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Evidence for modulation of GABAergic neurotransmission by nicotine
Brain Research, 453 (1988) 215-220 Elsevier
215
BRE 13678
Evidence for modulation of GABAergic neurotransmission by nicotine Ronald K. Freund 1, D a n a A. Jungschaffer l, Allan C. Collins 1'2 and Jeanne M. Wehner l'2 1Institutefor Behavioral Genetics and 2School of Pharmacy, University of Colorado, Boulder, CO 80309 (U.S.A.) (Accepted 5 January 1988) Key words: Nicotine; y-Aminobutyric acid; Hippocampus; Mouse; CA1; Population spike
Bath-application of nicotine (800/~M) to mouse hippocampal slices resulted in an increase in the amplitude of the population spike and the appearance of multiple population spikes in the CA1 pyramidal cell layer. Similar effects were observed after perfusion of the GABA A antagonist bicuculline methiodide (2 ~M) and the glutamate decarboxylase inhibitor L-C-allylglycine(4 mM). These apparently excitatory effects of nicotine (800 pM) could be reversed by bath-application of v-aminobutyric acid (GABA; 400/~M), as well as by the GABA uptake inhibitor nipecotic acid (5 mM) and the benzodiazepine flurazepam (4 ~M). Nicotine did not alter binding of [3H]GABA or [~H]flunitrazepam to whole brain plasma membranes. The results are consistent with the hypothesis that the electrophysiological effects of nicotine on CA1 pyramidal cell excitability is mediated by disruption of GABAergic transmission.
INTRODUCTION Nicotine produces a number of physiological and behavioral effects in mice, including the induction of clonic-tonic seizures, enhanced respiration, reduced Y-maze activity, and hypothermia 14'23'24'27. Since the hippocampus has been implicated as an epileptogenic fOCUS7'16'22, and since, in a genetic study in mice, a positive correlation was observed between the sensitivity to nicotine-induced seizures and the number of nicotinic receptors in the hippocampus, as measured by ct-bungarotoxin binding 27, the effects of nicotine on the electrophysiological activity of hippocampal cells from mice were investigated. Nicotine was found to increase the evoked CA1 population spike and to induce the appearance of multiple population spikes in a concentration-dependent and mecamylamine-inhibitable fashion l°. This profile of extracellular effects is similar to that observed by other agents (e.g. bicuculline and penicillin) which disinhibit the cell population under test 6"32"34. A m o n g potential inhibitory neurotransmitters in
the central nervous system, v-aminobutyric acid ( G A B A ) is thought to be prominent in the CA1 region of the hippocampus 2'3't~'29 and serve an important inhibitory function 37'3s. Therefore, the possibility that nicotine disinhibits CA1 pyramidal cells by interfering with G A B A e r g i c transmission was investigated. A number of G A B A - r e l a t e d agents were tested for the ability to produce effects similar to nicotine or to reverse effects of nicotine. The results are consistent with the hypothesis that nicotine acts, at least in part, by interfering with tonic G A B A e r g i c transmission. A preliminary report of this work has appeared s. MATERIALS AND METHODS Compounds t.-Nicotine, L-C-allylglycine, G A B A and (+)-nipecotic acid were obtained from Sigma (St. Louis, MO). Flurazepam was a generous gift from Hoffmann-La Roche (Nutley, N J). Stock solutions of these compounds were prepared in incubation medium
Correspondence: R.K. Freund, University of Colorado, Institute for Behavioral Genetics, Campus Box 447, Boulder, CO 80309, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
216 (see below). [3H]GABA was obtained from Amersham (Arlington Heights, IL), and [3H]flunitrazepam was purchased from Research Products International (Mount Prospect, ILL
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General procedure Experiments were performed on transverse slices (450 Hm thick) of the hippocampal formation from 60- to 90-day-old female DBA mice as previously described 9H°. Drugs were bath-applied to hippocampal slices submerged in incubation medium and maintained in a static perfusion chamber r. The composition of the incubation medium was (in mM): o-glucose 10, MgSO 4 2.4, CaCI 2 2.5, NaCI 124, KCI 3.3, KH2PO 4 1.2 and NaHCO 3 26.4 (pH 7.4). Population spikes were evoked using a bipolar stimulating electrode (0.2 Hz, 100 Hs duration) placed in the stratum radiatum of regio superior so as to activate Schaffer collateral fiber bundles (orthodromic) or in striatum oriens of regio superior (antidromic). A glass extracellular recording electrode (filled with 2 M NaCI; 2-15 MQ impedance) was placed in or near the CAI pyramidal cell layer for recording population spikes or in stratum radiatum for recording synaptic field potentials. Amplitudes of population spikes (PSAs) and synaptic field potentials were measured as described previously m. The maximum elicitable PSA was recorded prior to each experiment, and the stimulus intensity was then lowered so as to elicit a control PSA that was between 5 and 25% of this maximum. This stimulus intensity was then used as the standard for the remainder of the experiment. Three PSAs were averaged for each time point. For evoking synaptic field potentials, the stimulus intensity was maintained just subthreshold for the induction of population spikes. A test concentration of 800 ItM nicotine was selected since it was the lowest concentration that gave maximum effects m. Effects of 800 ttM nicotine could be largely inhibited by mecamylamine at a concentration (400 HM) that had no effect on evoked responses m. A fresh slice was used for each exposure to drug.
GABA and flunitrazepam binding Binding of [3H}GABA to mouse whole-brain membranes was performed as described by Olsen et al. 2s. Binding of [3H]flunitrazepam (FNZ) was performed as described by Marley and Wehner 25 with
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Fig. 1. Illustrative evoked responses before and after exposure to excitatory agents, a: effects of 800/xM nicotine on the CA1 synaptic field potential (SFP), the antidromic population spike (A-PS), and the orthodromic population spike (O-PS; 20 min exposure), b: effects of bicuculline (2 ,uM, 10 min). c: effects of allylglycine (4 raM, 60 min) on the orthodromic population spike. Calibration bars: vertical, 0.5 mV, horizontal 2 mV for SFP; vertical, 2 mV; horizontal, 5 ms for A-PS and all orthodromic population spikes.
slight modifications: membrane samples were incubated at 4 °C for 90 min, and the final concentration of [3H]FNZ was 0.5 nM. Nicotine was added in the concentration range of 10 -9 to 10 -3 M for both binding assays. RESULTS Bath-application of 800/~M nicotine resulted in an increase in the amplitude of the population spike measured in the CA1 pyramidal cell layer and the appearance of multiple population spikes (Fig. 1A and ref. 10). These effects of nicotine on evoked population spikes were not accompanied by marked alterations of synaptic field potentials or by effects on the antidromic population spike (Fig. 1A). To examine whether GABAergic transmission is involved in these effects of nicotine, slices were exposed to bicuculline methiodide, an antagonist of G A B A A receptors 5"15. This compound produced effects similar to those observed in the presence of nicotine (Fig. 1B). Allylglycine, an inhibitor of glutamate decarboxylase ~, the synthetic enzyme for GABA, also produced similar effects (Fig. 1C). Higher concentrations and longer time periods were required for effects of allylglycine, as might be ex-
217 1.00
[
A 0.80
TABLE I
Nicotine
J GABA "O
Effects of nicotine on binding of ff H]GABA and ff H]flunitrazepare (FNZ) to whole-brain membranes
O PSA 1
0.80
Values are expressed as the percent of specific binding in the absence of nicotine. For comparison a separate analysis of inhibition of [3H]GABA binding by GABA (10-* M) was included in each experiment. Under these conditions, average binding was 23.2 _+ 1.7 (mean _+ S.E.M.) % of the specific binding in the absence of GABA. Data are the mean _+S.E.M. for 2 ([3H]GABA binding) or 3 ([3H]FNZ binding) assays.
0.40 0.20 0.00
[' Nicotine I NipA
B
1.20 U') r~
0.80 E E E
Nicotine concentration (M)
% ff H]GABA bound
% flH]FNZ bound
10-9 10-7 10-5 10-3
99.4 + 23.3 103.5 _+4.2 111.5 + 19.7 106.7 + 5.2
108.5 _+5.4 97.5 _+4.5 98.2 + 6.7 113.6 _+6.0
0.40
"8 ¢O C) t_ tl=
0.00
C
I Nicotine
0.80 0.60
I Flur
m
0.40 0.20 0.0C
I -20
I
I 0
I
I 20
I
I 40
I
I 80
T i m e ( r a i n ) a f t e r nicotine oddition
Fig. 2. Reversal of nicotine effects by GABA-related agents. Hippocampal slices were exposed to 800 ~M nicotine for 22 min, after which (a) GABA (400 ,uM), (b) nipecotic acid (NipA; 5 mM), or (c) flurazepam (Flur; 4pM) was added to the perfusion medium while maintaining the nicotine concentration constant. The amplitudes of the first (PSA 1) and second (PSA 2) population spikes were measured, normalized, and averaged as described in Materials and Methods, Symbols represent the mean + S.E.M. for 4-6 slices.
pected for bath-application of an inhibitor of an intracellular enzyme. W h e n G A B A was a d d e d to slices which had been previously exposed to nicotine, the effects of nicotine were reversed, as evidenced by the time course of changes in the a m p l i t u d e s of the first and second population spikes (Fig. 2A). F u r t h e r m o r e , nipecotic acid, an inhibitor of G A B A u p t a k e , reversed the effects of nicotine (Fig. 2B). W h e r e a s exposure of nicotine-treated slices to 1 m M nipecotic acid (22 min) resulted in a slight depression of p o p u l a t i o n spikes
(data not shown), a shorter exposure (12 min) to a higher concentration (5 m M ) abolished population spikes completely (Fig. 2B). Washing with fresh medium resulted in recovery of e v o k e d activity, which was greater than control levels. Flurazepam (4 ~ M ) also reversed the nicotine-induced increase in P S A (Fig. 2C), indicating that enhanced G A B A e r g i c transmission can counteract electrophysiological effects of nicotine. Over the concentration range of 10 -9 to 10 -3 M, nicotine did not alter binding of [ 3 H ] G A B A to wholebrain plasma m e m b r a n e s (Table I). In addition, nicotine did not affect the binding of the benzodiazepine,
[3H]FNZ. DISCUSSION It was previously o b s e r v e d that nicotine produces an increase in the e v o k e d CA1 p o p u l a t i o n spike and induces the a p p e a r a n c e of multiple spikes in D B A mice l°. A l t h o u g h these effects required high concentrations of nicotine (400-1600/~M), they a p p e a r to be m e d i a t e d by nicotinic cholinergic receptors, since the actions of nicotine could be largely inhibited by bath-application of m e c a m y l a m i n e 1°. Since nicotine had no effect on synaptic field potentials or the antidromic spike, the r e p o r t e d effects of nicotine are not a consequence of increased synaptic activity, axonal conduction, or general excitability. The a p p e a r a n c e of multiple population spikes has been observed in h i p p o c a m p a l slices from rat and
218 guinea pig following bath-application of bicuculline and penicillin, compounds which inhibit GABAergic transmission 3i'32'3a. Similar effects of bicuculline were observed in mice (ref. 9 and this paper). Exposure of slices to allylglycine, an inhibitor of the synthetic enzyme (glutamic acid decarboxylase, G A D ) for G A B A 1, resulted in an increase in the population spike and the appearance of multiple spikes. It is clear, therefore, that nicotine induces effects which are qualitatively similar to those produced by agents known to interfere with GABAergic neurotransmission. Mechanisms by which nicotine might do this include: (1) direct interaction with G A B A receptors or the associated ion channels, (2) blockade of G A B A release, and (3) facilitated release and subsequent partial depletion of G A B A from presynaptic terminals. The possibility that nicotine may interact with G A B A receptors was investigated in t w o ways: nicotine was allowed to compete for binding of [3H]GABA to whole-brain plasma membranes and tested for the ability to alter binding of [~H]FNZ. Since nicotine (10 -9 to 10 3 M) did not affect binding of either ligand, it appears unlikely that nicotine binds to the G A B A recognition site or that nicotine acts to influence allosteric interactions between the G A B A and benzodiazepine receptor sites. It is conceivable, however, that nicotine inhibits G A B A function by some other direct mechanism, e.g. blockade of the GABA-gated chloride channel. Nicotine has been shown to enhance basal release of [3H]GABA in caudate nucleus slices via activation of a nicotinic cholinergic receptor 2~. Recent preliminary data indicate that this mechanism may also occur in the hippocampus. Nicotine was found to evoke the release of [3H]GABA from preloaded rat hippocampal synaptosomes 35'3~. Since this action of nicotine would have an inhibitory physiological consequence, it is clear that additional mechanisms are required to explain the present data. In view of the fact that the observed electrophysiological effects of nicotine require several minutes to occur, it may be that a nicotine-induced G A B A release/effect mechanism is inhibited over time, e.g. by desensitization of nicotinic or GABAergic receptors, or by nicotine-induced partial depletion of presynaptic G A B A stores. To test the hypothesis that nicotine interferes with GABAergic inhibition, exogenous G A B A was
added to nicotine-treated slices, and reversal of the effects of nicotine was observed. The G A B A uptake inhibitor nipecotic acid Is also reversed the effects ot nicotine as might be expected for an agent which increases synaptic G A B A levels. The benzodiazepine flurazepam, which is thought to increase GABAergic inhibition, also reversed the effects of nicotine. These results are consistent with the hypothesis that nicotine reduces presynaptic G A B A levels or that nicotine desensitizes nicotinic receptors responsible for modulation of G A B A release. The effectiveness of G A B A for eliciting inhibition in the presence of nicotine renders the possibility that G A B A receptors become desensitized unlikely, The effects of nipecotic acid and flurazepam indicate that nicotine may reduce, but does not completely block release of GABA, since the primary actions of these compounds require the presence of G A B A . It should be noted that slices exposed to nipecotic acid became hyperexcitable after washing the drug from the slices (see Fig. 2B). This was true whether nicotine had been present in addition to nipecotic acid (Fig. 2B) or whether nipecotic acid was perfused onto untreated slices (data not shown). These effects have been observed by others >, who suggest that nipecotic acid can be taken up by the G A B A uptake system, accumulated in nerve terminals, and released as a false transmitter. The effects of release of a false inhibitory transmitter would be increased excitation, as was observed. Whether nipecotic acid is taken up or not, perfusion with the drug would be expected to increase synaptic levels of G A B A , either by directly blocking G A B A uptake, or by effectively competing with G A B A for uptake, and our data are consistent with either mechanism. A number of suggestions have been put forth implicating the involvement of GABAergic inhibitory function in the actions of the transmitter acetylcholine (ACh). A disinhibitory role for ACh in the hippoeampus has been previously suggested 4"i~l'~'~°, but inhibition has also been observed 26'3~. Iontophoretic application of nicotine was found to produce d-tubocurarine-sensitive inhibition of firing of bursting (pyramidal) and non-bursting (presumably interneuronal) ceils 33. One of the actions of ACh in cortical pyramidal cells is to exert a fast inhibitory response which is due to excitation of inhibitory GABAergic interneurons 26. Although these effects were largely
219 muscarinic, dimethylphenylpiperazinium ( D M P P )
tion that the apparently excitatory effects of nicotine
also produced the hyperpolarizing response in pyra-
in mouse hippocampus are a consequence of decreased G A B A e r g i c transmission. These effects are
midal cells indicating a potential nicotinic component. Intracellular recording from G A B A e r g i c interneurons associated with cortical pyramidal cells revealed that these cells respond to application of A C h with an increase in m e m b r a n e conductance to depolarizing cations, i.e. Na + and/or Ca 2+26. O n e conse-
not thought to be due to interaction of nicotine with G A B A receptors, but are more likely to be a result of decreased release of G A B A . At least two mechanisms exist for the nicotine-induced blockade of G A B A release: nicotine may directly inhibit G A B A
quence of depolarization of these cortical interneu-
release via desensitization of nicotinic receptors
rons might be increased release of its transmitter,
which control G A B A release, or nicotine may facili-
G A B A . Nicotinic depolarization of cerebellar intern e u r o n s has also been reported 12. If a similar pattern
tate G A B A release to the extent that presynaptic G A B A levels are reduced, after which excitatory effects are observed. Experiments designed to delin-
exists in the hippocampus, both the reported inhibitory and disinhibitory effects could be accounted for by the following mechanism: nicotine may act via nicotinic receptors to depolarize i n t e r n e u r o n s resulting in enhanced release of G A B A . After several minutes G A B A may become partially depleted or nicotinic receptors may become desensitized, and disinhibitory effects would be observed. In the present study only effects of a disinhibitory phase could be observed. In summary, the present results lead to the sugges-
REFERENCES 1 Alberici, M., Rodriguez de Lores Arnaiz, G. and De Robertis, E., Glutamic acid decarboxylase inhibition and ultrastructural changes by the convulsant drug allylglycine, Biochem. Pharmacol., 18 (1969) 137-143. 2 Anderson, K.J., Maley, B.E. and Scheff, S.W., Immunocytochemical localization of gamma-aminobutyric acid in the rat hippocampal formation, Neurosci. Lett., 69 (1986) 7-12. 3 Barber, R. and Saito, K., Light microscopic visualization of GAD and GABA-T in immunocytochemical preparations of rodent CNS. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven, New York, 1976, pp. 113-132. 4 Ben-Ari, Y., Krnjevi6, K., Reinhardt, W. and Ropert, N., Intracellular observations on the disinhibitory action of acetylcholine in the hippocampus, Neuroscience, 6 (1981) 2475-2484. 5 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 6 Curtis, D.R., Felix, D. and MeLennan, H., GABA and hippocampal inhibition, Br. J. Pharmacol., 40 (1970) 881-883. 7 Dichter, M., Herman, C.J. and Seizer, M., Silent cells during interictal discharges and seizures in hippocampal penicillin foci. Evidence for the role of extracellular K + in the transition from the interictal state to seizures, Brain Research, 48 (1972) 173-183.
eate these possibilities are in progress. ACKNOWLEDGEMENTS This research was supported in part by grants from R.J. Reynolds, Inc., B R S G Grants RR-07013-19 and -20 awarded to the Biomedical Research Support Grant Program, Division of Research Resources, NIH to the University of Colorado, and N I D A training G r a n t DA-07043 to R . K . F .
8 Freund, R.K., Jungschaffer, D.A. and Wehner, J.M., Involvement of GABAergic transmission in the electrophysiological effects of nicotine, Soc. Neurosci. Abstr., 13 (1987) 145. 9 Freund, R.K., Marley, R.J. and Wehner, J.M., Differential sensitivity to bicuculline in three inbred mouse strains, Brain Res. Bull., 18 (1987) 657-662. 10 Freund, R.K. and Wehner, J.M., Strain-selective effects of nicotine on electrophysiological responses evoked in hippocampus from DBA/2Ibg and C3H/2Ibg mice, J. Neurogenet., 4 (1987) 75-86. 11 Gamrani, H., Onteniente, B., Seguela, P., Geffard, M. and Calas, A., Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies, Brain Research. 364 (1986) 30-38. 12 Garza, R. de la, Bickford-Wimer, C., Hoffer, B.J. and Freedman, R., Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study, J. Pharmacol. Exp. Ther., 240 (1987) 689-695. 13 Haas, H.L., Cholinergic disinhibition in hippocampal slices of the rat, Brain Research, 233 (1982) 200-204. 14 Hatchell, P.C. and Collins, A.C., The influence of genotype and sex on behavioral tolerance to nicotine, Psychopharmacology, 71 (1980)45-49. 15 Hill, D.R. and Bowery, N.G., 3H-Baclofen and 3H-GABA bind to bicuculline-insensitive GABA B sites in rat brain, Nature (Lond.), 290 ( 1981) 149-152. 16 Kandel, E.R. and Spencer, W.A., Electrophysiology of hippocampal neurons. II. Afterpotentials and repetitive firing, J. Neurophysiol., 24 (1961) 234-259.
220 17 Koerner, J.F. and Cotman, C.W., A micropcrfusion chamber for brain slice pharmacology, J. Neurosci. Methods', 7 (1983) 243-251. 18 Krogsgaard-Larsen, P. and Johnston, G.A.R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., 25 (1975) 797-802. 19 Krnjevid, K., Reiffenstein, R.J. and Ropert, N., Disinhibitory action of acetylcholine in the hippocampus, J. Physiol. (Lond.), 308 (1980) 73P-74P. 20 Lerma, J., Herreras, O. and Martin del Rio, R., Electrophysiological evidence that nipecotic acid can be used in vitro as a false transmitter, Brain Research, 335 (1985) 377-380. 21 Limberger, N., Spath, L. and Starke, K., A search for receptors modulating the release of gamma-[~H]aminobutytic acid in the rabbit caudate nucleus slices, J. Neurochem.. 46 (1986) 1109-1117. 22 Lothman, E.W. and Collins, R.C., Kainic acid induced limbic seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates, Brain Research, 218 (1981) 299-318. 23 Marks, M.J., Burch, J.B. and Collins, A.C., Genetics of nicotine response in four inbred strains of mice, J. Pharrnacol. Exp. Ther,, 226 (1983) 291-302. 24 Marks, M.J., Patinkin, D.M., Artman, L.D,, Butch, J.B. and Collins, A.C., Genetic influences on cholinergic drug response, Pharm. Biochem. Behav., 15 (1981)271-279. 25 Marley, R.J. and Wehner, J.M., GABA enhancement of flunitrazepam binding in mice selectively bred for differential sensitivity to ethanol, Alcohol Drug Res.. 7 (1986) 25-32. 26 McCormick, D.A. and Prince, D.A., Pirenzepine discriminates among ionic responses to acetylcholine in guinea-pig cerebral cortex and reticular nucleus of the thalamus, Trends Pharmacol. Sci., Suppl. Feb. (1986)72-77. 27 Miner, L.L., Marks, M.J. and Collins, A.C,, Classical genetic analysis of nicotine-induced seizures and nicotinic receptors, J. Pharmacol. Exp. Ther., 23I (1984)545-554. 28 OIsen, R.W., Bergman, M.O., Van Ness, P.C., Lummis,
S.C., Watkins, A.E., Napias, ( . and Glucnlcc, I ) . \ . Oamma-aminobutyric acid binding in manmlalian brain: heterogeneity of binding sites, Mol. Pharmaco/., I~ (I~Slt 217-227. 29 Ribak, C.E., Vaughn, ,I.E. and Saito, K., lmmunocytochemical localization of gtutamic acid decarboxylasc in neuronal somata following colchicine inhibition eft ztxonal transport, Brain Research, 140 (1978) 315-332. 30 Rovira, ('., Ben-Ari, Y., Cherubini, E., Krnjcvi~: and Ropert, N., Pharmacology of the dendritic action of acctylcholine and further observations on the somatic disinhibition in the rat hippocampus in situ, Neuroscience. 8 (1983) 97-- 10t~. 31 Schwartzkroin, P.A. and Prince, D.A., Changes in excitatory and inhibitory synaptic potentials leading to cpi[eptogenic activity, Brain Research, 183 (1980) 61 76, 32 Schwartzkroin, P.A. and Prince, D.A., Penicillin-induced epileptiform activity in the hippocampa[ in vitro preparation, Ann. Neurol., 1 (1977) 463-469. 33 Segal, M., The acetylcholine receptor in the rat hippocampus; nicotinic, muscarinic or both?, Neuropharmacologv, 17 (1978) 619-623. 34 Wheal. H.V., Ashwood, ].J. and Lancaster. B., A comparative in vitro study of the kainic acid lesioned and bicuculline treated hippocampus: chronic and acute models of focal epilepsy. In P.A. Schwartzkroin and H.V. Wheal (Eds.), Electrophysiology ¢~f'Epilepsy. Academic, London, 1984, pp. 173-200. 35 Wonnacott, S., Fryer, L. and Lunt, G.G., Nicotine-evoked [~H]GABA release from hippocampal synaptosomes, J. Neurochem., 48 Suppl. (1987) 72B. 36 Wonnacott. S., Fryer. L., Lunt, G.G., Freund, R.K., Jungschaffer, D.A. and Collins, A.C., Nicotinic modulation of GABAergic transmission in the hippocampus, Soc. Neurosci. Abstr., 17 (1987) 1352. 37 Wong, R.K.S. and Prince, D.A., Dendritic mechanisms underlying penicillin-induced cpileptiform activity, Science. 204 (1979) 1228-123 I. 38 Wong, R.K.S. and Watkins, D.J,, Cellular factors influencing GABA response in hippocampal pyramidal cells. J. Neurophysiol., 48 (1982) 938-95 l.
215
BRE 13678
Evidence for modulation of GABAergic neurotransmission by nicotine Ronald K. Freund 1, D a n a A. Jungschaffer l, Allan C. Collins 1'2 and Jeanne M. Wehner l'2 1Institutefor Behavioral Genetics and 2School of Pharmacy, University of Colorado, Boulder, CO 80309 (U.S.A.) (Accepted 5 January 1988) Key words: Nicotine; y-Aminobutyric acid; Hippocampus; Mouse; CA1; Population spike
Bath-application of nicotine (800/~M) to mouse hippocampal slices resulted in an increase in the amplitude of the population spike and the appearance of multiple population spikes in the CA1 pyramidal cell layer. Similar effects were observed after perfusion of the GABA A antagonist bicuculline methiodide (2 ~M) and the glutamate decarboxylase inhibitor L-C-allylglycine(4 mM). These apparently excitatory effects of nicotine (800 pM) could be reversed by bath-application of v-aminobutyric acid (GABA; 400/~M), as well as by the GABA uptake inhibitor nipecotic acid (5 mM) and the benzodiazepine flurazepam (4 ~M). Nicotine did not alter binding of [3H]GABA or [~H]flunitrazepam to whole brain plasma membranes. The results are consistent with the hypothesis that the electrophysiological effects of nicotine on CA1 pyramidal cell excitability is mediated by disruption of GABAergic transmission.
INTRODUCTION Nicotine produces a number of physiological and behavioral effects in mice, including the induction of clonic-tonic seizures, enhanced respiration, reduced Y-maze activity, and hypothermia 14'23'24'27. Since the hippocampus has been implicated as an epileptogenic fOCUS7'16'22, and since, in a genetic study in mice, a positive correlation was observed between the sensitivity to nicotine-induced seizures and the number of nicotinic receptors in the hippocampus, as measured by ct-bungarotoxin binding 27, the effects of nicotine on the electrophysiological activity of hippocampal cells from mice were investigated. Nicotine was found to increase the evoked CA1 population spike and to induce the appearance of multiple population spikes in a concentration-dependent and mecamylamine-inhibitable fashion l°. This profile of extracellular effects is similar to that observed by other agents (e.g. bicuculline and penicillin) which disinhibit the cell population under test 6"32"34. A m o n g potential inhibitory neurotransmitters in
the central nervous system, v-aminobutyric acid ( G A B A ) is thought to be prominent in the CA1 region of the hippocampus 2'3't~'29 and serve an important inhibitory function 37'3s. Therefore, the possibility that nicotine disinhibits CA1 pyramidal cells by interfering with G A B A e r g i c transmission was investigated. A number of G A B A - r e l a t e d agents were tested for the ability to produce effects similar to nicotine or to reverse effects of nicotine. The results are consistent with the hypothesis that nicotine acts, at least in part, by interfering with tonic G A B A e r g i c transmission. A preliminary report of this work has appeared s. MATERIALS AND METHODS Compounds t.-Nicotine, L-C-allylglycine, G A B A and (+)-nipecotic acid were obtained from Sigma (St. Louis, MO). Flurazepam was a generous gift from Hoffmann-La Roche (Nutley, N J). Stock solutions of these compounds were prepared in incubation medium
Correspondence: R.K. Freund, University of Colorado, Institute for Behavioral Genetics, Campus Box 447, Boulder, CO 80309, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
216 (see below). [3H]GABA was obtained from Amersham (Arlington Heights, IL), and [3H]flunitrazepam was purchased from Research Products International (Mount Prospect, ILL
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/
.......
General procedure Experiments were performed on transverse slices (450 Hm thick) of the hippocampal formation from 60- to 90-day-old female DBA mice as previously described 9H°. Drugs were bath-applied to hippocampal slices submerged in incubation medium and maintained in a static perfusion chamber r. The composition of the incubation medium was (in mM): o-glucose 10, MgSO 4 2.4, CaCI 2 2.5, NaCI 124, KCI 3.3, KH2PO 4 1.2 and NaHCO 3 26.4 (pH 7.4). Population spikes were evoked using a bipolar stimulating electrode (0.2 Hz, 100 Hs duration) placed in the stratum radiatum of regio superior so as to activate Schaffer collateral fiber bundles (orthodromic) or in striatum oriens of regio superior (antidromic). A glass extracellular recording electrode (filled with 2 M NaCI; 2-15 MQ impedance) was placed in or near the CAI pyramidal cell layer for recording population spikes or in stratum radiatum for recording synaptic field potentials. Amplitudes of population spikes (PSAs) and synaptic field potentials were measured as described previously m. The maximum elicitable PSA was recorded prior to each experiment, and the stimulus intensity was then lowered so as to elicit a control PSA that was between 5 and 25% of this maximum. This stimulus intensity was then used as the standard for the remainder of the experiment. Three PSAs were averaged for each time point. For evoking synaptic field potentials, the stimulus intensity was maintained just subthreshold for the induction of population spikes. A test concentration of 800 ItM nicotine was selected since it was the lowest concentration that gave maximum effects m. Effects of 800 ttM nicotine could be largely inhibited by mecamylamine at a concentration (400 HM) that had no effect on evoked responses m. A fresh slice was used for each exposure to drug.
GABA and flunitrazepam binding Binding of [3H}GABA to mouse whole-brain membranes was performed as described by Olsen et al. 2s. Binding of [3H]flunitrazepam (FNZ) was performed as described by Marley and Wehner 25 with
o- Ps ~-.-,J'-"~
, ~ - -
b. B I C U C U L L I N E
"~-~/~--
~/~/'~--
c. ALLYLGLYCINE
~
~-~JV
--
~ - -
Fig. 1. Illustrative evoked responses before and after exposure to excitatory agents, a: effects of 800/xM nicotine on the CA1 synaptic field potential (SFP), the antidromic population spike (A-PS), and the orthodromic population spike (O-PS; 20 min exposure), b: effects of bicuculline (2 ,uM, 10 min). c: effects of allylglycine (4 raM, 60 min) on the orthodromic population spike. Calibration bars: vertical, 0.5 mV, horizontal 2 mV for SFP; vertical, 2 mV; horizontal, 5 ms for A-PS and all orthodromic population spikes.
slight modifications: membrane samples were incubated at 4 °C for 90 min, and the final concentration of [3H]FNZ was 0.5 nM. Nicotine was added in the concentration range of 10 -9 to 10 -3 M for both binding assays. RESULTS Bath-application of 800/~M nicotine resulted in an increase in the amplitude of the population spike measured in the CA1 pyramidal cell layer and the appearance of multiple population spikes (Fig. 1A and ref. 10). These effects of nicotine on evoked population spikes were not accompanied by marked alterations of synaptic field potentials or by effects on the antidromic population spike (Fig. 1A). To examine whether GABAergic transmission is involved in these effects of nicotine, slices were exposed to bicuculline methiodide, an antagonist of G A B A A receptors 5"15. This compound produced effects similar to those observed in the presence of nicotine (Fig. 1B). Allylglycine, an inhibitor of glutamate decarboxylase ~, the synthetic enzyme for GABA, also produced similar effects (Fig. 1C). Higher concentrations and longer time periods were required for effects of allylglycine, as might be ex-
217 1.00
[
A 0.80
TABLE I
Nicotine
J GABA "O
Effects of nicotine on binding of ff H]GABA and ff H]flunitrazepare (FNZ) to whole-brain membranes
O PSA 1
0.80
Values are expressed as the percent of specific binding in the absence of nicotine. For comparison a separate analysis of inhibition of [3H]GABA binding by GABA (10-* M) was included in each experiment. Under these conditions, average binding was 23.2 _+ 1.7 (mean _+ S.E.M.) % of the specific binding in the absence of GABA. Data are the mean _+S.E.M. for 2 ([3H]GABA binding) or 3 ([3H]FNZ binding) assays.
0.40 0.20 0.00
[' Nicotine I NipA
B
1.20 U') r~
0.80 E E E
Nicotine concentration (M)
% ff H]GABA bound
% flH]FNZ bound
10-9 10-7 10-5 10-3
99.4 + 23.3 103.5 _+4.2 111.5 + 19.7 106.7 + 5.2
108.5 _+5.4 97.5 _+4.5 98.2 + 6.7 113.6 _+6.0
0.40
"8 ¢O C) t_ tl=
0.00
C
I Nicotine
0.80 0.60
I Flur
m
0.40 0.20 0.0C
I -20
I
I 0
I
I 20
I
I 40
I
I 80
T i m e ( r a i n ) a f t e r nicotine oddition
Fig. 2. Reversal of nicotine effects by GABA-related agents. Hippocampal slices were exposed to 800 ~M nicotine for 22 min, after which (a) GABA (400 ,uM), (b) nipecotic acid (NipA; 5 mM), or (c) flurazepam (Flur; 4pM) was added to the perfusion medium while maintaining the nicotine concentration constant. The amplitudes of the first (PSA 1) and second (PSA 2) population spikes were measured, normalized, and averaged as described in Materials and Methods, Symbols represent the mean + S.E.M. for 4-6 slices.
pected for bath-application of an inhibitor of an intracellular enzyme. W h e n G A B A was a d d e d to slices which had been previously exposed to nicotine, the effects of nicotine were reversed, as evidenced by the time course of changes in the a m p l i t u d e s of the first and second population spikes (Fig. 2A). F u r t h e r m o r e , nipecotic acid, an inhibitor of G A B A u p t a k e , reversed the effects of nicotine (Fig. 2B). W h e r e a s exposure of nicotine-treated slices to 1 m M nipecotic acid (22 min) resulted in a slight depression of p o p u l a t i o n spikes
(data not shown), a shorter exposure (12 min) to a higher concentration (5 m M ) abolished population spikes completely (Fig. 2B). Washing with fresh medium resulted in recovery of e v o k e d activity, which was greater than control levels. Flurazepam (4 ~ M ) also reversed the nicotine-induced increase in P S A (Fig. 2C), indicating that enhanced G A B A e r g i c transmission can counteract electrophysiological effects of nicotine. Over the concentration range of 10 -9 to 10 -3 M, nicotine did not alter binding of [ 3 H ] G A B A to wholebrain plasma m e m b r a n e s (Table I). In addition, nicotine did not affect the binding of the benzodiazepine,
[3H]FNZ. DISCUSSION It was previously o b s e r v e d that nicotine produces an increase in the e v o k e d CA1 p o p u l a t i o n spike and induces the a p p e a r a n c e of multiple spikes in D B A mice l°. A l t h o u g h these effects required high concentrations of nicotine (400-1600/~M), they a p p e a r to be m e d i a t e d by nicotinic cholinergic receptors, since the actions of nicotine could be largely inhibited by bath-application of m e c a m y l a m i n e 1°. Since nicotine had no effect on synaptic field potentials or the antidromic spike, the r e p o r t e d effects of nicotine are not a consequence of increased synaptic activity, axonal conduction, or general excitability. The a p p e a r a n c e of multiple population spikes has been observed in h i p p o c a m p a l slices from rat and
218 guinea pig following bath-application of bicuculline and penicillin, compounds which inhibit GABAergic transmission 3i'32'3a. Similar effects of bicuculline were observed in mice (ref. 9 and this paper). Exposure of slices to allylglycine, an inhibitor of the synthetic enzyme (glutamic acid decarboxylase, G A D ) for G A B A 1, resulted in an increase in the population spike and the appearance of multiple spikes. It is clear, therefore, that nicotine induces effects which are qualitatively similar to those produced by agents known to interfere with GABAergic neurotransmission. Mechanisms by which nicotine might do this include: (1) direct interaction with G A B A receptors or the associated ion channels, (2) blockade of G A B A release, and (3) facilitated release and subsequent partial depletion of G A B A from presynaptic terminals. The possibility that nicotine may interact with G A B A receptors was investigated in t w o ways: nicotine was allowed to compete for binding of [3H]GABA to whole-brain plasma membranes and tested for the ability to alter binding of [~H]FNZ. Since nicotine (10 -9 to 10 3 M) did not affect binding of either ligand, it appears unlikely that nicotine binds to the G A B A recognition site or that nicotine acts to influence allosteric interactions between the G A B A and benzodiazepine receptor sites. It is conceivable, however, that nicotine inhibits G A B A function by some other direct mechanism, e.g. blockade of the GABA-gated chloride channel. Nicotine has been shown to enhance basal release of [3H]GABA in caudate nucleus slices via activation of a nicotinic cholinergic receptor 2~. Recent preliminary data indicate that this mechanism may also occur in the hippocampus. Nicotine was found to evoke the release of [3H]GABA from preloaded rat hippocampal synaptosomes 35'3~. Since this action of nicotine would have an inhibitory physiological consequence, it is clear that additional mechanisms are required to explain the present data. In view of the fact that the observed electrophysiological effects of nicotine require several minutes to occur, it may be that a nicotine-induced G A B A release/effect mechanism is inhibited over time, e.g. by desensitization of nicotinic or GABAergic receptors, or by nicotine-induced partial depletion of presynaptic G A B A stores. To test the hypothesis that nicotine interferes with GABAergic inhibition, exogenous G A B A was
added to nicotine-treated slices, and reversal of the effects of nicotine was observed. The G A B A uptake inhibitor nipecotic acid Is also reversed the effects ot nicotine as might be expected for an agent which increases synaptic G A B A levels. The benzodiazepine flurazepam, which is thought to increase GABAergic inhibition, also reversed the effects of nicotine. These results are consistent with the hypothesis that nicotine reduces presynaptic G A B A levels or that nicotine desensitizes nicotinic receptors responsible for modulation of G A B A release. The effectiveness of G A B A for eliciting inhibition in the presence of nicotine renders the possibility that G A B A receptors become desensitized unlikely, The effects of nipecotic acid and flurazepam indicate that nicotine may reduce, but does not completely block release of GABA, since the primary actions of these compounds require the presence of G A B A . It should be noted that slices exposed to nipecotic acid became hyperexcitable after washing the drug from the slices (see Fig. 2B). This was true whether nicotine had been present in addition to nipecotic acid (Fig. 2B) or whether nipecotic acid was perfused onto untreated slices (data not shown). These effects have been observed by others >, who suggest that nipecotic acid can be taken up by the G A B A uptake system, accumulated in nerve terminals, and released as a false transmitter. The effects of release of a false inhibitory transmitter would be increased excitation, as was observed. Whether nipecotic acid is taken up or not, perfusion with the drug would be expected to increase synaptic levels of G A B A , either by directly blocking G A B A uptake, or by effectively competing with G A B A for uptake, and our data are consistent with either mechanism. A number of suggestions have been put forth implicating the involvement of GABAergic inhibitory function in the actions of the transmitter acetylcholine (ACh). A disinhibitory role for ACh in the hippoeampus has been previously suggested 4"i~l'~'~°, but inhibition has also been observed 26'3~. Iontophoretic application of nicotine was found to produce d-tubocurarine-sensitive inhibition of firing of bursting (pyramidal) and non-bursting (presumably interneuronal) ceils 33. One of the actions of ACh in cortical pyramidal cells is to exert a fast inhibitory response which is due to excitation of inhibitory GABAergic interneurons 26. Although these effects were largely
219 muscarinic, dimethylphenylpiperazinium ( D M P P )
tion that the apparently excitatory effects of nicotine
also produced the hyperpolarizing response in pyra-
in mouse hippocampus are a consequence of decreased G A B A e r g i c transmission. These effects are
midal cells indicating a potential nicotinic component. Intracellular recording from G A B A e r g i c interneurons associated with cortical pyramidal cells revealed that these cells respond to application of A C h with an increase in m e m b r a n e conductance to depolarizing cations, i.e. Na + and/or Ca 2+26. O n e conse-
not thought to be due to interaction of nicotine with G A B A receptors, but are more likely to be a result of decreased release of G A B A . At least two mechanisms exist for the nicotine-induced blockade of G A B A release: nicotine may directly inhibit G A B A
quence of depolarization of these cortical interneu-
release via desensitization of nicotinic receptors
rons might be increased release of its transmitter,
which control G A B A release, or nicotine may facili-
G A B A . Nicotinic depolarization of cerebellar intern e u r o n s has also been reported 12. If a similar pattern
tate G A B A release to the extent that presynaptic G A B A levels are reduced, after which excitatory effects are observed. Experiments designed to delin-
exists in the hippocampus, both the reported inhibitory and disinhibitory effects could be accounted for by the following mechanism: nicotine may act via nicotinic receptors to depolarize i n t e r n e u r o n s resulting in enhanced release of G A B A . After several minutes G A B A may become partially depleted or nicotinic receptors may become desensitized, and disinhibitory effects would be observed. In the present study only effects of a disinhibitory phase could be observed. In summary, the present results lead to the sugges-
REFERENCES 1 Alberici, M., Rodriguez de Lores Arnaiz, G. and De Robertis, E., Glutamic acid decarboxylase inhibition and ultrastructural changes by the convulsant drug allylglycine, Biochem. Pharmacol., 18 (1969) 137-143. 2 Anderson, K.J., Maley, B.E. and Scheff, S.W., Immunocytochemical localization of gamma-aminobutyric acid in the rat hippocampal formation, Neurosci. Lett., 69 (1986) 7-12. 3 Barber, R. and Saito, K., Light microscopic visualization of GAD and GABA-T in immunocytochemical preparations of rodent CNS. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven, New York, 1976, pp. 113-132. 4 Ben-Ari, Y., Krnjevi6, K., Reinhardt, W. and Ropert, N., Intracellular observations on the disinhibitory action of acetylcholine in the hippocampus, Neuroscience, 6 (1981) 2475-2484. 5 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 6 Curtis, D.R., Felix, D. and MeLennan, H., GABA and hippocampal inhibition, Br. J. Pharmacol., 40 (1970) 881-883. 7 Dichter, M., Herman, C.J. and Seizer, M., Silent cells during interictal discharges and seizures in hippocampal penicillin foci. Evidence for the role of extracellular K + in the transition from the interictal state to seizures, Brain Research, 48 (1972) 173-183.
eate these possibilities are in progress. ACKNOWLEDGEMENTS This research was supported in part by grants from R.J. Reynolds, Inc., B R S G Grants RR-07013-19 and -20 awarded to the Biomedical Research Support Grant Program, Division of Research Resources, NIH to the University of Colorado, and N I D A training G r a n t DA-07043 to R . K . F .
8 Freund, R.K., Jungschaffer, D.A. and Wehner, J.M., Involvement of GABAergic transmission in the electrophysiological effects of nicotine, Soc. Neurosci. Abstr., 13 (1987) 145. 9 Freund, R.K., Marley, R.J. and Wehner, J.M., Differential sensitivity to bicuculline in three inbred mouse strains, Brain Res. Bull., 18 (1987) 657-662. 10 Freund, R.K. and Wehner, J.M., Strain-selective effects of nicotine on electrophysiological responses evoked in hippocampus from DBA/2Ibg and C3H/2Ibg mice, J. Neurogenet., 4 (1987) 75-86. 11 Gamrani, H., Onteniente, B., Seguela, P., Geffard, M. and Calas, A., Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies, Brain Research. 364 (1986) 30-38. 12 Garza, R. de la, Bickford-Wimer, C., Hoffer, B.J. and Freedman, R., Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study, J. Pharmacol. Exp. Ther., 240 (1987) 689-695. 13 Haas, H.L., Cholinergic disinhibition in hippocampal slices of the rat, Brain Research, 233 (1982) 200-204. 14 Hatchell, P.C. and Collins, A.C., The influence of genotype and sex on behavioral tolerance to nicotine, Psychopharmacology, 71 (1980)45-49. 15 Hill, D.R. and Bowery, N.G., 3H-Baclofen and 3H-GABA bind to bicuculline-insensitive GABA B sites in rat brain, Nature (Lond.), 290 ( 1981) 149-152. 16 Kandel, E.R. and Spencer, W.A., Electrophysiology of hippocampal neurons. II. Afterpotentials and repetitive firing, J. Neurophysiol., 24 (1961) 234-259.
220 17 Koerner, J.F. and Cotman, C.W., A micropcrfusion chamber for brain slice pharmacology, J. Neurosci. Methods', 7 (1983) 243-251. 18 Krogsgaard-Larsen, P. and Johnston, G.A.R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., 25 (1975) 797-802. 19 Krnjevid, K., Reiffenstein, R.J. and Ropert, N., Disinhibitory action of acetylcholine in the hippocampus, J. Physiol. (Lond.), 308 (1980) 73P-74P. 20 Lerma, J., Herreras, O. and Martin del Rio, R., Electrophysiological evidence that nipecotic acid can be used in vitro as a false transmitter, Brain Research, 335 (1985) 377-380. 21 Limberger, N., Spath, L. and Starke, K., A search for receptors modulating the release of gamma-[~H]aminobutytic acid in the rabbit caudate nucleus slices, J. Neurochem.. 46 (1986) 1109-1117. 22 Lothman, E.W. and Collins, R.C., Kainic acid induced limbic seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates, Brain Research, 218 (1981) 299-318. 23 Marks, M.J., Burch, J.B. and Collins, A.C., Genetics of nicotine response in four inbred strains of mice, J. Pharrnacol. Exp. Ther,, 226 (1983) 291-302. 24 Marks, M.J., Patinkin, D.M., Artman, L.D,, Butch, J.B. and Collins, A.C., Genetic influences on cholinergic drug response, Pharm. Biochem. Behav., 15 (1981)271-279. 25 Marley, R.J. and Wehner, J.M., GABA enhancement of flunitrazepam binding in mice selectively bred for differential sensitivity to ethanol, Alcohol Drug Res.. 7 (1986) 25-32. 26 McCormick, D.A. and Prince, D.A., Pirenzepine discriminates among ionic responses to acetylcholine in guinea-pig cerebral cortex and reticular nucleus of the thalamus, Trends Pharmacol. Sci., Suppl. Feb. (1986)72-77. 27 Miner, L.L., Marks, M.J. and Collins, A.C,, Classical genetic analysis of nicotine-induced seizures and nicotinic receptors, J. Pharmacol. Exp. Ther., 23I (1984)545-554. 28 OIsen, R.W., Bergman, M.O., Van Ness, P.C., Lummis,
S.C., Watkins, A.E., Napias, ( . and Glucnlcc, I ) . \ . Oamma-aminobutyric acid binding in manmlalian brain: heterogeneity of binding sites, Mol. Pharmaco/., I~ (I~Slt 217-227. 29 Ribak, C.E., Vaughn, ,I.E. and Saito, K., lmmunocytochemical localization of gtutamic acid decarboxylasc in neuronal somata following colchicine inhibition eft ztxonal transport, Brain Research, 140 (1978) 315-332. 30 Rovira, ('., Ben-Ari, Y., Cherubini, E., Krnjcvi~: and Ropert, N., Pharmacology of the dendritic action of acctylcholine and further observations on the somatic disinhibition in the rat hippocampus in situ, Neuroscience. 8 (1983) 97-- 10t~. 31 Schwartzkroin, P.A. and Prince, D.A., Changes in excitatory and inhibitory synaptic potentials leading to cpi[eptogenic activity, Brain Research, 183 (1980) 61 76, 32 Schwartzkroin, P.A. and Prince, D.A., Penicillin-induced epileptiform activity in the hippocampa[ in vitro preparation, Ann. Neurol., 1 (1977) 463-469. 33 Segal, M., The acetylcholine receptor in the rat hippocampus; nicotinic, muscarinic or both?, Neuropharmacologv, 17 (1978) 619-623. 34 Wheal. H.V., Ashwood, ].J. and Lancaster. B., A comparative in vitro study of the kainic acid lesioned and bicuculline treated hippocampus: chronic and acute models of focal epilepsy. In P.A. Schwartzkroin and H.V. Wheal (Eds.), Electrophysiology ¢~f'Epilepsy. Academic, London, 1984, pp. 173-200. 35 Wonnacott, S., Fryer, L. and Lunt, G.G., Nicotine-evoked [~H]GABA release from hippocampal synaptosomes, J. Neurochem., 48 Suppl. (1987) 72B. 36 Wonnacott. S., Fryer. L., Lunt, G.G., Freund, R.K., Jungschaffer, D.A. and Collins, A.C., Nicotinic modulation of GABAergic transmission in the hippocampus, Soc. Neurosci. Abstr., 17 (1987) 1352. 37 Wong, R.K.S. and Prince, D.A., Dendritic mechanisms underlying penicillin-induced cpileptiform activity, Science. 204 (1979) 1228-123 I. 38 Wong, R.K.S. and Watkins, D.J,, Cellular factors influencing GABA response in hippocampal pyramidal cells. J. Neurophysiol., 48 (1982) 938-95 l.