85 Mv Respectively. When the Membranewas Depolarized, Th

J. Physiol. (1985), 360, pp. 161-185 161 With 14 text-figurem Printed in Great Britain

COMPARISON OF THE ACTION OF BACLOFEN WITH y-AMINOBUTYRIC ACID ON RAT HIPPOCAMPAL PYRAMIDAL CELLS IN VITRO BY N. R. NEWBERRY* AND R. A. NICOLLt From the Departments of Pharmacology and Physiology, University of California, San Francisco, CA 94143, U.S.A. (Received 13 June 1984)

SUMMARY 1. Intracellular recordings from CAI pyramidal cells in the hippocampal slice preparation were used to compare the action of baclofen, a y-aminobutyric acid (GABA) analogue, with GABA. 2. Ionophoretic application of GABA or baclofen into stratum (s.) pyramidale evoked hyperpolarizations associated with reductions in the input resistance of the cell. Baclofen responses were easier to elicit in the dendrites than in the cell body layer. 3. Blockade of synaptic transmission, with tetrodotoxin or cadmium, did not reduce baclofen responses, indicating a direct post-synaptic action. 4. (+ )-Bicuculline (10 ,UM) and bicuculline methiodide (100 /SM) had little effect on baclofen responses but strongly antagonized somatic GABA responses of equal amplitude. The bicuculline resistance of the baclofen response was not absolute, as higher concentrations of these compounds did reduce it. Pentobarbitone (100 /M) enhanced somatic GABA responses without affecting baclofen responses. (-)-Baclofen was approximately 200 times more potent than (+ )-baclofen. 5. The reversal potentials for the somatic GABA and baclofen responses were -70 mV and -85 mV respectively. When the membrane was depolarized, the baclofen response was reduced. This apparent voltage sensitivity was not seen with somatic GABA responses. 6. Altering the chloride gradient across the cell membrane altered the reversal potential of the somatic GABA response but not that ofthe baclofen response. It was extrapolated that a tenfold shift in the extracellular potassium concentration would cause a 48 mV shift in the reversal potential of the baclofen response. Barium ions reduced the baclofen response, but not the GABA response. 7. Orthodromic stimulation produced a fast inhibitory post-synaptic potential (i.p.s.p.) and a slow i.p.s.p. The properties of the fast and slow i.p.s.p.s were remarkably similar to those of the somatic GABA and baclofen responses, respectively. 8. Application of GABA to the pyramidal cell dendrites evoked, in addition to a depolarization, two types of hyperpolarization. One type of hyperpolarization was * Present address: Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR. t To whom reprint requests should be sent. 6 PHY 360 162 N. R. NEWBERR Y AND R. A. NICOLL bicuculline sensitive, had a reversal potential of about -65 mV and appeared to be chloride dependent. The other hyperpolarization was more easily observed in bicuculline methiodide (100 /M). This response was similar to that evoked by baclofen since it had a high reversal potential (about -90 mV), was relatively insensitive to changes in the chloride gradient across the cell membrane and was reduced by barium. 9. The bicuculline-sensitive hyperpolarization could be evoked by the dendritic or somatic ionophoresis of muscimol and THIP (4,5,6,7-tetrahydroisoxazolo- [5,4-c]pyridin-3(2H)-one. 10. These results suggest that baclofen, acting on dendritic GABAB-like receptors, opens voltage-sensitive potassium channels. It is possible that the fast and slow i.p.s.p.s in these cells are mediated by GABAA and GABAB receptors, respectively.

INTRODUCTION It is well established that the amino acid y-aminobutyric acid (GABA) is a ubiquitous inhibitory transmitter in the central nervous system (Roberts, Chase & Tower, 1976). It is also generally agreed that its inhibitory action is mediated primarily by an increase in chloride permeability of the membrane and that this action is prevented by the antagonists bicuculline and picrotoxin (Curtis & Johnston, 1974; Krnjevic, 1974). However, it has been inferred from recent studies with the f-(p-chlorophenyl) analogue of GABA, baclofen, that GABA may have additional roles (Bowery, 1982). Baclofen has been found to depress the excitability of central neurones (Curtis, Game, Johnston & McCulloch, 1974; Davies & Watkins, 1974) and to depress both peripheral (Bowery, Doble, Hill, Hudson, Shaw, Turnbull & Warrington, 1981) and central (Pierau & Zimmermann, 1973; Davidoff& Sears, 1974; Fox, Krnjevic, Morris, Puil & Werman, 1978) neurotransmission by a presynaptic action. The presynaptic inhibitory action, which may involve a reduction in calcium entry (Dunlap, 1981; Shapovalov & Shiriaev, 1982), can be mimicked by GABA (Bowery, Hill, Hudson, Doble, Middlemiss, Shaw & Turnbull, 1980; Bowery et al. 1981). However, the presynaptic action of GABA and baclofen is not blocked by GABA antagonists leading to the conclusion that baclofen acts at a bicuculline- insensitive GABA receptor. Studies on the binding ofbaclofen and GABA to synaptic membrane fragments have found that these two substances bind to a common site, referred to as a GABAB receptor, to distinguish it from the bicuculline-sensitive, GABAA, receptor (Bowery, Hill & Hudson, 1983). We have found that baclofen has a potent post-synaptic action on hippocampal pyramidal cells (see also Klee, Misgeld & Zeise, 1981; N. Ogata, personal communi- cation). We have compared this action of baclofen with the action of GABA and inhibitory post-synaptic potentials (i.p.s.p.s) in an attempt to find a physiological role for the response elicited by baclofen. Preliminary accounts of some of these results have appeared (Newberry & Nicoll, 1983, 1984a, b).

METHODS The methods used in this paper are similar to those previously described (Nicoll & Alger, 1981 a; Alger & Nicoll, 1982a; Newberry & Nicoll, 1984c). Briefly, rat hippocampal slices were cut and allowed to recover for 1-2 h. A single slice was then transferred to the recording chamber where BACLOFEN AND GABA 163 it was continually superfused with medium at 29-32 'C. The standard medium was an aqueous solution containing (in mM): NaCl, 116-4; KCl, 5-4; MgSO4, 1-3; CaCl2, 2-5; NaH2PO4, 10; NaHCO3, 26-2 and dextrose, 11. This was equilibrated with 95% 02/5 % CO2 before superfusion. Conventional intracellular recording techniques were employed. A calomel electrode was used for the indifferent electrode, to minimize junction potentials caused by low-chloride solutions. The impalements were made in stratum (s.) pyramidale of the CAI region of the slice. The recording electrodes were filled with either potassium methylsulphate (KMeSO4, 2 M, ICN) or KCI (3 M). They had resistances of 80-120 and 60-80 Mfl, respectively. The responses of GABA and baclofen were normally evoked by ionophoresis, although in a few experiments pressure ejection was used. The individual ionophoretic electrodes were filled with either GABA (1 M, pH 5, Sigma) or (±)-baclofen (20-50 mm, pH 3, Ciba-Geigy) dissolved in distilled water. The retaining current, usually around 20 nA for both GABA and baclofen, was adjusted so that there was no detectable resting leak. Often GABA responses were obtained by turning off the retaining current. This will be referred to as an ejecting current of 0 nA. The ionophoretic electrodes were positioned in either the pyramidal cell body layer (s. pyramidale) or the apical (s. radiatum) or basal (s. oriens) dendritic trees. Dendritic GABA applications were made in the mid-dendritic tree. Most of these experiments were performed in the apical dendrites approximately 150-200 ,um from s. pyramidale. To obtain a pure hyperpolarizing response to GABA in s. pyramidale, the ionophoretic electrode had to be positioned as close as possible to the tip of the intracellular recording electrode. This was considered necessary since it is thought that this response is evoked on the soma or initial segment (Andersen, Dingledine, Gjerstad, Langmoen & Mosfeldt Laursen, 1980; Alger & Nicoll, 1982b). For pressure ejection, pipettes, identical to those used for ionophoresis, were filled with GABA (1 mM) in standard perfusion medium and pressure pulses (50-200 ms in duration) were applied to the pipette using a Picospitzer. The individual, optical isomers of baclofen (Ciba-Geigy) were 'bath-applied' to the recording chamber, at known concentrations, via the superfusion system. The orthodromic synaptic responses, in particular the fast and slow i.p.s.p.s (Newberry & Nicoll, 1984c), were evoked by electrical stimulation (0-1 ms pulse width) of afferent fibres using bipolar stimulating electrodes situated in s. radiatum or occasionally s. oriens. A calcium-activated potassium hyperpolarization was evoked by a direct depolarizing current pulse delivered via the recording electrode, which evoked a given number of action potentials (Alger & Nicoll, 1980a; Hotson & Prince, 1980; Madison & Nicoll, 1982; Newberry & Nicoll, 1984c). The reversal potentials of the above-mentioned responses were usually determined by altering the cell membrane potential by passing constant direct current using a balanced bridge circuit (WPI 701) and repeating the responses at the altered membrane potentials. The reversal potential was determined from responses obtained from at least four different membrane potentials. Alternatively, the reversal potential was estimated using a step-pulse method (cf. Grafe, Mayer & Wood, 1980) using pulses of approximately 100 ms in duration. Here balanced, constant-current hyperpolarizing pulses were used to 'sample' the response of the neurone at a number of hyperpolarized membrane potentials. Using this method it was often possible to 'reverse' the polarity of a response which could not ordinarily be convincingly reversed by the 'direct current' method. Since the constant-current hyperpolarizing pulses used to measure input resistance during GABA responses usually shifted the membrane potential close to the reversal potential for the somatic GABA response, and since the amplitude of the response was linearly related to membrane potential, these pulses could be used to monitor the reversal potential for GABA during the response. Some of the responses analysed in this paper are generated in the dendrites, and therefore, the value obtained for the reversal potential could be further removed from the resting potential than the true value. To minimize this problem for the baclofen response, the baclofen was applied in s. pyramidale less than 50 ,m from the GABA electrode, unless otherwise stated. For various reasons it was necessary to alter the composition of the standard medium. For low-chloride solutions, 80 % of the total chloride (as NaCl) was replaced with equimolar sodium isethionate (ICN) or sodium methylsulphate (ICN). It was found that with the recording electrode in the bath there was a -4 to -5 mV potential produced when shifting to low-chloride solutions. A correction for this potential was made when determining reversal potentials in the low-chloride solution. When the concentration of KCI in the medium was halved or doubled, the osmolarity was corrected with equimolar NaCl. In order to superfuse BaCl2 or CdCl2, the NaH2PO4 was omitted and the MgSO4 replaced with equimolar MgCl2. Occasionally the spontaneous activity in the slice was prevented by the superfusion of 6-2 164 N. R. NEWBERRY AND R. A. NICOLL tetrodotoxin (TTX, 0-5 or 10 FM, Sigma or Calbiochem). This was especially necessary when using KCl-filled electrodes to prevent spontaneous depolarizing i.p.s.p. s (Alger & Nicoll, 1980b), or when using barium or GABA antagonists. A 10 mM-stock solution ofthe alkaloid, (+ )-bicuculline (Sigma) was prepared in 0-02 M-HCl and diluted just before use. The sodium salts of pentobarbitone (Abbott) and penicillin-G (Sigma), and bicuculline methiodide were dissolved directly into the medium just before use. The bicuculline methiodide was prepared from (+ )-bicuculline according to the method of Pong & Graham (1983). 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) was generously supplied by Dr P. Krogsgaard-Larsen. Numerical values are expressed as the mean +S.D.

RESULTS Baclofen and somatic GABA responses In agreement with previous reports, the ionophoresis of GABA, at the soma, evoked a hyperpolarization of the CAI pyramidal cell which had a fast onset and decay. Ionophoresis of baclofen into the pyramidal cell layer also hyperpolarized the cell but the onset and decay were considerably slower (Fig. 1A): full recovery of the membrane potential following large ionophoretic currents of baclofen took several minutes. The difference in time course is probably due to the fact that baclofen, unlike GABA (Iversen & Neal, 1968) is not actively taken up (Bowery et al. 1983) and may diffuse further into the tissue. The baclofen response was associated with a reduction in the input resistance of the cell. This can be seen in Fig. 1 A and B where the voltage deflexions produced by constant-current hyperpolarizing pulses were reduced during the baclofen response. The baclofen hyperpolarization could be elicited when ionophoresing in s. oriens, s. pyramidale or s. radiatum, and diminished very little in size with continued application. However, the responses were usually larger and had a faster onset when the ionophoretic electrode was positioned away from the pyramidal cell layer over the dendritic region (Fig. 1 B). The hyperpolarization to baclofen was a direct action on the pyramidal cell membrane since it could be evoked when synaptic transmission had been blocked with the calcium channel blocker, cadmium (n = three cells, e.g. Fig. 1C). Prolonged exposure to cadmium (30 min) did reduce the response but this occurred considerably after the complete blockade of synaptic transmission. The baclofen response could also be recorded after blockade of voltage-sensitive sodium channels with TTX, which would block action potential-dependent transmitter release (Fig. 2 and Newberry & Nicoll, 1984 a). Indeed, many of the experiments were done in the presence of tetrodotoxin to prevent cell discharge. Pharmacology. We have compared the sensitivity of pyramidal cells to the (+) and (-) optical isomers of baclofen by applying the drugs to the superfusate. In four experiments (e.g. Fig. 2) it was found that the (-) isomer was 180, 180, 180 and 200 times more potent than the (+) isomer in evoking a response. The potency ratio was determined at the level of the response to 3 SM-(-)-baclofen. In two of these experiments 0 3 /LM of the (-) isomer was effective in producing a response. Previous studies have shown that the action of baclofen, on transmitter release, is insensitive to GABA antagonists. We, therefore, compared the sensitivity of the baclofen response and the hyperpolarizing somatic GABA response to GABA antagonists. Since full dose-response curves were impractical because ofthe very long recovery time from large ionophoretic baclofen responses, submaximal GABA and BACLOFEN AND GABA 165 baclofen responses ofequal size were compared. The superfusion of 100 /SM-bicuculline methiodide (n = 8) or 10 /LM-( + )-bicuculline (in six out of seven experiments) for 10-20 min vas selective in depressingthe somatic GABA response. (In our experiments lower concentrations of (+ )-bicuculline were needed to depress the somatic GABA responses when compared to bicuculline methiodide; c.f. Simmonds, 1982; Olsen &

G B B B A - - B -p 20p Ogm 200 jurn A I

20 s Controli Cadmium Wash C i mV 10m Baclofen i JImV

40 s

Synaptic

400 ms Fig. 1. Baclofen hyperpolarizes hippocampal CAl pyramidal cells. A, comparison of the action ofGABA (G, 6 nA) ionophoresed at the soma with baclofen (B, 85 nA) ionophoresed into the pyramidal cell layer close by. Constant-current hyperpolarizing pulses (downward deflexions) were applied throughout. Resting potential was -55 mV. B, comparison of the responses evoked by baclofen (70 nA for 2 s) ionophoresed at the surface of s. pyramidale (0 jsm) and at the surface of s. radiatum 200 ,sm away from the cell layer. The pipette was then returned to s. pyramidale and gave an identical response to the one illustrated. Resting membrane potential was -60 mV. C, comparison of the action of ionophoresed baclofen (200 nA), into the mid-apical dendritic tree, with synaptic potentials evoked by stimulating via s. oriens (40 V). CdCl2 (100 #M) was superfused for 30 min and washed for 40 min. Resting potential was -53 mV for 'control' and 'cadmium' but the cell hyperpolarized 5 mV before the record in 'wash'. The drugs were ionophoresed for the periods indicated by the bars. B has the same gain as A and the same time calibration as in C (baclofen).

Snowman, 1983.) The bicuculline resistance ofthe baclofen hyperpolarization was not absolute since higher concentrations of these antagonists did depress the baclofen response (Fig. 3, BMI, BIC), possibly due to a non-specific action on potassium conductance (cf. Heyer, Nowak & Macdonald, 1982; see below). Therefore, in the rest of our studies we have used bicuculline methiodide (100 gLM) to determine if responses are sensitive to this antagonist. The superfusion of another GABA antagonist, penicillin-G (1 mM), was also selective in depressing GABA (n = 4). The baclofen response was also different from the GABA response in terms of its sensitivity to 166 N. R. NEWBERRY AND R. A. NICOLL

300 MM-(+) 3 JM-(-) A- 5 mV 5 min

B 20

> 15 E

.fQ 10 .0 5 I

0 1 0.1 1 10 100 1000 Baclofen (WM) Fig. 2. Comparison of the potencies of the individual optical isomers of baclofen. A, segment from a continuous record comparing 5 min superfusions of the two isomers of baclofen. B, a complete plot ofthe results from the experiment in A. The doses were applied in ascending order of their potency. Resting potential was -56 mV. Recording electrode was filled with KCL. TTX (1 uM) was present throughout experiment.

G B 0 AM _ 100 PM - _ 300mm BMI

0 _ 10 30

BIC

15 mv __O~~~~~~~I~~0 _ _ 100

10 mV PB 20 s jja_ h 17RJT1!I

Fig. 3. Pharmacological comparison ofGABA and baclofen. GABA was ionophoresed onto the soma and baclofen onto the pyramidal cell layer nearby. Three different experiments. Bicuculline methiodide (BMI), (+)-bicuculline (BIC) and pentobarbitone (PB) were superfused when recording from cells with resting potentials of -55, -53 and -57 mV respectively. During the superfusion of PB the cell hyperpolarized 1 mV. The GABA and baclofen ejecting currents (in nA) were respectively 8, 140 in BMI, 10, 70 in BIC and 10, 70 nA in PB respectively. BMI was superfused for 10 min (100 GEM) and 10 min (300 AM). BIC was superfused for 15 min (10 FM) and 15 min (30 FM). PB was superfused for 15 min (100,uM). TTX (0-5 uM) was present throughout the BMI and BIC experiments. All recording electrodes were filled with KMeSO4. BACLOFEN AND GABA 167 pentobarbitone. Pentobarbitone (100 /LM), which enhances GABA responses in many systems (Nicoll, 1975; Barker & Ransom, 1978; Alger & Nicoll, 1982b), failed to enhance the baclofen responses (Fig. 3) (n = 3). Reversal potentials. It was noted that, for equivalent hyperpolarizations, the baclofen response was associated with a smaller change in the input resistance of the

A G B B Response amplitude (mV) -46 - --8 -4 0 +4 +8 NM a a a a, 'a ' a I o 0 -50 -52 1~~~~~~~OObe A 0 0

4 -90 0 j5 mV ~~~~~~~~~-70Membrane potential (mV) 5 s 20 s Fig. 4. Response amplitudes to ionophoretic GABA and baclofen evoked at different membrane potentials. A shows responses to GABA ionophoresed at the soma and to baclofen ionophoresed into the pyramidal cell layer close by. The series of baclofen responses (B, 100 nA) was done before the series of GABA responses (G, 0 nA). B shows graph of responses partially illustrated in A. The reversal potential for GABA (0) in this cell was -64 mV and the extrapolated reversal potential for baclofen (@) was determined to be -85 mV. Incidentally, the extrapolated reversal potential for the after-hyperpolarization following three directly evoked spikes was determined to be -85 mV in this cell. The resting potential during the baclofen series was -56 mV and the resting potential during the GABA series was -52 mV. The gain for the original GABA records is the same as for baclofen. TTX not present. Recording electrode was filled with KMeSO4.

cell than was the somatic GABA response (Fig. 1 A). This suggested that the reversal potentials for the two responses were different. We therefore compared the effect of altering the membrane potential, with direct current, on the somatic GABA and baclofen responses. Although the somatic GABA response could be reversed in polarity with membrane hyperpolarization, the very negative reversal potential for the baclofen response required extrapolation. The presence of strong inward rectification presumably accounts for this difficulty (Halliwell & Adams, 1982). In every cell tested with both compounds the reversal potentials differed by at least 10 mV (n = 10) (e.g. Fig. 4). The extrapolated reversal potential for the baclofen response was, however, similar to that of the slow hyperpolarization following a fixed number of action potentials determined in the same cell (n = 3). This after- hyperpolarization is thought to be due to a potassium conductance activated by calcium (Alger & Nicoll, 1980a; Hotson & Prince, 1980; Schwartzkroin & Stafstrom, 1980). The average extrapolated reversal potential for the after-hyperpolarization was -85-3+227 mV (n = 7, cf. Alger & Nicoll, 1980a) and for the baclofen response it was - 85'1 + 2'6 mV (n = 8). In contrast, the average reversal potential for the 168 N. R. NEWBERR Y AND R. A. NICOLL somatic GABA response was - 70-5 + 3-8 mV (n = 33). Both muscimol (n = 2) and THIP (n = 3) hyperpolarized the soma with reversal potentials similar to the GABA response. Even though, in these experiments, baclofen was applied in the pyramidal cell layer, to ensure that the response was generated as close as possible to the soma, it A B

-60 E -65 0 40___s embra I(m-70I E

Fig. 5. Reversal potentials for GABA and baclofen are different. A, the effect of ionophoresing baclofen (B. 300 nA) into the pyramidal cell layer on the polarity of the response to repeated somatic GABA application (2 s applications, 0 nA). TTX present. The resting potential was -60 mV. B. using the step-pulse method with three different amplitudes of constant-current hyperpolarizing pulses, it can be seen that the response to baclofen (I100 nA into pyramidal cell layer) is 'reversed' with the highest current pulse. Resting membrane potential was-54 mV. The ordinate in the graph on the right gives the membrane potentials for the responses on the left. The recording electrodes were filled with KMeSO4. is possible that the receptors for baclofen may be concentrated on the dendrites (see Fig.rB). Therefore, the difference in the reversal potential for the GABA and baclofen responses could be more apparent than real. However, in six cells the GABA response could actually be reversed during a large baclofen response (Fig. 5A), strongly suggesting that the baclofen response does have a more negative reversal potential than the GABA response. Due to the negative reversal potential and slow time course ofthe baclofen response it was not possible to convincingly reverse e response by direct current. An alternative method for estimating the reversal potential is the step-pulse method (see Methods). Using this method it was possible to obtain a 'reversal' of the response. In Fig. 5B, the reversal potential for the baclofen response was determined to be -87 mV. Reversal potentials obtained with this method were similar to these obtained with the direct current method. BACLOFEN AND GABA 169 Ionic mechanisms. The difference in reversal potential for the GABA and baclofen response suggests that different ionic mechanisms may be involved. When intracellular recordings are made with micro-electrodes filled with 3 M-KCI the intracellular concentration of chloride increases and the chloride reversal potential becomes less negative than the resting membrane potential. This shift in the chloride gradient was

B2 A G B B 1 Amplitude of response (mV) iiijil! l. 1 11! 100% Cl- 20% Cl- -4 0 4 8 12 J55mV -50 --~ ~ X_50 o2000//ocCI -60 o~~~~--~o20% Cl-

C 100% Cl- 20% Cl-V70 G B G B 45 \-70 J5 m -75 ,Membrane potential (mV)

40 s Fig. 6. Chloride independence of the baclofen response. A, effect of reversing the chloride gradient of the pyramidal cell by recording with a KCl-filled electrode, on the responses to ionophoretic GABA (G) and baclofen (B). When recording with a KCl electrode the somatic GABA response (10 nA) is depolarizing whereas the response to baclofen (70 nA) ionophoresed into the pyramidal cell layer remains hyperpolarizing. Resting potential was -54 mV. B, effect ofreducing the external chloride concentration on the reversal potential ofa somatic GABA response. The low-chloride solution was superfused for 18 min. Sample records are shown in BI and plotted in B2. Recording electrode was filled with KMeSO4. Resting potential was -60 mV. C, effect of reducing the external chloride concentration on responses to GABA (2 nA) and baclofen (90 nA) recorded with a KMeSO4-filled electrode. GABA was applied to the soma and baclofen into the pyramidal cell layer. Superfusing a medium containing 20% chloride (isethionate substitution) for 12 min reverses the GABA (2 nA) but not the baclofen (90 nA) response. Resting potential was -62 mV. Constant-current hyperpolarizing pulses were applied throughout in both cases. Gains in A and B are the same. The upward deflexions in A are action potentials elicited by spontaneous depolarizing unitary i.p.s.p.s.

verified in each cell by observing spontaneous depolarizing i.p.s.p.s (Alger & Nicoll, 1980b). Fig. 6A shows a somatic GABA response which, as expected, was depolarizing. However, under these conditions the baclofen response was invariably hyperpolarizing (see also Fig. 2). The reversal potential obtained using KCl-filled micro-electrodes (- 86-2 + 3.3 mV, n = 7) (Fig. 7) was similar to that obtained using KMeSO4-filled micro-electrodes (-85-1 + 2-6 mV, n = 8). It is possible that the chloride leaking from the intracellular electrode failed to reach the site responsible for generating the baclofen response. As a consequence of this uncertainty, we examined the effects of reducing the chloride concentration in the extracellular medium. When the external chloride concentration was reduced to 20 % (replacing chloride with isethionate) the reversal potential of the somatic GABA response was shifted in a depolarized direction and sometimes reversed in polarity (Fig. 6B, C) while there was usually only a small depolarization of the cell. The 170 N. R. NEWBERRY AND R. A. NICOLL average shift in the reversal potential for the somatic GABA response, when reducing the extracellular chloride with isethionate, to 20 % was + 20-4 ± 6-0 mV (n = 12). This is clearly much less than the + 42 mV shift expected, from the Nernst equation, for a response generated solely by an increase in chloride conductance. However, in the experiment shown in Fig. 6C, it can be seen that the baclofen response was unaffected by the low-chloride solution, while the GABA response was reversed. - -59 mV - -83 mV A - -F- 5 mV 20 s

o -9 Ea 51//

CL 5~~~~~

-110 - -110~~~~~~~~ 2*7 54 10*8 Extracellular potassium (mM) Fig. 7. The effect of altering the external potassium concentration on the reversal potential of the baclofen response. A, reversal of the baclofen response in 10-8 mM-potassium. (Reversal potential was -73 mV.) B, graph of the actual ( 10-8) and extrapolated (5 4, 2 7) reversal potentials for baclofen obtained from seventeen determinations in eleven cells. The number on the S.D. bars indicates the number of determinations. The slope of the line is 48 mV per tenfold shift of potassium. (The ideal shift is 60 mY at this temperature.) These experiments (including that in A) were all done using KCl-filled electrodes to facilitate the passage of large currents. TTX (1 uM) was present in all experiments.

The amplitude of the baclofen response was frequently reduced in low-chloride solutions, but this was not accompanied by any detectable positive shift in the reversal potential of the response (using the step-pulse method). This depression appearedto be more pronounced when using methylsulphate, compared to isethionate, and consequently methylsulphate was not routinely used. Since there was no apparent shift in the reversal potential, it would apear that these chloride substitutes may have a non-specific depressant effect on the baclofen response. Such a depressant effect did not, however, apply to all responses presumed to be generated by an increase in potassium conductance, since the after-hyperpolarization following a train of action potentials was not reduced. The effects of changing the extracellular potassium concentration on the reversal BACLOFEN AND GABA 171 potential of the baclofen response were also examined (Fig. 7). Doubling the potassium concentration caused a depolarizing shift in the reversal potential and in this medium it was possible to reverse the baclofen response with hyperpolarizing current (Fig. 7A). Halving the potassium concentration caused a hyperpolarizing shift in the baclofen reversal potential. The slope of the line drawn in Fig. 7B was Barium B B B B B A

TVF 5mV 100 s

d.c.

B G G B G B

\ ~~~~~ ~ ~~~ ~~~~~ ~~~~~~ ~~20 s

Fig. 8. Effect of barium on the baclofen response. A, continuous record of the effect of BaCl2 (2 mM) on the response to baclofen (B, 200 nA) ionophoresed in the dendrites. Barium was superfused for the period indicated. Direct current (d.c.) was used to return the membrane potential to control. Constant-current hyperpolarizing pulses were applied while the barium was washed in and out of the bath. Resting potential was -59 mV at the end of the experiment. B, effect of barium (1 mM) on the response to ionophoresis of GABA (0 nA) at the soma and baclofen (80 nA) in the dendrites. Barium was superfused for 15 min and depolarized the cell by 5 mV. 40 min was allowed for recovery. The resting potential was restored to control in barium with the use ofdirect current. Resting potential was -55 nV. Gains in A and B are the same. Recording electrodes were filled with KMeSO4 and TTX (1 /M) was present. Although these particular examples are with ionophoresing baclofen in the dendrites, the same observations were made when ionophoresing it in the pyramidal cell layer.

48 mV for a tenfold change in the external potassium concentration. We have also examined the effect of changing the external potassium concentration on the hyperpolarizing somatic GABA response. In three experiments, we found that the reversal potential for this response shifted positive by 12, 12 and 13 mV when doubling the potassium concentration and 6, 7 and 10 mV negative when halving the potassium concentration. These shifts, at least in part, are to be expected because ofsecondary shifts in the chloride gradient (cf. Meyer, 1976; Matthews & Wickelgren, 1979; Aickin, Deisz & Lux, 1982). Barium ions are known to block a variety ofpotassium channels (cf. Dodd & Horn, 1982). In concentrations of 300 /IM to 3 mm, barium depolarized the membrane potential and increased neuronal input resistance (Fig. 8A). In eleven out of twelve cells examined, the baclofen response was substantially reduced in the presence of barium (Fig. 8A). Part of the reduction may be related to the apparent voltage sensitivity of the baclofen response in the depolarizing direction (see Fig. 4), since hyperpolarizing the membrane potential back to the control values partially restored the response. However, the baclofen response recovered more slowly than the 172 N. R. NEWBERRY AND R. A. NICOLL membrane potential when washing out the barium. While barium reduced baclofen responses, the somatic GABA responses were actually increased (Fig. 8B). On the basis of the above evidence it appears that baclofen may act by increasing potassium conductance of the cell membrane and that this effect is resistant to GABA antagonists. Amplitude of i.p.s.p. (mV) A _46 B _12 -8 -4 0 +4

o o 00 @ 0 -50 -55 POe

@0*so 0 0 -70 -64 % 0 0 o0 J 5 mV

-75 L-90 Membrane potential (mV) Fig. 9. The effect of altering the somatic membrane potential on the amplitudes of the fast and slow i.p.s.p. s evoked by stimulating s. radiatum. A shows responses evoked at different membrane potentials. The amplitudes of the i.p.s.p. s were measured at 40 and 200 ms (see arrows on response at -64 mV) and plotted in B. The reversal potential for the fast i.p.s.p. (0) and the extrapolated reversal potential for the slow i.p.s.p. (@) were determined to be -71 and -85 mV, respectively. Orthodromic stimulation was at 1/60 Hz. Resting potential was -55 mV. Recording electrode filled with KMeSO4.

Synaptic potentials The synaptically evoked late hyperpolarizing potential, which follows the fast, bicuculline-sensitive and chloride-dependent i.p.s.p. in hippocampal pyramidal cells (Nicoll & Alger, 1981 b) is now thought to be a bicuculline-resistant slow i.p.s.p. (Newberry & Nicoll, 1982, 1984c; Alger, 1984; Lancaster & Wheal, 1984; Thalmann, 1984). The evidence suggests that this potential may be due to an increase in potassium conductance (Nicoll & Alger, 1981 b; Thalmann & Ayala, 1982; Thalmann, 1984). Thus, this slow i.p.s.p. has properties similar to the hyperpolarizing baclofen response. We have now observed that the reversal potentials for the fast and slow i.p.s.p.s are similar to those of the somatic GABA and baclofen responses, respectively (Figs. 4 and 9). The average reversal potential for the fast, bicuculline-sensitive and presumably GABAA-mediated, i.p.s.p. (as measured at a latency of 40 ms) was - 71-4 + 3-3 mV (n = 12) and the average extrapolated reversal potential for the slow i.p.s.p. (as measured at 200 ms) was -91-2 + 6-0 mV (n = 12). Of particular interest, was the finding that the amplitudes ofboth the baclofen response and the slow i.p.s.p. BACLOFEN AND GABA 173 were reduced when the cell was depolarized (cf. Figs. 4 and 9). The slight voltage sensitivity of the i.p.s.p. measured at 40 ms is presumably due to some overlap between the two potentials. There appears, therefore, to be a remarkable similarity between the fast and slow i.p.s.p.s and the somatic GABA and baclofen responses, respectively. Since baclofen has been reported to act on a distinct class of GABA receptors, we investigated whether GABA might activate the same type of response as baclofen. The finding that the ionic mechanism for the somatic GABA response and baclofen response are different suggests that the receptors activated by baclofen are not concentrated on the somatic membrane. Evidence presented in Fig. 1 B suggests that the receptors for the baclofen response may be concentrated on the dendrites. We have therefore investigated the possibility that GABA may be able to mimic the action of baclofen when applied to the dendrites. Dendritic GABA responses The most obvious and prominent response to dendritic GABA application was a depolarization. This was associated with a reduction in the input resistance of the cell and it had an extrapolated reversal potential, -42+7 mV (n = 9) which was shifted + 12-8_± 1.1 (n = 3) in a solution containing 20 % chloride (isethionate substitution). The values for the reversal potential are subject to considerable error due to the strong rectification of the membrane. The depolarization was not the only type of response that could be recorded (see Fig. 10). Two types of hyperpolarizing response could be evoked by GABA which could be separated according to their reversal potentials and their time course. A small hyperpolarizing response could be recorded alone (Fig. 10A, D-0) or preceding the depolarizing response (Fig. 10A, D-10). This had a comparatively fast onset and decay and will be referred to as a 'fast' hyperpolarization. A larger hyperpolarization, of slower onset and decay could occur but this 'slow' hyperpolarization followed the depolarization in normal medium (Fig. 10A2, D-20). The resistance changes associated with these responses (cf. Fig. 10A, D-0 and A2, D-20) suggested that the 'fast' and 'slow' hyperpolarizations were associated with different reversal potentials. The depolarization and 'slow' hyperpolarization were usually recorded when using higher ejecting currents than those necessary to evoke an apparently pure 'fast' hyperpolarization. Occasionally all three responses were visible following ionophoresis of GABA at a single dendritic site (e.g. Fig. 11 A2, D-36; Fig. 13A, D-32). All of these responses could be evoked in both the apical and the basal dendrites but, for convenience, we usually studied the apical dendritic responses. In contrast to the dendrites, when the ionophoretic GABA electrode was carefully positioned at the soma, a pure hyperpolarization was obtained (Fig. 10B, S-0). However, it should be noted that if the ejecting current was increased, a shift in the reversal potential occurred during the hyperpolarization (Fig. 10B, S-40) and a depolarization sometimes followed the hyperpolarization. This biphasic response could be due to the spread of the GABA to a nearby dendrite. Alternatively, it could be due to a shift in the GABA reversal potential due to chloride redistribution. Care was taken in these and the previous experiments to obtain a 'pure' GABA hyperpolarization at the soma. 174 N. R. NEWBERRY AND R. A. NICOLL In the normal medium, the 'slow' hyperpolarization was usually evident as an 'off'response, i.e. it became evident after the ejection current had been turned off. It was possible to record this type of dendritic response from cells which had a reversed chloride gradient (at least at their soma) due to chloride leakage from a KCl-filled recording electrode (Fig. 10C). This was, however, more difficult and was

Al D-O D-10 A2 D-O D-20

15 mV Hl 5 s B S-0 S-40 C S-0 D-80

Fig. 10. Different responses evoked by the ionophoresis of GABA with different ejection currents through the same electrode on the hippocampal pyramidal cell. Responses to ionophoresis at the soma (S) or dendrites (D) at the indicated ejection currents. Al and A2 are responses following ionophoresis in the mid-apical dendritic tree. B, ionophoresis at the soma. C, ionophoresis at the soma and in the basal dendrites. Calibration applies to all records. TTX (1 gM) present in all records. Resting potentials were -53 (A 1), -55 (A2), -58 (B) and -56 mV (C). Recording electrodes filled with KMeSO4 except for C when KCI was used. not observed in a previous study (Alger & Nicoll, 1982b). When it was present the 'slow' hyperpolarizing response could be further revealed by the superfusion of bicuculline methiodide (100 /SM) which reduced the concomitant depolarizing response (Fig. 11). This concentration of bicuculline methiodide reduced the somatic hyperpolarization by over 80 % (n = 10). The resultant potential usually had evidence of the depolarizing response in it (e.g. Fig. 1 A3), but in cases where the 'slow' hyperpolarization was relatively pure (e.g. Fig. 11C), the resistance change was very similarto thatevokedbybaclofenejectedfrom the adjacent barrel ofa double-barrelled ionophoretic pipette (n = 4). Its extrapolated reversal potential, was -87+ 4 mV (n = 6) (Fig. 12 A). Although we were unable to convincingly reverse this response in normal medium, clear reversals could be obtained when the potassium concentration was doubled (Fig. 12B). The reversal potential shifted 17 mV in this experiment and 15 mV in another experiment, which are similar to the shifts obtained with baclofen responses. Like baclofen and the slow i.p.s.p., the 'slow' hyperpolarizations were also reduced when the cell was depolarized (n = 3). The observation of a relatively pure 'slow' hyperpolarization indicated that it was not induced by the preceding depolarization. As mentioned earlier, relatively pure dendritic hyperpolarizations to GABA could BACLOFEN AND GABA 175 be evoked in normal medium when using low ejection currents. This type of response was more often seen in cells with low membrane potentials (< -55 mV). On increasing the ionophoretic current this response was usually seen to precede the depolarization (see Fig. I1 Bi, D-40). When apparently uncontaminated (e.g. Fig. 1OA 1, D-O), the 'fast' hyperpolarization had an apparent reversal potential of

A 1 A2 Control A3 BMI G G S-18 D-36 S-18 D-36

5 mV B1 Control B2 BMI 10 S S-0 D-0 D-40 S-0 D-0 D-40

Ad ~~~~~~~~~~~~~~~~~~~~~~~~1111 Hmllt~m I10 mV Cl C2 Control C3 BMI 5 S G B G B G B

mV I |5

Fig. 11. Effect of bicuculline methiodide on somatic and dendritic GABA responses. Al, diagram of placement of ionophoretic electrodes. A2 and 3, somatic and dendritic GABA applications were repeated after superfusion with bicuculline methiodide (BMI) (100 /M) for 20 min. B, same as A except that a hyperpolarization evoked by low-current GABA ejection in the dendrites was also obtained. BMI (100 ,#M) superfused for 5 min before the second set of records. C1, diagram ofplacement ofdouble-barrelled ionophoretic electrode. C2 and 3, responses to GABA (G, 60 nA) and baclofen (B, 70 nA) before (C2) and 25 min after superfusing BMI (100 /M) (03). Resting potentials were -55 (A), -52 (B) and -62 mV (C). Recording electrodes filled with KMeSO4.

-66 + 3 mV (n = 4). In contrast to the 'slow' hyperpolarizing response, the 'fast' hyperpolarizing response was abolished by bicuculline methiodide (100 AM) (Fig. 11 B). The dendritic ionophoresis of two other GABA-mimetics, muscimol and THIP, which are thought to act primarly on GABAA receptors (Bowery, 1982), could elicit, with low ionophoretic current, hyperpolarizations with a reversal potential of -62 +4 mV (n = 14) (muscimol) and -64+ 5 mV (n = 12) (THIP). Depolarizations to these compounds could be evoked with higher ejecting currents (Fig. 13 A). In cells with resting potentials of greater than -65 mV only depolarizations were evoked by these GABA agonists. Both the hyperpolarizations (Fig. 13B) and depolarizations were sensitive to bicuculline methiodide (100 /UM) and, in contrast to dendritic GABA application, we have been unable, thus far, to evoke a bicuculline-resistant hyperpolarization that has a very negative reversal potential with these compounds. 176 N. R. NEWBERRY AND R. A. NICOLL Superfusion of the slice with low-chloride solution shifted the reversal potential for the hyperpolarizing responses to muscimol and THIP (Fig. 13C, n = 3). This procedure similarly affected the 'fast' hyperpolarizing response to GABA (Fig. 14A, n = 5). It should be noted that although muscimol and THIP evoke hyperpolarizations which have properties similar to those of the 'fast' hyperpolarization evoked by GABA, their responses were not 'fast' and this is presumably because they are poor substrates for the GABA-uptake system (Krogsgaard-Larsen, Falch & Jacobsen, 1984). A 1 A2 Response amplitude (mV) 6 4 -50 K^ I *I I .I 2I I-50 i - ~~~~~~~~~~~-50 -55 i

-65 -70

Membrane potential (mV) -60 rmV -85 mV < J 25 mV

Fig. 12. Effect ofaltering membrane potential on the bicuculline-resistant GABA response. A shows responses to the pressure ejection (20 lbf/in2 for 200 ms) of GABA onto the dendrites in the presence of bicuculline methiodide (100 /M). A2 is a graph of the response partially illustrated in A. In B the extracellular potassium was doubled to 10-8 mm. All responses are from the same cell. The calibration in B also applies to A. Resting potential = -65 mV.

Although GABA, muscimol and THIP appear to be able to evoke a chloride- and bicuculline-methiodide-sensitive hyperpolarization in the dendrites of some cells, the finding that GABA, in the presence of bicuculline methiodide, can also evoke a slow dendritic hyperpolarization with an apparent reversal potential similar to that of baclofen (see Fig. 12), suggests that GABA, like baclofen, may be able to increase potassium conductance. The effect of low-chloride solutions on the slow hyperpolarizing GABA response is shown in Fig. 14A and B. In Fig. 14A it can be seen that while the low-chloride solution reversed the fast hyperpolarization and increased the depolarization, the slow hyperpolarization remained hyperpolarizing. In another experiment this was done in the presence ofbicuculline methiodide so that the slow hyperpolarization could be examined in greater detail. The low-chloride solution caused some restoration of the depolarizing component, but the portion of BACLOFEN AND GABA 177 response following the cessation of the ionophoretic current was relatively unaffected (Fig. 14B). In this experiment the small somatic hyperpolarization that remained in bicuculline methiodide was unaffected by the low-chloride solution. In this cell, before the addition of bicuculline methiodide had depressed the somatic GABA response by over 80 %, the same low-chloride solution shifted the somatic GABA reversal potential by 15 mV.

A M-40 M-80 M 120

B Control BMI M G T

CM~~~~~~~~~~~~~~~~T 20 'lT C_ ~100% Cl- 20% Cl- t5 mV

Fig. 13. The actions of muscimol and THIP applied to pyramidal cell dendrites. A, effect of increasing ionophoretic current (indicated above bar) on response to muscimol. Resting potential was -55 mV. B, effect of bicuculline methiodide (BMJ, 100 /LM), superfused for 3 min, on responses to muscimol (M, 40 nA), GABA (G, 40 nA) and THIP (T, 70 nA). Drugs were applied from a triple-barrelled pipette. Same cell as A. C, effect of a 20% chloride medium, superfused for 8 mi on muscimol (M, 125 nA) and THIP (T, 125 nA) responses. Resting potential was -52 mV. Calibration in B also applies to A and C.

We have also examined the effect of barium ions on dendritic GABA responses. In four of six cells, barium (0A-OX8 mM) reduced the slow GABA response evoked in the presence of bicuculline methiodide (Fig. 140). However, barium never completely antagonized the response. In those cells in which a depolarizing component was present barium had little effect on the underlying conductance increase and the depolarization became larger.

DISCUSSION The principal findings of the present study are that (1) baclofen directly hyperpolarizes hippocampal CA1 pyramidal cells by a mechanism which is bicuculline resistant and probably involves an increase in potassium conductance; (2) ionophoresis ofGABA at the soma evokes a bicuculline-sensitive, chloride-dependent hyperpolarization; (3) ionophoresis of GABA, but not muscimol or THIP, into the 178 N. R. NEWBERRY AND R. A. NICOLL dendrites can evoke a bicuculline-resistant hyperpolarization which has a high reversal potential and other properties in common with the baclofen response; (4) dendritic application of GABA and the GABAA agonists, muscimol and THIP, can evoke a bicuculline-sensitive hyperpolarization with a relatively low reversal potential in addition to a bicuculline-sensitive depolarization; (5) there is a marked similarity

A 100% C- 20% C1- D 0 D-32

I5 mV B s-0 D-40 5 s

Control Barium Wash C D-24 D-24

!I III

Fig. 14. Effect of low external chloride solutions and barium ions on dendritic GABA responses. A, pure hyperpolarization and triphasic response (arrow indicates initial fast hyperpolarization). Low chloride (isethionate substitution) was superfused for 9 min. B, these responses were recorded in bicuculline methiodide (100#UM). Low chloride was superfused for 10 min. C, dendritic hyperpolarization evoked by GABA in the presence of bicuculline methiodide (100 #M). Barium (0-8 mM) was superfused for 9 min and then washed from the preparation for 20 min. Resting potentials were -52 (A), -58 (B) and -57 mV (C). Recording electrodes were filled with KMeSO4. between the hyperpolarization to GABAA receptor activation and the fast i.p.s.p., and the response to the GABAB receptor agonist, baclofen, and the slow i.p.s.p., respectively, in these cells. Baclofen evoked a potent, direct hyperpolarization of rat hippocampal CAl pyramidal cells. The response was largest and had the fastest onset when evoked in the dendrites and, consequently, the receptors may be concentrated there. Some of the earliest reports on the action of baclofen described a depression of neuronal excitability (Curtis et al. 1974; Davies & Watkins, 1974) which has recently been observed inthe hippocampus (Ault & Nadler, 1983). Since the responses to exogenously applied neuronal excitants were also depressed by baclofen (Fox et al. 1974; Curtis BACLOFEN AND GABA 179 et al. 1974; Davies, 1981) a post-synaptic action was proposed. In most cases, the post-synaptic action of baclofen has been reported to occur at higher concentrations than those required to depress synaptic transmission. However, the effective concentrations ofbaclofen which evoke a direct hyperpolarization ofhippocampal pyramidal cells (Fig. 2) are quite similar to those necessary for the depression of (1) hippocampal excitatory transmission (Ault & Nadler, 1982; Olpe, Baudry, Fagni & Lynch, 1982); (2) synaptic transmission in the peripheral nervous system (Bowery et al. 1981) and (3) displacement of bound [3H]baclofen from brain synaptic membranes (Bowery et al. 1983). It should also be pointed out that these concentrations are close to therapeutic levels found in the cerebrospinal fluid (Knuttson, Lindblom & Martensson, 1974). As with the presynaptic action of baclofen (Bowery et al. 1981; Ault & Nadler, 1982) and the depression ofneuronal firing (Olpe, Demieville, Baltzer, Bencze, Koella, Wolf & Haas, 1978), the direct action of baclofen reported here is stereoselective and not depressed by bicuculline methiodide (100 SM). In addition, we have found that it is not potentiated by pentobarbitone (100 /M). This response, therefore, may also be mediated by GABAB receptors (Bowery et al. 1981, 1983). In previous studies the presynaptic depressant action of baclofen was termed bicuculline insensitive when using 100 /M of one of the methohalide derivatives of (+ )-bicuculline (e.g. Bowery et al. 1981). In our experiments we have found that 100 #M-( + )-bicuculline depressed the baclofen response as did higher doses of bicuculline methiodide. This depression may be related to the ability ofthese compounds to block potassium channels at these concentrations (Heyer et al. 1982) or that pharmacological differences exist between the pre- and post-synaptic receptor activated by baclofen. The baclofen hyperpolarization was associated with a reduced neuronal input resistance. The hyperpolarization may be mediated by an increase in potassium conductance since its reversal potential (approximately -85 mV) and the shifts in that reversal potential, produced by changes in the extracellular potassium concentration, are similar to those of the known potassium hyperpolarization which follows a train of action potentials (see Alger & Nicoll, 1980a). Alterations in the chloride gradient across the cell membrane did not affect the reversal potential ofthe baclofen response. Finally, the baclofen response was depressed by barium which has been shown to depress a variety of potassium channels (e.g. Dodd & Horn, 1982). GABA ionophoresed at the pyramidal cell soma evoked a hyperpolarization of the cell which differed from the baclofen hyperpolarization in several ways: (1) it had a faster onset and decay; (2) it was severely reduced by GABA antagonists; (3) its reversal potential (-70 mV) was approximately 15 mV less negative than the baclofen response; (4) altering the chloride gradient across the cell membrane markedly affected its reversal potential; (5) the response was not reduced by barium; and (6) the reversal potential was less affected by changes in the external potassium concentration than was the baclofen reversal potential. The reversal potential shift could be explained by secondary shifts in the internal chloride gradient (cf. Meyer, 1976; Matthews & Wickelgren, 1979; Aickin et al. 1982). It is generally agreed that an increase in chloride permeability is involved in the bicuculline-sensitive action of GABA on hippocampal neurones (e.g. Ben-Ari, Krnjevic, Reiffenstein & Reinhardt, 1981; Alger & Nicoll, 1982b) and single-channel recordings from pyramidal cell soma have confirmed this (Bormann, Sakmann & Seifert, 1983). Our experiments agree 180 N. R. NEWBERR Y AND R. A. NICOLL with this, although, the extent of the shift of the GABA reversal potential when reducing the extracellular chloride concentration was less than that expected for a selective increase in chloride permeability. Possible explanations for this are, for example: (1) the low-chloride solution was not equilibrated in the slice; (2) the substitute, isethionate, may not be entirely impermeable to the somatic GABA- activated channel; (3) chloride may have leaked from the cell during its exposure to the low-chloride solution; and (4) another ion, e.g. potassium, may be involved in the hyperpolarization. This last possibility is supported by the finding that somatic GABA responses remaining in the presence of GABA antagonists had a reversal potential more negative than -70 mV. The presence of this component probably accounts for the relative resistance of the somatic GABA responses reported earlier (Alger & Nicoll, 1982b). The fast and slow i.p.s.p.s in these cells (Newberry & Nicoll, 1984c) have both similar sensitivities to bicuculline and similar ionic mechanisms to the somatic GABA and baclofen hyperpolarizations, respectively. In addition, there is a similar apparent voltage sensitivity in the depolarizing direction, shared by baclofen and bicuculline resistant GABA responses and the slow i.p.s.p. (see Figs. 4, 9 and 12) and all of these responses may originate primarily in the dendrites. The apparent voltage sensitivity of these responses may represent a true voltage sensitivity of the conductance mechanism or, alternatively, it may occur secondarily to the fall in membrane resistance which would have the effect ofshunting a dendritic response. Since baclofen has been found to be a selective agonist for GABAB receptors at presynaptic sites, we speculated that GABA might be able to mimic the action of baclofen and the slow i.p.s.p. transmitter on these cells. Indeed, it was recently reported that GABA could hyperpolarize hippocampal cell dendrites by a chloride-independent mechanism (Blaxter & Cottrell, 1982). We, therefore, searched for a GABA response in the dendrites which had the same pharmacological and ionic properties as the baclofen response. Dendritic GABA application, as reported previously (Andersen et al. 1980; Jahnsen & Mosfeldt-Laursen, 1981; Thalmann, Peck & Ayala, 1981; Alger & Nicoll, 1982b) usually evoked a depolarization of the pyramidal cell. However, with careful positioning of the ionophoretic electrode two types of hyperpolarization could be evoked (cf. Alger & Nicoll, 1982b). One of these hyperpolarizations had a slightly slower onset and decay, compared to the other, and in normal medium it was usually observed to follow the depolarizing response. This 'slow' hyperpolarization could be further revealed by the superfusion of bicuculline methiodide (100 sM), which depressed the depolarizing response. We cannot be sure that the 'slow' hyperpolarization has the same bicuculline resistance as baclofen, since its original amplitude (under the depolarization) is unknown, but it does have a markedly different apparent reversal potential to the 'fast' hyperpolarization (see below). The reversal potential ofthe 'slow' hyperpolarization was approximately -90 mV, which was similar to that of baclofen applied at the same dendritic location. Like the baclofen hyperpolarization and slow i.p.s.p., the 'slow' hyperpolarization was also voltage sensitive, i.e. it was reduced by depolarizing the cell. Elucidation of the ionic mechanism of the 'slow' hyperpolarization to GABA was limited by the co-occurrence of the depolarizing response since it was often not BACLOFEN AND GABA 181 possible to entirely eliminate this response with bicuculline methiodide. However, certain observations, when taken together, support the idea that GABA can evoke a bicuculline-methiodide-resistant increase in potassium conductance in these cells: (1) the shift in the reversal potential of the somatic GABA hyperpolarization, with low-chloride solutions, was less than expected; (2) bicuculline often caused a negative shift in the reversal potential of the somatic GABA response; (3) the 'slow' dendritic hyperpolarization to GABA, in bicuculline methiodide, had an apparent reversal potential and voltage sensitivity similar to that of the baclofen response. (Although the dendritic location ofthe response could influence the absolute value of the reversal potential and contribute to the apparent voltage sensitivity of the response, it is important to note that the bicuculline-sensitive responses evoked at the same dendritic location were very sensitive to altering the somatic membrane potential); (4) the apparent reversal potential for the late portion of the dendritic hyperpolarization, in bicuculline methiodide, was relatively resistant to low-chloride solutions (see also the bicuculline-resistant somatic GABA response in Fig. 14B, S-0); and (5) barium ions depressed the amplitude of the slow hyperpolarization. In addition to the depolarizing and 'slow' hyperpolarizing responses, low ionophoretic currents could, in some cells, evoke small hyperpolarizations which had a faster onset and decay than the slow hyperpolarization. This 'fast' dendritic hyperpolarization was similar to the somatic hyperpolarizing response in terms of its reversal potential, and its sensitivity to bicuculline methiodide and low-chloride- containing solutions. The ionophoresis of muscimol or THIP into the dendrites evoked, in addition to the depolarizing response, a bicuculline-sensitive, chloride- dependent hyperpolarization which had a similar reversal potential to the 'fast' hyperpolarization evoked by GABA. Although the reversal potential of the depolarizing responses was shifted when the chloride gradient was changed, it is possible that the 'fast' hyperpolarization may be present 'underneath' the depolarizing response which would make it difficult to determine the ionic mechanism of the depolarization. However, it is theoretically possible for both responses to be chloride mediated; for example, the main dendritic shafts which might be responsible for the 'fast' hyperpolarization could have a lower membrane potential (due perhaps to impalement of the soma) and/or a lower internal chloride concentration compared to the fine dendritic arbors which could be responsible for the depolarization. The problem concerning the chloride gradients across different parts of the pyramidal cell membrane remains. The observation that different agonists, e.g. baclofen and THIP seem to selectively activate one of the two types of hyperpolarizations evoked by GABA indicates that different subtypes of GABA receptor (Bowery, 1982) are being activated. We therefore propose that the 'fast' dendritic hyperpolarizing response, as well as the somatic response, being bicuculline sensitive and chloride dependent, are mediated by GABAA receptors and the 'slow' dendritic hyperpolarization, being bicuculline methiodide resistant and possibly potassium mediated, may be due to the activation of GABAB receptors. However, an important reservation to this conclusion is the observation that the baclofen response is reduced by high concentrations ofbicuculline (>10 #M) and bicuculline methiodide (> 100 #M). Based on the finding that THIP, a compound that has been reported to preferentially activate synaptic GABA 182 N. R. NEWBERRY AND R. A. NICOLL receptors (Allan, Evans & Johnston, 1980), can evoke dendritic hyperpolarizations, it was proposed that these responses might reflect the activation of synaptic receptors, whereas the depolarizing responses might reflect the activation of extra- synaptic receptors (Alger & Nicoll, 1982b). There is a remarkable similarity between the baclofen hyperpolarization, the 'slow' dendritic hyperpolarization evoked by GABA and the slow i.p.s.p. in these cells. Since synaptic profiles containing the synthesizing enzyme for GABA are present in s. radiatum (Somogyi, Smith, Nunzi, Gorio, Takagi & Wu, 1983) and GABA is known to be released onto CAI pyramidal cell dendrites (Alger & Nicoll, 1982a), GABA acting on dendritic GABAB-like receptors becomes a prime candidate for the slow i.p.s.p. transmitter. It is interesting to note that CA3 pyramidal and dentate granule cells are hyperpolarized by baclofen (Klee et al. 1981). There is evidence that these cells possess similar slow i.p.s.p.s to the CAl pyramidal cell (Thalmann & Ayala, 1982; Thalmann, 1984) and therefore similar receptors and ionic mechanisms may exist in all three cell types. Slow i.p.s.p.s, similar to those recorded in the hippocampus have been recorded in a number of regions in the brain, including the pyriform cortex (Constanti, Connor, Galvan & Nistri, 1980; Satou, Mori, Tazawa & Takagi, 1982), olfactory bulb (Mori, Nowycky & Shepherd, 1981; Jahr & Nicoll, 1982), and neocortex (Connors, Gutnick & Prince, 1982). The possibility that GABA may act by opening potassium channels gains additional support from invertebrate studies (Marder & Paupardin-Tritsch, 1978; Yarowski & Carpenter, 1978; Shimizu, Akaike, Oomura, Murahashi & Klee, 1983). The responses in the latter two studies were resistant to bicuculline and picrotoxin, respectively. Moreover, it has been shown, in Aplysia, that a single transmitter (e.g. acetylcholine), whether released synaptically or applied ionophoretically, can activate a fast, chloride-mediated response followed by a slower potassium-mediated response (Kehoe, 1972a, b). It is therefore not unreasonable to propose that GABA acts similarly on CAl pyramidal cells. In conclusion, baclofen hyperpolarizes rat hippocampal CAl pyramidal cells, possibly by an increase in potassium conductance. It appears that dendritic GABA application can mimic this action in addition to evoking bicuculline-sensitive responses. It is proposed that the fast and slow i.p.s.p.s in these cells may be mediated by GABAA and GABAB receptors, respectively. GABAA receptors are synaptically activated on the soma/initial segment but also in the dendrites, whereas GABAB receptors are activated mainly in the dendrites. However, further testing of this hypothesis, which relies on similarity of action, must await the development of selective GABAB receptor antagonists.

This research was supported by NIH Grant MH-38256 (RCDA), MH-38256 and the Klingenstein Fund to R. A. N. We thank Ciba-Geigy for the (± )-baclofen and its individual optical isomers and Dr Krogsgaard-Larsen for the generous supply of THIP. BACLOFEN AND GABA 183

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