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Na+ influx through Ca2+ channels can promote striatal GABA efflux in Ca2+-deficient conditions in response to electrical field depolarization
232
Brain Re~eantz, h32 ( 1993 ) 232 - 23~ i 1993 Elsevier Science Publishers B.V. All rights reserved 0(ll)f~-,~993/q3/Sl)6.(~l)
BRES 19566
Na + influx through C a 2 + channels can promote striatal G A B A effiux in C a 2 + - d e f i c i e n t conditions in response to electrical field depolarization Sandor Bernath a,,, Michael J. Zigmond a, Eric S. Nisenbaum a, E. Sylvester Vizi b, Theodore W. Berger c Department of Behavioral Neuroscience, Unicersity of Pittsburgh, 469 Crawford Hall, Pittsburgh, PA 15260, USA, t, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary c Department of Biomedical Engineering, Unit,ersity of Southern California, Los Angeles, CA, USA (Accepted 10 August 1993)
Key words: 7-Aminobutyric acid; Neostriatum; w-Conotoxin; BAPTA-AM; Antidromic stimulation; Na + permeability
Electrical field depolarization releases y-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca 2 +. w-Conotoxin GVIA (~o-CgTx; 1-50 nM), a neuronal Ca 2+ channel blocker, inhibits electrically evoked effiux of newly taken up [3H]GABA in a concentration-dependent m a n n e r in either normal or Ca2+-free medium. This suggests that ion influx occurs through Ca 2+ channels in the absence of external Ca 2 + and contributes to the effiux of GABA. Reducing external Na + concentration to 27.25 m M (low [Na + ]o medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked G A B A effiux depending on the external Ca 2 + concentrations. In normal Ca 2+ medium, electrically evoked G A B A effiux increases whereas, in CaZ+-free medium, it is greatly inhibited when [Na* ]o is reduced to 27.25 mM. In low [Na + ]o medium, G A B A effiux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na+-free medium, resting G A B A effiux increases 17-fold whereas evoked G A B A efflux diminishes. In Ca 2+-free medium, 70 rain of incubation with 1-2-bis-(2-aminophenoxy)ethane-N,N,N',N'tetraacetoxy methyl ester (BAPTA-AM, 1 p.M), an intracellular calcium chelator, increases both resting G A B A effiux and electrically evoked G A B A overflow by ~ 100%. These results suggest that: (1) in Ca2+-free conditions, Na + permeability of cells increases via Ca 2+ channels and this profoundly affects G A B A effiux. (2) Electrical field depolarization is likely to release G A B A by directly depolarizing axon terminals. (3) CaZ+-independent G A B A effiux is not promoted by an increase in intracellular free Ca 2+ concentration via N a + / C a 2+ exchange processes from internal pools.
INTRODUCTION It has been established that depolarization of nerve terminals increases Ca 2+ permeability and the subsequent Ca z+ influx promotes the release of neurotransmitter from synaptic vesicles 2. Although this process, exocytosis, is universal, it may not be the only way through which neurotransmitter can be released t'44. It has been proposed that GABA, and other putative amino acid neurotransmitters, can be released through a different mechanism as well 7'33'37. This alternative mechanism is Na+-sensitive and is thought to be mediated via carrier proteins tta4'3°. Uptake proteins cotransport amino acids and Na + and are sensitive to changes in membrane potential and in the extracellular/intracellular concentration gradient of Na+. It has been suggested that depolarization of nerve terminals
can result in a net effiux of transmitter from the cytoplasm into the synaptic cleft via the uptake system 11'32. Indeed, there have been observations that uptake inhibitors block the depolarization-evoked release of amino acids 35'43. We previously reported that nipecotic acid, a GABA uptake blocker, inhibited electrically evoked G A B A effiux in striatal slices in Ca 2*free conditions8C To study the nonexocytotic release of neurotransmitter, experiments are usually performed in cae+-de ficient medium. The various Ca2+-deficient conditions, however, have differential effects on the release of GABA 7. We previously observed the electrically evoked release of GABA in striatal slices was greatly inhibited in Ca2+-free medium when Mg 2+ concentration was elevated, and somewhat decreased when Ca e+ was reduced or omitted without further alterations in the
* Corresponding author. Fax: (1) (412) 624-9198 e-mail: [email protected].
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E l 1 5 0 - 2
233 buffer composition. In contrast, electrically evoked GABA efflux increased when ethyleneglycol-bis-(13aminoethylether)N,N'-tetra-acetic acid (EGTA) was administered to abolish the concentration gradient of Ca 2+8'9. In the present study, we explore this latter phenomenon by examining how Na ÷ influences electrically evoked GABA efflux in Ca2+-free conditions. MATERIALS AND METHODS
3-6 MO. The spike discharges of striatal neurons were amplified, filtered, displayed and stored on a computer.
Materials 1-2-bis-(2-Aminophenoxy)ethane-N,N,N',N' tetra-acetoxy methyl ester (BAPTA-AM) was obtained from Molecular Probes (Eugene, OR); o~-conotoxin GVIA, choline bicarbonate and choline chloride from Sigma Chemical (St Louis, MO); y-(2,3-3H)-aminobutyric acid (25 Ci/mmol, NET-191X) from New England Nuclear (Boston, MA) and TS-1 tissue solubilizer from Research Products International (Mt Prospect, IL). All other chemicals and reagents were obtained from Fisher Scientific (Pittsburgh, PA).
Analysis of data and statistics [ 3H] GABA efflux The term "GABA efflux" is used for the net release of GABA, i.e., the amount of GABA not captured by the uptake system after its release. In these studies, GABA efflux was measured as previously described 1°. Male Sprague-Dawley rats (200-400 g; ZivicMiller Laboratories, Allison Park, PA) were killed by decapitation and coronal slices (350/~m) of the rostral part of the striatum were made using a Mcllwain-Brinkmann tissue chopper (Brinkmann Instruments, Westbury, NY). The first two slices were discarded and the next four slices were preincubated at 25°C for 25 min in 2 ml of Ringer buffer containing 2.5 ~zCi [3H]GABA (0.1 /zM). Each slice then was transferred to perfusion chambers and perfused at a rate of 0.24 ml/min with the media at 37°C. Effluents were discarded during the first 60 min and thereafter 5-min fractions were collected and analysed by liquid scintillation spectroscopy. At the end of each experiment, slices were dissolved in 1 ml TS-1 tissue solubilizer and analysed to determine the tritium content. Unless otherwise noted, the medium for incubation and superfusion contained: 125 mM NaCI, 5 mM KCI, 1.5 mM MgCI~, 2 mM CaC12, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose and aminooxyacetic acid (0.1 mM). Two types of Na+-deficient conditions were used: external Na ÷ was either reduced to 27.25 mM by substituting sodium chloride with choline chloride (low [Na ÷ ]o buffer) or totally withdrawn by equimolar substitution of all sodium compounds with choline chloride, choline bicarbonate and potassium phosphate, respectively, according to the original composition (Na+-free buffer). When external Ca 2÷ was eliminated, the buffer contained either EGTA (1 mM) or, in a separate series of experiments, BAPTA-AM (1/~M). The buffer was gassed with 5% CO 2 and 95% 0 2 and the pH was maintained at 7.4. Tissue slices were stimulated once or twice using a constant current source at 10 Hz for 9 s during the collection of the third (S 0 and 12th (S 2) 5-min fractions. The stimulation consisted of biphasic square-wave pulses (20 mA, 3 ms pulse duration).
The amount of [3H]GABA present in the slice at any given time was estimated by adding all tritium collected in the superfusate in subsequent fractions to the amount present in the slice at the end of the experiment. Efflux of [3H]GABA was calculated by computing the amount of tritium present in each fraction as a percentage of the tritium estimated to be present in the slice at the beginning of the fraction. Basal efflux (B1, B 2) was estimated by determining the mean efflux during the 5-min fraction immediately before and after the fractions used to determine overflow. Evoked overflow ($1, S 2) was calculated by first determining efflux from the onset of stimulation until the prestimulation baseline was restored. Estimated basal efflux was then subtracted from this value. Means+_S.E.M. are given throughout this paper. Significant differences between means were calculated with a two-tailed Student's t test. Differences were regarded to be significant if P < 0.05.
RESULTS
Effects of to-CgTx The effects of to-CgTx, a neuronal Ca2+-channel blocker, were tested on spontaneous and electrically evoked efflux of GABA in the presence and absence of Ca 2÷. In normal conditions, administration of w-CgTx (1-50 nM) 20 min before the second stimulation ($2) inhibited evoked GABA efflux in a concentration-dependent manner whereas it did not affect the spontaneous efflux of GABA. The maximum inhibition (48%) was reached at 10 nM concentration (Fig. 1). Similarly,
In uitro extracellular recording In separate experiments, slices containing the striatum and globus pallidus were prepared from rat brain. Animals were decapitated and their brains removed. The brains were hemisected, blocked caudally at the level of the hippocampus and cut ~ 30° to the horizontal plate. Each block then was affixed to a glass slide with a cyanoacrylate compound and placed in oxygenated (95% 0 2 / 5 % CO 2) buffer contained in the slicing chamber of a Vibratome. Horizontal sections of 350 p.m thickness were cut containing the striatum and the adjacent globus pallidus. Slices were incubated in oxygenated buffer at room temperature for 1 h before recording. After the incubation period, a single slice was transferred to a submerged plexiglass recording chamber and superfused at 35°C at a rate of 1 ml/min. Bipolar stimulating electrodes were made from twisted, Teflon-coated silver wire (i.d. 50 /zm) and placed in the white fibers in the globus pallidus at the vicinity of the striatum to antidromically stimulate striatal cells. Antidromic responses were identified on the basis of short and stable latencies ( < 3 ms) and the ability of cells to respond to high-frequency stimulation (up to 200 Hz). Unipolar square-wave pulses (100/xs duration) were delivered using intensities of 0.1-1 mA. Evoked spike discharges of single striatal cells were recorded extracellularly using single-barrel glass micropipettes filled with 2 M NaCI and having input impedances of
• × D .,~ m .< (_9
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0
Ca-free (lmM EGTA)
60
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20
°
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1o
2 0'
3 'o
4 0'
5o
conotoxin (nM) Fig. I. Inhibitory effects of w-CgTx on electrically evoked GABA effiux in presence and absence of external Ca 2+. Ca2+-free buffer (1 mM EGTA added) was administered throughout superfusion. Slices were stimulated twice with electrical field pulses (10 Hz, 20 mA, for 9 s) and co-CgTxwas administered 20 min before second stimulation.
234
0
normal
•
low
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[Na+]o
E <
=< 2.5 T'2.0
% 1.5 ×
E
Co-free + low [No*]o
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Co-free
%
I I l L I I I J I I I I 1.0 15 20 25 30 35 5 10 15 20 25 30 35 40 0 Time (min) Time (min) Fig. 2. Effects of reduced Na ~ content (27.25 mM) on fractional efflux of [3H]GABA in normal (a) and Ca 2 '-free conditions (b). In low [Na ' L, buffer, choline chloride was substituted for NaCI. Where indicated, ion-deficient conditions were administered throughout superft, sion. Each point represents mean ± S.E.M. of four (a) and six (b) experiments, respectively. * P < 0.05 when compared with corresponding control. I
I
5
10
in Ca 2+-free conditions (1 mM E G T A added), ¢o-CgTx inhibited evoked GABA effiux but did not influence spontaneous GABA effiux. The maximum inhibition (52%) was reached at 10 nM concentration (Fig. 1).
Effects oJ" low [Na *],, concentration on antidromically ecoked spike firing of striatal cells Electrically evoked GABA efflux is TTX-sensitive in normal medium ~ and the above results demonstrate that it remains TTX-sensitive in low [Na ~],, buffer as well. The involvement of voltage-sensitive Na ~ channels in the release process suggests that electrical field pulses promote action potential-like activity thereby releasing GABA. A reduced [Na+]o content, however, is expected to affect the excitability of neuronal membranes z2. Therefore, we tested the effects of low [Na + ],, buffer on the action potential activity of striatal cells. Striatopallidal projecting fibers of striatal cells were stimulated to evoke spike discharges antidromically (n = 8). Cells were stimulated 10 times with six-pulse trains and traces were overlayed by computer to detect changes in latency. Antidromic responses were identified by their short latency ( < 3 ms) and verified by the ability of cells to follow high-frequency stimulation (up to 200 Hz) with no change in latency. Fig. 3 shows a representative example of spike discharges of a striatal cell evoked by a six-pulse train delivered antidromically
Effects of Na +-deficient conditions on GABA e]flux The effects of reduced [Na ' ]o content were tested on spontaneous and electrically evoked effiux of GABA. In low [Na + ]o buffer (27.25 raM), spontaneous effiux of radiolabelled GABA increased more than three-fold and electrically evoked GABA overflow increased more than two-fold (Fig. 2a, Table I). In low [Na+],, conditions, TTX (5 /,tM) inhibited evoked GABA overflow by 78% (Table II) whereas spontaneous GABA effiux was not affected. In low [Na+]o buffer, electrically evoked GABA effiux was highly dependent on the presence of Ca 2+. In Ca2+-free and low [Na + ],, medium, electrically evoked GABA effiux was depressed by 88% (Fig. 2b, Table 1). In Na+-free conditions, spontaneous GABA effiux was elevated ~ 17-fold whereas evoked GABA overflow was depressed by 76% (Table I).
TABLE 1
Effects ¢~f Na +-deficient conditions on spontaneous (B) and electrically evoked (S) GABA ej]hcr in presence and absence qf ¢:wernal Ca 2 Ca2%free a n d / o r Na'-deficient buffers were administered throughout superfusion. Where indicated, NaCI was replaced with equimolar concentration of choline CI. When using Na +-free buffer, NaCI, NaHCO 3 and Nail 2PO4 were replaced by equimolar concentrations of choline CI, choline HCO~ and KH2PO 4, respectively. * P < 0.05 when compared with buffer with normal Ca 2+ and Na ~. ** P < 0.05 when compared with buffer with normal Na + in absence of Ca 2+. See Materials and Methods for details.
Buffer
Fractional efflux Ofl [3H]GABA (%)
lCa2+],,
[Na+],,
B
S
Normal
Normal Low (27.25 mM) Na+-free Normal Low (27.25 mM)
0.44 ± 0.03 1.54+_0.12 * 7.82±0.15 * 2.45 ± (I.25 * 2.81 ± 0.29
0.59 4- 0.08 1.36±0.18 0.14±_0.05 3.09 ± 0.58 0.38 + II. 11
Ca 2 +-free + EGTA ( 1 mM)
n
* * * ~*
4 4 4 6 6
235
0
TABLE II
normal
•
C a - f r e e + BAPTA-AM (1 #M)
2.0
Effect of TTX (5 I~M) on spontaneous (B2/B1) and electrically euoked ($2/S1) GABA release in presence of reduced external Na + <
NaCl was replaced with choline Cl and was administered throughout superfusion. TTX (5 ~M) was added to buffer 20 min before second stimulation. * P < 0.05 when compared with control.
1.5 !
% "5
Control TTX (5 ~M)
B2/B1
$2/S1
n
1.06 _+0.11 1.14_+0.07
0.73 _+0.10 0.16_+0.04*
4 4
1.0
-~ o.5 D
©
0.0 0
I 5
, 10
, 15
, 20
I 25
, 30
I ,35
Time (rain)
Fig. 5. Effects of BAPTA-AM on [3H]GABA efflux in rat striatal slices. Ca2+ was omitted and BAPTA-AM (1/zM) was administered throughout superfusion. Each point represents mean_+S.E.M. of six experiments. * P < 0.05 when compared with corresponding control. See Materials and Methods for details.
I
0.2 mV
5.0
ms
Fig. 3. Representative example of spike firing of a striatal cell elicited by antidromic stimulation of striatopallidal projecting fibers at 200 Hz delivered by six-pulse trains. Unipolar square-wave pulses were delivered using intensities of 0.1-1.0 mA. This procedure was used to verify antidromic nature of electrical stimulation. Antidromic responses were identified by their short latencies (< 3 ms) and verified by ability of cells to respond to high-frequency stimulation without changes in latency. See Materials and Methods for details.
at 200 Hz. R e p l a c e m e n t of n o r m a l buffer with low [Na ÷]o m e d i u m gradually i n h i b i t e d s t i m u l a t i o n - e v o k e d spike discharges (Fig. 4, traces B,C). T h e total inhibition of spike discharge was p r e c e d e d by an increase in latency (Fig. 4, trace B). S u p r a m a x i m a l c u r r e n t (2 m A ) did not i n d u c e spike discharge in low [Na÷]o buffer (Fig. 4, trace D), however, spike firing was restored in n o r m a l m e d i u m (Fig. 4, trace E).
Effects o f B A P T A - A M T h e effects of B A P T A - A M , a n i n t r a c e l l u l a r Ca 2÷ chelator, were tested to e x a m i n e w h e t h e r i n t r a c e l l u l a r Ca z+ p r o m o t e d G A B A efflux in Ca2÷-free conditions. In n o r m a l buffer, slices released 0.44 + 0.6% of their
A
radiolabelled G A B A c o n t e n t in response to electrical
B
field d e p o l a r i z a t i o n (Fig. 5). I n Ca2+-free conditions, after 70 m i n of i n c u b a t i o n in the p r e s e n c e of B A P T A -
C
D E
mV 2 ms Fig. 4. Representative example of effects of reduced [Na + ]o content (27.25 mM) on spike discharges of striatal neurons in response to antidromic stimulation (n = 8). Trace A: response to a single pulse delivered at 0.7 mA in normal superfusion medium. Trace B: response 8 min after replacing normal buffer with low [Na + ]o solution. Trace C: next response 6 s after previous response shown as trace B. Trace D: response to a supramaximal stimulus (2 mA) 9 min after replacing normal buffer with low [Na + ]o solution. Trace E: response 20 min after replacing Na+-deficient buffer with normal medium.
A M (1 /~M), evoked G A B A overflow = 100% (S1 = 0.84 + 0.11; Fig. 5).
increased
DISCUSSION A n i m p o r t a n t criterion for n e u r o t r a n s m i t t e r function is that an e n d o g e n o u s substance is released from nerve t e r m i n a l s as a result of action p o t e n t i a l - d r i v e n C a 2 + - d e p e n d e n t exocytosis. It appears, however, that G A B A , the major inhibitory n e u r o t r a n s m i t t e r in the CNS, only partially satisfies this r e q u i r e m e n t since it can be released, to some extent, in the a b s e n c e of external Ca 2+ (ref. 7). P a r a d o x i c a l l y , d e p o l a r i z a t i o n - e v o k e d efflux of G A B A can be a n t a g o n i z e d by verapamil, a Ca 2+ channel blocker, even in Ca2+-free c o n d i t i o n s 1°,t5,34,38,39 sug-
236 gesting that Ca 2 + channels influence G A B A effiux in the absence of external Ca 2+. Verapamil, originally introduced as a cardiac Ca ~+ channel blocker 2°, has been reported to block neuronal Ca 2+ channels to some extent 3~. Verapamil, however, has multiple effects, especially, at higher concentrations ( > 10 /xM). It has been reported to inhibit (a) the N a + / C a 2~ exchange Is'24, (b) Na ÷ channels and inward Na + current 1~'~'~4 and (c) neurotransmitter uptake~S'26'2'( To avoid these ambiguities, we used o)-CgTx which has been reported to block both N and L type neuronal Ca 2+ channels 2s'4L. It has also been suggested that w-CgTx might affect Na +-channels at higher concentrations ~.23. w-CgTx, like verapamil, inhibits the electrical field depolarization-evoked effiux of G A B A in Ca2+-free conditions. This confirms previous observations with verapamil and suggests that ion influx, most likely Na + influx, occurs through Ca 2+ channels and contributes to the effiux of GABA. This hypothesis is consistent with observations that Na + enters nerve terminals through Ca 2+ channels when Ca 2+ is omitted from the extracellular medium 3'4'12'2~'25. Indeed, it has been suggested that E G T A , a Ca 2+ chelator, which removes the channel-bound Ca 2+ in Ca2+-free medium, facilitates G A B A effiux in synaptosomes by increasing Na + permeability 5,~4. The present results indicate that G A B A can be released with electrical field depolarization of striatal slices when either Na + or Ca 2+ is present at normal concentrations. However, the impact of electrical field pulses on G A B A effiux is greatly diminished in buffers which are deficient in both ions. These observations suggest that G A B A can be released via both Ca 2+-dependent and Na+-dependent mechanisms and the presence of at least one of the two cations is necessary for electrical field depolarization-evoked effiux of GABA. It remains to be elucidated whether Na + influx through Ca 2+ channels is directly related to a putative carrier-mediated release or movement of charged particles through those channels influences the release process via other ways. Although it is not clear whether the Na+-dependent mechanism contributes to the release of G A B A under physiological conditions, it has important implications from an experimental point of view. Manipulations which alter Na + influx through excitable membranes are likely to modify the overall release of G A B A and this calls for caution in evaluating experimental data. We observed, for example, that electrically evoked release of G A B A increases in either Ca 2 +- or Na+-deficient conditions. These results, however, may not reflect an absolute increase in G A B A release. Instead, it is likely to be the result of inhibition of the electro-
genic G A B A uptake when the electrochemical gradient for Na + decreases due to either Na+-deficient conditions or an increased Na + permeability. The present results demonstrate that evoked G A B A efflux is TTX-sensitive in low [Na +],, buffer. This suggests that electrical field pulses promote G A B A effiux by eliciting action potential-like activity of striatal cells. However, spike firing of striatal cells is inhibited under such conditions, suggesting that the 27.25-mM external Na + concentration is insufficient to support a self-sustained propagation of action potentials. These observations suggest that electrical field pulses can directly depolarize axon terminals, thereby, promoting transmitter efflux. In this respect, electrical field depolarization has similar characteristics to those of high K + depolarization. Since electrically evoked G A B A effiux is TTX-sensitive in slice preparations under conditions where action potential activity of striatal cells is inhibited maT, it appears that Na + influx at the vicinity of the terminals is sufficient to promote the release of GABA. This suggests that the effiux of G A B A evoked by electrical field pulses may not be influenced by events occurring at somatodendritic sites. This further suggests that slice preparations may not be appropriate for exploring postsynaptic effects of neuronal interactions disregarding the extent to which certain neuronal networks are preserved intact. It has been proposed that the release of G A B A in Ca2+-free conditions does not reflect real Ca 2 ~ independence because intracellular Ca 2~ released from internal pools through a Na + / C a 2+ exchange system may promote G A B A efflux 17'27'31"~.Our present observations, however, that G A B A effiux can be evoked by electrical field depolarization in Ca2+-free conditions after a 70-min incubation in the presence of BAPTA, an intracellular calcium chelator, are not consistent with this proposal. BAPTA, like E G T A , is highly selective for Ca 2+ over Mg 2+ and other cations and its acetomethoxy ester form (BAPTA-AM) can penetrate cell membranes. Within the cell, the AM groups are cleaved by intracellular esterases yielding a cytoplasmtrapped compound (BAPTA) that is able to bind Ca-" ~, thereby, antagonizing an increase in intracellular Ca 2~ concentration (Cai)31'41L42. There have been other observations which also argue against the idea that a N a + / C a 2+ exchange system would be involved in Ca2+-independent G A B A efflux. (a) G A B A can be released from synaptosomes in the absence of extracellular Ca ?+ under conditions where they cannot maintain large intracellular Na + pools for the exchange process 6. (b) Veratridine-evoked G A B A efflux does not show correlation with the synaptosomal Ca 2+ content 3s.
237 In summary, it has been accepted that the depolarization-evoked release of transmitter which is neuronal of origin is Ca2+-dependent. To test this hypothesis, Ca 2÷ has to be omitted from the extracellular fluid or Ca 2+ influx must be prevented by Ca 2÷ blockers. Neither manipulation, however, can abolish the depolarization-evoked efflux of G A B A or other amino acid neurotransmitters 7. It appears that G A B A can be released by depolarizing stimuli via a Na÷-dependent mechanism as well although it remains to be elucidated whether it contributes to the overall release of G A B A under physiological conditions. The extent of this contribution may be overestimated under experimental conditions designed to test this due to an increased Na ÷ permeability and a subsequently augmented G A B A efflux. Nonetheless, sudden changes in the extracellular ion environment occur as a result of certain pathological processes and these can have profound physiological importance. Acknowledgements. We would like to thank A. Balla for skillful assistance. The research was supported by USPHS Grants MH45156, MH00343 and NS19608 and the Tourette Syndrome Association Permanent Research Fund.
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onist D600 in human heart cell segments, Pfluegers Arch., 403 (1985) 225-227. 14 Carvalho, C.M., Bandeira-Duarte, C., Ferreira, I.L. and Carvalho, A.P., Regulation of carrier-mediated and exocytotic release of [3H]GABA in rat brain synaptosomes, Neurochem. Res., 16 (1991) 763-772. 15 Carvalho, C.M., Santos, S.V. and Carvalho, A.P., ~,-Aminobutyric acid release from synaptosomes as influenced by Ca 2÷ and Ca 2+ channel blockers, Eur. J. Pharmacol., 131 (1986) 1-12. 16 Clay, J.R. and Shrier, A., Effects of D-600 on sodium current in squid axons, J. Membr. Biol., 79 (1984) 211-214. 17 Cunningham, J. and Neal, M.J., On the mechanism by which veratridine causes a calcium-independent release of ~/-aminobutyric acid from brain slices, Br. J. Pharmacol., 73 (1981) 665-667. 18 Erdreich, A., Spanier, R. and Rahamimoff, H., The inhibition of Na-dependent Ca uptake by verapamil in synaptic plasma membrane vesicles, Eur. J. Pharmacol., 90 (1983) 193-202. 19 Feldman, D.H., Olivera, B.M. and Yoshikami, D., Omega Conus geographus toxin: a peptide that blocks calcium channels, FEBS Lett., 214 (1987) 295-300. 20 Fleckenstein, A., Specific inhibitors and promoters of calcium action in the excitation-contraction coupling of heart muscle and their role in the prevention or production of myocardial lesions. In P. Harris and L. Opie (Eds.), Calcium and the Heart, Academic Press, London, UK, 1971, pp. 135-188. 21 Fukuda, J. and Kameyama, M., Tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in tissue-cultured spinal ganglion neurons from adult mammals, Brain Res., 182 (1980) 191-197. 22 Hodgkin, A.L. and Katz, B., The effect of sodium ions on the electrical activity of the giant axon of the squid, Z Physiol. (London), 108 (1949) 37-77. 23 Huang, H.-Y., w-Conotoxin GVIA inhibits release of noradrenaline from rat hippocampal slices in the absence of extracellular calcium, Neuropharmacology, 32 (1993) 133-137. 24 Kaczorowski, G.J., Slaughter, R.S., King, V.F. and Garcia, M.L., Inhibitors of sodium calcium exchange - identification and development of probes of transport activity, Biochim. Biophys. Acta, 988 (1989) 287-302. 25 Kostyuk, P.G., Mironov, S.L. and Shuba, Y.M., Two ion-selecting filters in the calcium channel of the somatic membrane of mollusc neurons, J. Membr. Biol., 76 (1983) 83-93. 26 Liron, Z., Roberts, E. and Wong, E., Verapamil is a competitive inhibitopr of -,/-aminobutyric acid and calcium uptakes by mouse brain subcellular particles, Life Sci., 36 (1985) 321-327. 27 Lockerbie, R.O. and Gordon-Weeks, P.R., Further characterization of [3H]-y-aminobutyric acid release from isolated neuronal growth cones: role of intracellular Ca 2÷ stores, Neuroscience, 17 (1986) 1257-1266. 28 McCleskey, E.W., Fox, A.P., Feldman, D.H., Cruz, L.J., Olivera, B.M., Tsien, R.W. and Yoshikama, D., to-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle, Proc. Natl. Acad. Sci. USA, 84 (1987) 4327-4331. 29 McGee, R. and Schneider, J.E., Inhibition of high affinity synaptosomal uptake systems by verapamil, Mol. Pharmacol., 16 (1979) 877-885. 30 McMahon, H.T., Rosenthal, L., Meldolesi, J. and Nicholls, D.G., o~-Latrotoxin releases both vesicular and cytoplasmic glutamate from isolated nerve terminals, J. Neurochem., 55 (1990) 20392047. 31 Negulescu, P.A., Reenstra, W.W. and Machen, T.E., Intracellular Ca requirements for stimulus-secretion coupling in parietal cell, Am. J. Physiol., 256 (1989) C241-C251. 32 Nelson, M.T. and Blaustein, M.P., GABA efflux from synaptosomes: effects of membrane potential, and external GABA and cations, J. Membr. Biol., 69 (1982) 213-223. 33 Nicholls, D.G., Release of glutamate, aspartate, and ,/-aminobutyric acid from isolated nerve terminals, J. Neurochem., 52 (1989) 331-341. 34 Norris, P.J., Dhaliwal, D.K., Druce, D.P. and Bradford, H.F., The suppression of stimulus-evoked release of amino acid neurotrans-
238
35
36
37 38
39
millers from synaptosomes by verapamil, J, Neurochem., 40 ( 19831 514-521. Pin, J.-P., Yasumoto, T. and Bockaert, J., Maitotoxin-evoked y-aminobutyric acid release is due not only to the opening of calcium channels, J. Neurochem., 5(1 (19881 1227-1232. Sandoval, M.E., Studies on the relationship between Ca 24 efflux from mitochondria and the release of amino acid neurotransmitters, Brain Res., 181 (1980) 357-367. Saransaari, P. and Oja, S.S., Release of G A B A and taurine from brain slices, Prog. Neurobiol., 38 (1992) 455-482. Sihra, T.S., Scott, I.G. and Nicholls, D.G., lonophore A23187, verapamil, protonophores, and veratridine influence the release of T-aminobutyric acid from synaptosomes by modulation of the plasma membrane potential rather than the cytosolic calcium, J. Neurochem., 43 (1984) 1624-1630. Sitges, M., Effect of organic and inorganic calcium channel blockers on gamma-amino-n-butyric acid release induced by monensin and veratrine in the absence of external calcium, J. Neurochern., 53 (1989) 436-441.
4/) "Fanabe, N. and Kijima, tt., Transmitter release at frog end-plat,: loaded with a Ca ~ %chelator, BAPTA: hypertonicity and erylhrosin B augment the release independently of internal Ca 2 . Neurosci. Lett., 92 (19881 52 ~57. 41 Tsien, R.W., Lipscombe, D.. Madison, D.\:., Bley, K.R. and Fox, A.P., Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci., 11 (1988)431 437. 42 Tsien, R.Y., A non-disruptive technique f~r loading calcium buffers and indicators inh~ cells, Nature (I,~mdon), 2q(} (1981) 527-528. 43 Turner, T.J. and Goldin, S.M., Multiple components of synapto~ somal [3H]-y-aminobutyric acid release resolved by a rapid superfusion system, Biochemistry, 28 (19891 586-593. 44 Vizi, E.S., Somogyi, G.T., ttarsing, L.G. and Zimanyi, 1., External Ca-independent release of norepinephrine by sympathomimetics and its role in the negative feedback modulation, Proc. Natl Acad. Sci. USA, 82 (19851 8775-8779.
Brain Re~eantz, h32 ( 1993 ) 232 - 23~ i 1993 Elsevier Science Publishers B.V. All rights reserved 0(ll)f~-,~993/q3/Sl)6.(~l)
BRES 19566
Na + influx through C a 2 + channels can promote striatal G A B A effiux in C a 2 + - d e f i c i e n t conditions in response to electrical field depolarization Sandor Bernath a,,, Michael J. Zigmond a, Eric S. Nisenbaum a, E. Sylvester Vizi b, Theodore W. Berger c Department of Behavioral Neuroscience, Unicersity of Pittsburgh, 469 Crawford Hall, Pittsburgh, PA 15260, USA, t, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary c Department of Biomedical Engineering, Unit,ersity of Southern California, Los Angeles, CA, USA (Accepted 10 August 1993)
Key words: 7-Aminobutyric acid; Neostriatum; w-Conotoxin; BAPTA-AM; Antidromic stimulation; Na + permeability
Electrical field depolarization releases y-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca 2 +. w-Conotoxin GVIA (~o-CgTx; 1-50 nM), a neuronal Ca 2+ channel blocker, inhibits electrically evoked effiux of newly taken up [3H]GABA in a concentration-dependent m a n n e r in either normal or Ca2+-free medium. This suggests that ion influx occurs through Ca 2+ channels in the absence of external Ca 2 + and contributes to the effiux of GABA. Reducing external Na + concentration to 27.25 m M (low [Na + ]o medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked G A B A effiux depending on the external Ca 2 + concentrations. In normal Ca 2+ medium, electrically evoked G A B A effiux increases whereas, in CaZ+-free medium, it is greatly inhibited when [Na* ]o is reduced to 27.25 mM. In low [Na + ]o medium, G A B A effiux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na+-free medium, resting G A B A effiux increases 17-fold whereas evoked G A B A efflux diminishes. In Ca 2+-free medium, 70 rain of incubation with 1-2-bis-(2-aminophenoxy)ethane-N,N,N',N'tetraacetoxy methyl ester (BAPTA-AM, 1 p.M), an intracellular calcium chelator, increases both resting G A B A effiux and electrically evoked G A B A overflow by ~ 100%. These results suggest that: (1) in Ca2+-free conditions, Na + permeability of cells increases via Ca 2+ channels and this profoundly affects G A B A effiux. (2) Electrical field depolarization is likely to release G A B A by directly depolarizing axon terminals. (3) CaZ+-independent G A B A effiux is not promoted by an increase in intracellular free Ca 2+ concentration via N a + / C a 2+ exchange processes from internal pools.
INTRODUCTION It has been established that depolarization of nerve terminals increases Ca 2+ permeability and the subsequent Ca z+ influx promotes the release of neurotransmitter from synaptic vesicles 2. Although this process, exocytosis, is universal, it may not be the only way through which neurotransmitter can be released t'44. It has been proposed that GABA, and other putative amino acid neurotransmitters, can be released through a different mechanism as well 7'33'37. This alternative mechanism is Na+-sensitive and is thought to be mediated via carrier proteins tta4'3°. Uptake proteins cotransport amino acids and Na + and are sensitive to changes in membrane potential and in the extracellular/intracellular concentration gradient of Na+. It has been suggested that depolarization of nerve terminals
can result in a net effiux of transmitter from the cytoplasm into the synaptic cleft via the uptake system 11'32. Indeed, there have been observations that uptake inhibitors block the depolarization-evoked release of amino acids 35'43. We previously reported that nipecotic acid, a GABA uptake blocker, inhibited electrically evoked G A B A effiux in striatal slices in Ca 2*free conditions8C To study the nonexocytotic release of neurotransmitter, experiments are usually performed in cae+-de ficient medium. The various Ca2+-deficient conditions, however, have differential effects on the release of GABA 7. We previously observed the electrically evoked release of GABA in striatal slices was greatly inhibited in Ca2+-free medium when Mg 2+ concentration was elevated, and somewhat decreased when Ca e+ was reduced or omitted without further alterations in the
* Corresponding author. Fax: (1) (412) 624-9198 e-mail: [email protected].
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E l 1 5 0 - 2
233 buffer composition. In contrast, electrically evoked GABA efflux increased when ethyleneglycol-bis-(13aminoethylether)N,N'-tetra-acetic acid (EGTA) was administered to abolish the concentration gradient of Ca 2+8'9. In the present study, we explore this latter phenomenon by examining how Na ÷ influences electrically evoked GABA efflux in Ca2+-free conditions. MATERIALS AND METHODS
3-6 MO. The spike discharges of striatal neurons were amplified, filtered, displayed and stored on a computer.
Materials 1-2-bis-(2-Aminophenoxy)ethane-N,N,N',N' tetra-acetoxy methyl ester (BAPTA-AM) was obtained from Molecular Probes (Eugene, OR); o~-conotoxin GVIA, choline bicarbonate and choline chloride from Sigma Chemical (St Louis, MO); y-(2,3-3H)-aminobutyric acid (25 Ci/mmol, NET-191X) from New England Nuclear (Boston, MA) and TS-1 tissue solubilizer from Research Products International (Mt Prospect, IL). All other chemicals and reagents were obtained from Fisher Scientific (Pittsburgh, PA).
Analysis of data and statistics [ 3H] GABA efflux The term "GABA efflux" is used for the net release of GABA, i.e., the amount of GABA not captured by the uptake system after its release. In these studies, GABA efflux was measured as previously described 1°. Male Sprague-Dawley rats (200-400 g; ZivicMiller Laboratories, Allison Park, PA) were killed by decapitation and coronal slices (350/~m) of the rostral part of the striatum were made using a Mcllwain-Brinkmann tissue chopper (Brinkmann Instruments, Westbury, NY). The first two slices were discarded and the next four slices were preincubated at 25°C for 25 min in 2 ml of Ringer buffer containing 2.5 ~zCi [3H]GABA (0.1 /zM). Each slice then was transferred to perfusion chambers and perfused at a rate of 0.24 ml/min with the media at 37°C. Effluents were discarded during the first 60 min and thereafter 5-min fractions were collected and analysed by liquid scintillation spectroscopy. At the end of each experiment, slices were dissolved in 1 ml TS-1 tissue solubilizer and analysed to determine the tritium content. Unless otherwise noted, the medium for incubation and superfusion contained: 125 mM NaCI, 5 mM KCI, 1.5 mM MgCI~, 2 mM CaC12, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose and aminooxyacetic acid (0.1 mM). Two types of Na+-deficient conditions were used: external Na ÷ was either reduced to 27.25 mM by substituting sodium chloride with choline chloride (low [Na ÷ ]o buffer) or totally withdrawn by equimolar substitution of all sodium compounds with choline chloride, choline bicarbonate and potassium phosphate, respectively, according to the original composition (Na+-free buffer). When external Ca 2÷ was eliminated, the buffer contained either EGTA (1 mM) or, in a separate series of experiments, BAPTA-AM (1/~M). The buffer was gassed with 5% CO 2 and 95% 0 2 and the pH was maintained at 7.4. Tissue slices were stimulated once or twice using a constant current source at 10 Hz for 9 s during the collection of the third (S 0 and 12th (S 2) 5-min fractions. The stimulation consisted of biphasic square-wave pulses (20 mA, 3 ms pulse duration).
The amount of [3H]GABA present in the slice at any given time was estimated by adding all tritium collected in the superfusate in subsequent fractions to the amount present in the slice at the end of the experiment. Efflux of [3H]GABA was calculated by computing the amount of tritium present in each fraction as a percentage of the tritium estimated to be present in the slice at the beginning of the fraction. Basal efflux (B1, B 2) was estimated by determining the mean efflux during the 5-min fraction immediately before and after the fractions used to determine overflow. Evoked overflow ($1, S 2) was calculated by first determining efflux from the onset of stimulation until the prestimulation baseline was restored. Estimated basal efflux was then subtracted from this value. Means+_S.E.M. are given throughout this paper. Significant differences between means were calculated with a two-tailed Student's t test. Differences were regarded to be significant if P < 0.05.
RESULTS
Effects of to-CgTx The effects of to-CgTx, a neuronal Ca2+-channel blocker, were tested on spontaneous and electrically evoked efflux of GABA in the presence and absence of Ca 2÷. In normal conditions, administration of w-CgTx (1-50 nM) 20 min before the second stimulation ($2) inhibited evoked GABA efflux in a concentration-dependent manner whereas it did not affect the spontaneous efflux of GABA. The maximum inhibition (48%) was reached at 10 nM concentration (Fig. 1). Similarly,
In uitro extracellular recording In separate experiments, slices containing the striatum and globus pallidus were prepared from rat brain. Animals were decapitated and their brains removed. The brains were hemisected, blocked caudally at the level of the hippocampus and cut ~ 30° to the horizontal plate. Each block then was affixed to a glass slide with a cyanoacrylate compound and placed in oxygenated (95% 0 2 / 5 % CO 2) buffer contained in the slicing chamber of a Vibratome. Horizontal sections of 350 p.m thickness were cut containing the striatum and the adjacent globus pallidus. Slices were incubated in oxygenated buffer at room temperature for 1 h before recording. After the incubation period, a single slice was transferred to a submerged plexiglass recording chamber and superfused at 35°C at a rate of 1 ml/min. Bipolar stimulating electrodes were made from twisted, Teflon-coated silver wire (i.d. 50 /zm) and placed in the white fibers in the globus pallidus at the vicinity of the striatum to antidromically stimulate striatal cells. Antidromic responses were identified on the basis of short and stable latencies ( < 3 ms) and the ability of cells to respond to high-frequency stimulation (up to 200 Hz). Unipolar square-wave pulses (100/xs duration) were delivered using intensities of 0.1-1 mA. Evoked spike discharges of single striatal cells were recorded extracellularly using single-barrel glass micropipettes filled with 2 M NaCI and having input impedances of
• × D .,~ m .< (_9
5O
g
3O
~
normal
0
Ca-free (lmM EGTA)
60
4O
20
°
0'
1o
2 0'
3 'o
4 0'
5o
conotoxin (nM) Fig. I. Inhibitory effects of w-CgTx on electrically evoked GABA effiux in presence and absence of external Ca 2+. Ca2+-free buffer (1 mM EGTA added) was administered throughout superfusion. Slices were stimulated twice with electrical field pulses (10 Hz, 20 mA, for 9 s) and co-CgTxwas administered 20 min before second stimulation.
234
0
normal
•
low
0
[Na+]o
E <
=< 2.5 T'2.0
% 1.5 ×
E
Co-free + low [No*]o
F i
£3.5~
H/ \'
I
~3.0" % 2× 2 . 5 ~ ,7_ i ® 2.0 L sc i •2 1.5i-
1.0
B g o.5 to o
O~ 0.0
•
4.0"-
m ,(
g
Co-free
%
I I l L I I I J I I I I 1.0 15 20 25 30 35 5 10 15 20 25 30 35 40 0 Time (min) Time (min) Fig. 2. Effects of reduced Na ~ content (27.25 mM) on fractional efflux of [3H]GABA in normal (a) and Ca 2 '-free conditions (b). In low [Na ' L, buffer, choline chloride was substituted for NaCI. Where indicated, ion-deficient conditions were administered throughout superft, sion. Each point represents mean ± S.E.M. of four (a) and six (b) experiments, respectively. * P < 0.05 when compared with corresponding control. I
I
5
10
in Ca 2+-free conditions (1 mM E G T A added), ¢o-CgTx inhibited evoked GABA effiux but did not influence spontaneous GABA effiux. The maximum inhibition (52%) was reached at 10 nM concentration (Fig. 1).
Effects oJ" low [Na *],, concentration on antidromically ecoked spike firing of striatal cells Electrically evoked GABA efflux is TTX-sensitive in normal medium ~ and the above results demonstrate that it remains TTX-sensitive in low [Na ~],, buffer as well. The involvement of voltage-sensitive Na ~ channels in the release process suggests that electrical field pulses promote action potential-like activity thereby releasing GABA. A reduced [Na+]o content, however, is expected to affect the excitability of neuronal membranes z2. Therefore, we tested the effects of low [Na + ],, buffer on the action potential activity of striatal cells. Striatopallidal projecting fibers of striatal cells were stimulated to evoke spike discharges antidromically (n = 8). Cells were stimulated 10 times with six-pulse trains and traces were overlayed by computer to detect changes in latency. Antidromic responses were identified by their short latency ( < 3 ms) and verified by the ability of cells to follow high-frequency stimulation (up to 200 Hz) with no change in latency. Fig. 3 shows a representative example of spike discharges of a striatal cell evoked by a six-pulse train delivered antidromically
Effects of Na +-deficient conditions on GABA e]flux The effects of reduced [Na ' ]o content were tested on spontaneous and electrically evoked effiux of GABA. In low [Na + ]o buffer (27.25 raM), spontaneous effiux of radiolabelled GABA increased more than three-fold and electrically evoked GABA overflow increased more than two-fold (Fig. 2a, Table I). In low [Na+],, conditions, TTX (5 /,tM) inhibited evoked GABA overflow by 78% (Table II) whereas spontaneous GABA effiux was not affected. In low [Na+]o buffer, electrically evoked GABA effiux was highly dependent on the presence of Ca 2+. In Ca2+-free and low [Na + ],, medium, electrically evoked GABA effiux was depressed by 88% (Fig. 2b, Table 1). In Na+-free conditions, spontaneous GABA effiux was elevated ~ 17-fold whereas evoked GABA overflow was depressed by 76% (Table I).
TABLE 1
Effects ¢~f Na +-deficient conditions on spontaneous (B) and electrically evoked (S) GABA ej]hcr in presence and absence qf ¢:wernal Ca 2 Ca2%free a n d / o r Na'-deficient buffers were administered throughout superfusion. Where indicated, NaCI was replaced with equimolar concentration of choline CI. When using Na +-free buffer, NaCI, NaHCO 3 and Nail 2PO4 were replaced by equimolar concentrations of choline CI, choline HCO~ and KH2PO 4, respectively. * P < 0.05 when compared with buffer with normal Ca 2+ and Na ~. ** P < 0.05 when compared with buffer with normal Na + in absence of Ca 2+. See Materials and Methods for details.
Buffer
Fractional efflux Ofl [3H]GABA (%)
lCa2+],,
[Na+],,
B
S
Normal
Normal Low (27.25 mM) Na+-free Normal Low (27.25 mM)
0.44 ± 0.03 1.54+_0.12 * 7.82±0.15 * 2.45 ± (I.25 * 2.81 ± 0.29
0.59 4- 0.08 1.36±0.18 0.14±_0.05 3.09 ± 0.58 0.38 + II. 11
Ca 2 +-free + EGTA ( 1 mM)
n
* * * ~*
4 4 4 6 6
235
0
TABLE II
normal
•
C a - f r e e + BAPTA-AM (1 #M)
2.0
Effect of TTX (5 I~M) on spontaneous (B2/B1) and electrically euoked ($2/S1) GABA release in presence of reduced external Na + <
NaCl was replaced with choline Cl and was administered throughout superfusion. TTX (5 ~M) was added to buffer 20 min before second stimulation. * P < 0.05 when compared with control.
1.5 !
% "5
Control TTX (5 ~M)
B2/B1
$2/S1
n
1.06 _+0.11 1.14_+0.07
0.73 _+0.10 0.16_+0.04*
4 4
1.0
-~ o.5 D
©
0.0 0
I 5
, 10
, 15
, 20
I 25
, 30
I ,35
Time (rain)
Fig. 5. Effects of BAPTA-AM on [3H]GABA efflux in rat striatal slices. Ca2+ was omitted and BAPTA-AM (1/zM) was administered throughout superfusion. Each point represents mean_+S.E.M. of six experiments. * P < 0.05 when compared with corresponding control. See Materials and Methods for details.
I
0.2 mV
5.0
ms
Fig. 3. Representative example of spike firing of a striatal cell elicited by antidromic stimulation of striatopallidal projecting fibers at 200 Hz delivered by six-pulse trains. Unipolar square-wave pulses were delivered using intensities of 0.1-1.0 mA. This procedure was used to verify antidromic nature of electrical stimulation. Antidromic responses were identified by their short latencies (< 3 ms) and verified by ability of cells to respond to high-frequency stimulation without changes in latency. See Materials and Methods for details.
at 200 Hz. R e p l a c e m e n t of n o r m a l buffer with low [Na ÷]o m e d i u m gradually i n h i b i t e d s t i m u l a t i o n - e v o k e d spike discharges (Fig. 4, traces B,C). T h e total inhibition of spike discharge was p r e c e d e d by an increase in latency (Fig. 4, trace B). S u p r a m a x i m a l c u r r e n t (2 m A ) did not i n d u c e spike discharge in low [Na÷]o buffer (Fig. 4, trace D), however, spike firing was restored in n o r m a l m e d i u m (Fig. 4, trace E).
Effects o f B A P T A - A M T h e effects of B A P T A - A M , a n i n t r a c e l l u l a r Ca 2÷ chelator, were tested to e x a m i n e w h e t h e r i n t r a c e l l u l a r Ca z+ p r o m o t e d G A B A efflux in Ca2÷-free conditions. In n o r m a l buffer, slices released 0.44 + 0.6% of their
A
radiolabelled G A B A c o n t e n t in response to electrical
B
field d e p o l a r i z a t i o n (Fig. 5). I n Ca2+-free conditions, after 70 m i n of i n c u b a t i o n in the p r e s e n c e of B A P T A -
C
D E
mV 2 ms Fig. 4. Representative example of effects of reduced [Na + ]o content (27.25 mM) on spike discharges of striatal neurons in response to antidromic stimulation (n = 8). Trace A: response to a single pulse delivered at 0.7 mA in normal superfusion medium. Trace B: response 8 min after replacing normal buffer with low [Na + ]o solution. Trace C: next response 6 s after previous response shown as trace B. Trace D: response to a supramaximal stimulus (2 mA) 9 min after replacing normal buffer with low [Na + ]o solution. Trace E: response 20 min after replacing Na+-deficient buffer with normal medium.
A M (1 /~M), evoked G A B A overflow = 100% (S1 = 0.84 + 0.11; Fig. 5).
increased
DISCUSSION A n i m p o r t a n t criterion for n e u r o t r a n s m i t t e r function is that an e n d o g e n o u s substance is released from nerve t e r m i n a l s as a result of action p o t e n t i a l - d r i v e n C a 2 + - d e p e n d e n t exocytosis. It appears, however, that G A B A , the major inhibitory n e u r o t r a n s m i t t e r in the CNS, only partially satisfies this r e q u i r e m e n t since it can be released, to some extent, in the a b s e n c e of external Ca 2+ (ref. 7). P a r a d o x i c a l l y , d e p o l a r i z a t i o n - e v o k e d efflux of G A B A can be a n t a g o n i z e d by verapamil, a Ca 2+ channel blocker, even in Ca2+-free c o n d i t i o n s 1°,t5,34,38,39 sug-
236 gesting that Ca 2 + channels influence G A B A effiux in the absence of external Ca 2+. Verapamil, originally introduced as a cardiac Ca ~+ channel blocker 2°, has been reported to block neuronal Ca 2+ channels to some extent 3~. Verapamil, however, has multiple effects, especially, at higher concentrations ( > 10 /xM). It has been reported to inhibit (a) the N a + / C a 2~ exchange Is'24, (b) Na ÷ channels and inward Na + current 1~'~'~4 and (c) neurotransmitter uptake~S'26'2'( To avoid these ambiguities, we used o)-CgTx which has been reported to block both N and L type neuronal Ca 2+ channels 2s'4L. It has also been suggested that w-CgTx might affect Na +-channels at higher concentrations ~.23. w-CgTx, like verapamil, inhibits the electrical field depolarization-evoked effiux of G A B A in Ca2+-free conditions. This confirms previous observations with verapamil and suggests that ion influx, most likely Na + influx, occurs through Ca 2+ channels and contributes to the effiux of GABA. This hypothesis is consistent with observations that Na + enters nerve terminals through Ca 2+ channels when Ca 2+ is omitted from the extracellular medium 3'4'12'2~'25. Indeed, it has been suggested that E G T A , a Ca 2+ chelator, which removes the channel-bound Ca 2+ in Ca2+-free medium, facilitates G A B A effiux in synaptosomes by increasing Na + permeability 5,~4. The present results indicate that G A B A can be released with electrical field depolarization of striatal slices when either Na + or Ca 2+ is present at normal concentrations. However, the impact of electrical field pulses on G A B A effiux is greatly diminished in buffers which are deficient in both ions. These observations suggest that G A B A can be released via both Ca 2+-dependent and Na+-dependent mechanisms and the presence of at least one of the two cations is necessary for electrical field depolarization-evoked effiux of GABA. It remains to be elucidated whether Na + influx through Ca 2+ channels is directly related to a putative carrier-mediated release or movement of charged particles through those channels influences the release process via other ways. Although it is not clear whether the Na+-dependent mechanism contributes to the release of G A B A under physiological conditions, it has important implications from an experimental point of view. Manipulations which alter Na + influx through excitable membranes are likely to modify the overall release of G A B A and this calls for caution in evaluating experimental data. We observed, for example, that electrically evoked release of G A B A increases in either Ca 2 +- or Na+-deficient conditions. These results, however, may not reflect an absolute increase in G A B A release. Instead, it is likely to be the result of inhibition of the electro-
genic G A B A uptake when the electrochemical gradient for Na + decreases due to either Na+-deficient conditions or an increased Na + permeability. The present results demonstrate that evoked G A B A efflux is TTX-sensitive in low [Na +],, buffer. This suggests that electrical field pulses promote G A B A effiux by eliciting action potential-like activity of striatal cells. However, spike firing of striatal cells is inhibited under such conditions, suggesting that the 27.25-mM external Na + concentration is insufficient to support a self-sustained propagation of action potentials. These observations suggest that electrical field pulses can directly depolarize axon terminals, thereby, promoting transmitter efflux. In this respect, electrical field depolarization has similar characteristics to those of high K + depolarization. Since electrically evoked G A B A effiux is TTX-sensitive in slice preparations under conditions where action potential activity of striatal cells is inhibited maT, it appears that Na + influx at the vicinity of the terminals is sufficient to promote the release of GABA. This suggests that the effiux of G A B A evoked by electrical field pulses may not be influenced by events occurring at somatodendritic sites. This further suggests that slice preparations may not be appropriate for exploring postsynaptic effects of neuronal interactions disregarding the extent to which certain neuronal networks are preserved intact. It has been proposed that the release of G A B A in Ca2+-free conditions does not reflect real Ca 2 ~ independence because intracellular Ca 2~ released from internal pools through a Na + / C a 2+ exchange system may promote G A B A efflux 17'27'31"~.Our present observations, however, that G A B A effiux can be evoked by electrical field depolarization in Ca2+-free conditions after a 70-min incubation in the presence of BAPTA, an intracellular calcium chelator, are not consistent with this proposal. BAPTA, like E G T A , is highly selective for Ca 2+ over Mg 2+ and other cations and its acetomethoxy ester form (BAPTA-AM) can penetrate cell membranes. Within the cell, the AM groups are cleaved by intracellular esterases yielding a cytoplasmtrapped compound (BAPTA) that is able to bind Ca-" ~, thereby, antagonizing an increase in intracellular Ca 2~ concentration (Cai)31'41L42. There have been other observations which also argue against the idea that a N a + / C a 2+ exchange system would be involved in Ca2+-independent G A B A efflux. (a) G A B A can be released from synaptosomes in the absence of extracellular Ca ?+ under conditions where they cannot maintain large intracellular Na + pools for the exchange process 6. (b) Veratridine-evoked G A B A efflux does not show correlation with the synaptosomal Ca 2+ content 3s.
237 In summary, it has been accepted that the depolarization-evoked release of transmitter which is neuronal of origin is Ca2+-dependent. To test this hypothesis, Ca 2÷ has to be omitted from the extracellular fluid or Ca 2+ influx must be prevented by Ca 2÷ blockers. Neither manipulation, however, can abolish the depolarization-evoked efflux of G A B A or other amino acid neurotransmitters 7. It appears that G A B A can be released by depolarizing stimuli via a Na÷-dependent mechanism as well although it remains to be elucidated whether it contributes to the overall release of G A B A under physiological conditions. The extent of this contribution may be overestimated under experimental conditions designed to test this due to an increased Na ÷ permeability and a subsequently augmented G A B A efflux. Nonetheless, sudden changes in the extracellular ion environment occur as a result of certain pathological processes and these can have profound physiological importance. Acknowledgements. We would like to thank A. Balla for skillful assistance. The research was supported by USPHS Grants MH45156, MH00343 and NS19608 and the Tourette Syndrome Association Permanent Research Fund.
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