Release of GABA and taurine from brain slices

Progressin NeurobiologyVol. 38, pp. 455 to 482, 1992 Printed in Great Britain.All rightsreserved

0301-0082/92/$15.00 © 1992PergamonPress plc

RELEASE OF GABA A N D T A U R I N E FROM BRAIN SLICES PIRJO SARANSAARIa n d S. S. OJA Tampere Brain Research Center, Department of Biomedical Sciences, University of Tampere, Box 607, SF.33101 Tampere, Finland

(Received 23 August 1991)

CONTENTS Abbreviations 1. Introduction 2. Methodological considerations 2.1. Release mechanisms 2.2. Release experiments 2.3. Calculation of results 2.4. Tissue preparations 2.5. Release of endogenous vs exogenous compounds 3. Metabolism and release 3.1. Taurine 3.2. Metabolism of GABA 3.3. Releasable pools 3.4. Relation of metabolism to release of GABA 4. Unstimulated release 4.1. GABA 4.2. Taurine 5. Stimulated release 5.1. Electrical stimulation 5.2. Potassium stimulation 5.3. Sodium ion effects 5.4. Calcium dependence 6. Effects of various compounds and drugs 6.1. Excitatory amino acids and agonists 6.2. Drug effects 7. Modulation of GABA and taurine release 7.1. Presynaptic autoreceptors 7.2. Presynaptic heteroreceptors 7.3. Modulation by catecholamines 8. Changes during developing and ageing 9. Taurine and regulation of cell volumes I0. Summary References

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ABBREVIATIONS

1. INTRODUCTION

AOAA AMPA

aminooxyacetate ct-amino-3-hydroxy-5-methylisooxazole-4proprionate ATPase adenosinetriphosphatase 5(2.cyclohexylidineethyl)-5-ethylbarbiturate CHEB CNS central nervous system DADLE D-Ala2-D-leuS-enkephalin D-AP5 o-2-Amino-5-phusphonovalerate DIDS 4,4'-diisothiocyanostilbene-2,2'-disulphonate EDTA ethylenediaminetetraacetate GABA ),-aminobutyrate GABA-T GABA transaminase GAD glutamate decarboxylase NMDA N-methyl-D-aspartate SSAD succinatesemialdehydedehydrogenase TAG taurine antagonist (6-aminomethyl-3-methyi4H- 1,2,4-benzothiadiazine-1,l-dioxide) THA 1,2,3,4-tetrahydro-9-aminoacridine

In this article we review in vitro studies on the release processes of two structurally related amino acids, y-aminobutyrate (GABA, 4-aminobutyrate) and 2-aminoethanesulfonate (taurine) from brain slices. GABA is an established neurotransmitter acting in a great majority of inhibitory synapses in the central nervous system (CNS). The functions of taurine are at present less well characterized. It does not appear to be a classical transmitter in the CNS in the proper connotation of this term but rather a modulator of synaptic activities, a membrane stabilizer and a factor in cell volume regulation (Oja and Kontro, 1983; Huxtable, 1989). Since the literature on GABA is voluminous, and several books and reviews have also appeared on 455

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taurine, we shall focus only on the narrow scope outlined in the title. A glance through the literature on the release of GABA and taurine from brain slices soon reveals a matter which severely hampers any attempt to review the available information. The data originate from different research groups who have chosen their own solutions to many methodological matters. Such a diversity is of definite value as such but, on the other hand, any inferences must rely on fragmentary knowledge obtained by varying means. In particular, comparisons of the properties of GABA and taurine release from brain slices must rely too heavily on studies originating from our laboratory. We are perhaps the only research group who have systematically endeavored to characterize the release of both amino acids in different brain areas at various stages of brain development in experiments carried out with essentially similar techniques. Both GABA and taurine are zwitterions at physiological pH. They have equally ionizable Nterminal amino groups, but the sulfonyl group of taurine is even more strongly ionized than the carboxyl group of GABA. Hence taurine is more hydrophilic and less lipid-soluble than GABA. The voluminous sulfonyl group in the taurine molecule also greatly impedes the passage of taurine across biological membranes. The rates of penetration of taurine into brain cells are therefore generally smaller than those of GABA in different preparations tested. Both compounds are, however, able to generate very high intracellular/ extracellular concentration gradients across cell membranes (Oja et al,, 1977). They are thus effectively taken up by brain slices upon in vitro incubation from the surrounding medium (Iversen and Neal, 1968; Oja, 1971; Bond, 1973). Demonstration of the enrichment of GABA in synaptic structures in vivo has not generally been very successful, whereas taurine is clearly enriched in synaptosomes, in synaptic vesicles in particular, when compared to the concentrations obtained elsewhere in neural tissue (De Belleroche and Bradford, 1973; Kontro et al.. 1980). A further salient feature of taurine is that it tends to be tightly bound to brain membranes, being removable only by effective treatments with detergents (Kontro and Oja, 1987a). As no doubt prevails as to the importance of GABA in the CNS and its role as a neurotransmitter, a thorough comparison of the properties of the release of taurine to those of GABA may also offer clues to the physiological functions of taurine in nervous tissue. In this article we discuss the release processes of both in concert, emphasizing similarities and dissimilarities. Our further aim is to summarize the present pertinent data on the release of GABA and taurine in tissue slices obtained from different regions of the CNS, and to some extent relate the findings on tissue slices to those obtained with other preparations from the CNS. Since the release of GABA and taurine has also to some extent been dealt with in a number of earlier review articles or monographs, we shall concentrate, when possible, mainly on the most recent studies.

2. METHODOLOGICAL CONSIDERATIONS 2.1. RELEASEMECHANISMS Neurotransmitters and neuromodulators are released from preparations of the CNS by several mechanisms in vitro. One of these is spontaneous leakage through intact or damaged cell membranes in the specimens. One may assume that this is a purely physical phenomenon influenced solely by the prevailing concentration and electrical gradients and the physicochemical status of the bordering membranes. A second mechanism involves extrusion of compounds by the agency of specific transport sites in a process reminiscent of the carrier-mediated uptake but acting in the opposite direction. This type of efflux is subject to interference with competing molecules on both cis and trans sides of the membranes, exhibiting homo- and hetero-inhibition or -stimulation. For instance, h o m o - t r a m - s t i m u l a t i o n of the release of taurine or GABA results in homoexchange in which the overall concentrations in the intra- and extracellular compartments remain the same, but any exchange occurring is detectable if either pool is radioactively labeled. In such a case detection of a flux of radioactivity does not indicate the actual occurrence of any mass transfer of substances across cell membranes, a fact which should be borne in mind in interpretation of results, Furthermore, as a third mechanism, depolarization of cell membranes also calls forth a prompt and massive release of transmitter molecules. In most studies depolarization is effected by high extracellular K + concentrations. Demonstration of such a depolarization-evoked release has been considered an indication of the transmitter nature of the compounds studied. In particular, the dependence of the stimulated release on extracellular Ca 2+ has been considered characteristic of a transmitter, since exocytotic emptying of synaptic vesicles is dependent on Ca 2+. Release upon depolarization is generally taken a priori to imply that it originates from neurons, from synaptic terminals in particular, since non-neuronal elements in the brain are widely considered nonexcitable. Such a presumption is not valid in general, however. Moreover, neuromodulators can also be released under the same experimental conditions either from synaptoplasmic or vesicular compartments. Furthermore, when one is experimenting in vitro on tissue slices it is often extremely difficult to demarcate exactly which type of the release process is in question and if all the above processes are in operation simultaneously-as is often the case--to know exactly how the different types of processes are affected by changes in experimental set-up. 2.2. RELEASEEXPERIMENTS The obvious reason for studying the release of neurotransmitters and modulators in vitro is the simplicity of modification of experimental conditions, greater flexibility of experimental design and simpler interpretation of data obtained. Probably the most straightforward way to study the release is to incubate slices in medium periodically changed at short intervals and preserved for analysis. The method is

GABA AND TAURINERELEASE

somewhat laborious, demanding frequent suction of medium out of the incubation vessels or regular transfers of specimens from one vessel to another containing new medium. The advantage is that the preparation can always be subjected to a completely new medium and thus all alterations in medium composition are abrupt. A great disadvantage is the mechanical strain on the slices, which often results in tissue damage or even loss of fragments from the specimens. The majority of release studies on slices have been carried out using superfusion. In this method the slices are kept in a continuous flow of medium in tiny superfusion chambers. The outflowing medium is collected for analysis either manually or preferably by means of a fraction collector. Several superfusion experiments can be carried out simultaneously in parallel, thus permitting studies in which the experiment-to-experiment variation is minimized. Almost all investigators except ourselves have kept the studied slices between nylon nets in order to immobilize the preparations during superfusions. This immobilization minimizes mechanical damage to the preparations, since the nets prevent loss of all macroscopic tissue fragments during the experiments. The method can also be applied to studies on small cell fragments such as synaptosomes, which can be sandwiched as synaptosome beds between the nets. The superfusion medium flows through the nets, passing the slices, and carries along the compounds released. We have been concerned, however, for the sufficient oxygenation of preparations in such an assembly, since oxygen is delivered to them only by the preoxygenated flowing medium. As an alternative, the slices can be kept freely floating under agitation in a small volume of medium (Vahvelainen and Oja, 1972; Korpi and Oja, 1979). Indeed, in such a system the viability of the slices seems to be better preserved and the ionic gradients and the typical properties of transmitter release are well maintained (Korpi and Oja, 1984a). Therefore we have preferred to use it continuously. The reuptake of G A B A and taurine molecules which have been released from the slices into medium often complicates interpretation of results. Both amino acids possess active carrier-mediated transport mechanisms which are concentrative in nature. In the course of experiments the amino acids released gradually accumulate in medium, even though medium may have initially been amino acid-free. It must be kept in mind that in effiux experiments GABA and taurine recovered in medium only represent an 'overflow' of molecules released and then escaped reuptake. There are several ways of minimizing difficulties which arise from the increasing re-entry of G A B A and taurine molecules released from the slices (Oja and Vahvelainen, 1975): (1) The volume of the medium should be kept large in relation to the volume of the slices: (2) Efficient mixing is essential for the rapid dispersion of the released amino acid molecules in medium. Mixing also reduces the thickness of stationary layer of medium adjacent to slice surfaces. The released molecules must traverse this layer in addition to the extracellular spaces in slices to be recognized as 'release': (3) The incubation time should be kept as short as possible. In particular, in

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the case of taurine long-term experiments are however often unavoidable: (4) The medium should be renewed as fast as possible, either by transferring the slices frequently to fresh medium or by changing the surrounding medium effectively (flushes or superfusion): (5) The reuptake can be inhibited by inhibitors of G A B A and taurine transport. It must be borne in mind, however, that such inhibitors cannot be considered inert with respect to release. They affect at least the carrier-mediated component in release which invariably operates in slices in parallel with diffusion and exocytosis. 2.3. CALCULATIONOF RESULTS There occurs slow spontaneous leakage of transmitters from slices upon incubation. Any stimulusevoked release must be extracted from this baseline release in order to assess its absolute or relative magnitude. The simplest straightforward way is to express the stimulated release of a transmitter as excess over this baseline efflux, using percentages or some other quantitative units. However, it is a rule that spontaneous release gradually declines upon prolonged incubation if the studied slices preserve their viability well enough. It may thence prove difficult to estimate the share of spontaneous release during the stimulation period. The magnitude of stimulation will be underestimated if the stimulusevoked release is simply related to spontaneous release during the prestimulation period. We have sought to overcome this difficulty in experiments on the release of labeled G A B A and taurine by taking the logarithm of the amount of label remaining in the specimens at each time point of superfusion and plotting these values as a function of superfusion time (e.g. Kontro and Oja, 1987b,c). An approximate linear relationship then obtains in the plot, and the slope of a straight line fitted to the data by means of least squares estimates the so-called fractional efflux rate constants for the release (Korpi and Oja, 1979). In this process random experimental errors are partially eliminated and the calculated constants can easily be used in quantitative comparisons and subjected to statistical analyses. 2.4. TISSUEPREPARATIONS The release of G A B A and taurine has been studied in vitro with a number of preparations other than

slices, for example tissue pieces, isolated cerebellar glomeruli, nerve ending-enriched fractions (synaptosomes), isolated neurons or fractions enriched in giial cells, cultured neurons and glial cells, mechanically chopped or minced brain tissue or subcellular organelles. We shall discuss results obtained from such preparations insofar as they are pertinent to the present topic, brain slices. Even though synaptosomes and cultured cells have also been frequently used as experimental material, brain slices probably represent the preparations most widely used in studies on the release of G A B A and taurine. They can be easily cut from all brain regions without any timeconsuming manipulations. Moreover, they represent a preparation that may most closely reflect relations prevailing in in vivo conditions, since the tissue

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structure and cellular integrity are largely preserved. Neuronal networks are also potentially in partial working conditions and electrically and chemically excitable. The main disadvantage, on the other hand, is the same structural and cellular complexity of this type of preparation. The partially preserved neuronal circuits often complicate interpretation of the results, for instance, from studies in which the actions of a single type of amino acid receptor have been the subject. On the other hand, slices are by no means equivalent to brain tissue in vivo. Cutting creates artificial interfaces, disrupts a number of tissue elements and damages cellular and subcellular me branes. There is a narrow extracellular space in the brain in vivo, but in incubated slices this tends to increase (Laakso and Oja, 1976) and the slices commonly swell both intracellularly and extracellularly during the course of incubations in vitro. The swelling is greatly influenced by incubation conditions and depends on the source of slices; for instance, slices from the adult brain swell considerably more than slices from immature brain. The latter may under some circumstances even shrink upon incubation (Oja and Vahvelainen, 1975). The origin of transmitter release cannot be defined in a slice preparation. The release may originate from synaptic terminals, but other neuronal cell parts may contribute, to say nothing of the possibility that the studied release originates in part or entirely in glial cells. The mechanisms of release from neurons and glial cells are not similar, however (cf. Jaff6 and Cuello, 1981). Even synaptosome-enriched fractions may be misleading preparations in release experiments. In addition to the transmitter heterogeneity of the isolated synaptic terminals, these fractions often tend to be heavily contaminated by glial cell fragments, gliosomes (Henn, 1976). Well-characterized cultured neurons and glial cells offer a good opportunity to assess separately the release processes of transmitters. However, their behavior may be completely different when the cells are residing in situ in neural tissue. The simultaneous use of slices and some other less complex neural preparations must also be strongly advocated when one attempts to draw firm inferences on characteristics of the release of GABA and taurine. 2.5. RELEASE OF ENDOGENOUS vs EXOGENOUS COMPOUNDS

The vast majority of studies on the release of GABA and taurine have been done using exogenous preloaded labels. The obvious reason is the more time-consuming and laborious nature of the detection methods needed in assessing the release of endogenous compounds. High-pressure liquid chromatography with a high sensitivity is needed to measure endogenous amino acids released. When using radiolabeled GABA and taurine a question of utmost importance must be considered, namely whether the results obtained truly reflect the behavior of the studied endogenous compound or not (of. Figs 1 and 2). The key question is the distribution of the label in tissue and cellular compartments. If the label uniformly enters pools identical to those occupied by the endogenous compound, the exogenous labeled

compound may indeed mimic the behavior of endogenous GABA and taurine. This has not so far been shown to be the case. For example, it appears that exogenous GABA is not released in the same manner as endogenous GABA (Szerb, 1983). The existence has been demonstrated of several GABA pools which are differently fed by different precursors and consequently do not exhibit similar properties with regard to release processes (see Tapia, 1983; Levi, 1984). It appears that the problem of whether or not the recently preloaded exogenous GABA in brain slices is released by depolarizing stimuli similarly to the endogenous pool of GABA is still largely unsettled. In the case of taurine the studies are very scant. Any investigator in the field must be very careful when drawing conclusions on an assumption that the behavior of endogenous and exogenous GABA or taurine is truly similar in CNS preparations,

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FIG. 1. Release of endogenous GABA (--O--) and taurine (--O--) from cerebral cortical slices prepared from young adult, 3 month-old (A) and 3 day-old (B) mice. The slices were first preincubated for 30 min and then superfused as in Oja and Kontro (1989) for 50 min, first 30 rain in normalK + (5 mM) medium and then for 20 min in medium in which 50 mMNa ÷ were replaced by equimolar K +, as indicated by the bar. Mean values of 5 experiments with SEMs. 2.4

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FIG. 2. Release of preloaded labeled GABA ( - - 0 - - ) and taurine (--©--) from cerebral cortical slices prepared from young adult, 3 month-old (A) and 3 day-old (B) mice. The slices had been preloaded for 30 rain with 10 #M GABA or taurine labeled with 3H. They were then superfused as in Oja and Kontro (1987) for 50rain, first 30rain in normal-K + (5 raM) medium and then for 20 rain in medium in which 50 mMNa + were replaced by equimolar K + , as indicated by the bar. Mean values of 8 experiments with SEMs.

GABA ANDTAURINERELEASE 3. METABOLISM AND RELEASE

3.1. TAURINE Taurine seems to be metabolically relatively inert in the brain. It is likely that the formation of taurine in the brain of most species is slow, if it exists at all (Oja et al., 1977). At least, taurine is not broken down in the brain (Oja and Kontro, 1983). Metabolic interconversions in the brain can thus be relatively safely ignored in the case of this compound. 3.2. METABOLISMOF GABA The synthesis route for GABA in the brain originates from glutamate, which is decarboxylated to GABA by the enzyme glutamate decarboxylase (EC 4.1.1.15, GAD). GABA is subsequently broken down by GABA-aminotransferase (EC 2.6.1.19, GABA-T) and suceinatesemialdehyde dehydrogenase (EC 1.2.1.24, SSAD). The turnover of GABA in the brain is fast and must also thence be considered in studies on the release of GABA. The very active GABA-T in many brain regions is a factor which often greatly complicates interpretation of data from experiments on the release of GABA. In general, it is not possible to obtain correct results on the release of labeled GABA from neural tissue preparations unless either GABA-T is completely inhibited or the radioactive authentic GABA is properly separated from all possible radioactive metabolites generated during experiments. In order to block the action of GABA-T, brain preparations have been exposed to various appropriate inhibitors. In the majority of cases this has been aminooxyacetic acid (AOAA). A concentration of 0.01 mM of AOAA has often been considered sufficient to block the metabolism of GABA (see Bedwani et al., 1984, for references). However, this concentration of AOAA has not been large enough to completely inhibit GABA-T in synaptosomal preparations from the rat cerebral cortex (Gardner and Richards, 1981). In slices from the cerebral cortex, cerebellum, striatum and brain stem from adult mice a concentration of 0.01 mM has likewise proved inadequate (Oja and Kontro, 1988). Only in developing mice, in which the enzyme activity is relatively low, was complete inhibition obtained, except in the cerebellum where the activity of GABA-T is highest. In all cases and at all ages 0.1 mM AOAA was able to block GABA breakdown in our studies. It should be noted that if radioactive GABA is separated from labeled metabolites in thin-layer chromatography, one gets a wrong impression of the extent of breakdown of radioactive GABA, since often an overwhelming proportion of radioactivity resides in tritiated water which is volatile and escapes detection in thin-layer chromatography. On the other hand, it should also be noted that even though a very large percentage of the radioactivity released from unstimulated brain preparations is in compounds other than GABA, all stimuli which greatly increase the release of radioactivity preferentially induce the release of radioactive GABA but not that of radioactive metabolites (Oja and Kontro, 1988). Therefore a large proportion of the stimulusinduced release of radioactivity truly represents reJPN 38/5--42

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lease of labeled GABA (Gardner and Richards, 1981; Bedwani et al., 1984; Oja and Kontro, 1988). The use of high concentrations of AOAA cannot be strongly advocated for other reasons. GABA-T inhibitors also often significantly inhibit GAD, though less effectively. Thence, in their presence the intracellular GABA pools may change in size. On the other hand, it has been suggested that the intraterminal concentration and synthesis of GABA could be regulated via product inhibition (Lid6n et al., 1987), wherefore alterations in the synaptic GABA pools may be self-limiting. In cerebral cortex slices 0.05 mM AOAA has increased excitability and induced anomalies in the stimulus-evoked release of GABA (Orrego and Miranda, 1976), and 0.01 mM AOAA considerably reduces the Ca 2÷ dependence of GABA release (Bedwani et al., 1984). The apparent magnitude of K + stimulation of GABA release has also been significantly smaller in the presence than in the absence of AOAA (Oja and Kontro, 1988). In this respect AOAA concentrations of 0.01 and 0.1 mM have caused almost the same reduction in the depolarization-induced release of GABA. Moreover, the spontaneous unstimulated release of GABA seems to be significantly diminished by both 0.01 and 0.1 mM AOAA and in addition the uptake of GABA is significantly inhibited upon prolonged exposure to AOAA (Snodgrass and Iversen, 1973). AOAA may thus also secondarily affect the apparent magnitude of the release of GABA through inhibition of reuptake of GABA already once released from preparations studied. Two other GABA-T inhibitors tested, gabaculine and 7-vinyl-GABA, are likewise not without effects on the K ÷-stimulated release of GABA from rat cerebral cortex slices (Szerb, 1982a; Bedwani and Mehta, 1987). Also the electrically stimulated release of GABA has been shown to be increased by AOAA, gabaculine and 7-vinyl-GABA in slices from the rat medulla oblongata (Kihara et al., 1988). The characteristics of release of labeled GABA are obviously not identical in the presence and absence of GABA-T inhibitors and, moreover, they are differently affected by different inhibitors (Bedwani and Mehta, 1987). GABA-T inhibitors should possibly be totally omitted in future studies, since an arrest of GABA metabolism may distort relations between different releasable GABA pools. 3.3. RELEASABLEPOOLS

Several studies have suggested that even in nerve endings there exist at least two GABA pools (Abe and Matsuda, 1983; Wood et al., 1988). These pools apparently have different metabolic functions. One involves GABA newly synthesized from glutamate by GAD. It is bound to be released into synaptic clefts upon stimulation in a Ca2+-dependent manner. GABA in this pool is not affected by GABA-T. The other comprises GABA recently taken up from the synaptic clefts to be degraded by GABA-T. GABA is released from this pool in a Ca 2+-independent manner (Asakura and Matsuda, 1990). Incubation of cerebral cortex slices with radioactive GABA or taurine apparently labels well their releasable intracellular pools (Oja and Kontro, 1989). The relative amounts of labeled and endogenous

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GABA or taurine released from slices upon potassium depolarization, together with the time courses of the release, were fairly similar in these studies. The only difference was that in those cases when the stimulated release was very large, i.e. in adult mice in the case of GABA and in developing mice in the case of taurine, the release of the label started to decline faster than that of the corresponding endogenous compounds during prolonged stimulation. This discernible difference can be seen when comparing the curves shown in Figs 1 and 2. This may be a sign of preferential labeling of the readily releasable pools, as a clearly greater fraction of radioactivity than that of endogenous total GABA or taurine is released from the slices upon stimulation. Exogenous preloaded GABA has also been shown to be more readily released than endogenous GABA from slices from the rat dentate gyrus, though the effects of various treatments on the release were otherwise qualitatively similar (Nadler et al., 1977). However, the general qualitative parallelism of exogenous and endogenous release validates most of the studies done under similar conditions on the release of exogenous preloaded labels. They seem to admix with the intracellular pools and mimic the behavior of the endogenous compounds. The properties of the release of labeled and endogenous GABA have also been markedly similar in slices from the rat medulla oblongata (Kihara et al., 1989a). On the other hand, some investigators have suggested that they may obtain certain differences in the behavior of labeled and endogenous GABA. For instance, Green et al. (1987) proposed that some convulsion-induced changes in the endogenous releasable pools of GABA may remain undetected if only the release of preloaded labeled GABA from slices from the rat cerebral cortex, hippocampus and striatum are studied. Figures 1 and 2 also demonstrate another aspect of the potential delusiveness of studies using labeled GABA and taurine. For instance, in adult mice the apparent magnitude of K +-stimulated release of labeled GABA seems overwhelmingly great in comparison to that of taurine, even though the molar amounts of endogenous compounds are almost equal. The reason is that the release of labeled compounds indicates only the relative magnitude of the release, not the absolute amount released. Furthermore, the K + stimulated release of taurine is particularly striking in 3 day-old mice due to the very slow efflux of taurine from unstimulated slices from the immature cerebral cortex. At that age the K ÷ stimulation of release of preloaded labeled GABA also appears appreciable, though only negligible molar amounts of endogenous GABA are released. For reasons of this kind we strongly recommend that data obtained with radioactively labeled GABA or taurine be frequently checked and verified by concomitant study of the behavior and responses of their endogenous counterparts.

1977), glutamate (Ryan and Roskoski, 1975; Tapia and Gonzales, 1978; Canzek and Reubi, 1980), glucose (Minchin, 1977; Bradford et aL, 1978; Potashner, 1978) and glutamine. The depolarizationinduced, Ca 2÷-dependent release of GABA derived from glutamine has been shown in several in vitro preparations (Tapia and Gonzales, 1978; Reubi et al., 1978; Reubi, 1980; Szerb, 1984), glutamine being in some reports a better precursor than glutamate (Minchin and Beart, 1975; Canzek and Reubi, 1980). Glutamine is also a good precursor for GABA in vivo (Gauchy et al., 1980). Since newly synthesized GABA is preferentially released upon stimulation (Ryan and Roskoski, 1975; Gauchy et al., 1977), a close relationship must exist between the synthesis and release of GABA, In cerebral cortical slices the spontaneous release of GABA derived from glutamate has been greater than that of GABA synthesized from glutamine (Tapia and Gonzales, 1978) suggesting that endogenous glutamate feeds a GABA pool directly connected with the synthesis-release coupling. Tapia (1983) also further postulates that under resting conditions the activity of G A D is coupled to the release of GABA. This opinion seems to find support from other experimental results. In cerebral cortical slices the degree of inhibition of G A D has shown a high positive correlation with the decrease in spontaneous release of GABA synthesized from glutamate (Tapia and Gonzales, 1978). In rat hippocampal slices the synthesis of GABA occurs in a pool of glutamate which is derived from both glucose and glutamine, the rate of formation of GABA being regulated by the activity of G A D (Szerb and O'Regan, 1986). The electrically evoked release of endogenous GABA is inhibited in slices from the rat medulla oblongata by 3-mercaptopropionate, a G A D inhibitor, whereas the release of preloaded radioactive GABA is not affected (Kihara et al., 1989a). This suggests that inhibition of synthesis can reduce the release of recently synthesized G A B A without affecting the release process per se. Furthermore, superfusion of brain slices with glutamine has failed to increase the evoked release of GABA, while the release of glutamate has been greatly enhanced (Hamberger et al., 1978; Szerb and O'Regan, 1984; t985). Furthermore, the in vivo studies of van der Heyden et al. (1979) have demonstrated that the release of endogenous GABA decreases immediately after superfusion with 3-mercaptopropionate. 4. UNSTIMULATED RELEASE In the matter of definition, we discuss here as 'unstimulated release' the release processes of GABA and taurine in the absence of any specific agent which could cause membrane depolarization, i.e. homoexchange and heteroexchange are dealt with here among other processes which are not purely spontaneous in nature.

3.4. RELATION OF METABOLISM TO RELEASE OF G A B A

The relationships between the metabolism and release of G A B A have been previously extensively discussed by Tapia (1983). Precursors of releasable GABA in the brain include pyruvate (Gauchy et al.,

4.1. GABA The spontaneous efflux of GABA is very slow, being in this respect similar to that of taurine (Korpi et al., 1981). About 81 percent of labeled preloaded

GABA AND TAURINE RELEASE

GABA still remains in cerebral cortical slices after a 50min superfusion with physiological medium (Kontro and Oja, 1987b). In a number of studies on various brain preparations, including slices from different brain regions, the effiux of GABA has been shown to be fomented by exogenous GABA (OIsen et al., 1977; Brennan and CantriU, 1978; Do Nascimento and De Mello, 1985; Kontro and Oja, 1987b,c). These studies have thus unanimously demonstrated that the release of GABA is stimulated by homoexchange. The magnitude of stimulation of the release of GABA by homoexchange is distinctly greater under the same experimental conditions than that of the release of taurine, and apparently dependent on the extracellular concentration of GABA (Kontro and Oja, 1987b). The release of GABA is also strongly stimulated by nipecotate and homotaurine, and moderately by hypotaurine, fl-alanine and 2-guanidinoethanesulfonate, but the efficacy of taurine is very small (Baba et al., 1984; Kontro and Oja, 1987b). The enhancement by nipecotate has been shown to be pronounced (Johnston et al., 1976; Olsen et al., 1978; Brown et al., 1980; Moscowitz and Cutler, 1980; Jaff6 et al., 1984), especially in slices from the cerebral cortex (Szerb, 1982b; Kontro and Oja, 1987b), hippocampus (Jonzon and Fredhom, 1985), caudate nucleus (Limberger et al., 1986a) and medulla oblongata (Kihara et al., 1989a). Only in rat striatal slices has a relatively low extracellular concentration of nipecotate been reported to enhance neither the basal nor the K ÷evoked release of radioactive GABA (Kuriyama et al., 1984). The potentiation of GABA release has been reported to disappear in the absence of extracellular Ca 2+ or in the presence of tetrodotoxin (Szerb, 1982b). This author takes the phenomenon to signify that the enhancement of the stimulation-induced release results from reduced uptake rather than from heteroexchange. It remains open, however, whether or not the ionic requirements of heteroexchange could mimic those of the uptake. The much higher affinity of the transport system for exogenous GABA than for exogenous taurine (Kontro and Oja, 1983) is the obvious reason for the difference in the efficacies of taufine and GABA in the exchange processes. Nipecotate has also in vivo elevated the extracellular levels of GABA and taurine in the hippocampus (Lerma et al., 1984), which is in line with the vast majority of results on brain slices. The efflux of GABA has been enhanced by hypotaurine, which appears to share the same uptake system with GABA in brain slices (Kontro and Oja, 1983). fl-Alanine also employs the same system (Kontro, 1983; Holopainen and Kontro, 1986). A moderate acceleration of the effiux of GABA by fl-alanine from slices has indeed been observed (Hammerstad and Lytle, 1976; Hammerstad et al., 1979; Jaff6 et al., 1984; Kontro and Oja, 1987b), whereas the release of taurine is even more augmented (Kontro and Oja, 1987b). This may be explained by differences in the affinities of the uptake carriers for GABA, taurine and fl-alanine, but it has also been suggested that fl-alanine is exchanged only with glial GABA (Hammerstad and Lytle, 1976; Brennan and Cantrill, 1978). This is not likely, since it has been shown that fl-alanine also inhibits the

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uptake of GABA in cultured neurons (Larsson et al., 1986). Likewise, the uptake of taurine is strongly inhibited by fl-alanine in neuronal (Borg et al., 1979) and synaptosomal preparations (Kontro, 1984). The efflux of both GABA and taurine is generally enhanced by a great number of structural analogs. Most potent here are those compounds which are simultaneously the most potent inhibitors of the uptake (Kontro and Oja, 1981; Kontro, 1984). It has been suggested for GABA that inhibitors activate the membrane carriers which transport GABA from its storage sites to the extracellular space (Johnston et al., 1976; Early et al., 1981) or displace stored GABA by heteroexchange (Levi et al., 1978). The close correlation between inhibition of uptake and stimulation of efflux by structural analogs indicates that the effiux of both amino acids is at least partly mediated by the carriers operating in an outward direction. In keeping with this, GABA has been shown to stimulate the release of taurine in vivo in the rat hippocampus, probably by counter-transport (Lerma et al., 1985).

4.2. TAURINE The spontaneous efflux of taurine is slow from slices from all brain regions so far studied (Oja, 1971; Oja et al., 1981); for example about 74 percent of the original preloaded radiolabeled taurine still remains in cerebral cortical slices after a 50 min superfusion with physiological media (Kontro and Oja, 1987b). Spontaneous efflux from rat brain synaptosomes has been divided into two exponential components with widely differing half-lives, the slower component probably representing release from intrasynaptosomal compartments (Kontro, 1979). The more complex efflux from brain slices cannot, however, be similarly resolved into components (Korpi et al., 1981). The release of taurine can be elicited by exogenous taurine via homoexchange from brain slices (Korpi and Oja, 1984b; Kontro and Oja, 1987b, 1989) and purified synaptosomes (Kontro, 1979), but the response has been generally small and discernible only at relatively high concentrations of extracellular taufine. This apparently results from the rather high transport constant, Kin, in the low-affinity uptake of taurine (Kontro and Oja, 1981, 1983). A number of structural analogs of taurine (hypotaurine, fl-alanine, GABA, 2-guanidinoethanesulfonate, homotaurine and glycine) or uptake inhibitors of GABA (e.g. nipecotate) also enhance the effiux of taurine from synaptosomes and slices, but the proposed taurine antagonist 6-aminomethyl-3-methyl-4H- 1,2,4-benzothiadiazine-l,l-dioxide (TAG) (Yarbrough et al., 1981) is not effective (Kontro and Oja, 1987b). In synaptosomes the stimulation is greater in magnitude when the intrasynaptosomai pools have been flooded by preioading in a medium with a fairly high concentration of taurine. This may mean that the excess of taurine is located predominantly in synaptoplasma and hence more prone to exchange with exogenous taurine than the smaller amounts accumulated from a medium with a low taurine concentration. It is likely--but not experimentally shown--that similar

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relationships exist when brain slices are loaded in media with differing taurine concentrations. At least, exchange with exogenous taurine or taurine analogs (homo- and hetero-trans-stimulation, respectively) is very similar in slices and synaptosomes. Only the baseline efflux rates are several times greater in synaptosomes (Korpi et al., 1983) because these are more 'leaky' preparations, obviously owing to more extensive mechanical damage of cell membranes during preparation. Potassium stimulation increases the release of taufine from brain slices (Oja et al., 1981; Korpi et al., 1981; Kontro and Oja, 1987b,c), but the stimulated release is no further enhanced by taurine analogs (Kontro and Oja, 1989). On the contrary, there may even obtain a slight inhibition of release if slices are exposed to high-K + medium in the presence of taurine analogs. Does this mean that a part of the K ÷ -stimulated release of taurine is in fact accelerated outward-directed, carrier-mediated efflux? We have not yet been able to prove this conclusively. There may also be only a small fraction of the total taurine that is releasable by either K ÷ stimulation or by taurine analogs, since the slices are only partially depleted of taurine even after prolonged experiments (Oja, 1971; Oja et al., 1981; Oja and Kontro, 1989).

5. STIMULATED RELEASE Depolarizing stimuli may increase release of amino acid neurotransmitters and -modulators by facilitating the mechanisms of spontaneous release and/or initiating new specific processes, such as opening of synaptic vesicles and emptying of their contents into synaptic clefts. The abrupt torrent of Ca 2÷ ions into nerve endings caused by depolarization is believed to be the triggering mechanism for the release of neurotransmitters. A demonstration of the depolarizationinduced, Ca 2÷-dependent release of a compound is thus considered the most valid neurochemical criterion of a neurotransmitter function. This aspect of the release of GABA and taurine has been the most intensively studied during recent years. However, the results obtained on this important issue are still partly contradictory and at present somewhat confusing, in particular in the case of taurine. The discrepancies are in our opinion largely due to certain methodological matters, i.e. they result from differences in experimental conditions such as the type of stimulation used, rates of superfusion, composition of incubation media, frequency of sample collection, duration of stimulation periods, and calculation and presentation of results. The demonstration that a release process is Ca 2÷-dependent has created most confusion, since there is generally no easy way to show the 'calcium dependency'. An omission of Ca2 ÷ ions from incubation media, adding Ca 2÷ chelators or Ca 2÷ blocking agents, all have their disadvantages and can make interpretation of results complicated or even impossible (see below). In release studies of GABA and taurine, high concentrations of potassium ions in medium, electrical stimulation or veratridine alkaloids have been used most frequently as depolarizing stimuli. The validity of these agents in mimicking physiological

events has been previously expertly discussed by Levi (1984). The method most often used, K + -stimulation, has been claimed to cause a relatively nonspecific release of transmitters and neuromodulators. Veratridine is known to depolarize excitable cells by opening Na + channels with a concomitant influx of Na + and Ca 2÷ and effiux of K +, which effects are all abolished by tetrodotoxin. These ionic fluxes evoked by veratridine may more closely resemble physiological depolarization than the effects of high K ÷ concentrations.

5.1. ELECTRICALSTIMULATION Electrical stimulation has been frequently used in the past to elicit release of GABA and taurine from brain slices and synaptosomes. Electrical stimulation of brain slices is experimentally rather difficult to accomplish properly and there is so far no unanimity on the characteristics of applied stimulation. These have varied considerably from study to study. Electrical stimulation has generally been a successful means to elicit release of GABA from brain slices (Levi, 1984). The electrical parameters for the optimal stimulation of different transmitters are not alike. To date, with electrical field stimulation using rectangular pulses, 4 V/cm at 3 Hz has been sufficient to cause a maximal release of norepinephrine, whereas 8 V/cm was needed to cause a maximal release of GABA from rat hippocampal slices (Jonzon and Fredholm, 1985) and 9 V/cm at 5 Hz from rabbit caudate nucleus slices (Limberger et al., 1986a). On the other hand, electrical field stimulation with steeply rising and exponentially falling pulses has failed to elicit any release of taurine from cerebral (L/ihdesm~iki and Oja, 1972) and spinal cord slices from adult rats (Collins and Topiwala, 1974) but has elicited release from cerebral slices (Kaczmarek and Davison, 1972). Electrical stimulation increases several-fold the release of GABA from slices from different brain areas (see Oja et al., 1977, for references). In some more recent studies electrical stimulation has induced release of GABA, for example, from hippocampal (Jonzon and Fredholm, 1985) and medullar slices (Kihara et al., 1989b). These electrical effects may not be strictly specific for neurotransmitters. No induced release is observed from non-neural tissue, e.g. slices of liver and kidney (Katz et al., 1969), but the efflux of lysine, proline and cycloleucine from rat spinal cord slices (Hammerstad et al., 1971) and that of valine from chopped guinea-pig cerebral cortex (Jones and Banks, 1970) can be enhanced by electrical pulses. However, electrical stimulation has failed to affect the release of leucine, ~-aminoisobutyrate and urea from cerebral cortical slices (Srinivasan et al., 1969), or leucine and glycine from the substantia nigra (Okada and Hassler, 1973). Although there are no significant regional differences in the spontaneous efflux of GABA from slices from various brain areas, the electrically evoked release is considerably greater from slices from the striatum, diencephalon and cerebellum than from frontal cortical slices (Katz et al., 1969). These regional variations in the efflux of GABA parallel the endogenous concentrations of this amino acid (Oja et al., 1977), as was also shown with

GABA ANDTAUmr~ RELEASE the spinal cord, cerebral cortex and cerebellum of the rat (Hammerstad et al., 1971). With regard to the type of electrical pulses used, it has been reported that rectangular pulses evoke a Ca2+-dependent release of radioactive GABA from brain slices, whereas sine-wave field stimulation induces the release of noradrenaline but not that of GABA (Srinivasan et al., 1969; Orrego and Miranda, 1976). On the other hand, sine-wave stimulation has induced Ca2+-dependent release of endogenous GABA from brain slices, while the release evoked by rectangular pulses has been reported to be Ca 2÷-independent (Valdes and Orrego, 1978). The omission of Ca 2÷ from the incubation medium reduces the response of GABA to electrical pulses in many brain areas (see Oja et al., 1977). The dependence of the electrically induced release of GABA on Ca 2÷, however, may manifest itself only after sufficient depletion of endogenous Ca 2÷ (Hammerstad et al., 1971). The enhancement in the efflux of GABA by electrical stimuli from brain slices (Srinivasan et al., 1969) and from the isolated spinal cord (Roberts and Mitchell, 1971) gradually diminishes when the concentration of Mg 2÷ in medium is increased (up to 1 x 10 -2 mol/1). Mg 2÷ ions probably compete with Ca 2÷ ions in the release of neurotransmitters. Superfusion of spinal cord slices with media containing high K ÷ or low Na ÷ concentrations has no effect on the electrically evoked release of GABA (Hammerstad et al., 1971). Such agents as tetrodotoxin and lidocaine, which block the influx of Na ÷ into rat cortical slices, do not generally diminish the electrically stimulated release of GABA (Hammerstad and Cutler, 1972). However, electrical field pulses have elicited Ca2+-dependent and tetrodotoxin-sensitive neuronal release of GABA from rabbit caudate nucleus slices (Limberger et al., 1986a). The electrically induced release of taurine from slices of the cerebral cortex has been claimed to be Ca2+-dependent (Collins and Topiwala, 1974; Bradford et al., 1976). The replacement of Ca 2÷ with Mg 2+ results in a somewhat slower release of taurine from rat brain slices (Kaczmarek and Davison, 1972). However, in another study the electrically stimulated release of taurine from slices from the striatum was not found to be altered by the omission of Ca 2÷ from perfusion medium (Katz et al., 1969). 5.2. POTASSIUMSTIMULATION The release of neurotransmitters elicited in vitro by high K + may less closely reflect the physiological action potential-evoked release than the release elicited by electrical pulses. For instance, the electrically induced release, but not in general the K+-evoked release, is tetrodotoxin-sensitive. Moreover, the high K + concentrations may release GABA not only from neurons but from glial cells as well (see Levi, 1984, for references). On the other hand, veratridine has commonly been used as a tool to show the neuronal origin of release. Some recent reports, however, indicate that veratridine also depolarizes cultured astrocytes (Enkvist et al., 1988), which also possess functional Na ÷ channels (Bowman et al., 1984), responding to veratridine similarly to the respective channels in neurons (Nowak et al., 1987).

463

The K+-stimulated release of GABA from slices from different brain areas has been extensively characterized (see Levi, 1984). The K+-stimulated release of taurine has also been demonstrated in brain slices from various areas in a number of reports (e.g. Benjamin and Quastel, 1977; Vargas et al., 1977; Bergez et al., 1978; L6pez-Garcia et al., 1990). A salient feature of the stimulated release of taurine is a slow and prolonged time course. This was first reported by us in vitro in cerebral cortical slices (Korpi et al., 1981) and later confirmed by others in different brain preparations (e.g. Pin et al., 1986; Philibert et al., 1988). It is discernible in cerebellar, striatal, hippocampal and brain stem slices (Kontro and Oja, 1987d) and also in vivo in the striatum (Girault et al., 1986a) and hippocampus (Lehmann et al., 1986). In the retina K+-evoked release of taurine has also occurred only after the cessation of the light stimulus (Smith and Pycock, 1982). This kind of behavior differs strikingly from the prompt stimulation response in the release of GABA (cf. Kontro and Oja, 1987b,c). There are, however, certain similarities. The potassium dependence curves of the release of GABA and taurine are very similar (Kontro and Oja, 1987c) and proportional to the membrane depolarization. In the mouse cerebral cortex the delayed time course of the release of exogenous taurine has been confirmed by measuring the release of endogenous taurine (Oja and Kontro, 1989). Here again, the K÷-stimulated release has a strikingly slower onset than the stimulated release of GABA. Also the basal prestimulation level of release is reattained considerably more slowly in the case of taurine (cf. also Figs 1 and 2). Spontaneous release of taurine is often greater than that of GABA, but only in the adult brain. This is the case if either the release of exogenous preloaded or endogenous compounds have been under study (Kontro and Oja, 1987b,c; Oja and Kontro, 1989). K ÷ stimulation enhances the release of preloaded taurine from cerebral cortical slices only about twofold, whereas the increment in the release of GABA from the same slices has been more than 10-fold (Kontro and Oja, 1987b). The stimulation of the release of taurine is only negligible in the cerebellar cortex (Oja and Kontro, 1989), wherefore it has sometimes escaped the notice of some earlier investigators (L6pez-Colom6 et al., 1978; Foster and Roberts, 1980; Flint et al., 1981; McBride et al., 1983). In rat striatal slices K + stimulation evokes about a two-fold enhancement, whereas the release of GABA increases more than 14-fold (Kontro and Oja, 1988a), again the release of taurine being very slow at the onset. 5.3. SODIUMION EFFECTS The release of GABA has been found to be increased by omission of Na ÷ ions from the incubation medium of brain slices, as shown by ourselves (Heredero et al., 1983; Oja and Kontro, 1987) and others (see Oja and Korpi, 1983, for references). In this respect the response of brain slices differs from that of synaptosomes, which fail to respond to the omission of Na + (see Levi, 1984, for references). This could on one hand be interpreted to indicate that the

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release in the absence of Na + ought to originate from cell compartments other than nerve endings but, on the other hand, the nerve endings have been shown to be depleted of vesicles upon exposure to Na +-free media (Pitk/inen et al., 1985). Na +-free media greatly increase the influx of Ca 2÷ into synaptic terminals (Vargas et al., 1976) through the Na +/Ca 2+ exchangers in plasma membranes (Coutinho et al., 1984) and hence facilitate extrusion of transmitters into synaptic clefts. An elevation of the intracellular Na + content accelerates the spontaneous efflux of GABA but inhibits its electrically stimulated release from rat cerebral cortical slices (Hammerstad and Cutler, 1972). On the other hand, the spontaneous efflux of GABA from spinal cord slices is reduced when the slices are first preincubated and then superfused with Na +-free media (Cutler et al., 1971). The conditions which diminish the transmembrane Na + gradient thus seem to enhance the spontaneous efflux of GABA. Totally Na +-free media have in our experiments caused the maximal release of taurine from rat cerebral cortical slices (Korpi and Oja, 1983; Heredero and Oja, 1987; Oja and Kontro, 1987). The responses in the release of taurine have even been relatively larger than those in the release of GABA under the same experimental conditions. The responses of taurine and GABA to Na + omission are not likely to result from any permanent damage to cell membranes, since the effects can be quickly and totally reversed by reintroducing Na ÷ ions into the medium (Korpi and Oja, 1983). We have also suggested that Na + gradients can affect the orientation of unoccupied membrane taurine transporters favoring in Na + free media extrusion of taurine molecules from the cells. Thus, the enhanced release of taurine is not mediated by any exocytotic process (Korpi and Oja, 1983). The drugs which affect the plasma membrane Na + channels or Na ÷/K+-ATPase also influence the release of GABA from brain slices. For instance, veratridine has stimulated the release of GABA from slices from the rat dentate gyrus (Nadler et al., 1977). It causes an inward torrent of Na + with a concomitant influx of K +, and induces the release of GABA from neurons (Levi et al., 1980) proportionally to the degree of plasma membrane depolarization (Sihra et al., 1984). Consequently, veratridine has been claimed to be the tool of choice in evoking transmitter release from neurons, since glial cells respond only poorly to the drug (see Levi, 1984). Ouabain itself induces a large release of taurine from cerebral cortical slices, which is not further enhanced by the omission of Na + (Korpi and Oja, 1983). Ouabain also increases the efflux of GABA from slices from the cerebral cortex (Benjamin and Quastel, 1972; Hammerstad and Cutler, 1972; Tappaz and Pacheco, 1973), striatum (Brosemer, 1985) and spinal cord (Cutler et aL, 1971). These results indicate the involvement of Na +/K +-ATPase in the regulation of transport. Tetrodotoxin, which blocks the opening of Na ÷ channels, completely suppresses the stimulated release of transmitter amino acids, for example the protoveratrine-evoked release of GABA from cerebral cortical slices (Benjamin and Quastel, 1972), the veratridine-evoked release from rat olfactory bulb

slices (Jaff6 and Cuello, 1981) and the electrically evoked release from rat striatal slices (Bernath et al., 1989), but not the release of taurine from rat cerebral slices induced by the omission of Na + ions from superfusion medium (Korpi and Oja, 1983). 5.4. CALCIUMDEPENDENCE

Ca: ~ ions are considered a prerequisite for exocytotic emptying of the storage granules of transmitters into synaptic clefts, though the stimulation-evoked release of transmitters may originate in cytoplasma by a mechanism independent of Ca :+ (Vizi, 1984). The release of GABA is apparently only partially Ca: + -dependent, since there is a Na+-dependent release component which is independent of Ca 2+ (Levi et al., 1978; Sandoval, 1980). The nonstimulated release of GABA is even somewhat enhanced in media low in Ca 2+, e.g. as shown in striatal slices from rats (Bernath and Zigmond, 1988). We have seen a relatively great enhancement of spontaneous release of GABA from mouse cerebral cortical slices in the absence of Ca 2+, but the effect was obviously accentuated by the presence of a Ca 2+ chelator, ethylenediaminetetraacetate (EDTA), in medium (Oja and Kontro, 1987). The membrane-bound positive Ca 2~ ions which screen the negative surface charges are apparently chelated by EDTA. Since the enhancement of spontaneous release of GABA was dependent on Na + ions we suggested that it resulted from increased influx of Na ÷ across destabilized plasma membranes. In rat hippocampal slices both spontaneous and veratridine-induced release of preloaded GABA have also been shown to be increased by Ca: ~ withdrawal (Minc-Golomb et al., 1988). The enhancement of spontaneous release was totally blocked by tetrodotoxin, wherefore the authors likewise suggested that the enhanced release of GABA caused by Ca 2+ withdrawal is mediated by voltage-dependent Na ~ channels (Minc-Golomb et al., 1988). There is to date an abundance of reports on the Ca: +-dependence of the release of GABA in various preparations from nervous tissue. A certain amount of earlier material has been compiled by Levi (1984) in his review on the release of putative transmitter amino acids. According to these data the release of GABA from brain slices has been only partially Ca 2~ -dependent. In most studies the major part of K÷-stimulated release has been Ca2+-dependent, whereas the electrically evoked or veratridine-induced release has shown less Ca: + dependency. Many more recent papers have also focused on this matter. For instance, the Ca 2+ dependency of the electrically induced release of GABA has been studied with slices from the rat and guinea-pig olfactory cortex (Collins et al., 1981), cat red nucleus (Kubota et al., 1983) and rat medulla oblongata (Kihara et al., 1989b) and with bovine cerebellar glomeruli (Terrian et al., 1987). The K ÷-evoked release has been similarly analyzed with slices from the rat hippocampus (Jonzon and Fredholm, 1985), rabbit caudate nucleus (Limberger et al., 1986a), guinea-pig cochlear nucleus (Potashner et al., 1985), and rat cerebral cortex (Waldmeier et al., 1988a). In all cases the release has been found to be more or less Ca2+-dependent, but not completely.

GABA

AND TAURINE RELEASE

If we assume that the transmitter pool of GABA is vesicular in nerve endings and that the exocytotic emptying of synaptic vesicles is strictly dependent on extracellular Ca 2÷, then the Ca 2÷-independent part of the release of GABA from brain slices must represent something other than vesicular release. The release mechanisms of GABA from neurons and glial cells of the CNS are likely to be different; the release from glial cells showing no Ca 2÷ dependency and also not being influenced by agents that interfere with the transmembrane movements of Ca 2÷ ions (Jaff6 and Cuello, 1981). There is evidence that the release of GABA, which is independent of Ca 2÷ and may co-occur with the Ca2÷-dependent release, takes place via the same carrier system as utilized for the high-affinity uptake of GABA (Bernath and Zigmond, 1988; Bernath et al., 1989). These investigators also suggest that the Ca 2+ channels serve to permit an influx of Na ÷, which in turn promotes the Ca 2÷-independent release through an influence on the high-affinity transport system of GABA (Bernath and Zigmond, 1990a). Moreover, even the Ca 2÷-dependent release may not represent only one mechanism. It has been shown that the release of endogenous GABA from rat cortical slices probably originates in different cellular pools activated by different types of stimulation, i.e. by depolarizing concentrations of K ÷ ions and by electrical pulses at varying frequencies (Waldmeier et al., 1989a,b). The stimulated release of GABA shows generally more Ca 2÷ dependence in adult than in developing mice (Kontro and Oja, 1987c). These results of ours are in keeping with the studies of Balcar et al. (1986), who reported that most of the stimulated release of GABA from slices from the rat cerebral cortex is Ca 2+ -independent during the neonatal period and an important Ca 2÷-dependent component appears only at later stages of development. Some Ca 2÷ -dependent K+-stimulated release of GABA has been earlier noted in cerebral cortical slices from neonatal rats (Davies et al., 1975; Schousboe et al., 1976), though Levi et al. (1979) and Redburn et al. (1978) report that in brain synaptosomes Ca 2÷-dependent release is discernible only in rats older than 8 and 14 days, respectively. In keeping with this, the K ÷-stimulated release of both labeled and endogenous GABA has been shown to be independent of Ca 2÷ in the immature brain. It is assumed to be effected by reversal of the plasma membrane GABA transporter in neuronal growth cones (Taylor and Gordon-Weeks, 1991). On the other hand, the antagonistic effects of Ca 2+ and Mg 2÷ ions on the stimulated release of GABA from cerebral cortical slices have been reported to be more prominent in 3 day-old than in adult mice (Kontro and Oja, 1987c). Nor have we been able to confirm the fundamental differences reported by others in the Ca 2+ dependency of the K ÷-evoked release from the mature and immature brain (Fig. 3). Depending on the preparation studied and on the experimental design, the release of taurine in the adult brain has been claimed to be more or less Ca 2+dependent (Bergez et al., 1978; Okamoto and Namima, 1978; Placheta et al., 1979; Korpi and Oja, 1983; Oja et al., 1985), unaffected by extracellular Ca 2÷ (Flint et al., 1981; McBride et al., 1983) or even

465 I0-I

8

A

~

2

"

0

1

N

Ca z÷ EDTA

B

4"

~

--

+

--

--

-

-

+

-

-

+

FIG. 3. Calcium dependency of the K ÷-stimulated release of labeled GABA (A) and taurine (B) in cerebral cortical slices from young adult, 3 month-old mice. The experiments were done as in Fig. 2, except that, when indicated in the graph, medium was Ca2÷-free from the beginning of superfusion and contained 2 mM EDTA. The fractional efflux rate constants for the stimulation periods were calculated as in Oja and Kontro (1987) for the time intervals of 32-40 min (GABA) and 34-50min (taurine). Mean values of 6 experiments with SEMs.

enhanced in the absence of Ca 2÷ (L6pez-Colom6 et al., 1978; Korpi and Oja, 1984b). Evaluation as to whether or not the K÷-stimulated release is dependent on Ca 2÷ is hampered more with taurine than with GABA by the great enhancement of the basal efflux in Ca2+-free media (Kontro and Oja, 1987b). This circumstance is also depicted in Fig. 3. Moreover, Ca 2÷ chelators such as EDTA, needed for total depletion of cytoplasmic Ca 2+ (Vizi, 1984), destabilize plasma membranes by chelating endogenous membrane-bound Ca 2+ ions which screen the negative surface charges (Hille, 1968). The permeability of membranes to Na ÷ ions increases, Na ÷ flows in and taurine out. Consequently, K ÷ stimulation also fails to work under these circumstances (Korpi and Oja, 1984b). However, a high Mg 2÷ concentration diminishes the stimulated release of taurine more in developing than in adult mice, and verapamil is effective in the former but not in the latter age group (Kontro and Oja, 1987c). All this may indicate that the Ca2+-dependence of stimulated taurine release is greater in the immature than in the mature brain. In this respect the release of taurine in developing mice resembles that of GABA in adults. Verapamil, a blocker of the voltage-sensitive Ca 2÷ channels (Schramm and Towart, 1985), prevents the K ÷-stimulated release of both GABA and taurine from cerebral cortical slices, though the release of GABA is more sensitive to this drug (Oja and Kontro, 1987). The antagonistic effects of Mg 2÷ ions on the Ca2+-dependent K÷-stimulated release of GABA and taurine have been demonstrated with mouse cerebral cortical (Oja and Kontro, 1987) and rat cerebellar slices (Toggenburger et al., 1983), as well as on the Ca2+-dependent electrically evoked release of GABA from pigeon optic tectal slices (Toggenburger et al., 1982). The ability of the Ca 2+ ionophore A23187 to enhance the release of GABA from cerebral slices from rats (Frieder and Rapport,

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1987) is further evidence of the role of Ca 2+ in stimulus-evoked release. When slice preparations are under study one cannot be certain which cells and cellular organelles the release of GABA and taurine originates from. It is thus conceivable that the release of both GABA (Levi, 1984) and taurine (Oja and Kontro, 1983) has been reported to be only partially Ca 2÷ -dependent in slices. In subcellular particles, the exchangeable nontransmitter pools of GABA have been depleted by exposing the preparations to nonlabeled extracellular G A B A after preloading with radiolabeled GABA in an attempt to elucidate more clearly the release originating in the synaptic transmitter pools (e.g. Terrian et al., 1987). There is not sufficient experience to decide whether or not the same procedure could expose the release of G A B A or taurine originating in slices from the synaptic structures. At least some taurine released by K ÷ stimulation may originate from synaptic vesicles (Kontro, 1979) which are also enriched in taurine, though there is also an abundance of both taurine and GABA in the synaptoplasma (Kontro et aL, 1980). The mechanisms of the release of GABA have been studied in greater detail than those of taurine, which are known only superficially. In general, the stimulated release of taurine and GABA show qualitatively similar characteristics with respect to their Ca 2÷ dependence in spite of the greater magnitude of the stimulation in the release of GABA. The importance of the Ca 2÷ dependency of taurine release greatly depends on the still not definitively settled question of the role of taurine in the CNS; i.e. to what extent the compound is confined only to the regulation of cell volumes also in terrestial animals (see below) and to what extent taurine serves as a synaptic effector.

6. EFFECTS OF VARIOUS C O M P O U N D S AND DRUGS 6.1. EXCITATORYAMINOACIDS AND AGONISTS Activation of synaptic excitatory amino acid receptors by glutamate or by various other agonists has been shown to modulate the release of GABA from various brain tissue preparations, including slices (see Levi, 1984; Saransaari and Oja, 1991). Glutamate potentiates the basal release of both G A B A and taurine from mouse cerebral cortical slices, but has no effect on the K +-stimulated release (Kontro and Oja, 1987b). Aspartate increases the K+-stimulated release of GABA and also the basal release of taurine in the above preparations. Cysteinesulfinate induces release of GABA from rat hippocampal slices (MincColomb et al., 1989). Kainate evokes the release of both G A B A and taurine from the cerebral cortex of immature mice (Kontro and Oja, 1987c). Glutamate, aspartate and their agonists liberate massively more taurine from cerebral cortical slices from developing than from adult mice, thus suggesting that cell membranes could be more sensitive to this kind of excitation in immature than in mature brain tissue (Kontro and Oja, 1988b; Saransaari and Oja, 1991). In brain slices from adult animals kainate has been unable to stimulate the release of GABA (Ferkany

and Coyle, 1983; Kontro and Oja, 1987b), although there is a report of kainate-stimulated release of endogenous GABA from rat brain slices (Connick and Stone, 1986). On the other hand, kainate and glutamate stimulate the release of GABA from the chick retina. This release is due to a Na +-dependent, carrier-mediated mechanism that responds to the entry of Na ÷ produced by the interactions of kainate and glutamate with retinal membranes (Tapia and Arias, 1982). The effects of the glutamate agonists on the release of endogenous and exogenous taurine have recently been thoroughly investigated by Saransaari and Oja (1991) in cerebral cortical slices. The N-methyl-Daspartate- (NMDA) and quisqualate-evoked release of taurine was blocked by o-2-amino-5-phosphonovalerate (D-AP5) and glutamatediethylester, respectively, in immature mice, suggesting that the corresponding receptor subtypes are likely to be involved in the responses. Such a regulation of the release of taurine appears to be extinct in the mature brain, since these antagonists were not effective in adults. The kainate-stimulated release of taurine, though very pronounced in the developing cerebral cortex, is apparently not mediated via kainate-sensitive receptors, since 7-D-glutamylglycine and 7-D-glutamyltaurine, which exhibit some selectivity as antagonists of the kainate receptor (Watkins, 1984), had no effect on the kainate-stimulated release of taurine from cerebral cortical slices (Saransaari and Oja, 1991). As mentioned earlier, the basal unstimulated release of taurine is greatly enhanced in Ca 2+ -free medium. The kainate-evoked release of taurine is apparently not dependent on Ca 2÷ ions, since the amount of taurine released by kainate is the same in the absence and presence of Ca 2÷ (Saransaari and Oja, 1991), differing from the kainate-stimulated release of glutamate and aspartate, which has been shown to be Ca 2÷-dependent in hippocampal synaptosomes (Poli et al., 1985). Furthermore, the density of kainate binding sites increases during postnatal development in the rat cerebral cortex (Miller et al., 1990) which corroborates the assumption that kainate receptors are not involved in this type of taurine release. The K ÷-stimulated release of taurine is qualitatively similarly modified by the excitatory amino acids and their agonists in both adult and developing cerebral cortex (Saransaari and Oja, 1991). The inhibitory action of D-AP5 on the NMDA-potentiated release suggests that N M D A receptors could also modulate the stimulated release of taurine. The kainate effects were also markedly antagonized by ]'-D- and ),-L-glutamyltaurine. Kainate may evoke taurine release in brain slices from two different pools with different mechanisms. In this case the K +stimulated release of taurine could be modulated by an activation of presynaptic kainate receptors in both adult and developing brain. The K+-stimulated release of taurine has been significantly inhibited by a number of dipeptides, which are present in low amounts in brain tissue in vivo. 7-L-Glutamyltaurine has been the most potent of them, but also 7-L-glutamylaspartate, ~t-L-glutamyltaurine and Ct-L-glutamylaspartate have caused a significant inhibition (Varga et al., 1987). Of these

GABA AND TAURINE RELEASE

dipeptides only V-L-glutamyltaurine has slightly inhibited the K +-stimulated release of GABA, whereas ?-L-glutamylhomotaurine somewhat enhances the stimulated release of G A B A (Varga et al., 1988). The physiological significance of these endogenous dipeptides is not known, although they also interfere with the excitatory amino acid receptors, but are not very potent or very selective for the various subtypes of receptors (Varga et al., 1989). Kainate has increased the extracellular G A B A levels in vitro (Young et al., 1988), but this has not been confirmed in in vivo experiments, probably due to the low detection level of G A B A (Ferkany and Coyle, 1983; Lehmann et al., 1983; Wade et al., 1987). The response to a pretreatment with a GABA-transaminase inhibitor, ),-vinyl-GABA has, however, shown that kainate administration to rats increases the hippocampal levels of G A B A also in vivo (Zhang et al., 1990). A systemic injection of kainate is known to destroy neurons in the hippocampus, inducing limbic-type seizure activity (Ben-Aft, 1985). The K +stimulated release of G A B A from hippocampal slices from kainate-treated rats has decreased at the onset of convulsions, the decrease persisting several days after the treatment (Arias et al., 1990). The release of taurine has been shown in vivo to increase in the hippocampus in response to direct local and systemic administrations of kainate (Lehmann et al., 1983; Wade et al., 1987), to direct local administration of N M D A (Lehmann et al., 1985) and during periods of ischemia/hypoxia or cardiac arrest (Benveniste et al., 1984; Korf et al., 1988), which suggests that endogenous taurine may have a role in the maintenance of homeostasis during hyperexcitability. Exposure of brain slices to the excitatory amino acids and their agonists causes cellular swelling of the slices. The relation of this to the release of taurine will be discussed later. 6.2. DRUG EFFECTS The search for drugs specifically affecting the release of G A B A or taurine has so far met with no success. The fragmentary information currently available is briefly discussed below. Ouabain increases the spontaneous efflux of GABA from slices from the cerebral cortex (Benjamin and Quastei, 1972; Hammerstad and Cutler, 1972; Tappaz and Pacheco, 1973) and spinal cord (Cutler et al., 1971). The other drugs that interact with Na + movements across membranes also influence the efflux of GABA. Besides ouabain, protoveratrines, particularly in Ca 2+-free media, can increase the release of GABA. The stimulated release of putative neurotransmitter amino acids is generally abolished by tetrodotoxin, a very potent neurotoxin which abolishes the generation of action potentials in excitable tissues by blocking the opening of Na ÷ channels. Tetrodotoxin only partially suppresses the effect of ouabain, but completely that of protoveratrine (Benjamin and Quastel, 1972). Hammerstad and Cutler (1972) report that tetrodotoxin likewise only partially prevents the Na + and K + shifts induced by ouabain. Protoveratrine is therefore thought to act only on neurons and ouabain on both neurons and

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glia (Benjamin and Quastel, 1972). p-Hydroxymercurybenzoate inhibits the Na ÷ pump, eventually reversing the transmembrane Na + gradient. It enhances the spontaneous efflux but inhibits the stimulus-induced release of G A B A from the spinal cord (Collins, 1974). An activation of protein kinase C has been shown to enhance the Ca2+-dependent release of neurotransmitters, particularly that of catecholamines (Feuerstein et al., 1987; Malenka et al., 1987). Activation of protein kinase C has also stimulated the K +-evoked release of G A B A from slices from the deep cerebellar nuclei of the guinea-pig in the presence of tetrodotoxin (Suntoh et al., 1989). In the same experiments the ouabain-evoked release of G A B A was not potentiated by a phorbol ester, which gave these authors reason to suggest that protein kinase C potentiates the vesicular release of G A B A from nerve endings. Also cyclic AMP, presumably by initiating phosphorylation of some specific component, has a remarkable potentiating effect on the release of G A B A induced by cysteinesulfinate from rat hippocampal slices (Minc-Golomb et al., 1989). Pentobarbital, the barbiturate that has been studied most, inhibits both basal and K÷-stimulated GABA release from brain slices (Cutler et al., 1974; Cutler and Dudzinski, 1975; Cutler and Young, 1979; Waller and Richter, 1980). The inhibitory effect of pentobarbital on the Ca 2÷ -dependent K ÷ -stimulated release of G A B A has also been reported to be only very slight (Olsen et al., 1977). Moreover, pentobarbital has even potentiated the release of G A B A from electrically stimulated cerebral cortical slices (Cutler and Dudzinski, 1974; Collins, 1980). Pentobarbital not only affects the stimulated release of GABA, since the release of taurine from slices from the olfactory cortex is likewise increased (Collins, 1980). Phenobarbital has no effect on the release of G A B A from brain slices, whereas a convulsant barbiturate, 5(2cyclohexylidineethyl)-5-ethylbarbiturate(CHEB), enhances the spontaneous effiux of GABA, simultaneously strongly inhibiting the K ÷-evoked release (Holtman and Richter, 1983; Skerritt et al., 1983). The convulsant barbiturates have some GABAenhancing and GABA-mimetic actions, possibly due to activation of presynaptic G A B A autoreceptors which depress the release of GABA. Baclofen is the well-known agonist of G A B A B binding sites, which are assumed to regulate the release of transmitters by means of activation of presynaptic receptors (Bowery et al., 1980; Zhu and Chuang, 1987). However, baclofen at concentrations from 0.001 to 0.1 mM has had no effects on the basal or K+-stimulated release of G A B A from cerebral cortical, cerebellar and hippocampal slices (Kontro and Oja, 1987b; Burke and Nadler, 1988; Kontro and Oja, 1989, 1990). Similarly, the release of taurine from mouse cerebral cortical slices has been unaffected by baclofen (Kontro and Oja, 1987b, 1990), whereas in cerebellar slices the K ÷-stimulated release of taurine has been strongly depressed by baclofen (Kontro and Oja, 1989), which suggests the possibility that baclofen-sensitive receptors could modulate the cerebellar release of taurine. Moreover, in cerebral cortical slices fi-aminovalerate, a G A B A B receptor antagonist (Muhyaddin et al., 1982),

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stimulates the release of taurine. This effect is partially abolished by baclofen, indicating modification of the cortical release of taurine as well (Kontro and Oja, 1990). Of the convulsant antagonists of G A B A and glycine (bicuculline, picrotoxin, strychnine), only strychnine significantly inhibits the spontaneous and evoked release of GABA from the spinal cord and cerebral cortical slices (Johnston and Mitchell, 1971; Tappaz and Pacheco, 1973; Collins, 1974; Kontro and Oja, 1987b). Pentetrazole depresses the spontaneous efflux of GABA without affecting either the influx or the electrically evoked release of GABA (Johnston and Mitchell, 1971). This may result from a lowering of the threshold to spontaneous firing of the GABA-containing neurons. However, Tappaz and Pacheco (1973) have not been able to confirm this finding. In their studies, pentetrazole significantly depressed only the K+-stimulated efflux. Of the above alkaloids, picrotoxin and strychnine inhibit the K +-stimulated release of taurine from cerebral cortical slices without any effect on the spontaneous efflux (Kontro and Oja, 1987b). Penicillin has reduced the K +-evoked, Ca 2+-dependent release of endogenous GABA from cerebral cortical slices (Cutler and Young, 1979). Some anticonvulsants, e.g. phenytoin, trimethadione, phenacemide and ethosuximide, prolong or increase the K+-induced efflux of GABA. Phenacemide also increases the spontaneous efflux (Tappaz and Pacheco, 1973). Phenacemide, but not trimethadione, is antagonistic to the strychnine-induced suppression of GABA release. In cerebral cortical slices, the anticonvulsants thus appear to potentiate the inhibitory action of GABA by enhancing its release (Tappaz and Pacheco, 1973). On the other hand, ethosuximide, methsuximide, carbamazepine, sulthiame and dipropylacetate have failed to alter the K +-evoked release of G A B A from rat cerebral cortical minislices (Skerritt and Johnston, 1983). Acute administration of ~-vinyl-GABA potentiates the K +stimulated release of G A B A from rat cerebral cortex and spinal cord slices, the enhancement being reduced to one half in rats chronically treated with this drug (Neal and Shah, 1990). The K +-stimulated release of taurine from cerebral cortical slices is not affected by either acute or chronic treatment with 7-vinylGABA. In the context of an attempt to develop new anticonvulsant drugs based on taurine (Kontro et al., 1983; Lind6n et al., 1983; Oja et aL, 1983), we have analyzed the effects of some novel derivatives of taurine on the release of GABA and taurine from mouse cerebral cortical slices. Taltrimide (2phthalimidoethanesulfon-N-isopropylamide) and its biologically active dealkylated metabolite, 2-phthalimidoethanesulfonamide, both clearly enhance the K +-stimulated release of GABA but slightly inhibit the K÷-stimulated release of taurine from slices, whereas the unstimulated release of both compounds is only marginally, if at all, affected (Kontro and Oja, 1987e). The inhibition of taurine release is discernible in both adult and developing mice (Kontro and Oja, 1987c). In acute experiments tetanus toxin has inhibited in vitro the release of GABA from slices prepared

from the rat hippocampus (Collingridge et al., 1981) and substantia nigra (Collingridge and Davies, 1982). Pretreatment of rats in vivo with tetanus toxin also inhibits the subsequent release of GABA from rat cerebral cortical slices in vitro evoked by both highK + and Na ÷ -free media, but not the release induced by ouabain or veratrine (Heredero et al., 1983). The release of taurine is not affected by the toxin under identical experimental conditions (Heredero and Oja, 1987). Ruthenium red impairs the inhibition of GABA release by tetanus toxin, possibly by interfering with the fixation of toxin to its membrane binding sites, though the dye itself elicits some release of GABA from rat cerebral cortical slices (Heredero and Oja, 1985). Tetanus toxin also affects the release of other transmitters, e.g. that of dopamine from striatal slices, although some specificity for GABA may exist (Collingridge et al., 1980). Repeated injections of tetanus toxin over a prolonged period into the rat hippocampus in vivo as a model of limbic epilepsy significantly reduce the Ca2+-dependent release of GABA from hippocampal slices in vitro (Jefferys et al., 1991). After several weeks the release shows signs of recovery to the control levels, thus providing a likely basis for seizure remission in this chronic epileptic syndrome. After a series of electroconvulsive shocks the K+-evoked release of GABA has been inhibited in slices from the rat hippocampus and striatum but not in those from the cerebral cortex (Green et al., 1987; Green and Vincent, 1987). A low concentration of diazepam stimulates the release of exogenous GABA, but a high concentration is inhibitory in cerebral cortical slices (Mitchell and Martin, 1978). The K+-stimulated release of GABA and taurine from cortical slices has been unaffected by diazepam in our experiments (Kontro and Oja, 1987b). An antianxiety compound 5- {3- [4-(4-fluorophenyl)-1-piperazinyl]-propoxy}indan (BP-528) has not affected the binding of GABA or diazepam but has inhibited the K ÷-induced release of GABA from hippocampal slices (Ohta et al., 1987). Haloperidol has significantly potentiated the release of both G A B A and taurine from cerebral cortical slices in our studies (Kontro and Oja, 1987b), which is apparently at variance with the results of Olsen et al. (1977, 1978). In their experiments this dopamine antagonist inhibited the Ca2+-dependent depolarization-induced release of GABA from synaptosomal fractions. In keeping with our studies, haloperidol, as well as the serotonin-uptake inhibitor imipramine, has in t,ivo enhanced the release of endogenous GABA from the rat thalamus (Korpf and Venema, 1983). The strong depression of the evoked release of GABA from cerebral cortical slices by imipramine (Kontro and Oja, 1987b), however, is consonant with the above studies of Olsen et al. On the other hand, the stimulated release of taurine has been considerably enhanced and the spontaneous release suppressed by imipramine (Kontro and Oja, 1987b). Haloperidol and imipramine are rather effective inhibitors of the uptake of GABA (Olsen et al., 1978; Harris et al., 1973) and taurine (Schmid et al., 1975; Schousboe et al., 1976), which could at least, to some extent, explain their enhancing actions on the stimulated release of these amino acids. The monoaminergic drugs seem to be able to modify the

GABA AND TAURINE RELEASE

K+-stimulated release of both G A B A and taurine, but the mechanisms of their action and the significance of the effects are still undefined. Ethanol has been found to inhibit the K ÷-stimulated release of G A B A from rat cerebral cortical slices and the spontaneous release from cerebellar slices (Howerton and Collins, 1984). However, ethanol has been ineffective in the case of the electrically stimulated release of G A B A from cortical slices (Strong et aL, 1987). Lead acetate inhibits both uptake and stimulated release but facilitates the spontaneous etflux of G A B A in rat brain slices (Drew et al., 1989). Spontaneous release of taurine has not been affected in any brain area studied in experimental hepatic encephalopathy in rats injected with thioacetamide, and the only significant change in the K+-stimulated release was an enhancement in the striatum (Wysmyk et al., 1991).

7. M O D U L A T I O N OF GABA AND TAURINE RELEASE 7.1. PRESYNAPTICAUTORECEPTORS The conception that the depolarization-induced release of neurotransmitters is subject to a negative feedback regulation mediated by presynaptic autoreceptors has lately been the object of intensive studies. The existence of presynaptic autoreceptors for G A B A has been presumed in synaptosomal preparations (Snodgrass, 1978; Brennan et al., 1980) and also in slices from various brain regions (Arbilla et al., 1979; Collins, 1980; Namima et al., 1983; Kuriyama et al., 1984). The finding that low concentrations of G A B A agonists are able to inhibit the depolarization-induced release of GABA is considered to bespeak the action of these presynaptic autoreceptors. However, in mouse cerebral cortical slices, the K +-stimulated release of G A B A has not been suppressed by G A B A or muscimol at low concentrations (Kontro and Oja, 1987b). In contrast, a high concentration of G A B A in medium more than tripled the K + -stimulated efflux in the above study of ours. This effect obviously results from homoexchange. Furthermore, the GABA antagonists picrotoxin and bicuculline were ineffective, thus confirming the apparent absence of presynaptic G A B A autoreeeptors in the cerebral cortex in these experimental conditions. In keeping with this, no evidence has been found for G A B A A autoreceptors in rabbit caudate nucleus slices (Limberger et al., 1986b). Also in the cerebellum the K+-stimulated release of G A B A has not been suppressed but greatly potentiated by G A B A and muscimol in both adult and developing mice, which suggests that conventional G A B A A receptors may not be involved (Kontro and Oja, 1989). On the other hand, bicuculline stimulated the release of GABA in cerebellar slices, as was also observed by Namima et al. (1983). Both taurine and hypotaurine are able to counter the potentiation of bicuculline in the cerebellum of adult mice, indicating that taurine could modulate the release of G A B A through bicuculline-sensitive receptors (Kontro and Oja, 1989). This modulation is not yet evident in cerebellar slices from 7 day-old mice.

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The possible involvement of baclofen-sensitive GABA B receptors in the regulation of GABA release was briefly discussed in Section 6.2. We have found no evidence of modulation by baciofen of the 50 mM K +-stimulated release of GABA from cerebral cortical slices (Kontro and Oja, 1990), in agreement with the results of others on rat substantia nigra slices (Waldmeier et al., 1989a). Bonanno et al. (1989) recently reported a concentration-dependent inhibition of G A B A release by baclofen from synaptosomes from the rat cerebral cortex, suggesting the existence of terminal G A B A s receptors, which were disclosed only by using rather small concentrations of K + to evoke the release. Similar findings have been reported by others in synaptosomes and brain slices (Anderson and Mitchell, 1985; Pittaluga et al., 1987; Waldmeier et al., 1988b; Deitz and Prince, 1989). The evidence consists mainly of a suppression of GABA release by baclofen under appropriate stimulation conditions, e.g. certain types of electrical pulses have been effective (Maurin, 1988; Raiteri et al., 1989; Baumann et al., 1990). The inhibition has also been sensitive to a novel GABAB receptor antagonist, phaclophen, [fl-(p-chlorphenyl)-3-aminopropylphosphonic acid], which itself has potentiated the electrically stimulated release of G A B A from slices from the rat temporoparietal cortex (Raiteri et al., 1989). The presence of G A B A uptake inhibitors may also be essential in improving the electrically evoked release. In rats treated with 7-vinyl-GABA the K +-evoked release of GABA from rat cerebral cortex and spinal cord slices has been reduced by baclofen, which is antagonized by phaclofen, further strongly indicating the involvement of G A B A 8 receptors (Neal and Shah, 1989). Furthermore, there are also results which show that the electrically evoked release of GABA from rat cerebral cortical slices could be modulated by a receptor-mediated mechanism sensitive to baclofen, bicuculline and picrotoxin but not to GABA or muscimol (Maurin, 1988). This suggests that both GABAB and GABA A receptor subtypes might participate in the regulation of GABA release (Anderson and Mitchell, 1985; Maurin, 1988). The existence of two separate releasable neuronal pools of GABA in rat cerebral cortical slices has recently been proposed, one of them being regulated by autoreceptors of the GABA B type (Waldmeier et al., 1989a). These results were obtained by comparing the effects of certain types of electrical and K + stimulations. So far there is no evidence for regulation of the release of taurine by presynaptic receptors, although in cerebellar slices a 1 ~M concentration of taurine has reduced the K +-stimulated release (Kontro and Oja, 1989). The proposed taurine antagonist T A G has depressed the evoked release of taurine (Kontro and Oja, 1987b,c), which is at variance with the general conception of a negative feedback regulation.

7.2. PRESYNAPTICHETERORECEPTORS There~are a mounting number of reports which show that the release of a neurotransmitter can be stimulated or inhibited by other neurotransmitters or -modulators. These interactions appear to involve activation of presynaptic receptors located in the

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membrane of the nerve ending which releases the neurotransmitter subject to modulation. A slice preparation has been used in most studies on these heteroreceptors. The structural complexity of the preparation greatly hampers the analysis of effects of a given transmitter on the release of another (cf. Levi, 1984). However, in synaptosomal preparations, which are structurally simpler, the necessary connections for this kind of study are often totally lost, though the membrane may still contain the receptor sites under study. Taurine could modulate the K ~-stimulated release of GABA via GABAergic receptors, as briefly discussed above. Taurine has been found to potentiate the K ÷-induced release of GABA from rat cerebral cortical slices (Leach, 1979) and also in insect synaptosomes (Whitton et al., 1988), whereas no effect has been observed in cerebellar (Kontro and Oja, 1989) or striatal slices (Kuriyama et al., 1984). Glycine and fl-alanine have had no effects on the evoked release of GABA from rat striatal slices (Kuriyama et al., 1984). An opioid agonist, D-Ala2-D-LeuS--enkephalin (DADLE), has only at low concentrations decreased the K ÷-stimulated release of GABA from pallidal slices prelabeled with radioactive GABA, suggesting the presence of 6-receptors on the terminals of GABAergic neurones in the globus pallidus (Dewar et al., 1987). Somatostatin-14 has also direct and indirect modulatory effects on the release of GABA from slices of the rat caudatoputamen, suggesting that somatostatin is a modulator of the activity of striatonigral GABA-neurons (Meyer et al., 1989). In rat corticoparietal slices cholecystokinin octapeptide and its analog caerulein facilitate the K÷-evoked release of GABA, the enhancement being reversed by proglumide, a proposed competitive antagonist of the cholecystokinin receptor (Sheehan and De Belleroche, 1983). 1,2,3,4-Tetrahydro-9-aminoacridine (THA), an acetylcholinesterase inhibitor used in the treatment of Alzheimer's disease, has dose-dependently inhibited the K ÷-stimulated release of GABA from rat cerebral cortical slices (De Belleroche and Gardiner, 1988). GABA, muscimol and baclofen have equally depressed the K ÷-stimulated release of taurine in the cerebral cortex and cerebellum, suggesting that GABAergic substances could modify the release (Kontro and Oja, 1989; Neal and Shah, 1989). The release was in cerebellar slices further depressed by bicuculline (Kontro and Oja, 1989). This inhibition is a peculiar finding not in line with inhibition of the above GABA agonists. Baclofen has been shown to reduce the release of neurotransmitters from several brain areas through an action at the GABA B receptor. Thus, it is possible that these baclofen-sensitive receptors could also modulate the release of taurine.

7.3. MODULATIONBY CATECHOLAMINES Modulation of the release of GABA by eatecholaminergic systems has been extensively studied because of the close connections between catecholaminergic and GABAergic nerve terminals in many brain regions. The results from different release ex-

periments are frequently conflicting and firm inferences are extremely difficult to draw. In the striatum there is an extensive distribution of GABA-containing neurons, some of which project to distal nuclei, e.g. to the globus pallidus and substantia nigra, and some of which are local interneurons. The importance of dopamine as a modulator of GABA release has been studied mostly in the substantia nigra and striatum in vitro, though the results are largely contradictory. For example, it has been reported that dopamine directly liberates newly acquired GABA from slices from the rat substantia nigra (Reubi et al., 1977), while this finding has been refuted (Kelly et al., 1985). Dopamine and apomorphine have inhibited the K÷-stimulated release of GABA from rat striatal slices (Brase, 1980; De Belleroche and Gardiner, 1983; Kontro and Oja, 1988a; Umeda and Sumi, 1989) and carp retina (Kato et al., 1985). Only high concentrations of dopamine agonists have reduced the K÷-evoked release of GABA from rat substantia nigra slices (Kelly et al., 1985). However, in another study, activation of dopamine D2-receptors by low concentrations of antagonistic drugs has had no effect on the striatal release of GABA, whereas the release of dopamine is suppressed (Stoof et al., 1982). Kuriyama et aL (1984) failed to show any effect of apomorphine on the K÷-stimulated release of GABA from rat striatal slices. The spontaneous efflux of GABA has been potentiated by both dopamine and apomorphine (Kontro and Oja, 1988a). The basal effiux was also enhanced by 10-100/.tM dopamine but not by apomorphine in the studies of Limberger et al. (1986b). In the substantia nigra dopamine and apomorphine have exerted opposite effects on the release of endogenous GABA, which effects, moreover, depend on the drug concentrations (van der Heyden et al., 1980). Treatment with haloperidol could be expected to affect the release of GABA from striatal slices since it antagonizes the effects of dopamine. However, the results obtained have been inconsistent. Haloperidol has had no effect on (Kuriyama et al., 1984) or suppressed (Kontro and Oja, 1988a; Umeda and Sumi, 1989) the striatal release of GABA. Selective dopaminergic compounds have recently been used to provide explanations for the complex interactions in the release processes of GABA and dopamine. In in vivo studies dopamine has been shown to exert excitatory influences on the striatal release of GABA through a dopamine D~ subtype of receptor and inhibitory effects via a D 2 receptor (Girault et al., 1986b; Reid et al., 1990). Similar results have been obtained on slices from the rat substantia nigra (Starr, 1987) and striatum (Bernath and Zigmond, 1989). In addition to these mechanisms, a third type of dopamine effect on the efflux of GABA has been proposed, which is mediated through neither type of receptor (Bernath and Zigmond, 1989, 1990b). Dopamine has been shown to enhance both spontaneous and electrically evoked release of GABA from striatal slices via a mechanism involving the high-affinity GABA uptake system (Bernath and Zigmond, 1990b). This assumption is based mainly on results showing that the excitatory effect of dopamine on both spontaneous efflux and evoked release of GABA is abolished by nipecotate,

GABA AYe,TAURINERELEASE a proposed selective inhibitor of the neuronal uptake of GABA, whereas fl-alanine, thought to be an inhibitor of glial uptake of GABA, potentiates the effects of dopamine on the spontaneous efflux of GABA. Noradrenaline has been considered to inhibit GABAergic transmission via ~2-adrenoceptors, as clonidine, an agonist of ct2-adrenergic receptors, has reduced the release of GABA (Dolphin, 1982), albeit at a high concentration. The Ca 2+-dependent release of GABA evoked by electrical stimulation from rat and mouse cerebral cortex and brain stem slices has been inhibited by yohimbine and other ~2-adrenoceptor antagonists, effects which have not been antagonized by clonidine (Maurin et al., 1985). Thus the observed inhibition appears to be independent of the blockade of ~t2-adrenoceptors and unspecific under these conditions. In rat cortical slices an ~t2-receptor antagonist, rauwolscine, has increased only the Ca 2+dependent release of GABA, whereas the agonists guanabenz and clonidine have inhibited only the Ca2+-independent release (Peris et al., 1987). In contrast, noradrenaline has significantly enhanced the K + -stimulated release of GABA from slices from the rat medial preoptic area in ovariectomized and in estrogen-primed and ovariectomized rats (Herbison et aL, 1989). Since the effects of noradrenaline were blocked by phenoxybenzamine, an ~t-adrenergic blocker, it was suggested that the release of GABA is modulated by both estrogens and noradrenergic systems in the medial preoptic area. It has been shown that the release of taurine from the retina is reduced by dopamine concentrations of 0.8 and 4mM (Pycock and Smith, 1983). However, the effects of catecholamines on the release of taurine have been studied mainly in the striatum, which contains high amounts of this sulfur amino acid (Perry et al., 1972; Lombardini, 1976). There is some evidence that taurine could modulate dopaminergic transmission in the striatum (see Kontro and Oja, 1988a). In rats, dopamine, at concentrations prevailing in the rat striatum in vivo (about 100/,mol/kg; Sharp et al., 1986) has inhibited the evoked release of taurine and altered the time-course of the release in both immature and mature striatum, although the effects were somewhat weaker in 7 day-old rats than in adults (Kontro and Oja, 1988a). Furthermore, the dopaminergic systems were able to modify the stimulated release of taurine in a manner which probably implicates activation of presynaptic dopamine receptors. Apomorphine mimicked the action of dopamine and the dopamine effect was also partially antagonized by haloperidol in both age groups. Dopamine and apomorphine also increased the spontaneous efflux of taurine from striatal slices, which further bespeaks modification of the release of taurine by dopaminergic receptors. On the other hand, these effects may not be fully specific, since the stimulated release of taurine was also depressed by the other monoamines, noradrenaline and serotonin. Haloperidol and butaclamol also failed to enhance significantly the stimulated release of taurine in the striatum, a finding at variance with data obtained from the mouse cerebral cortex (Kontro and Oja, 1987b).

471

8. CHANGES DURING DEVELOPMENT AND AGEING The concentration of taurine is very high in the developing brain, decreasing towards adulthood, a pattern which is in striking contrast to those of other neuroactive amino acids. Their concentrations tend to increase during development (Oja et al., 1968). The concentration of GABA also clearly increases with maturation of the brain (Kontro et al., 1984) and with the appearance of inhibitory synapses (B/ihr and Wolff, 1985). It has therefore been suggested that taurine may have a specific regulatory role in brain excitability in developing animals during the early periods of life (Oja, 1966). Certain aspects of GABA and taurine release from slices of the immature brain have already been discussed in previous sections. During ontogeny the potassium-stimulated release of GABA is initially very low and increases strikingly during the second and third postnatal weeks in rodents (Balcar et al., 1983, 1986). The spontaneous effiux of exogenous GABA from mouse cerebral cortical slices increases with the cerebral GABA content during postnatal development, but the spontaneous effiux of taurine is approximately the same in both neonatal and adult mice, in spite of a several-fold higher cerebral taurine content in the former (Kontro and Oja, 1987c). The K + stimulation of release of preloaded labeled taurine has a strikingly slow time course in adults, but even more so in developing mice in all brain areas studied, cerebral cortex, cerebellum, striatum, hippocampus and brain stem (Kontro and Oja, 1987c,d, 1988a, 1989). This finding is also confirmed by measuring the release of endogenous taurine from cerebral cortical and cerebellar slices (Oja and Kontro, 1989). The responses in the release of GABA were also fairly slow in neonates, almost reminiscent of the responses in the release of taurine. K + stimulation evokes a large release of GABA in the cerebral cortex in adults but not in developing mice (Kontro and Oja, 1987c). The evoked release of taurine is several times greater in developing mice than ,the evoked release of GABA in every brain area studied; this relationship drastically changes with age (Kontro and Oja, 1987c, 1988a, 1989). As discussed above, the K +-stimulated release of both amino acids is only partially Ca 2+-dependent in cerebral cortical slices, more so with GABA in adults and with taurine in neonates, but a high Mg 2+ concentration inhibits the release of both amino acids more strongly in the latter age group. Verapamil (0. l mM) has almost abolished the K + stimulation in the release of GABA in both adult and neonatal mice. It is more effective in the case of taurine release in neonatal mice (Kontro and Oja, 1987c). The major differences in the properties of the evoked release of GABA and taurine apparently reflect an important functional difference between the two amino acids, contradicting the possibility that taurine could act as any major conventional fast-acting inhibitory transmitter in adults. On the other hand, the prolonged time course of the K +-stimulated release of taurine has been even more pronounced in cerebral cortical slices from 3 day-old mice than in slices from adults. Morphological

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(Ahman, 1972a,b,c; Purves and Lichtman, 1985), electrophysiological (Shimono et al., 1976), and biochemical and receptor-binding studies (Coyle and Enna, 1976; Palacios and Kuhar, 1982; Frostholm and Rotter, 1987) all indicate that the major proliferation of GABAergic synaptic connections is on the point of inception at from one to two postnatal weeks in the cerebral cortex and cerebellum. It is known that the number of postsynaptic GABA receptors also increases in the rodent brain during postnatal development (Skerrit and Johnston, 1982). Although taurine exhibits some interaction with them, its efficacy is not very pronounced (Greenlee et al., 1978; Malminen and Kontro, 1986). Neither does GABA strongly interfere with the Na +-independent specific binding of taurine to mouse brain synaptic membranes (Kontro and Oja, 1987f), a binding that seems to decline with age (Kontro et al., 1984). We have earlier suggested that taurine could act as a ubiquitous neuromodulator that switches off any prolonged excitation in neuronal networks (Kontro and Oja, 1987c). It may be particularly active in the developing brain, where the established inhibitory mechanisms using GABA, lagging behind the excitatory ones in development, have not yet functionally matured (Sedlfi6ek, 1978) and cannot account for any major inhibition. Obviously no striking changes occur in the release of GABA and taurine during ageing (Oja et al., 1990). Figure 4 shows how the potassium-stimulated release of GABA and taurine changes during ageing in different brain areas in mice. Only in the cerebral cortex is there a somewhat decreasing trend in the magnitude of the efflux rate constants for GABA during ageing, whereas in the striatum and hippocampus the release is temporarily higher at the ages of 12 and 18 months, but decreases again at 24 months. It should be kept in mind, however, that these results were only obtained with preloaded exogenous GABA, and the relative magnitudes of the transmitter pools of GABA in slices may change during ageing and these changes could influence the results. The only striking change in the K +-stimulated release of taurine is the transient increase in the hippocampus at the ages of 12 and 18 months. 1600

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FIG. 4. Changes in the K+-stimulated release of labeled GABA (A) and taurine (B) from mouse brain slices during ageing. The experiments and calculations were done as in Fig. 3. Cerebral cortex ( - - 0 - - ) , striatum ( - m - ) , hippocampus (--IS]--), cerebellum ( - - 0 - - ) , and brain stem (--A ). Mean values of 4 to 8 experiments with SEMs.

Nothing is known of any alterations in the release of GABA or taurine from brain slices in pathological states during development and ageing.

9. TAURINE AND REGULATION OF

CELL VOLUMES Brain cells should be capable of maintaining a relatively constant volume despite variations in the composition or osmolarity of the surrounding extracellular fluid, since the brain is situated inside a rigid bone cavity. The role of taurine in osmoregulation of marine animals has been known for a number of years (e.g. Simpson et al., 1959). Some free amino acids could participate in adjustments of cell volumes also in terrestial animals (see Fugelli, 1980, for references). Recently taurine was suggested to have an osmoregulatory function in the mammalian brain (Wade et al., 1987; Walz and Allen, 1987). It has subsequently been seriously considered to play a dominant role in regulation of cell volumes in the CNS (Pasantes-Morales and Schousboe, 1988; Schousboe and Pasantes-Morales, 1989; Martin et al., 1990; Olson and Goldfinger, 1990; PasantesMorales et aL, 1990; Schousboe et al., 1990). In these studies hypo-osmotic media have been shown to evoke release of taurine from cultured astrocytes and glutamatergic and GABAergic neurons and hyperosmotic media to prevent K ÷ stimulation in the release. Furthermore, K + stimulation evokes swelling of astrocytes concomitant with the release of taurine, and both processes are inhibited when the cells are stimulated in media in which the K + × C1- product is maintained at a constant (Pasantes-Morales and Schousboe, 1989). The above results, obtained with cultured astrocytes and neurons, may not as such be applicable to brain slices in which the structural integrity and cell-to-cell interactions are to some extent preserved. So far, we have been the only investigators to study the dependence of taurine release, with brain slices, upon hypo- and hyperosmotic conditions. Hypoosmotic media enhance the release of taurine from cerebral cortical slices in both developing and adult mice (Oja et al., 1990). Exposure of slices to excitatory amino acids and their agonists evokes release of taurine from cerebral cortical slices from both adult and 3 day-old mice; N M D A being the most potent stimulator (Saransaari and Oja, 1991). However, simultaneously the slices swell more than unstimulated ones. The swelling is considerably more pronounced in adults, whereas the magnitude of taurine release evoked by K ÷ ions, N M D A and kainate is considerably greater in 3 day-old mice. Taurine is also released from cerebral cortical slices by K ÷ ions in media in which the K + x CI ionic product is kept constant and in hyperosmotic high-K ÷ media (Fig. 5). The K ÷ stimulation was totally preserved in adult mice when the C I - deficit was compensated by a permeant anion, acetate, and partially when the compensation was effected by an impermeant anion, gluconate, The K + stimulation was not significantly different in hyperosmotic medium. In 3 day-old mice, the preservation was partial in all these media.

G A B A AND TAURINE RELEASE

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FIG. 5. The K ÷-stimulation in the release of taurine from cerebral cortical slices from young adult, 3 month-old (A) and 3 day-old (B) mice in media of varying ionic composition. The experiments were done as in Fig. 3. The average K + stimulation was calculated by relating the estimated fractional efflux rate constants (Oja and Kontro, 1989) for the stimulated release during the superfusion period of 34-50 min to the corresponding constants for the baseline control efflux of taurine during the same period in slices superfused continuously with normal-K + (5 mM) medium. (1) Open columns: media in which 50 mM Na ÷ were equimolarly replaced by K +; (2) cross-hatched columns: media which contained 50 mM K + but in which the ionic product K ÷ x C1- was kept constant by replacing the Na ÷ omitted with a permeant anion, acetate; (3) hatched columns: as in (2) but the ionic product K ÷ × CI - was kept constant by using an impermeant anion, gluconate; (4) solid columns: media directly supplemented by 50 mM KC1 (hyperosmotic media). Mean values with SEMs are shown. The number of experiments is indicated below each column. The asterisks indicate a significantly(p < 0.01) smaller stimulation than in the experiments in which 50 mM Na + were replaced by K +. Most studies on the effects of anions on the release of taurine and GABA have been done with preparations other than tissue slices, for example cultured cells, synaptosomes and isolated retinae. For instance, Pasantes-Morates et al. (1988) have shown that the resting release of GABA and taurine is not markedly modified when CI- ions in medium are replaced by gluconate, but the K ÷-stimulated release of both amino acids is diminished. The anion transport blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) inhibited the K ÷ -stimulated release of taurine as much as C1 omission, but the DIDS inhibition was, relatively, even more pronounced in the release of GABA. Furosemide, an inhibitor of C1- transport, reduced the K+-evoked release of GABA even more. The C1 -dependent component in the release of taurine has been shown to be related to cell volume changes, since it is markedly reduced when cell swelling is prevented (Dominquez et al., 1989). CI- ions seem to be essential for swelling and release of taurine in astrocytes, since both processes are inhibited in low-C1- media or in the presence of furosemide (Kimelberg and Frangakis, 1985; Pasantes-Morales et al., 1990). On the other hand, swelling of astrocytes causes depolarization of their membranes proportionally to the degree of swelling (Kimelberg and O'Connor, 1988). However, also in crude synaptosomal fractions from the rat cerebral cortex the K +-evoked release of taurine has been shown to be abolished in media in which CI- ions have been replaced by gluconate (S~inchez Olea and Pasantes-Morales, 1990). With regard to brain slices, Turner et al. (1987) have shown that the release of preloaded GABA from rat cerebral cortical slices, in contrast to 5-hydroxytryptamine and noradrenaline, the other putative neurotransmitters studied, is not markedly increased when C1- ions are replaced by a number of different

impermeant anions. We have found that the replacement of C1- ions by a permeant anion, acetate, evokes release of taurine from slices from the mouse cerebral cortex. The evoked release is markedly greater in 3 day-old mice than in adults. The effect is also discernible, though somewhat smaller in magnitude, when an impermeant anion, gluconate, is used as replacement (Oja and Saransaari, 1992). These results indicate that only a part of the induced release of taurine is unquestionably due to swelling of the preparations. In feline cerebral cortical slices the K +-induced swelling has been attributed to the K +stimulated release of transmitters (Bourke et al., 1983). In in vivo experiments weak organic acids such as acetate have been shown to induce release of taurine through an osmotic-sensitive process in the rat hippocampus (Solis et al., 1990). Our above results indicate that acetate similarly induces some release of taurine also in vitro from slices in the absence of chloride. In this respect taurine apparently differs from GABA, since no significant difference has been observed between the effects of propionate, isethionate, gluconate and methylsulfate on the release of GABA from rat cerebral cortical slices (Turner et al., 1987). In order to establish whether or not the release of taurine in our experiments could be due to cell volume changes during superfusion and stimulation, the extent of intracellular swelling of the slices has been assessed and related to the magnitude of taurine release under the same experimental conditions. A positive correlation is obtained between these two parameters in both 3 day-old and adult mice, but it is not perfect (Saransaari and Oja, 1991). We interpret this to show that taurine is indeed released as a consequence of cell volume adjustments from cerebral cortical slices, but a part of the release could arise directly from membrane depolarization.

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10. SUMMARY In brain slices the mechanisms of release of GABA have been extensively studied, but those of taurine markedly less. The knowledge acquired from studies on GABA is, nevertheless, still fragmentary, not to speak of that obtained from the few studies on taurine, and firm conclusions are difficult, even impossible, to draw. This is mainly due to methodological matters, such as the diversity and pitfalls of the techniques applied. Brain slices are relatively easy to prepare and they represent a preparation that may most closely reflect relations prevailing in vivo, since the tissue structure and cellular integrity are largely preserved. In our opinion the most recommendable method at present is to superfuse freely floating agitated slices in continuously oxygenated medium. Taurine is metabolically rather inert in the brain, whereas the metabolism of GABA must be taken into account in all release studies. The use of inhibitors of GABA catabolism is discouraged, however, since a block in GABA metabolism may distort relations between different releasable pools of GABA in tissue. It is not known for sure how well, and homogeneously, incubation of slices with radioactive taurine labels the releasable pools but at least in the case of GABA there may prevail differences in the behavior of labeled and endogenous GABA. It is suggested therefore that the results obtained with radioactive GABA or taurine should be frequently checked and confirmed by analyzing the release of respective endogenous compounds. The spontaneous efflux of both GABA and taurine from brain slices is very slow. The magnitude of stimulation of GABA release by homoexchange is greater than that of taurine under the same experimental conditions. However, the release of both amino acids is generally enhanced by a great number of structural analogs, the most potent being those which are simultaneously the most potent inhibitors of uptake. This may result in part from inhibition of reuptake of amino acid molecules released from slices but the findings may also signify that the efflux of GABA and taurine is at least partially mediated by the membrane carriers operating in an outward direction. It is thus advisable not to interpret that stimulation of release in the presence of uptake inhibitors solely results from the block of reuptake of exocytotically released molecules, since changes in the carriermediated transport are also likely to occur upon stimulation. The electrical and K ÷ stimulation evoke the release of both GABA and taurine. The evoked release of GABA is several-fold greater than that of taurine in slices from the adult brain. During ontogeny, the K÷-stimulated release of GABA is initially very low, increasing strikingly towards adulthood. In contrast, the response of taurine release to K ÷ is slow but strikingly large in the immature brain, decreasing gradually during development. In fact, the evoked release of taurine has been in the developing brain several-fold greater than the evoked release of GABA in every brain area studied. This relationship is drastically altered with advancing age. These results emphasize the importance of taurine in regulation of excitability in the immature brain. No marked changes occur in the

basal or evoked release of GABA and taurine in different brain areas during ageing. A salient feature of the stimulated release of taurine is a slow and prolonged time course, while the responses in the release of GABA are prompt. The K+-dependence curves are similar for GABA and taurine, however. An omission of Na ÷ ions causes a more pronounced release of taurine than that of GABA from slices. The membrane Na + channels and Na÷/K+-ATPases are apparently involved in the regulation of the release. In most brain regions the major part of the K +-stimulated release of GABA has been dependent on Ca 2+, whereas the electrically or veratridine-evoked release has shown less Ca 2+ dependency. The Ca: ~ -independent release may occur concomitantly with the Ca 2+-dependent release and take place via the high-affinity transport sites of GABA. The stimulated release of GABA is generally more Ca 2~ dependent in the adult than developing brain. The stimulated release of taurine is likewise Ca" ÷ dependent, the dependency being greater in the immature than mature brain. The evaluation as to whether or not the K ÷-stimulated release is dependent on Ca 2 + is hampered by the great enhancement of basal release in Ca 2+-free media, more so in the case of taurine than GABA. Excitatory amino acids and their agonists evoke release of GABA and taurine from brain slices. The stimulated release of taurine from cerebral cortical slices is massively greater in developing than in adult mice. Furthermore, the N M D A and A M P A (quisqualate) subtypes of glutamate receptors could be involved in the stimulation of taurine release in low-K + media, whereas both N M D A - and kainatesensitive receptors may regulate the K ÷-stimulated release. Effects of drugs have been tested almost exclusively on the release of GABA. The drugs that interact with Na + movements across plasma membranes affect the release of GABA as well. Other effective drugs include barbiturates, convulsant alkaloids, anticonvulsants, tetanus toxin, diazepam, haloperidol and imipramil, to mention only those that have been reliably shown to exert an influence. No drugs that affect specifically only the release of GABA or taurine are known at present. Taurine has been demonstrated to interfere with both GABAA and GABAB receptors. This may be the reason why taurine is able to modulate the K ÷-stimulated release of GABA. On the other hand, GABAergic substances modify the release of taurine from brain slices. The importance of dopamine as a modulator of the release of GABA has been extensively studied, though the results are largely contradictory. The excitatory and inhibitory effects of dopamine on the release of GABA may be explained by the actions of the different subtypes of dopamine receptors. The release of taurine from striatal slices is also modified by presynaptic dopamine receptors. It has been suggested recently that the release of taurine solely results from cell volume regulation and water movements across plasma membranes. These ideas mainly stem from data obtained in experiments carried out with cultured astrocytes and cerebellar granule cells. In brain slices the matter is not so simple, since intracellular swelling in slices cannot be the only factor responsible for the enhanced release

GABA AND TAURINE RELEASE

of taurine under the influence of depolarizing agents. The general properties of the evoked release of taurine show, however, that a major part of it must be due to processes other than exocytosis of synaptic vesicles.

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