interfaces A, B, C and D formed over 0.9, 1.1, 1.2 and 1.3 m sucrose, with highest amounts of label in fractions C and D for both lipids. Carbamylcholine induced the greatest increment in these two labelled lipids in fractions A and B. This distribution correlated with the presence of acetylcholinesterase activity and membrane ghosts. No correlation was found among the four fractions between the induced increase in labelling and succinic dehydrogenase activity or with the abundance of mitochondria, synaptic vesicles, or cytoplasmic fragments identified by electron microscopy. In contrast with the increases seen in phosphatidylinositol and phosphatidate labelling, carbamylcholine caused a decrease in ³²P-labelling of the polyphosphoinositides, and this effect was seen primarily in the heavier subsynaptosomal fractions, C and D.     

METABOLISM OF BEDS OF MAMMALIAN CORTICAL SYNAPTOSOMES: RESPONSE TO DEPOLARIZING INFLUENCES

Abstract— The metabolic properties of synaptosome beds (deposits positioned between nylon gauzes) were studied. They respired, glycolysed, produced ATP and phosphocreatine, and metabolized [U-¹⁴C]glucose to glutamate, aspartate, alanine and GABA at similar rates to synaptosome suspensions. Metabolic inhibitors caused massive loss of amino acids from the beds. Synaptosome beds also responded metabolically to electrical pulses; respiration and lactate production increasing by 40 per cent. Differential release of glutamate, aspartate and GABA occurred during electrical stimulation, maximum release being after 10–15 min of stimulation. This differential release also occurred when medium potassium was increased. Omitting and chelating calcium reduced or abolished this response with both forms of stimulation. Including amino acid analogues (β-aminobutyric acid, α, γ-diaminobutyric acid and N -acetyl glutamic acid) in the incubation medium changed the patterns of amino acids present in the medium, indicating that under normal conditions active amino acid uptake processes are occurring in synaptosomes. Tetrodotoxin and ouabain also interfered with amino acid release without greatly affecting the response to stimulation. Cerebral cortex slices incubated between gauzes also showed a glycolytic response to electrical stimulation. GABA was the only amino acid showing a significant increase in the amount released with both potassium and electrical stimulation of the slices.     

POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

PROXIMO-DISTAL TRANSPORT OF [14C]NOR-ADRENALINE AND PROTEIN IN SYMPATHETIC NERVES

Abstract— —Both [¹⁴C]noradrenaline and [¹⁴C]leucine were injected into the coeliac ganglia of cats in an attempt to label the noradrenaline and protein of the granular vesicles, so that their movement in the splenic nerves could be followed.
When a constriction was placed on the nerves, labelled noradrenaline and protein accumulated just proximal to it, but there was no such accumulation below it, nor above a second, more distal constriction placed on the same nerve. This indicated that a neural transport mechanism, rather than uptake from the circulation, was responsible for the accumulation.
Peaks of labelled noradrenaline and protein were observed to move down the axon at about 5 mm/hr. In addition a slow moving component of axonal protein, advancing at about 1 mm/day, was detected.
The results demonstrate a rapid proximo-distal movement of noradrenaline and protein which could represent the transport of granular synaptic vesicles from their site of manufacture in the cell body to their site of storage in the nerve terminals within the spleen.     

ACTIONS OF NEUROHUMORAL AGENTS AND CEREBRAL METABOLITES ON OUTPUT OF ADENINE DERIVATIVES FROM SUPERFUSED TISSUES OF THE BRAIN

—Adenine nucleotides of guinea-pig neocortical tissues were labelled by prior incubation with [¹⁴C]adenine and excess of adenine was then removed by superfusion with precursor-free media. During continued superfusion labelled adenine derivatives were released at a stable rate of about 0·05 per cent of the tissue ¹⁴C/min and this rate was increased about five-fold by electrical stimulation. Various compounds, including some known to increase the cyclic AMP content of cerebral tissues, were examined for action on the release of [¹⁴C]adenine derivatives from the tissue and also on the rates of lactate production by the tissue, both before and during electrical excitation. The tissue content of adenine nucleotides following exposure of the tissue to these compounds was also determined. Noradrenaline, γ-aminobutyrate and acetylcholine together with carbamoylcholine at the concentrations examined were without effect on the release of ¹⁴C compounds from the tissue. Also, noradrenaline and γ-aminobutyrate caused no alteration in lactate production but brought about some decrease in the adenylate energy charge of the tissue. Histamine, 100 μm , brought about a small but consistent increase (35 per cent) both in release of ¹⁴C-compounds and lactate output, while reducing the adenylate energy charge of the tissues. l -Glutamate at 5 mm decreased the tissue adenylate energy charge to a greater extent than did histamine; it increased the release of ¹⁴C-compounds seven to eight-fold and similarly increased the tissues' rates of lactate production. Lower concentrations of glutamate had smaller effects. In those cerebral tissues whose cyclic AMP content is increased by l -glutamate, the increase is probably brought about by intermediation of released adenosine.     

SODIUM AND THE FLUX OF CALCIUM IONS IN ELECTRICALLY-STIMULATED CEREBRAL TISSUE

Abstract— The rate of efflux of ⁴⁵Ca²⁺ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of ⁴⁵Ca²⁺ from the more stable pool and the time course for the redistribution of Na⁺ and K⁺ paralleled that for the increased efflux of Ca²⁺. This effect of stimulationwas dependent on the presence of Na⁺ in the incubation medium. Lack of Na⁺ in the medium during loading of the slices with ⁴⁵Ca²⁺ increased uptake but on subsequent transfer to a medium containing Na⁺, electrical pulses failed to increase the rate of efflux of ⁴⁵Ca²⁺. In unstimulated slices, the rate of efflux of ⁴⁵Ca²⁺ was dependent upon the concentration ratio of Na⁺ to Ca²⁺ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of ⁴⁵Ca²⁺ that occurred during electrical stimulation but exerted no effect on Ca²⁺-Ca²⁺ exchange. Our results suggest that there is a Na⁺-dependent turnover of Ca²⁺ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na⁺-Ca²⁺ interaction in the regulation of neurotransmitter function is discussed.     

Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [¹⁴C] leucine into protein, and accumulation of⁴⁵Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassiumor ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. These effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called reversed Pasteur effect of brain slices incubated in high-potassium media.     

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 (K_m= 0.7 mm ) and high affinity (K_m= 27?30 μM) processes, the uptake of d -glutamate occurs only by a low affinity (K_m= 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 RIBOSOMAL MATERIAL IN INCUBATED AND ELECTRICALLY STIMULATED CEREBRAL SLICES

Abstract— The distribution of ribosomal fractions has been examined in fresh cerebral cortex tissue and in slices maintained in vitro both with and without electrical stimulation. The electrical stimulation used was of a type that has previously been shown to diminish amino acid incorporation into protein.
Membrane-bound and free fractions were obtained and the ratio of their RNA contents were, for the control tissue 3, and for the electrically stimulated tissue 1 , 8. Electrical stimulation was found to decrease the Mg²⁺ binding affinity of the free. fraction but was without effect on the bound fraction. Stimulation was also found to increase the leakage of soluble protein and RNA from the tissue and its accumulation in the incubation medium.     

ON THE INVOLVEMENT OF INORGANIC IONS IN THE EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND NOREPINEPHRINE

Abstract— The loss of GABA, norepinephrine and serotonin and the uptake of GABA (in the presence of 1 mM-GABA) and the effect of GABA on the loss of norepinephrine and serotonin were investigated in rat midbrain slices incubated in media of various compositions. In a medium of low Na⁺ concentration the loss of serotonin from incubated slices was markedly inhibited while that of norepinephrine and GABA was significantly increased. Conversely the most pronounced loss of serotonin from slices was observed on the addition of ouabain to a medium of a balanced ionic composition. Whereas the loss of serotonin from slices increased in a medium of high K⁺ content, it was significantly reduced after 45 min incubation in a high K⁺-low Na⁺ medium. In all the modified media used, a significant loss of norepinephrine was observed while that of GABA was not affected by the omission of Ca2⁺ and was slightly reduced in the absence of K⁺. GABA enhanced the loss of norepinephrine and inhibited that of serotonin in a high-K⁺ medium and in one with a balanced ionic composition. A deficiency of Na⁺ in the medium had a differential effect on the loss of norepinephrine and serotonin similar to that observed with 1 mM-GABA. These results suggest that Na⁺ may be of crucial importance in the release of serotonin from midbrain slices and that an enhancement of the Na+ extrusion mechanism at the synaptosomal level may be involved in the effect of GABA on brain monoamines.