Baclofen: Effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice

Brahz Research, 238 (1982) 371-383

371

Elsevier Biomedical Press

BACLOFEN: EFFECTS ON EVOKED FIELD POTENTIALS AND AMINO ACID N E U R O T R A N S M I T T E R RELEASE 1N T H E RAT O L F A C T O R Y CORTEX SLICE

G. G. S. COLLINS, J. ANSON and E. P. KELLY Department of Pharmacology, University ~f Sheffield, Sheffield SIO 2TN (U.K.)

(Accepted September 17th, 1981) Key words: baclofen -- synaptic transmission -- amino acid neurotransmitter release -- olfactory

cortex

SUMMARY A study has been made of the in vitro effects of ( ± ) - and (--)-baclofen on the evoked field potentials and release of endogenous amino acid neurotransmitter candidates (aspartate, glutamate, GABA and possibly taurine) which accompany electrical stimulation of the excitatory input to the olfactory cortex slice, the lateral olfactory tract. Baclofen appears to reduce the excitatory input to the GABA-utilizing inhibitory interneurones; this action was manifest as a drug-induced abolition of the field potential known as the P-wave (1C50 for (--)-baclofen, !.7 _-~:0.4/~M) together with a simultaneous reduction in the synaptically evoked release of aspartate and glutamate from the cut surface of slices. Both these actions of baclofen exhibited concentration dependence and stereospecificity and were not antagonized by picrotoxin (25 /~M) thereby suggesting that they are directly related. The consequence of this action of baclofen was the abolition of GABA-mediated presynaptic and postsynaptic inhibition together with their respective field potential correlates, the late N- and 1waves. (±)-Baclofen (5 and 25/zM) also inhibited the potassium-evoked release of aspartate and glutamate from small cubes of tissue but, except at a high concentration (1 mM), had no effect on GABA release. Baclofen (up to 1 raM) did not affect transmission either at the lateral olfactory tract-superficial pyramidal cell synapse, a site where aspartate is the likely neurotransmitter 9-16, or at the superficial pyramidal cell collateral-deep pyramidal cell excitatory synapse. It is proposed that: (i) the actions of baclofen on the olfactory cortex are the result of inhibition of aspartate and glutamate release, probably from deep pyramidal cell collaterals; and (ii) not all neurones utilizing excitatory amino acids as their neurotransmitters are subject to the inhibitory action of baclofen.

0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

372 INTRODUCTION Baclofen (fl-(4-chlorophenyl)-~-aminobutyrate) is well known as a potent, centrally acting muscle retaxanteffective in the treatment of certain types of spasticity (refs. 3. 5). Both neurochemica128,ag,40 and electrophysiologica118,19,21,37 investigations are consistent with the proposal that at therapeutic concentrations, baclofen reduces the release of certain central neurotransmitters by a mechanism insensitive to antagonists of y-aminobutyric acid (GABA) receptors 17,19,~°. Preliminary findings indicate that release of aspartate and glutamate is particularly sensitive to inhibition by baclofen2s,39,40. The ( )-isomer of baclofen is significantly more potent than the (+)-isomer in depressing neuronal activitylS.19,zl and in reducing the potassiumevoked release of [aH]D-aspartate2S although there is no clear evidence that these two effects are directly related. At somewhat higher concentrations, baclofen also depresses the excitability of postsynaptic membranes to a number of excitant compounds (refs. 17, 19. 20. 21.32). The isolated rat olfactory cortex slice is a useful preparation for studying the sites and mechanisms of action of centrally acting drugs, since electrical stimulation of its excitatory input, the lateral olfactory tract, evokes a characteristic series of surface field potentials a4,35,41,42 together with the release of a number of amino acid neurotransmitter candidatesS,9,11,1L A considerable body of evidence has accumulated implicating both aspartate and glutamate as mediators of excitatory neurotransmission in the olfactory cortex. For example, chronic denervation9.~°,16 and release experiments9,11,14,16 suggest that aspartate may be the transmitter utilized by at least some fibres of the lateral olfactory tract whereas other release studies 1~ together with an investigation of the depth distribution of amino acid neurotransmitter candidates1° indicate that the olfactory cortex pyramidal cell populations may utilize aspartate and glutamate as transmitters. In addition, both electrophysiologicat and neurochemical studies support the proposed role of GABA as the mediator of both presynaptic33-3'~ and postsynaptic35,43 inhibition in this brain region. The primary aim of the present investigation was to monitor simultaneously the effects of baclofen on the electrical activity and amino acid neurotransmitter release evoked by stimulation of the lateral olfactory tract in order to provide evidence whether any depression of synaptic transmission was a direct consequence of a drug-induced failure of neurotransmitter release. Some of the present results have been published in preliminary form ta MATERIALSAND METHODS

Electrical recordings Slices of rat olfactory cortex were cut, preincubated for 2 h at ambient temperature and perfused using the experimental protocol described by Pickles and Simmonds (ref. 34). The perfusion solution (pH 7.3-7.4) was bubbled continuously with 5 ~o CO2 in O~ and contained NaC1 (118.1 raM), CaCI~ (2.5 mM), MgSOa (2.2 mM), KC1 (2.l mM), KHzPO4 (0.93 mM), NaHCO~ (25 mM) and glucose (1t.1 raM). The lateral olfactory tract of slices was stimulated electrically by way of a bipolar platinum

373 electrode (pulse width of 50 #s and supramaximal voltage) at a stimulus frequency of l every 5 rain; such a low frequency was used because of the marked frequency-sensitivity of the late N- and I-waves33,34. Field potentials (500 ms sweep time) were recorded from the pial surface of preparations by means of a silver/silver chloride electrode, amplified using a DC preamplifier and the signals stored in a Datalab DL905 transient recorder. Permanent records were obtained by way of a chart recorder. An indifferent silver/silver chloride electrode was present in the perfusion medium. A typical recording of the field potentials evoked by single supramaximal stimulus is shown in Fig. lA. In all experiments, the amplitudes of the N- and late Nwaves were measured at their peaks irrespective of latency whereas the I-wave amplitude was measured at a fixed latency of 400 ms. In the presence ofpicrotoxin (25 /~M), a modified series of field potentials was recorded (Fig. 2A); in these experiments, the N'a'-, N'b'- and P-waves were all measured at their peak amplitudes. Both (--)and (-±)-baclofen was applied dropwise to the pial surface of slices as insufficient compound was available for inclusion in the perfusion medium. However, application of drug was continued until a plateau response was achieved (20 rain), thereby eliminating the possibility that drug penetration was affecting the experimental measurements. Analysis of inhibition. In 3 experiments, presynaptic and postsynaptic inhibition was monitored using the procedures developed by Pickles and Simmonds~. This involved presenting preparations with pairs of stimuli of varying interstimulus delays. When the second stimulus is given during the period of the late N-wave generated by the first, the reduction in amplitudes of the tract action potential and N-wave of the second stimulus is thought to be the consequence of presynaptic inhibition35. Postsynaptic inhibition was assessed by monitoring changes in the latency of the population spike evoked by the second of the stimulus pair (for example, see refs. 14 and 35).

Release experiments The release of the endogenous amino acid neurotransmitter candidates of the olfactory cortex (aspartate, glutamate, GABA and taurine) was monitored by means of two different procedures (for full experimental details, see ref. 15). ~n the first, a cortical cup technique was used to collect amino acids released from either the pial or cut surfaces of slices. The lateral olfactory tract was stimulated over a 20-rain time period (50 /~s pulse width, supramaximal voltage, 5 stimuli/min) and amino acid release monitored until it returned to prestimulation resting levels. Results of such experiments have been expressed in terms of the mean total amino acid released in excess of the prestimulation resting efflux15 (Table I). Where appropriate, drugs were present both in the perfusion solution and also in the solution within the cup. In the second type of procedure, the potassium-evoked release of endogenous amino acids from small cubes of tissue was measured. Freshly prepared olfactory cortex slices were cut into 0.5 mm sided cubes, transferred to a small perfusion vessel through which the warmed (37 °C) oxygenated perfusion solution was pumped at a rate of 0.5 ml/min. The tissue slices were depolarized by exposing them to a perfusion

)-bac~fen ~n the synaptica~y-evoked re~ease ~ f end~gen~us arnin~ acids fr~m s~ices ~ f rat ~fact~ry c~rtex

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Freshly cut slices were either perfused immediately (experiments in which release f r o m the cut surface was measured ~) or preincubated for 2 h (experiments in which release from the pial surface was measured~Sl in either drug-free tcontrol) solutions or in solution containing the appropriate drug(s) (for composition, see Methods section 1. A m i n o acid release was estimated using a cortical cup techniquO 5. The lateral olfactory tract of each preparation was stimulated electrically (pulse width 50 ffsec, supramaximal voltage, 5 pulses rain x) for 20 rain. Each value is a mean {~ S.E.) total a m o u n t of a m i n o acid released (in pmol) in excess o f or below (negative values) prestimulation resting efflux (not shown). The n u m b e r o f experiments is s h o w n in parentheses For full experimental procedure and calculation of results, see ref. 15. Release of glutamine, which was also monitored, showed no significant changes.

Effects o f ( j- - a n d (

TABLE 1

4~

375 solution containing 50 mM potassium chloride over a 12-rain time period. Where appropriate, (±)-baclofen was dissolved in the perfusion medium and was present throughout the experiment. Results have been expressed as the mean total amount of each amino acid (in pmol) released/rag tissue in excess of the pre-potassium resting efflux (Fig. 3). In both series of experiments, the amounts of endogenous aspartate, glutamate, glutamine, GABA and taurine released were estimated using a sensitive double-label modifications of the radiochemical microdansylation procedure described by Briel and Neuhoff7. Briefly, internal standards of 50,000 dpm each of [l~C]-labelled amino acids were added to every sample prior to reaction with [3H]dansyl chloride (6.0 × 106 dpm per sample): by such means,the recovery of the amino acids in all samples was monitored. The dansyl amino acid derivatives were separated by thin-layer chromatography using 3 different solvent systems, visualized under UV light and extracted prior to counting in a scintillation spectrometer. Using this procedure, 10 pmol of aspartate, glutamate and GABA may be reliably assayed with a coefficient of variance of less than 8 ~i (n -= 10)s.

Materials 4-Amino-n-[U-14C]butyric acid (226 mCi/mmol), L-[U-14C]glutamic acid (295 mCi/mmol), L-[U-14C]aspartic acid (231 mCi/mmol), [u-a4C]taurine (114 mCi/mmol), L-[U-t4C]glutamine (50 mCi/mmol) and [G-3H]dansyl chloride (13.7 Ci/mmol) were all purchased from the Radiochemical Centre, Amersham, U.K. Polyamide thin-layer chromatography plates for the microdansylation of amino acids were purchased from British Drug Houses, Poole, U.K. Both (--)- and (~)-baclofen were the generous gift of CIBA-Geigy, Basle, Switzerland. RESULTS

The peak plasma concentration of (±)-baclofen following a single, large subsedative dose is approximately 4/~M3°; the results of the present experiments should be considered with this in mind.

E[fects Qf baclofen on evoked field potentials When the lateral olfactory tract of slice preparations is stimulated by a single supramaximal shock, a characteristic series of field potentials may be recorded from the pial surface (see Fig. IA; for a full explanation, see ref. 14). The synchronously evoked massed EPSP (N-wave), which reflects the depolarization of the apical dendrites of the superficial pyramidal cells by the excitatory transmitter released from the lateral olfactory tract terminals24,%,41,42, is followed by two polysynaptic postsynaptic potentials which give rise to the surface late N- and I-waves34,3~: these two field potentials reflect GABA-mediated presynaptic34,35 and postsynaptic35 inhibition respectively. (--)-Baclofen (0.195 to 6.25 /~M with a drug contact time of 20 rain) caused a readily reversible, concentration dependent reduction in both late N- and lwave amplitudes (see Fig. 1) with ICs0 values of 3.1 ± 0.5 and 0.9 ~ 0.2 #M, respectively (mean ~ S.E., n -- 6). However, it should be noted that the effect of

376

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Fig. I. Effect of ( )-baclofen on evoked field potentials of slices perfused with drug-flee solution. Olfactory cortex slices were pre-incubated for 2 h prior to perfusion. The lateral olfactory tract of preparations was stimulated once every 5 rain (50/~s pulse width, supramaximal voltage) and the evoked field potentials recorded by a silver~silver chloride electrode over a 500ms sweep. Above: A, pre-drug control; B, (--)-baclofen (5 ~tM), 20 rain contact time; C, recovery following 15 rain washing. Negativity is upward and the dashed line indicates the recording baseline. Note the effect of baclofen in abolishing the late N. and I-waves. Below: graphical representation of the effects Of (--)baclofen on the evoked field potentials (drug contact time, 20 rain). Each point is the mean of 6 experimental values. The mean ICa0 values from the graphs were 3.1 ~ 0.5 ~M for the late N-wave and 0.9 ± 0.2/~M for the l-wave (mean ~- S.E. ). For a full explanation of the synaptie basis of the evoked field potentials, see Results section and ref. 14. baclofen o n the a m p l i t u d e of the late N-wave is not a simple f u n c t i o n of concentration. suggesting that a complex series o f effects may be involved. W h e n the (:~) racemate was used, the p a t t e r n of effects was identical (not s h o w n ) : the IC50 c o n c e n t r a t i o n s ( # M ) for suppression o f the late N- a n d I-waves in this case were 9.1 ~ 1.2 a n d 2.5 ~_ 0.2. respectively ( m e a n ~ S.E., n ~- 5). Thus the ( - - ) - i s o m e r i s significantly more p o t e n t t h a n the racemic mixture (Student's t-test. P < 0.01) in depressing the amplitudes o f these field potentials. I n 3 experiments, the effects o f 2 5 / t M ( ~ ) - b a c l o f e n o n evoked i n h i b i t i o n was assessed (see M e t h o d s section). The increased delay in the

377

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Fig. 2. Effect of (--)-baclofen on evoked field potentials of slices perfused with solution containing picrotoxin (25 pM). The experimental procedure is described in the IResults section and in the legend to Fig. 1. Above: A, pre-drug control (compare with Fig. 1A); B, (--)-baclofen (5/~ M), 20 min contact time; C, recovery following 15 min washing. Negativity is upward and the dashed line indicates the recording baseline. Note the specific effect of baclofen on the P-wave. Below: graphical representation of the effects of (--)-baclofen on the evoked field potentials (drug contact time, 20 min). Each point is the mean of 6 experimental values. Open circles, N'a'-wave; open square, N'b'-wave. The mean IC50 value for the depression of P-wave amplitude was 1.7 ± 0.4/zM (mean ± S.E.). For an explanation of the synaptic basis of the evoked field potentials, see Results section.

p o p u l a t i o n spike latency o f the second o f a stimulus p a i r which is seen in drug-free solutions14, a5 was c o m p l e t e l y and reversibly abolished by baclofen (not shown) suggesting that baclofen abolishes p o s t s y n a p t i c inhibition ~5. Similarly, the ratio o f the r e d u c e d a m p l i t u d e o f the N - w a v e evoked b y the second stimulus relative to that e v o k e d by the first o f the stimulus p a i r seen in drug-free solutions14, ~5 was reversed in the presence o f baclofen (not shown); this suggests that baclofen also reduces p r e s y n a p t i c inhibition 34. These results d e m o n s t r a t e that the b a c l o f e n - i n d u c e d reduct i o n in the late N- a n d I-wave a m p l i t u d e s was a c c o m p a n i e d by decreases in p r e s y n a p t i c a n d p o s t s y n a p t i c inhibition respectively.

378 In a small number of slices (3 of 6), low concentrations (0.195-0.78/~M) of (-=:)baclofen caused a small but definite potentiation of N- and late N-wave amplitudes which :ranged between 6.5 and 31.2%, and 7.7 and 16.4%, respectively. Note that at very high concentrations of bactofen (up to 1 m M, not shown), there was no significant reduction of the N-wave amplitude. When slices of olfactory cortex are incubated and perfused in the presence of GABA receptor antagonists such as picrotoxin or bicuculline, the late N- and l-waves are abolished and replaced by a positive-going field potential (P-wave; see Fig. 2A and refs. 35 and 44). This P-wave probably consists of polysynaptic massed EPSP generated deep within the slice25, 26,44. In addition, a prominent 'shoulder' (N'b',wave) appears on the N'a'-wave (see Fig. 2A): this N'b'-wave refleCts dendritic: depolarization of the deep pyramidal cells evoked by the excitatory transmitter released from superficial pyramidal cell collaterals 22 (the deep pyramidal cells are not directly innervated by the lateral olfactory tract fibres2). Both (--)- and (:~:)-baclofen (0.197-12.5/~M) reversibly reduced the amplitude of the P-wave in a concentration dependent manner (see Fig. 21) with ICs0 values of 1.7 =~- 0.4 and 4.3 ~: 013 #M respectively (mean :~= S.E., n := 6; significant difference, Student's t-test, P 0.00t). In contrast, baclofen had no effect on the amplitudes of the N'a'- and N'b'-waves (see Fig. 2).

Effects of baclofen on evoked amino acid neurotransmitter release Synaptically evoked release When the lateral olfactory tract of slices is stimulated electiically, the evoked release of aspartate, glutamate and GABA is almost entirely calcium-dependent 9,~5 suggesting that the site of release is neuronal. In the present experiments, when amino acid release from the cut surface of slices was monitored (±)-baclofen significantly attenuated the evoked release of aspartate, glutamate and GABA in a concentrationdependent manner. ( )-Baclofen (5/zM) produced the same pattern of effects and was approximately equal in potency to 25 ,uM of the racemic mixture. Although picrotoxin alone (25/zM) significantly increased the evoked release of aspartate, glutamate and GABA it did not antagonize the inhibitory effect of (zL)-baclofen (25/~M) on amino acid release (Table I). When release from the pial surface o! slices was monitored, neither (r_F}-baclofen (5 and 25 /~M) nor (--)-baclofen (5 ,uM) affected the evoked release of aspartate whereas GABA release was significantly reduced. Picrotoxin alone (25/~M) had no significant effect on amino acid release from the pial surface ~2. neither did it antagonize the baclofen-induced fall in GABA release. No significant changes in the synaptically evoked release of taurine were observed. Potassium-evoked release In order to identify the direct effects of baclofen on synaptically evoked amino acid release, a second series of experiments was performed in which the potassiumevoked release of amino acids was monitored (see Methods section). Exposure of small

379 400

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Fig. 3. Effects of (+_)-baclofen (5 /~M, 25 /tM and 1 mM) on the potassium-evoked release of endogenous amino acids from small cubes of olfactory cortex tissue. Freshly prepared olfactory cortex slices were cut into 0.5 ram-sided cubes using a mechanical chopper and between 40 and 80 mg transferred to a small perfusion vessel through which oxygenated solution at 37 °C was pumped at 0.5 ml/min (for composition of solution, see Methods section). Release of amino acids was evoked by changing the perfusion solution to one containing 50 mM potassium chloride. Using this procedure, the evoked release of glutamate, aspartate and GABA is almost entirely calcium-dependentaS. Each histogram shows the mean ( ± S.E., n 6-12) amount of amino acid (in pmol mg ~) released by the potassium chloride in excess of prestimulation resting efflux (not shown). An asterisk above a column indicates a significant difference (Student's t-test, P < 0.05) when compared to release into baclofenfree solution (columns containing a zero). Where appropriate, baclofen was present throughout the perfusion. For full experimental details, see Methods section and refs. 15 and 16, c h o p p e d cubes o f olfactory cortex tissue to 50 m M p o t a s s i u m chloride induces a c a l c i u m - d e p e n d e n t release o f aspartate, g l u t a m a t e and G A B A (see ref. 15). ( ~ ) Baclofen (25 # M ) s i g n i f i c a n t l y a t t e n u a t e d the evoked release o f a s p a r t a t e and g l u t a m a t e but not G A B A whereas at a c o n c e n t r a t i o n o f 5/~M, only the effect on aspartate release was statistically significant (see Fig. 3). Increasing the baclofen c o n c e n t r a t i o n to 1 m M did not further significantly reduce the evoked release o f a s p a r t a t e and g l u t a m a t e but in this case, the release o f G A B A was significantly reduced to 51.4 ± 10.0% o f control (mean ~ S.E., n = 3, P < 0.05). These findings suggest that when small c h o p p e d slices o f olfactory cortex are exposed to high p o t a s s i u m - i o n concentrations, first, the release o f G A B A is not mediated as a consequence o f the release o f a s p a r t a t e a n d g l u t a m a t e and second, that baclofen has no direct effect in inhibiting G A B A release except at very high concentrations. DISCUSSION W h e n a p p l i e d to the rat olfactory cortex slice, the most d r a m a t i c effect o f baclofen is to abolish G A B A - m e d i a t e d presynaptic and p o s t s y n a p t i c inhibition: this

380 action is manifest as a concentration-dependent, stereospecific depression of the late N- and I-waves, respectively. The mechanisms by which baclofen produces this effect might conceivably involve: (i) antagonism of the receptors at which the GABA is acting; (ii) inhibition of GABA release; or (iii) reduction in the excitatory drive to the GABA-releasing cells. Each of these possibilities will be discussed in turn. Notwithstanding the close structural similarities between baclofen and GABA, it is unlikely that at therapeutically relevant concentrations, baclofen interacts with the classical bicuculline-sensitive GABA receptod 7,19,2o for the striking insensitivity of its depressant actions on both monosynaptic and polysynaptic transmission lsee, for example, refs. 1, 19, 2t, 29. 36-38) to GABA receptor antagonists is welt known17-19~ At rather higher concentrations, baclofen possesses weak GABA-mimetic activity2L However, this cannot provide the basis of the effects of baclofen in the present study where GABA-mediated inhibition is abolished. On the other hand, inhibition of GABA release by baclofen would be expected to abolish the field potential correlates of GABA-mediated presynaptic and postsynaptic inhibition. Although the results ot' electrophysiological experiments have been adduced to support such a hypothesisis, direct measurement of GABA release has shown baclofen at therapeutic concentrations to be inactive in this respect. For example, Potashner a9,4°, in an elegant series of experiments, showed that (±)-baclofen at a concentration (4 #M) which dramatically inhibited the potassium-evoked release of excitatory amino acids had no effect on evoked GABA release. Moreover, in the present experiments, 25 uM (:&)-baclofen failed to significantly depress the potassium-evoked release of GABA (Fig. 3) from small chopped tissue slices although at a concentration of I mM, baclofen did significantly depress release by some 50~/~ (see Results). Therefore, inhibition of GABA release by a direct action on GABA-releasing terminals would appear to be a most unlikely effect of baclofen in the present study. The current experimental results, however, do support the possibility that the primary action of baclofen is to reduce the excitatory drive to the GABA-releasing inhibitory interneurones with a consequent secondary failure of GABA release, GABA-mediated presynaptic and postsynaptic inhibition and their field potential correlates. The evidence for such a proposal is two-fold. First, baclofen reduces the amplitude (and duration, not shown) of the P-wave in a stereospecific, concentrationdependent manner (see Fig. 2). This field potential, which is the surface manifestation of excitation of deep-lying structures 25,28,44 including the cell bodies of the GABAutilizing inhibitory interneurones, is usually masked by the late N- and 1-waves and its duration severely limited by recurrent inhibition; by antagonizing GABA-mediated inhibition, drugs such as picrotoxin and bicucullinea5,44 reveal the P-wave. That baclofen depresses the P-wave amplitude in the presence of picrotoxin (25 pM) strongly suggests that the mechanism does not involve an interaction with 'classical' GABA receptors. It is also important to note that baclofen does not depress excitatory neurotransmission at other sites in the olfactory cortex, for the field potentials known as the N'a'- and N'b'-waves, which reflect excitatory transmission at the lateral olfactory tract-superficial pyramidal cell22,24,25,41,42 and superficial pyramidal cell

381 collateral-deep pyramidal cell 22 synapses, respectively, are unaffected even by high (1 mM) drug concentrations. The second piece of evidence which supports the proposal that baclofen reduces excitation of the GABA-releasing inhibitory interneurones concerns the experiments in which amino acid release was monitored (Table 1 and Fig. 3). Current neuroanatomica145 and electrophysiological2,~, 24 evidence suggests that these inhibitory interneurones receive their excitatory input from pyramidal cell collaterals and preliminary studies 1°,15,16 implicate aspartate and glutamate as the neurotransmitters invclved. In those experiments in which the synaptically-evoked release of amino acids from the cut surfaces of slices was monitored, it is likely that the origin of the amino acids was neurones 9,15 lying close to the surface, including the pyramidal cell collaterals and the cell bodies or processes of the inhibitory interneurones 15. In the presence of baclofen, there is a concentration-dependent, stereospecific inhibition in the synaptically-evoked release of aspartate and glutamate from the cut surface of slices which is picrotoxin-insensitive (Table l); not unexpectedly, this manifestation of reduced excitation of the inhibitory interneurones is accompanied by a reduced release of GABA. That the action of baclofen on excitatory amino acid release is a direct effect is confirmed by the ability of baclofen to significantly reduce the potassium-evoked release of aspartate and glutamate from small cubes of olfactory cortex tissue (see Fig. 3 and refs. 28, 39 and 40). It should be noted that the concentration of baclofen required to inhibit the synaptically-evoked release of glutamate and aspartate is closely similar to those which suppress the I-, late N- and P-wave amplitudes. However, in addition to reducing the excitatory drive to the GABA-utilizing interneurones by inhibiting excitatory amino acid transmitter release there are several reports which suggest that at concentrations little above those achieved during therapy, baclofen will also diminish the excitability of the cells to excitants such as aspartate and glutamatelS-'~a ; it is not possible to ascertain whether baclofen is exerting such an effect in the present experiments. Three further points are worthy of note. First, it has been known for some years that the neurophysiological actions of baclofen are not mediated by 'classical' GABA receptors 17,a9 and more recently it has been proposed that atypical, bicucullineinsensitive receptors may be involved4, 27. The present results are consistent with such a proposal and, in addition, clearly demonstrate that the effects of baclofen on aspartate and glutamate release are picrotoxin-insensitive. Second, the present experiments demonstrate that the effects of baclofen in reducing the synaptically-evoked excitatory amino acid release and depressing synaptic transmission occur simultaneously within the same drug concentration range and are both stereospecific, thereby demonstrating that the two effects are likely to be directly related. Third, the present results provide indirect evidence that not all neurones utilizing aspartate as their neurotransmitter are subject to the actions of baclofen. For example, there is much evidence implicating aspartate as the neurotransmitter of the lateral olfactory tract-superficial pyramidal cell synapseg-~2,14-~6 and yet transmission at this synapse is unaffected directly by 1 mM baclofen (see Results). Moreover, the synaptically-evoked release of aspartate from the pial surface of slices, which probably largely reflects release from the tract

382 t e r m i n a l s 15. is n o t significantly affected by c o n c e n t r a t i o n s o f b a c l o f e n w h i c h a l m o s t a b o l i s h release o f a s p a r t a t e a n d g l u t a m a t e f r o m the cul s u r f a c e o f slices. T h i s f i n d i n g m a y g o s o m e w a y to e x p l a i n t h e relatively selective a c t i o n s o f b a c l o f e n on synaptJc transmission. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d by g r a n t s f r o m the M e d i c a l R e s e a r c h C o u n c i l ( U . K . ) a n d t h e U n i v e r s i t y o f Sheffield M e d i c a l R e s e a r c h F u n d to G . G . S . C . T h e gift o f ( ± ) and (

)-baclofen f r o m C I B A - G e i g y L t d . , S w i t z e r l a n d is g r a t e f u l l y a c k n o w l e d g e d .

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