Dopamine Transporter-Dependent and -Independent Endogenous Dopamine Release from Weaver Mouse Striatum In Vitro

Abstract: The weaver mutant mouse (wv/wv) has an ～70% loss of nigrostriatal dopamine (DA) neurons, but the fractional DA release evoked by amphetamine (but not a high potassium level) has been shown to be greater from striatal slices of the weaver compared with +/+ mice. In the present work we tested the hypothesis that fractional DA release from weaver striatum would be greater when release was mediated by the DA transporter. Serotonin (5-HT)-stimulated fractional DA release was greater from weaver than from +/+ striatum. The release evoked by 5-HT in the presence of 10 µM nomifensine (an antagonist of the DA transporter) was less than in its absence, but the difference between weaver and +/+ striatum remained. In the presence of nomifensine, 1-(m-chlorophenyl)biguanide, classified as a 5-HT₃ agonist, also induced a greater fractional release from weaver compared with +/+ striatum. When veratridine was used at a low concentration (1 µM), the fractional evoked release of DA was higher from the weaver in the presence and absence of nomifensine. These findings suggest that the reason for the difference in the responsiveness of the two genotypes to these release-inducing agents is not related to DA transporter function.     

The Tyrosine Kinase Inhibitor Genistein Increases Endogenous Dopamine Release from Normal and Weaver Mutant Mouse Striatal Slices

Abstract: Protein tyrosine kinases that are known to have major roles in the control of cell growth and transformation are abundant and have numerous phosphoprotein substrates in the adult CNS. Although less well characterized than serine/threonine kinases, tyrosine kinases are also concentrated in the synapse. The effect of genistein, a selective inhibitor of tyrosine kinase activity, was examined on the in vitro release of endogenous dopamine (DA) from superfused mouse striatal slices. Fractional release of DA was significantly increased over basal release levels by genistein (100 and 200 µ M ). The effect was concentration dependent and rapidly reversible on washout of the kinase inhibitor. No significant change from basal release levels was observed with two structural analogues of genistein that do not inhibit tyrosine kinase activity at the same concentration. We have previously described alterations in basal and evoked DA release from the striatum of the weaver ( wv/wv ) mutant mouse, and genotypic differences in fractional release were also observed with genistein stimulation. The total evoked release was 25–50% greater from the wv/wv striatum. These results suggest a modulatory role for tyrosine kinase activity in neurotransmitter release and perhaps an alteration of kinase-regulated mechanisms in the DA-deficient wv/wv striatum.     

Characterization of the effects of serotonin on the release of [3H]dopamine from rat nucleus accumbens and striatal slices

The effect of serotonin agonists on the depolarization (K⁺)-induced, calcium-dependent, release of [³H]dopamine (DA) from rat nucleus accumbens and striatal slices was investigated. Serotonin enhanced basal³H overflow and reduced K⁺-induced release of [³H]DA from nucleus accumbens slices. The effect of serotonin on basal³H overflow was not altered by the serotonin antagonist, methysergide, or the serotonin re-uptake blocker, chlorimipramine, but was reversed by the DA re-uptake carrier inhibitors nomifensine and benztropine. With the effect on basal overflow blocked, serotonin did not modulate K⁺-induced release of [³H]DA in the nucleus accumbens or striatum. The serotonin agonists, quipazine (in the presence of nomifensine) and 5-methoxytryptamine, did not significantly affect K⁺-induced release of [³H]DA in the nucleus accumbens. This study does not support suggestions that serotonin receptors inhibit the depolarization-induced release of dopamine in the nucleus accumbens or striatum of the rat brain. The present results do not preclude the possibility that serotonin may affect the mesolimbic reward system at a site which is post-synaptic to dopaminergic terminals in the nucleus accumbens.     

cDNA approaches to isolation of the mouse mutant weaver gene

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a subtracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a subtracted cDNA library.Special issue dedicated to Dr. Sidney Ochs.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

(Na++K+)-adenosinetriphosphatase in the brain of Shiverer (Shi/Shi) mice

The myelin-deficient Shiverer (Shi/Shi) mutant mouse may be a useful model in assessing the dependence of brain (Na⁺+K⁺)-ATPase concentration and composition on myelin membrane formation. Brain microsomal membranes from age-matched control (+/+) and Shiverer (Shi/Shi) mice were fractionated by differential centrifugation and sucrose gradient sedimentation. No reduction in (Na⁺+K⁺)-ATPase specific activity was measured in whole homogenates, high-and low-speed fractions or gradient fractions from brains of Shi/Shi mice as compared to those of +/+ mice. In addition, sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antisera specific for mouse brain (Na⁺+K⁺)-ATPase revealed no significant difference in catalytic subunit composition between fractions of +/+ and Shi/Shi brains. The similar results obtained for both +/+ and myelin-deficient Shi/Shi mice suggest that myelin contributes little to total brain (Na⁺+K⁺)-ATPase.     

Taurine Release Is Enhanced in Cell-Damaging Conditions in Cultured Cerebral Cortical Astrocytes

The release of preloaded [³H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K⁺ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2-disulphonate) and SITS-(4-acetamido-4-isothiocyanostilbene-2,2-disulphonate) sensitive CI^– channels, since these blockers were able to reduce both basal and K⁺ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K⁺ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.     

Characterization of Tritiated Noradrenaline Release from the Rat Preoptic Area with Microdialysis In Vivo

Abstract: Present techniques are unable to provide a sensitive and accurate index of noradrenergic activity in the rat preoptic area. In this study, we have examined the brainstem A₁ noradrenergic input to the preoptic area using a new technique whereby [³H]noradrenaline is preloaded into the preoptic area and release of radioactivity from this region is measured subsequently using microdialysis in vivo. Electrical stimulation of the ipsilateral A₁ area for 20 min at 5, 10, and 15 Hz evoked significant increases in dialysate radioactivity that were repeatable and frequency-dependent. After removal of calcium from the perfusion medium, basal release of radioactivity was markedly reduced and the effect of A₁ stimulation abolished. Changing to a 100 mM K⁺ medium evoked an increase in the release of radioactivity that was sixfold greater than that seen after A₁ stimulation. Separation of the dialysate with HPLC showed that 33% of the increase in measured radioactivity after A₁ stimulation was directly attributable to [³H]noradrenaline and the remainder to the metabolites vanillylmandelic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol. In contrast, the increase in radioactivity after K⁺ depolarization was due almost completely to [³H]noradrenaline. Addition of 10 μM clonidine to the perfusion medium markedly reduced basal release of radioactivity, but had no effect on evoked release following A₁ stimulation. Conversely, perfusion with 10 μM yohimbine had no effect on basal release, but significantly increased evoked release after A₁ stimulation. These results now provide a characterization of noradrenergic activity in the preoptic area and indicate the importance of the A₁ noradrenergic input to this region. The technique of measuring radioactivity with microdialysis after preloading with [³H]noradrenaline provides a relatively simple, sensitive index of noradrenergic activity in vivo with good temporal resolution.     

Nigral and Striatal Comparative Study of the Neurotoxic Action of 1-Methyl-4-Phenylpyridinium Ion: Involvement of Dopamine Uptake System

Abstract: Microdialysis was used in a comparative study of the neurotoxic action of MPP⁺ in the absence or presence of nomifensine (20 µM) in the striatum and substantia nigra. Three different concentrations of MPP⁺ (1, 2.5, and 5 mM) were perfused for 15 min at 24 (day 1) and 48 h (day 2) after surgery. The dopamine basal value in the striatum was ～17 fmol/min. Nomifensine (20 µM) stimulated dopamine release to ～170 fmol/min. The increase of dopamine extracellular output in the striatum after MPP⁺ perfusion on day 1 was independent of the concentration of MPP⁺ perfused and of the absence or presence of nomifensine (20 µM), being ～2,500 fmol/min. The dopamine basal value in the substantia nigra was below the detection limit of our HPLC equipment. Nomifensine (20 µM) stimulated dopamine release to ～6.3 fmol/min. The increase of dopamine extracellular output in the substantia nigra was MPP⁺ dose-dependent (1 mM, 75 fmol/min; 2.5 mM, 150 fmol/min; and 5 mM, 250 fmol/min) and independent of the presence or absence of nomifensine. On day 2, the presence of nomifensine on day 1 produced a total protection against MPP⁺ (1 mM) perfusion in the striatum, which was not observed against MPP⁺ (5 mM). MPP⁺ (1 mM) did not produce any neurotoxic action in the substantia in the absence or presence of nomifensine. The MPP⁺ (2.5 mM) effect on dopamine extracellular output in the absence of nomifensine (20 µM) in the substantia nigra on day 2 was similar to that of MPP⁺ (1 mM) in the striatum. The presence of nomifensine (20 µM) partially prevented the neurotoxic effect of MPP⁺ (2.5 mM) on dopaminergic cell bodies/dendrites in the substantia nigra. The MPP⁺ (5 mM) effect on dopamine extracellular output was similar in both structures studied in the absence or presence of nomifensine on day 2. These results suggest that terminals in the striatum are more sensitive to the neurotoxicity of MPP⁺ than cell bodies/dendrites in the substantia nigra.     

Potassium-Stimulated Taurine Release and Nitric Oxide Synthase Activity During Quinolinic Acid Lesion of the Rat Striatum

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K⁺-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K⁺-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K⁺-induced taurine release. We conclude that a substantial part of K⁺-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K⁺-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.     

Effects of systematically administered lithium on tryptophan transport and exchange in plasma-membrane vesicles isolated from rat brain

The effect of lithium on the sodium-dependent high-affinity system for tryptophan uptake was examined in plasma membrane vesicles derived from rat brain. We demonstrated that Na⁺ could be replaced by lithium in the external medium and the presence of lithium produced an increase in theV _max of the tryptophan transport whereas it had no significant effect on theK _m for the substrate. Plasma membrane vesicles derived from synaptosomes obtained from long-term lithium-treated rats are able to accumulate tryptophan to a greater extent than normal rats and maintain a more negative membrane potential than controls. Our data support the idea that the stimulation by lithium of the high-affinity uptake system for tryptophan by maintaining adequate membrane potentials across the membrane, could lead to the stabilization of serotonin production, as has been demonstrated in long termlithium treatment.     

Characteristics of GABA Release in Mouse Brain Stem Slices under Normal and Ischemic Conditions

GABA is known to be the inhibitory neurotransmitter in the majority of brain stem nuclei. The release of GABA has been extensively studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release in the brain stem have not been systemically characterized. The properties of preloaded [³H]GABA were now investigated in mouse brain stem slices, using a superfusion system. The basal release was enhanced by K⁺ stimulation (50 mM K⁺) and under various cell-damaging conditions (ischemia, hypoglycemia, the presence of free radicals and metabolic poisons). No K⁺-stimulated release was discernible in the absence of Ca²⁺, indicating that the release was at least partly Ca²⁺-dependent. Moreover, the release was increased when Na⁺ or Cl⁻ was omitted from the superfusion medium. GABA and β-alanine stimulated the release, confirming the involvement of the reversed function of GABA transporters. Incubation of the slices with the anion channel inhibitors diisothiocyanostilbene and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate and with the Cl⁻ uptake inhibitor 9-anthracenecarboxylic acid also reduced GABA release, demonstrating that a part of it comprises leakage through anion channels. All these mechanisms were involved in the ischemia-induced GABA release, which was over 4-fold greater than the release in normoxia. Contrary to the other brain areas, GABA release in the brain stem was not affected by ionotropic glutamate receptors but may be modulated by metabotropic receptors. This ischemia-induced GABA release might constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions. Special issue dedicated to Dr. Simo S. Oja     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Morphine Activates ω-Conotoxin-Sensitive Ca2+ Channels to Release Adenosine from Spinal Cord Synaptosomes

Abstract: Morphine-induced release of adenosine from the spinal cord is believed to contribute to spinal antinociception. Although this release is Ca²⁺ dependent, little is known of the nature of this dependence. In this study, the effects of the dihydropyridine L-type Ca²⁺ channel agonist Bay K 8644 and the antagonist nifedipine, the N-type Ca²⁺ channel antagonist ω-conotoxin, and ruthenium red, a blocker of Ca²⁺ influx induced by capsaicin, on release of adenosine evoked by morphine were determined. The effect of partial depolarization with a minimally effective concentration of K⁺ on morphine-evoked release of adenosine also was examined. Morphine 10^?5-10^?4M produced a dose-dependent enhancement of adenosine release from dorsal spinal cord synaptosomes. Following the addition of 6 mM K⁺ (total K⁺ concentration of 10.7 mM), 10^?6M morphine also enhanced release, and an additional component of action at 10^?8M was revealed. Release was Ca²⁺-dependent as it was not observed in the absence of Ca²⁺ and presence of EGTA. Bay K 8644 (10 nM) and nifedipine (100 nM) had no effect on the release of adenosine evoked by morphine, but ω-conotoxin (100 nM) markedly reduced such release in both the absence and the presence of the additional 6 mM K⁺. Morphine-evoked adenosine release was not altered in the presence of a partially effective dose of capsaicin, nor by ruthenium red. These results indicate that morphine can stimulate two distinct phases of adenosine release from the spinal cord (nanomolar and micromolar), and that both phases of release are due to Ca²⁺ entry via ω-conotoxin-sensitive N-type Ca²⁺ channels.     

Neuronal differentiation in cultures of weaver (wv) mutant mouse cerebellum

In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7- and 8-day-old normal ( + / + ), heterozygous weaver ( + /wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly-L-lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by “birthday” radiolabeling techniques) survived by Day 3. Cell death was less extensive in + /wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in + /wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class with time; (c) the lengths of the longest processes formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 μm for + / +, + /wv, and wv/wv granule cells, respectively.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

Distribution of Glutamate Receptors of the NMDA Subtype in Brains of Heterozygous and Homozygous Weaver Mutant Mice

In weaver mice, mutation of an G-protein inwardly rectifying K⁺ channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) were examined by autoradiography with [³H]MK-801 in 36 brain regions from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild type (+/+) mice. In wv/+ and wv/wv mutants labelling decreased in cortical regions, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus and in the cerebellar granular layer. The reductions in [³H]MK-801 binding were particularly specific in the cerebellar granular layer of wv/wv mutants, but an ubiquitous altered NMDA receptor topology was revealed in other brain regions. Abnormal developmental signals, or aberrant cellular responses, may underlie widespread NMDA receptor reductions, while in cerebellar cortex they could be lacking due to the massive loss of cerebellar granule cells.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

Effects of des-tyr1 -γ-endorphin on dopamine release from various rat brain regions in vitro

In rat striatum, nucleus accumbens and frontal cortex slices 6×10^?8M of the potential neuroleptic peptide des-Tyr-γ-endorphin (DTγE) did not affect basal dopamine release but depressed K⁺-evoked release. Haloperidol at 5×10^?6M increased both basal and K⁺-induced release in striatal and nucleus accumbens slices whereas it increased only basal dopamine release in frontal cortex slices. At 5×10^?8M haloperidol, however, had no effect. It is concluded that DTγE may decrease dopaminergic activity in the brain by depressing depolarization-induced dopamine release, possibly via a presynaptic mechanism.