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.     

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.     

HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY AND SOURCES OF RELEASE

Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na⁺ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca²⁺-dependent in cortical slices. When barium ions replaced Ca²⁺ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca²⁺ -free hyperosmolal medium did not induce stimulation. The increased release of α-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not Ca²⁺-dependent.
GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with β-alanine or L-2,4-diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.
The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K⁺-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca²⁺ -dependent response to osmotic equilibration. The observation that pre-treatment with β-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

Protection and Potentiation of 1-Methyl-4-Phenylpyridinium-Induced Toxicity by Cytochrome P450 Inhibitors and Inducer May Be Due to the Altered Uptake of the Toxin

Abstract: Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP⁺). Incubation of mouse brain slices with various concentrations of MPP⁺ (1–100 µ M ) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP⁺-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP⁺. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP⁺ into the slices. In contrast, MPP⁺ uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP⁺ in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices.     

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.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

UPTAKE OF CALCIUM BY SUBCELLULAR FRACTIONS ISOLATED FROM OUABAIN-TREATED CEREBRAL TISSUES

Abstract— Slices of cerebral cortex were incubated in medium containing 0·75 or 2·8 mM ⁴⁵CaCl₂, in the presence or absence of 0·01–0·1 m m -ouabain. Ouabain induced accumulation of calcium by slices to a maximum of 4 μmoles/g of tissue/hr (0·75 m m -CaCl₂ in the medium) and to 8 μmoles/g of tissue/hr (2·8 m m -CaCl₂ in the medium). Accumulation of Ca²⁺ occurred more slowly than loss of K⁺ from the slices and more closely resembled the pattern of Na⁺ uptake.
Mitochondrial fractions isolated from ouabain-treated slices contained significantly more calcium than controls. Inclusion of EDTA in the homogenization medium resulted in decreased amounts of particulate-bound calcium.
The effect of ouabain on accumulation of calcium is discussed with regard to possible relationships to processes of active and passive transport.     

Maturation of Potassium-Stimulated Respiration in Rat Cerebral Cortical Slices

Abstract: Maximal dinitrophenol-stimulated respiration and K⁺-stimulated respiration were measured polarographically in cerebral cortical slices taken from rats aged 2–60 days. Increasing K⁺ concentrations produced an increase in respiration in slices from animals aged 15 days and older, but not in slices from animals aged 10 days and younger. Dinitrophenol-stimulated respiration, or the maximal respiratory capacity of the tissue, showed a similar increase between 10 and 15 days of age. At each age the maximal respiratory capacity was 6–8 ng at 0/mg protein min greater than the maximal K⁺-stimulated respiration.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Enhanced potassium uptake and phosphatidylinositol -phosphate turnover by hypertonic mannitol shock

A hypertonic mannitol shock enhanced K⁺ uptake by Beta vulgaris L. (cv. early flat Egyptian) storage tissue slices and also increased the inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃) content of the slices as well as of Sorghum bicolor L. (cv. Hazera) and Vigna radiata L. (cv. unknown) roots. K⁺ uptake by B. vulgaris slices could be enhanced, in the absence of mannitol, by application of effectors that mimic products of the phosphatidylinositol 4,5-bisphosphate (PIP₂) turnover cycle. Maximal Ins (1,4,5)P₃ content was found 10 min after hypertonic induction and maximal K⁺ uptake was obtained 10 min later. The hypertonic mannitol shock, administered to intact B. vulgaris slices, also enhanced the phosphorylation of a 39 kDa protein in the plasmalemma.     

Muscarinic Receptors and Hydrolysis of Inositol Phospholipids in Rat Cerebral Cortex and Parotid Gland

Abstract: Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [³H]inositol, this response could be monitored by measuring the formation of [³H]inositol phosphates. Accumulation of inositol 1,4-biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li⁺ (10 m M ) in the medium completely blocked the hydrolysis of inositol 1-phosphate, partially inhibited inositol 1,4bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5-trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [³H]inositol 1-phosphate in the presence of 10 m M Li⁺. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 ± 0.12. Dose-response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low-affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.     

Acetylcoenzyme A and Acetylcholine in Slices of Rat Caudate Nuclei Incubated with (-)-Hydroxycitrate, Citrate, and EGTA

Abstract: The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 m M K⁺, 0 or 2.5 mM Ca²⁺, and 0, 1, or 10 m M EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used: in experiments with 2.5 m M OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 m M K⁺ and 1 m M EGTA, they had different effects on the content of ACh during incubations in the presence of Ca²⁺. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca²⁺ and 31.2 or 6.2 m M K⁺, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca⁺², is interpreted to be a consequence of the chelation of intracellular Ca²⁺; this interpretation is supported by the observation of a similar effect caused by 10 m M EGTA.     

UPTAKE AND RELEASE OF TAURINE FROM RAT BRAIN SLICES

Abstract— Rapid efflux of [³⁵S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca²⁺ content of the perfusion medium was replaced with Mg²⁺. Uptake of [³⁵S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [³⁵S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed.     

Basic and Regulatory Mechanisms of In Vitro Release of Met-Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K⁺ (28 and 56 m M instead of 5.6 m M ). The K ⁺ -evoked release was totally dependent on the presence of Ca²⁺ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca²⁺-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K⁺-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K⁺ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K⁺-evoked release of MELM.     

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.     

Dual Effects of Anandamide on NMDA Receptor-Mediated Responses and Neurotransmission

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ⁹-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (ΔCa²⁺_NMDA) in rat brain slices. The presence of anandamide reduced ΔCa²⁺_NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa²⁺_NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa²⁺_NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa²⁺_NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.     

Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin-500–750 mouse lethal doses (MLD)-injected into the SN 6 h before preparing the slices significantly reduced the calcium-dependent, potassium-evoked release of [³H]GABA. Endogenous GABA levels in the SN and [³H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium-dependent, potassium-evoked release of [¹⁴C]GABA and also [³H]dopamine, but had no effect on the K⁺-evoked release of [³H]5-hydroxytryptamine or [¹⁴C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.