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Ethanol-induced inhibition of GABA release from LS and SS mouse brain slices
Ah'ohol, Vol. 1, pp. 471-477, 1984. © Ankho International Inc. Printed in the U.S.A.
0741-8329/84 $3.00 + .00
Ethanol-Induced Inhibition of GABA Release From LS and SS Mouse Brain Slices THOMAS
C. H O W E R T O N
AND ALLAN
C. C O L L I N S 2
Institute f o r Behavioral Genetics and School o f Pharmacy University o f Colorado, BouMer, CO 80309 R e c e i v e d 14 J u n e 1984 HOWERTON, T. C. AND A. C. COLLINS. Ethanol-induced inhibition of GABA releasej?om LS and SS mouse brain slices. ALCOHOL 1(6) 471-477, 1984.--The effects of ethanol on K+-stimulated and spontaneous release of gammaaminobutyric acid (GABA) were studied in cortical and cerebellar brain slices obtained from long-sleep (LS) and shortsleep (SS) mice. A superfusion technique was used. Tissue slices were perfused with ethanol (0--515 raM) for 20 rain. Ethanol inhibited K+-stimulated 3H-GABA release at lower concentrations in LS cortical slices than in SS slices. Little or no inhibition of K+-stimulated aH-GABA release was seen in both LS and SS cerebellar slices. The spontaneous release of :~H-GABA was inhibited to equal degrees in LS and SS cerebellar slices but was unaffected in cortical slices, at the concentrations used. The possibility that ethanol inhibits 3H-GABA release by stimulating prostaglandin production was partially tested by assessing the effects of prostaglandin F.,,~ (PGF~) on :~H-GABA release in cortical and cerebellar slices. No effect of PGF2, on 3H-GABA release was seen in either brain region. These results support the notion that ethanol may elicit some of its actions by inhibiting neurotransmitter release. This effect appears to be influenced by genotype and brain region. Ethanol
GABA release
Genetics
Cortex
Cerebellum
A considerable literature indicates that ethanol alters the fluidity of biological m e m b r a n e s as measured by electron spin resonance spectroscopic and fluorescence polarization techniques [4, 9, 10, 12, 13, 22]. E v i d e n c e which suggests that this effect is related to the behavioral actions of ethanol includes the observation that ethanol's ability to fluidize m e m b r a n e s is reduced in animals that are tolerant to ethanol [22] and the observation that ethanol is less effective in fluidizing neuronal m e m b r a n e s obtained from short-sleep (SS) mice than it is in fluidizing m e m b r a n e s obtained from long-sleep (LS) mice [10]. The SS and LS mouse lines were selectively bred for differences in duration of ethanolinduced anesthesia ~sleep time) following the intraperitoneal injection of ethanol [25]. These animals differ in response to an acute ethanol dose largely because o f differing central nervous system sensitivity to ethanol [14]. While many investigators would agree that ethanol alters m e m b r a n e fluidity, little a g r e e m e n t exists as to precisely what membrane-related functions are critically involved in eliciting the behavioral actions o f ethanol. Ethanol is known to affect a number of m e m b r a n e - b o u n d e n z y m e s and m e m b r a n e - m e d i a t e d processes. For example, ethanol inhibits the e n z y m e N a K - A T P a s e [19, 20, 21], it alters ion fluxes across m e m b r a n e s [11, 20, 21, 26, 35, 43], and it inhibits neurotransmitter uptake [16, 17, 18, 28] and stimulated release [3, 5, 15, 22, 36]. Ethanol also appears to increase spontaneous release of norepinephrine [6,15]. Precisely
which of these effects, or o t h e r unstudied actions, underlie e t h a n o l ' s behavioral effects remains to be determined. The LS and SS mice offer a powerful tool for testing hypotheses related to e t h a n o l ' s behavioral actions. Presumably, i f a neurochemical action of ethanol is related to e t h a n o l ' s soporific effects, the LS mice should be affected more, or at a lower ethanol concentration, than are the SS. Recently, we reported that ethanol inhibited the K ÷stimulated release o f norepinephrine from LS cortical slices to a greater degree, and at l o w e r concentrations, than from SS cortical slices [15]. N o difference was seen b e t w e e n the two m o u s e lines for ethanol-induced inhibition of K ÷stimulated release from cerebellar slices, thereby indicating a regional specificity in e t h a n o l ' s actions. The e x p e r i m e n t s reported here are an extension o f this analysis. The effects of ethanol on K+-stimulated and spontaneous release of (3H)G A B A from LS and SS cortical and cerebellar slices were e x a m i n e d in an attempt to test further the hypothesis that inhibition of transmitter release is related to the soporific actions of ethanol. In addition, because prostaglandins are known to inhibit neurotransmitter release [33, 34, 42] and because previous studies from our laboratory have indicated that some of the behavioral effects of ethanol may involve an interaction between ethanol and one or more of the prostaglandins [7,27], the effects of prostaglandin Fe, (PGF.,~) on 3H-GABA release were examined.
1Supported, in part, by a predoctoral fellowship (AA-05158) to TCH, a Research Scientist Development Award (AA-00029) to ACC, and grant AA-03527. Partial support was also provided by grant RR-07013-16 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health. ZRequest for reprints should be addressed to Allan C. Collins. Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder. CO 80309. 471
472
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FIG. 1. Initial washout of 3H-GABA from cerebellar slices of LS (solid lines) and SS (dashed lines). Cerebellar slices were preloaded with 3H-GABA as described in the Method section• Slices were perfused with buffer and aliquots of each 4-rain fraction taken for scintillation counting. Effects of ions, ethanol and PGF2 were assessed on slices after a stable baseline had been attained.
METHOD Animals Male and female LS and SS mice, 60-80 days of age, were used in these experiments. The animals were from selected generations 33-36 and were housed with like-sexed littermates with food (Wayne Sterilizable Lab Biox) and water available ad lib. The animals were maintained in a colony room with a 12-hr light/dark cycle (lights on 0700-1900 hr) and a temperature of 23_ + 1°. Release Studies After an animal was sacrificed by cervical dislocation, the brain was quickly removed and washed with 2.0 ml of icecold isotonic saline. The cortex and cerebellum were dissected free from the rest of the brain and 0.3-ram slices were prepared with the aid of a McIlwain tissue chopper. The sliced tissue was transferred to 3.0 ml of Krebs-Ringer buffer (NaC1, 121 raM; KC1, 5.0 raM; CaCl2, 1.2 raM; KH2PO., 1.0 raM; MgC12, 2.0 raM; glucose, 11 raM; and NaHCO:~, 25 raM) in a stoppered flask. The tissue was continually gassed with a 95/5% mixture of oxygen and carbon dioxide to maintain a pH of 7.4. The slices were preloaded with exogenous 3HGABA by incubating the tissue for 30 rain, at 37°, in the presence of 30/~M 3H-GABA. The release of 3H-GABA was studied using the methods described by Arbilla et al. [1]. Three to four slices (6-10 mg) were transferred to one of four individual glass-jacketed superfusion chambers maintained at 25°. This temperature was used because preliminary experiments indicated that a stable baseline effiux of 3H-GABA was not attained by 45 rain when the slices were superfused at 37°. This is consistent with the published observations of Arbilla and coworkers [ 1,24]. The slices were supported by nylon netting in a volume of 0.75 ml of Krebs-Ringer buffer. A Buchler fourchannel polystaltic pump provided a constant flow rate of 0.5 ml/min to all four chambers, thus permitting the simultaneous stimulation of all four chambers. After an initial 45-rain washout period (see Fig. 1), a stable
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Min. Superfusion FIG. 2. Elution profiles of:~H-GABA release fi'om cerebellum of LS (upper panel) and SS (lower panel) mice. The SI and $2 responses were elicited by stimulating the release with 30 mM K~. Data are reported as %S1 values. These were calculated by comparing individual S1 values with the SI which contained the greatest number of CPM's. In the majority of experiments this was the first S1 fraction. The time shown includes the 45-rain washout period, fN=6).
baseline was attained. Thereafter. a total of 19 4-rain fractions was collected. Thus. the total superfusion time was 121 mm Stimulation of release was elicited by superfusing for two l-rain periods with high potassium buffer at 16-17 min (SI) and 56-57 rain ($2) after the 45-rain washout period had been completed: i.e.. SI and $2 were measured at 61-62 and 101-102 rain after the initiation of superfusion. The Supeffusion with the high potassium buffer resulted in elevated release of preloaded 3H-GABA and allowed the calculation of an $2/S1 ratio [1,2]. $2/S1 ratios provide an excellent method to assess drug effects on release since control {S 1) and drug effect ($2) can be measured in the same tissue under virtually identical conditions. Ethanol- or PGF~ocontaining buffers were superfused for 20 rain before $2 in order to determine their effects on K+-stimulated release. Atiquots (0.5 ml) of each fraction were taken for determination of radioactivity by liquid scintillation counting. In addition, an aliquot of the solubilized tissue slices was also taken for radioactivity determination. Effiux coefficients were calculated for each fraction. This was achieved in the following fashion: The number of CPMs of 3H-GAGA released in the last fraction was added to the total number of counts remaining in the tissue. The CPM's released during the last 4-min superfusion period was then divided by the total number of counts available for release during this superfusion period {tissue counts - CPM's released) to yielded the effiux coefficient. This procedure was repeated for each fraction. Baseline values for each stimulation were determined by averaging two fractions prior and two fractions after each release event These baselines were then subtracted from the coefficients determined during the stimulated release. The $2/S1 ratio was calculated using the efflux coefficients. In some experiments, the spontaneous release (Sp) was also estimated by calculating the ratio of the two baseline values (Sp2/Spl). Ionic Requirements Preliminary studies were carried out to assess the doseresponse relationships for 3H-GABA release with respect to
E T H A N O L INHIBITION OF GABA R E L E A S E
473
potassium and calcium ion. Buffers containing different calcium concentrations were superfused over the tissue slices for 20 min prior to exposure to 30 mM K + during the $2 stimulation. The release of 3H-GABA during this $2 stimulation was compared with a 1.0 mM Ca ++, 30 mM K + SI stimulation. The K + dose-response curve was constructed by comparing an initial 30 mM K + SI stimulation to lower concentrations of K + during the $2 stimulation.
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Chromatography GABA is deaminated as part of its metabolic fate. Therefore, in order to ascertain whether parent compound or deaminated metabolite was being released, aliquots of the superfusate were passed across a Dowex-50X (H ÷) cation exchange column [31]. Deaminated material passes through the column, whereas acid (1.0 N HCI) is required to elute GABA from the column. Acidified fractions of baseline and stimulated release effiux were passed over a 3×5-mm Dowex-50X column. The eluate was collected and the amount of deaminated metabolite released was measured by determining the radioactivity in this fraction. For both the LS and SS mice, greater than 95% of the added radioactivity was recovered from the column with the addition of 3.0 ml of 1.0 N HCI. Less than 5% passed through the column in those fractions expected to contain neutral and acidic compouL, ds. It was therefore concluded that the released radioactivity is due largely (approximately 95%) to :~H-GABA.
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Data Analysis Dose-response curves were analyzed by a two-way ANOVA program using a TI-59 programmable calculator. The release event was determined to be significant by a comparison of baseline coefficients and stimulated release coefficients using a Student's t-test. Whenever a significant F value was obtained with the ANOVA, data were analyzed further using the Tukey's B test for post hoc comparisons. RESULTS These studies were designed to ascertain whether in vitro ethanol alters the release of 3H-GABA from cortical and cerebellar slices obtained from LS and SS mice and to assess whether any effect seen might possibly mimic that elicited by PFG2,,.
Sp2/ C Spl ETHANOL (raM) FIG. 3. Effects of ethanol on K+-stimulated (30 mM) 3H-GABA release from LS (closed circles) and SS (open circles) cortical slices. The upper panel presents $2/S 1 ratios (stimulated release) calculated as described in the Method section. The lower panel presents Sp2/Spl ratios (spontaneous release). Each point represents the mean_+S.E.M, of 6 observations.
Ionic Requirements No differences were found in 3H-GABA release between LS and SS mice as ion concentrations were changed. Preliminary experiments showed K+-stimulated release of 3HGABA to be concentration dependent from 10-75 mM. At no K + concentration was any differential response seen between the LS and SS mouse lines. Substantial release was seen at 30 mM K + in cerebellum in both lines (Fig. 2). Similar release profiles were obtained with cortical slices (data not shown). Ca ++ ion dependence was also identical for the two lines. As a result, all further studies used 30 mM K + and 1.0 mM Ca ++.
Effects of Ethanol on :~H-GABA Release Figure 3 presents the results of experiments that assessed the effects of ethanol (0-515 mM) on the release of 3H-GABA from cortical slices. K+-stimulated release of 3H-GABA from both LS and SS slices was inhibited by ethanol
F(3,40)=22.5, p<0.001. The LS line is affected to a greater degree as is evidenced by the main effect of mouse line F(1,40)=3.6,p<0.05. The results depicted in Fig. 3 also indicate that ethanol did not affect the spontaneous release of 3H-GABA from cortical slices in either LS or SS mice. The effects of ethanol on the K+-stimulated and spontaneous 3H-GABA release from cerebellar slices are illustrated in Fig. 4. Ethanol (0--515 mM) did not have a significant effect on K+-stimulated release. However, these same concentrations of ethanol did affect spontaneous release. Significant F(3,40)=7.0, p<0.01 inhibition of 3H-GABA release was detected in both lines of mouse.
Effects of PGF2~ on 3H-GABA Release Figures 5 and 6 present the results of those experiments in which the potential effects of PGF.,~ on 3H-GABA release
474
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FIG. 4. Effects of ethanol on K+-stimulated (30 mM) 3H-GABA release from LS (closed circles) and SS (open circles) cerehellar slices. The upper panel presents $2/S1 ratios (stimulated release) calculated as described in the Method section. The lower panel presents Sp2/Spl ratios (spontaneous release). Each point represents the mean+_S.E.M, of 6 observations.
FIG. 5. Effects of PGFz~ on K~-stimulated (30 raM) release of :~HG A B A from LS (closed circles) and SS fopen circles)corticat slices. The upper panel presents the effects on $2/S1 ratios istimu!ated release) while the lower panel presents the effects on Spe/Spl ratios (spontaneous release). Each point represents the mean+_S.E.M, of 6 observations.
from cortical (Fig. 5) and cerebellar (Fig. 6) slices from LS and SS mice were examined. PGF~, concentrations of 0.01-1 /zM were used. N o effect of PGF2,, on spontaneous or K +stimulated 3I-I-GABA release was detected at any concentration in cortical or cerebellar slices.
from LS mice were more sensitive to ethanol-induced inhibition of release than were SS slices. Thus, it appears that the effects of ethanol on neurotransmitter release are probably influenced by the genotype o f the test organism and by the tissue being tested. Should this be true, further studies of the neurochemical actions of ethanol would best be done by carefully analyzing various brain regions and by carefully controlling the genotype of the test animal. The ethanol concentrations required to inhibit the release of 3H-GABA in the present study are clearly much higher than any that would be attained in vivo following the administration of even the most heroic of ethanol doses. However, this should not be used as evidence that ethanol does no,, inhibit the release o f G A B A in vivo. The method used to elicit G A B A release in the present studies, high extracettutar K +. may be affected less by ethanol than is the normal
DISCUSSION
The most notable f'mdings of the present study are the observations that ethanol inhibits K+-stimulated release of 3H-GABA from cortical but not cerebellar slices and that LS cortical slices are approximately three times more sensitive to this inhibition of release than are SS slices. We [15] have reported previously that ethanol inhibits the K+-stimulated release of norepinephrine from c o n i c a l but not cerebellar slices. In addition, we noted that cortical slices obtained
E T H A N O L INHIBITION OF GABA RELEASE
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stimulus-secretion process. Although we observed inhibition of release at high ethanol concentrations, it seems possible that ethanol could inhibit GABA release in vivo at much lower concentrations if the normal stimulation-secretion coupling process is more sensitive to ethanol than is K ÷stimulated release. Clearly, the observation that LS cortical slices were more sensitive to the inhibitory effects of ethanol on aH-GABA release following stimulation with 30 mM K ÷ argues that further studies of the effects of ethanol on the release of this transmitter substance may be fruitful. Several recent studies indicate that more than one pool of GABA may exist within the neuron [27,41]. Szerb and coworkers [38, 39, 40] have demonstrated that the specific activity of 3H-GABA released by high K + or veratridine is greater than that of nonreleased GABA, that a greater percent of exogenous than endogenous GABA is released following
475 stimulation and that GABA formed from glutamine is preferentially released over GABA formed from a glucose precursor. Szerb concludes from these studies that more than one GABA pool may exist and that the pool labelled with exogenous 3H-GABA probably represents the pool used for transmitter release. Thus, in the present study it is likely that the effects of ethanol on the physiologically important GABA pool were being examined. When the results of the present study are compared with those of our earlier study of the effects of ethanol on norepinephrine release [15], several interesting conclusions can be drawn. Firstly, as noted above, ethanol seems to inhibit transmitter release more readily in cortical than in cerebellar slices. This seems to be the case for both norepinephrine and GABA. Secondly, norepinephrine release was inhibited in LS cortical slices at concentrations of ethanol that are attained in vivo, while much higher concentrations were required to inhibit GABA release. This suggests that the release of different transmitters is differentially sensitive to ethanol's actions. Carmichael and Israel [3] have reported a similar finding. These investigators noted that electrically stimulated release of norepinephrine from rat brain cortical slices was inhibited by much lower concentrations of ethanol than was GABA release. This supports the notion that ethanol has some degree of specificity in its inhibitory effects on transmitter release. This specificity may involve the transmitter and the brain region being studied, as well as the genotype of the test animal. Other studies have indicated that the brain region being examined influences the results obtained with ethanol. For example, Sorenson and coworkers have examined the effects of ethanol on electrical activity in the cerebellum and hippocampus of LS and SS mice [32]. These investigators noted that Purkinje cell firing in LS cerebellum was inhibited at much lower concentrations of ethanol than was Purkinje cell firing in SS cerebellum. Differential sensitivity to the inhibitory actions of ethanol on firing rate was not seen in hippocampus. At the biochemical level, we [16] have reported that the uptake of norepinephrine and GABA into synaptosomes from cortex of LS and SS mice is inhibited by ethanol concentrations that are without effect on uptake into cerebellar synaptosomes. Ethanol inhibited uptake of these transmitters into synaptosomes at nonphysiological concentrations (5% w/v and above). While this earlier finding concerning the effects of ethanol on uptake suggests that an effect on this process does not relate to the mechanism by which ethanol elicits its behavioral effects, the fact that differences in sensitivity between cortex and cerebellum were seen is consistent with the argument that different brain regions are differentially sensitive to ethanol. The cortex does not appear to be more sensitive to all of the actions of ethanol, however. For example, Stokes and Harris [35] have reported that K+-stimulated uptake of Ca ++ into cerebellar synaptosomes is inhibited to a greater extent by ethanol than is Ca ++ uptake into cortical or brain stem synaptosomes. These results argue that no single brain region is uniquely sensitive to ethanol. The electrophysiological study of Sorenson et al. [32] argues that the most precise analysis of the effects of ethanol will probably be obtained by examining specific cell types. In summary, the results presented here indicate that ethanol inhibits the release of aH-GABA and that this inhibition is influenced by brain region and genotype. The fact that the LS mice are more sensitive to the effects of ethanol on 3H-GABA release lends support to the hypothesis that inhi-
476
HOWERTON
b i t i o n o f r e l e a s e u n d e r l i e s s o m e o f the b e h a v i o r a l a c t i o n s o f e t h a n o l . Since the L S a n d SS mice w e r e s u b j e c t e d to a found a t i o n effect early in the s e l e c t i o n p r o c e s s (the n u m b e r o f b r e e d i n g pairs w a s r e d u c e d in s e v e r a l early g e n e r a t i o n s ) , the c o n c l u s i o n s d r a w n f r o m t h e d a t a r e p o r t e d h e r e s h o u l d not be t a k e n as a n u n e q u i v o c a l d e m o n s t r a t i o n t h a t e t h a n o l e x e r t s its b e h a v i o r a l effects b y inhibiting n e u r o t r a n s m i t t e r release. S u p p o r t i v e e v i d e n c e m u s t b e o b t a i n e d u s i n g o t h e r selected genomic populations. T h e o b s e r v a t i o n t h a t PFGz,~ did not a l t e r G A B A release i n d i c a t e s t h a t e t h a n o l d o e s n o t a l t e r G A B A release by a m e c h a n i s m i n v o l v i n g t h e p r o d u c i n g o f PGF2~. A p r e l i m i n a r y r e p o r t f r o m o u r l a b o r a t o r y [8] h a s i n d i c a t e d t h a t e t h a n o l inc r e a s e s b r a i n PGE~ a n d PGF=,,~ c o n t e n t . O u r o b s e r v a t i o n s
AN D COLLINS
that LS and SS mice do n o t differ in PGE,~-induced inhibition o f n o r e p i n e p h r i n e release [15] a n d the c u r r e n t finding relating to the lack of effect o f PGF~,~ on '~H-GABA release suggest no s t r a i g h t f o r w a r d i n t e r a c t i o n b e t w e e n e t h a n o l a n d n e u r o t r a n s m i t t e r r e l e a s e , i n v o l v i n g p r o s t a g l a n d i n s , exists~ F u r t h e r studies will b e r e q u i r e d to fully e l u c i d a t e the m e c h a n i s m w h e r e b y e t h a n o l inhibits t r a n s m i t t e r release and the role s u c h an effect h a s in the b e h a v i o r a l effects o f e t h a n o l
ACKNOWLEDGEMF~NTS The assistance of M. L. Dubcovici~ in developing the release methodology and the editorial assistance of Rebecca Miles are appreciated.
REFERENCES
I. Arbilla, S., L. A. Kamal and S. Z. Langer. Inhibition by apomorphine of the potassium-evoked release of ['~H]-GABA from the rat substantia nigra in vitro. Br J Pharmacol 74: 38% 397, 1981. 2. Brennan, M. J. W. and R. C. Cantrill. Presynaptic membrane phosphorylation modulates the release of GABA from preloaded synaptosomes. J Neurochem 35: 506-508, 1980. 3. Carmichael, F. J. and Y. Israel. Effects of ethanol on neurotransmitter release from brain cortical slices. J Pharmacol Exp Ther 193: 824-834, 1975. 4. Chin, J. H. and D. B. Goldstein. Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes. Mol Pharmacol 13: 435-441, 1977. 5. Clark, J. W., H. Kalant and F. J. Carmichael. Effect of ethanol tolerance on release of acetylcholine and norepinephrine by rat cerebral cortical slices. Can J Physiol Pharmacol 55: 758-768, 1977. 6. Degani, N. D., E. M. Sellers and K. Kadzielawa. Ethanol induced spontaneous norepinephrine release from rat vas deferens. J Pharmacol Exp Ther 210: 22-26, 1979. 7. George, F. R. and A. C. Collins. Prostaglandin synthetase inhibitors antagonize the depressant effects of ethanol. Pharmacol Biochem Behav 10: 865-869, 1979. 8. George, F. R., G. I. Elmer, M. C. Ritz and A. C. Collins. Ethanol differentially increases in viw~ brain prostaglandin levels in LS vs. SS mice: A dose response analysis. Behav Genet 13: 534-535, 1983. 9. Goldstein, D. B. and J. H. Chin. Disordering effect of ethanol at different depths in the bilayer of mouse brain lipids. A lcoholism. Clin Exp Res 5: 256-258, 1981. 10. Goldstein, D. B., J. H. Chin and R. C. Lyon. Ethanol disordering of spin-labeled mouse brain membranes: Correlations with genetically determined ethanol sensitivity in mice. Proc Nail Acad Sci USA 79: 4231-4233, 1982. 11. Harris, R. A. and W. F. Hood. Inhibition of synaptosomal calcium uptake by ethanol. J Pharmacol Exp Ther 213: 562-568, 1980. 12. Harris, R. A. and F. Schroeder. Ethanol and the physical properties of brain membranes: Fluorescence studies. Mol Phurmacol 20: 128--137, 1981. 13. Harris, R. A. and F. Schroeder. Effects of barbiturates and ethanol on the physical properties of brain membranes. J Pharmacol Exp Ther 223: 424-431, 1982. 14. Heston, W. D. W., V. G. Erwin, S. M. Anderson and H. A. Robbins. A comparison of effects of alcohol on mice selectively bred for differences in ethanol sleep time. Lift, Sci 14: 365-370. 1974. 15, Howerton, T. C. and A. C. Collins. Ethanol-induced inhibition of norepinephrine release from brain slices obtained from LS and SS mice. Alcohol 1: 47-53, 1984.
16. Howerton, T. C., M. J. Marks anti A. C. Collins. Norepinephfine, gamma-aminobutyric acid and choline reuptake kinetics and the effects of ethanol in long-sleep and short-sleep mice. Subst Alcohol Actions Misuse 3: 8%99, 1982. 17. Hunt, W. A., E. Majchrowicz and T. K. Dalton. Alterations in high affinity choline uptake in brain after acute and chronic ethanol treatment. J Pharmacol Exp Ther 210: 25%263, 1979. 18. Israel, Y., F. J. Carmichael and J. A. MacDonald. Effects of ethanol on norepinephrine uptake and electrically stimulated release in brain tissue. Ann N Y Acad Sci 215: 38-48, 1973. 19. Israel, Y., H. Kalant and I. Laufer. Effects of ethanol on Na, K, Mg-stimulated microsomal ATPase activity. Biochem Pharmacol 14: 1803-1814, 1%5. 20. Israel, Y., H. Kalant and A. E. LeBlanc. Effects of lower alcohols on potassium transport and microsomal adenosinetriphopshatase activity of rat cerebral cortex. Biochem J 100: 27-33, 1966. 21. Israel, Y., H. Kalant, A. E. LeBianc, J. C. Bernstein and 1. Salazar. Changes in cation transport and (Na + + K+)-activated adenosine triphosphatase produced by chronic administration of ethanol. J Pharmacol Exp Ther 174: 330-336, 1970. 22. Johnson, D. A., N. M. Lee, R. Cooke and H. H. Loh. Ethanolinduced fluidization of brain lipid bilayers: Required presence of cholesterol in membranes for the expression of tolerance. ,'v/ot Pharmacol 15: 73%746, 1979. 23. Kalant. H. and W. Grose. Effects of ethanol and pentobarbital on release of acetytcholine from cerebral cortex slices..IPharmacol Exp Ther 158: 386-393. 1967, 24. Kamal. L. A.. S. Arbilla and S, Z Langer. Effects of GABAreceptor agonists on the potassium-evoked release ofaH-GABA from the rat substantia nigra. In: Presynaptic Receptors. Adwinces m the Biosciences. vo] 18. edited by S. Z Langer, K. Starke and M. L. Dubcovich. New York: Pergamon Press. 1979. pp. 193-197. 25. McClearn. G E. and R. Kakihana Selective breeding for ethanol sensitivity: Short-sleep and long-sleep mice. In: Develc~pment o f Animal Models as Pharmacogenetic Tools. edited by G. E. McClearn. R. A. Deitrich and V. G. Erwin. DHHS Publication No. ADM 81-1133. Washington. DC: U.S. Government Printing Office. 1981. pp. 147-160. 26. Michaelis. M. L.. E. K. Michaelis and T. Tehan. Alcohol effects on synaptic membrane calcium ion fluxes. Pharmaeol Biochem Behav Suppl, 18: 1%23. 1983. 27. Reubi. A. J.. L. L. Iversen and T. M. Jessell. Dopamine selectively increases 3H-GABA release from slices of rat substanti~ mgra in vitro. Nature 268: 652-654. 1977. 28. Ritz. M. C.. F. R. George and A. C. Collins. Indomethacm antagonizes ethanol-induced but not pentobarbitat-induced behavioral activation. Subst Alcohol Actions Misuse 2: 28%299. 1981.
ETHANOL INHIBITION OF GABA RELEASE
29. Roach, M. K., D. L. Davis, W. Pennington and E. Nordyke. Effects of ethanol on the uptake by rat brain synaptosomes of 3H-5-hydroxytryptamine, 3H-GABA and 3H-glutamate. Life Sci 12: 433-441, 1973. 30. Roberts, E. and R. Hammerschlag. Amino acid transmitters. In: Basic Neurochemistry. 2nd edition, edited by G. J. Siegel, R. W. Albers, R. Katzman and B. W. Agranoff. Boston: Little Brown and Company, 1976, pp. 218-245. 31. Rutledge, C. O. and J. Jonason. Metabolic pathways of dopamine and norepinephrine in rabbit brain, in vitro. J Pharmacol Exp 7"her 157: 493-502, 1967. 32. Sorenson, S., T. Dunwiddie, G. McClearn, R. Freeman and B. Hoffer. Ethanol-induced depressions in cerebellar and hippocampal neurons of mice selectively bred for differences in ethanol sensitivity: An electrophysiological study. Pharmacol Biochem Behav 14: 227-234, 1981. 33. Starke, K. Regulation of noradrenaline release by presynaptic receptor systems. Rev Physiol Biochem Pharmacol 17: 1-124, 1977. 34. Stjarne, C. Inhibitory effect of PGEz on noradrenaline secretion from sympathetic nerves as a function of external calcium, Prostaglandins 3: 105-109, 1973. 35. Stokes. J. A. and R. A. Harris. Alcohols and synaptosomal calcium transport. Mol Pharamcol 22: 9%104, 1982. 36. Sun, A. Y. Alcohol-membrane interactions in the brain: Norepinephrine release. Res ('ommltn Chem Pathol Pharmacol 15: 705-719, 1976.
477
37. Sun, A. Y., R. N. Seaman and C. C. Middleton. Effects of acute and chronic alcohol on brain membrane transport systems. Adv Exp Med Biol 85A: 123-138, 1977. 38. Szerb, J. C. Relationship between calcium-dependent and independent release of [~H]-GABA evoked by high K +, veratridine or electrical stimulation from rat cortical slices. J Neurochem 32: 1565-1573, 1979. 39. Szerb, J. C., E. T. Ross and L. Gurevich. Compartments of labeled and endogenous gamma-aminobutyric acid giving rise to release evoked by potassium or veratridine in rat cortical slices. J Neurochem 37:1186-1192, 1981. 40. Szerb, J. C. Storage and release of endogenous and labeled GABA formed from [:~H]-glutamine and [~C]-glucose in hippocampal slices: Effects of depolarization. Brain Res 293: 293303, 1984. 41. van der Hayden, J. A. M., K. Venema and J. Korf. Biphasic and opposite effects of dopamine and apomorphine on endogenous GABA release in the rat substantia nigra..I Neuro¢hem 34: 11%125, 1980. 42. Westfall, T. C. Local regulation of adrenergic neurotransmission. Physiol Rev 57: 65%727, 1977. 43. Yamamoto, H.-A. and R. A. Harris. Calcium-dependent "6Rb effiux and ethanol intoxication: Studies of human red blood cells and rodent synaptosomes. Eur J PharmacM 88: 357-363, 1983.
0741-8329/84 $3.00 + .00
Ethanol-Induced Inhibition of GABA Release From LS and SS Mouse Brain Slices THOMAS
C. H O W E R T O N
AND ALLAN
C. C O L L I N S 2
Institute f o r Behavioral Genetics and School o f Pharmacy University o f Colorado, BouMer, CO 80309 R e c e i v e d 14 J u n e 1984 HOWERTON, T. C. AND A. C. COLLINS. Ethanol-induced inhibition of GABA releasej?om LS and SS mouse brain slices. ALCOHOL 1(6) 471-477, 1984.--The effects of ethanol on K+-stimulated and spontaneous release of gammaaminobutyric acid (GABA) were studied in cortical and cerebellar brain slices obtained from long-sleep (LS) and shortsleep (SS) mice. A superfusion technique was used. Tissue slices were perfused with ethanol (0--515 raM) for 20 rain. Ethanol inhibited K+-stimulated 3H-GABA release at lower concentrations in LS cortical slices than in SS slices. Little or no inhibition of K+-stimulated aH-GABA release was seen in both LS and SS cerebellar slices. The spontaneous release of :~H-GABA was inhibited to equal degrees in LS and SS cerebellar slices but was unaffected in cortical slices, at the concentrations used. The possibility that ethanol inhibits 3H-GABA release by stimulating prostaglandin production was partially tested by assessing the effects of prostaglandin F.,,~ (PGF~) on :~H-GABA release in cortical and cerebellar slices. No effect of PGF2, on 3H-GABA release was seen in either brain region. These results support the notion that ethanol may elicit some of its actions by inhibiting neurotransmitter release. This effect appears to be influenced by genotype and brain region. Ethanol
GABA release
Genetics
Cortex
Cerebellum
A considerable literature indicates that ethanol alters the fluidity of biological m e m b r a n e s as measured by electron spin resonance spectroscopic and fluorescence polarization techniques [4, 9, 10, 12, 13, 22]. E v i d e n c e which suggests that this effect is related to the behavioral actions of ethanol includes the observation that ethanol's ability to fluidize m e m b r a n e s is reduced in animals that are tolerant to ethanol [22] and the observation that ethanol is less effective in fluidizing neuronal m e m b r a n e s obtained from short-sleep (SS) mice than it is in fluidizing m e m b r a n e s obtained from long-sleep (LS) mice [10]. The SS and LS mouse lines were selectively bred for differences in duration of ethanolinduced anesthesia ~sleep time) following the intraperitoneal injection of ethanol [25]. These animals differ in response to an acute ethanol dose largely because o f differing central nervous system sensitivity to ethanol [14]. While many investigators would agree that ethanol alters m e m b r a n e fluidity, little a g r e e m e n t exists as to precisely what membrane-related functions are critically involved in eliciting the behavioral actions o f ethanol. Ethanol is known to affect a number of m e m b r a n e - b o u n d e n z y m e s and m e m b r a n e - m e d i a t e d processes. For example, ethanol inhibits the e n z y m e N a K - A T P a s e [19, 20, 21], it alters ion fluxes across m e m b r a n e s [11, 20, 21, 26, 35, 43], and it inhibits neurotransmitter uptake [16, 17, 18, 28] and stimulated release [3, 5, 15, 22, 36]. Ethanol also appears to increase spontaneous release of norepinephrine [6,15]. Precisely
which of these effects, or o t h e r unstudied actions, underlie e t h a n o l ' s behavioral effects remains to be determined. The LS and SS mice offer a powerful tool for testing hypotheses related to e t h a n o l ' s behavioral actions. Presumably, i f a neurochemical action of ethanol is related to e t h a n o l ' s soporific effects, the LS mice should be affected more, or at a lower ethanol concentration, than are the SS. Recently, we reported that ethanol inhibited the K ÷stimulated release o f norepinephrine from LS cortical slices to a greater degree, and at l o w e r concentrations, than from SS cortical slices [15]. N o difference was seen b e t w e e n the two m o u s e lines for ethanol-induced inhibition of K ÷stimulated release from cerebellar slices, thereby indicating a regional specificity in e t h a n o l ' s actions. The e x p e r i m e n t s reported here are an extension o f this analysis. The effects of ethanol on K+-stimulated and spontaneous release of (3H)G A B A from LS and SS cortical and cerebellar slices were e x a m i n e d in an attempt to test further the hypothesis that inhibition of transmitter release is related to the soporific actions of ethanol. In addition, because prostaglandins are known to inhibit neurotransmitter release [33, 34, 42] and because previous studies from our laboratory have indicated that some of the behavioral effects of ethanol may involve an interaction between ethanol and one or more of the prostaglandins [7,27], the effects of prostaglandin Fe, (PGF.,~) on 3H-GABA release were examined.
1Supported, in part, by a predoctoral fellowship (AA-05158) to TCH, a Research Scientist Development Award (AA-00029) to ACC, and grant AA-03527. Partial support was also provided by grant RR-07013-16 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health. ZRequest for reprints should be addressed to Allan C. Collins. Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder. CO 80309. 471
472
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FIG. 1. Initial washout of 3H-GABA from cerebellar slices of LS (solid lines) and SS (dashed lines). Cerebellar slices were preloaded with 3H-GABA as described in the Method section• Slices were perfused with buffer and aliquots of each 4-rain fraction taken for scintillation counting. Effects of ions, ethanol and PGF2 were assessed on slices after a stable baseline had been attained.
METHOD Animals Male and female LS and SS mice, 60-80 days of age, were used in these experiments. The animals were from selected generations 33-36 and were housed with like-sexed littermates with food (Wayne Sterilizable Lab Biox) and water available ad lib. The animals were maintained in a colony room with a 12-hr light/dark cycle (lights on 0700-1900 hr) and a temperature of 23_ + 1°. Release Studies After an animal was sacrificed by cervical dislocation, the brain was quickly removed and washed with 2.0 ml of icecold isotonic saline. The cortex and cerebellum were dissected free from the rest of the brain and 0.3-ram slices were prepared with the aid of a McIlwain tissue chopper. The sliced tissue was transferred to 3.0 ml of Krebs-Ringer buffer (NaC1, 121 raM; KC1, 5.0 raM; CaCl2, 1.2 raM; KH2PO., 1.0 raM; MgC12, 2.0 raM; glucose, 11 raM; and NaHCO:~, 25 raM) in a stoppered flask. The tissue was continually gassed with a 95/5% mixture of oxygen and carbon dioxide to maintain a pH of 7.4. The slices were preloaded with exogenous 3HGABA by incubating the tissue for 30 rain, at 37°, in the presence of 30/~M 3H-GABA. The release of 3H-GABA was studied using the methods described by Arbilla et al. [1]. Three to four slices (6-10 mg) were transferred to one of four individual glass-jacketed superfusion chambers maintained at 25°. This temperature was used because preliminary experiments indicated that a stable baseline effiux of 3H-GABA was not attained by 45 rain when the slices were superfused at 37°. This is consistent with the published observations of Arbilla and coworkers [ 1,24]. The slices were supported by nylon netting in a volume of 0.75 ml of Krebs-Ringer buffer. A Buchler fourchannel polystaltic pump provided a constant flow rate of 0.5 ml/min to all four chambers, thus permitting the simultaneous stimulation of all four chambers. After an initial 45-rain washout period (see Fig. 1), a stable
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baseline was attained. Thereafter. a total of 19 4-rain fractions was collected. Thus. the total superfusion time was 121 mm Stimulation of release was elicited by superfusing for two l-rain periods with high potassium buffer at 16-17 min (SI) and 56-57 rain ($2) after the 45-rain washout period had been completed: i.e.. SI and $2 were measured at 61-62 and 101-102 rain after the initiation of superfusion. The Supeffusion with the high potassium buffer resulted in elevated release of preloaded 3H-GABA and allowed the calculation of an $2/S1 ratio [1,2]. $2/S1 ratios provide an excellent method to assess drug effects on release since control {S 1) and drug effect ($2) can be measured in the same tissue under virtually identical conditions. Ethanol- or PGF~ocontaining buffers were superfused for 20 rain before $2 in order to determine their effects on K+-stimulated release. Atiquots (0.5 ml) of each fraction were taken for determination of radioactivity by liquid scintillation counting. In addition, an aliquot of the solubilized tissue slices was also taken for radioactivity determination. Effiux coefficients were calculated for each fraction. This was achieved in the following fashion: The number of CPMs of 3H-GAGA released in the last fraction was added to the total number of counts remaining in the tissue. The CPM's released during the last 4-min superfusion period was then divided by the total number of counts available for release during this superfusion period {tissue counts - CPM's released) to yielded the effiux coefficient. This procedure was repeated for each fraction. Baseline values for each stimulation were determined by averaging two fractions prior and two fractions after each release event These baselines were then subtracted from the coefficients determined during the stimulated release. The $2/S1 ratio was calculated using the efflux coefficients. In some experiments, the spontaneous release (Sp) was also estimated by calculating the ratio of the two baseline values (Sp2/Spl). Ionic Requirements Preliminary studies were carried out to assess the doseresponse relationships for 3H-GABA release with respect to
E T H A N O L INHIBITION OF GABA R E L E A S E
473
potassium and calcium ion. Buffers containing different calcium concentrations were superfused over the tissue slices for 20 min prior to exposure to 30 mM K + during the $2 stimulation. The release of 3H-GABA during this $2 stimulation was compared with a 1.0 mM Ca ++, 30 mM K + SI stimulation. The K + dose-response curve was constructed by comparing an initial 30 mM K + SI stimulation to lower concentrations of K + during the $2 stimulation.
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Chromatography GABA is deaminated as part of its metabolic fate. Therefore, in order to ascertain whether parent compound or deaminated metabolite was being released, aliquots of the superfusate were passed across a Dowex-50X (H ÷) cation exchange column [31]. Deaminated material passes through the column, whereas acid (1.0 N HCI) is required to elute GABA from the column. Acidified fractions of baseline and stimulated release effiux were passed over a 3×5-mm Dowex-50X column. The eluate was collected and the amount of deaminated metabolite released was measured by determining the radioactivity in this fraction. For both the LS and SS mice, greater than 95% of the added radioactivity was recovered from the column with the addition of 3.0 ml of 1.0 N HCI. Less than 5% passed through the column in those fractions expected to contain neutral and acidic compouL, ds. It was therefore concluded that the released radioactivity is due largely (approximately 95%) to :~H-GABA.
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Data Analysis Dose-response curves were analyzed by a two-way ANOVA program using a TI-59 programmable calculator. The release event was determined to be significant by a comparison of baseline coefficients and stimulated release coefficients using a Student's t-test. Whenever a significant F value was obtained with the ANOVA, data were analyzed further using the Tukey's B test for post hoc comparisons. RESULTS These studies were designed to ascertain whether in vitro ethanol alters the release of 3H-GABA from cortical and cerebellar slices obtained from LS and SS mice and to assess whether any effect seen might possibly mimic that elicited by PFG2,,.
Sp2/ C Spl ETHANOL (raM) FIG. 3. Effects of ethanol on K+-stimulated (30 mM) 3H-GABA release from LS (closed circles) and SS (open circles) cortical slices. The upper panel presents $2/S 1 ratios (stimulated release) calculated as described in the Method section. The lower panel presents Sp2/Spl ratios (spontaneous release). Each point represents the mean_+S.E.M, of 6 observations.
Ionic Requirements No differences were found in 3H-GABA release between LS and SS mice as ion concentrations were changed. Preliminary experiments showed K+-stimulated release of 3HGABA to be concentration dependent from 10-75 mM. At no K + concentration was any differential response seen between the LS and SS mouse lines. Substantial release was seen at 30 mM K + in cerebellum in both lines (Fig. 2). Similar release profiles were obtained with cortical slices (data not shown). Ca ++ ion dependence was also identical for the two lines. As a result, all further studies used 30 mM K + and 1.0 mM Ca ++.
Effects of Ethanol on :~H-GABA Release Figure 3 presents the results of experiments that assessed the effects of ethanol (0-515 mM) on the release of 3H-GABA from cortical slices. K+-stimulated release of 3H-GABA from both LS and SS slices was inhibited by ethanol
F(3,40)=22.5, p<0.001. The LS line is affected to a greater degree as is evidenced by the main effect of mouse line F(1,40)=3.6,p<0.05. The results depicted in Fig. 3 also indicate that ethanol did not affect the spontaneous release of 3H-GABA from cortical slices in either LS or SS mice. The effects of ethanol on the K+-stimulated and spontaneous 3H-GABA release from cerebellar slices are illustrated in Fig. 4. Ethanol (0--515 mM) did not have a significant effect on K+-stimulated release. However, these same concentrations of ethanol did affect spontaneous release. Significant F(3,40)=7.0, p<0.01 inhibition of 3H-GABA release was detected in both lines of mouse.
Effects of PGF2~ on 3H-GABA Release Figures 5 and 6 present the results of those experiments in which the potential effects of PGF.,~ on 3H-GABA release
474
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FIG. 4. Effects of ethanol on K+-stimulated (30 mM) 3H-GABA release from LS (closed circles) and SS (open circles) cerehellar slices. The upper panel presents $2/S1 ratios (stimulated release) calculated as described in the Method section. The lower panel presents Sp2/Spl ratios (spontaneous release). Each point represents the mean+_S.E.M, of 6 observations.
FIG. 5. Effects of PGFz~ on K~-stimulated (30 raM) release of :~HG A B A from LS (closed circles) and SS fopen circles)corticat slices. The upper panel presents the effects on $2/S1 ratios istimu!ated release) while the lower panel presents the effects on Spe/Spl ratios (spontaneous release). Each point represents the mean+_S.E.M, of 6 observations.
from cortical (Fig. 5) and cerebellar (Fig. 6) slices from LS and SS mice were examined. PGF~, concentrations of 0.01-1 /zM were used. N o effect of PGF2,, on spontaneous or K +stimulated 3I-I-GABA release was detected at any concentration in cortical or cerebellar slices.
from LS mice were more sensitive to ethanol-induced inhibition of release than were SS slices. Thus, it appears that the effects of ethanol on neurotransmitter release are probably influenced by the genotype o f the test organism and by the tissue being tested. Should this be true, further studies of the neurochemical actions of ethanol would best be done by carefully analyzing various brain regions and by carefully controlling the genotype of the test animal. The ethanol concentrations required to inhibit the release of 3H-GABA in the present study are clearly much higher than any that would be attained in vivo following the administration of even the most heroic of ethanol doses. However, this should not be used as evidence that ethanol does no,, inhibit the release o f G A B A in vivo. The method used to elicit G A B A release in the present studies, high extracettutar K +. may be affected less by ethanol than is the normal
DISCUSSION
The most notable f'mdings of the present study are the observations that ethanol inhibits K+-stimulated release of 3H-GABA from cortical but not cerebellar slices and that LS cortical slices are approximately three times more sensitive to this inhibition of release than are SS slices. We [15] have reported previously that ethanol inhibits the K+-stimulated release of norepinephrine from c o n i c a l but not cerebellar slices. In addition, we noted that cortical slices obtained
E T H A N O L INHIBITION OF GABA RELEASE
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stimulus-secretion process. Although we observed inhibition of release at high ethanol concentrations, it seems possible that ethanol could inhibit GABA release in vivo at much lower concentrations if the normal stimulation-secretion coupling process is more sensitive to ethanol than is K ÷stimulated release. Clearly, the observation that LS cortical slices were more sensitive to the inhibitory effects of ethanol on aH-GABA release following stimulation with 30 mM K ÷ argues that further studies of the effects of ethanol on the release of this transmitter substance may be fruitful. Several recent studies indicate that more than one pool of GABA may exist within the neuron [27,41]. Szerb and coworkers [38, 39, 40] have demonstrated that the specific activity of 3H-GABA released by high K + or veratridine is greater than that of nonreleased GABA, that a greater percent of exogenous than endogenous GABA is released following
475 stimulation and that GABA formed from glutamine is preferentially released over GABA formed from a glucose precursor. Szerb concludes from these studies that more than one GABA pool may exist and that the pool labelled with exogenous 3H-GABA probably represents the pool used for transmitter release. Thus, in the present study it is likely that the effects of ethanol on the physiologically important GABA pool were being examined. When the results of the present study are compared with those of our earlier study of the effects of ethanol on norepinephrine release [15], several interesting conclusions can be drawn. Firstly, as noted above, ethanol seems to inhibit transmitter release more readily in cortical than in cerebellar slices. This seems to be the case for both norepinephrine and GABA. Secondly, norepinephrine release was inhibited in LS cortical slices at concentrations of ethanol that are attained in vivo, while much higher concentrations were required to inhibit GABA release. This suggests that the release of different transmitters is differentially sensitive to ethanol's actions. Carmichael and Israel [3] have reported a similar finding. These investigators noted that electrically stimulated release of norepinephrine from rat brain cortical slices was inhibited by much lower concentrations of ethanol than was GABA release. This supports the notion that ethanol has some degree of specificity in its inhibitory effects on transmitter release. This specificity may involve the transmitter and the brain region being studied, as well as the genotype of the test animal. Other studies have indicated that the brain region being examined influences the results obtained with ethanol. For example, Sorenson and coworkers have examined the effects of ethanol on electrical activity in the cerebellum and hippocampus of LS and SS mice [32]. These investigators noted that Purkinje cell firing in LS cerebellum was inhibited at much lower concentrations of ethanol than was Purkinje cell firing in SS cerebellum. Differential sensitivity to the inhibitory actions of ethanol on firing rate was not seen in hippocampus. At the biochemical level, we [16] have reported that the uptake of norepinephrine and GABA into synaptosomes from cortex of LS and SS mice is inhibited by ethanol concentrations that are without effect on uptake into cerebellar synaptosomes. Ethanol inhibited uptake of these transmitters into synaptosomes at nonphysiological concentrations (5% w/v and above). While this earlier finding concerning the effects of ethanol on uptake suggests that an effect on this process does not relate to the mechanism by which ethanol elicits its behavioral effects, the fact that differences in sensitivity between cortex and cerebellum were seen is consistent with the argument that different brain regions are differentially sensitive to ethanol. The cortex does not appear to be more sensitive to all of the actions of ethanol, however. For example, Stokes and Harris [35] have reported that K+-stimulated uptake of Ca ++ into cerebellar synaptosomes is inhibited to a greater extent by ethanol than is Ca ++ uptake into cortical or brain stem synaptosomes. These results argue that no single brain region is uniquely sensitive to ethanol. The electrophysiological study of Sorenson et al. [32] argues that the most precise analysis of the effects of ethanol will probably be obtained by examining specific cell types. In summary, the results presented here indicate that ethanol inhibits the release of aH-GABA and that this inhibition is influenced by brain region and genotype. The fact that the LS mice are more sensitive to the effects of ethanol on 3H-GABA release lends support to the hypothesis that inhi-
476
HOWERTON
b i t i o n o f r e l e a s e u n d e r l i e s s o m e o f the b e h a v i o r a l a c t i o n s o f e t h a n o l . Since the L S a n d SS mice w e r e s u b j e c t e d to a found a t i o n effect early in the s e l e c t i o n p r o c e s s (the n u m b e r o f b r e e d i n g pairs w a s r e d u c e d in s e v e r a l early g e n e r a t i o n s ) , the c o n c l u s i o n s d r a w n f r o m t h e d a t a r e p o r t e d h e r e s h o u l d not be t a k e n as a n u n e q u i v o c a l d e m o n s t r a t i o n t h a t e t h a n o l e x e r t s its b e h a v i o r a l effects b y inhibiting n e u r o t r a n s m i t t e r release. S u p p o r t i v e e v i d e n c e m u s t b e o b t a i n e d u s i n g o t h e r selected genomic populations. T h e o b s e r v a t i o n t h a t PFGz,~ did not a l t e r G A B A release i n d i c a t e s t h a t e t h a n o l d o e s n o t a l t e r G A B A release by a m e c h a n i s m i n v o l v i n g t h e p r o d u c i n g o f PGF2~. A p r e l i m i n a r y r e p o r t f r o m o u r l a b o r a t o r y [8] h a s i n d i c a t e d t h a t e t h a n o l inc r e a s e s b r a i n PGE~ a n d PGF=,,~ c o n t e n t . O u r o b s e r v a t i o n s
AN D COLLINS
that LS and SS mice do n o t differ in PGE,~-induced inhibition o f n o r e p i n e p h r i n e release [15] a n d the c u r r e n t finding relating to the lack of effect o f PGF~,~ on '~H-GABA release suggest no s t r a i g h t f o r w a r d i n t e r a c t i o n b e t w e e n e t h a n o l a n d n e u r o t r a n s m i t t e r r e l e a s e , i n v o l v i n g p r o s t a g l a n d i n s , exists~ F u r t h e r studies will b e r e q u i r e d to fully e l u c i d a t e the m e c h a n i s m w h e r e b y e t h a n o l inhibits t r a n s m i t t e r release and the role s u c h an effect h a s in the b e h a v i o r a l effects o f e t h a n o l
ACKNOWLEDGEMF~NTS The assistance of M. L. Dubcovici~ in developing the release methodology and the editorial assistance of Rebecca Miles are appreciated.
REFERENCES
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