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Journal of Physiology (1992), 451, pp. 329-345 With 6 figures Printed in Great Britain

COMPARISON OF THE ACTIONS OF BACLOFEN AT PRE- AND POSTSYNAPTIC RECEPTORS IN THE RAT HIPPOCAMPUS IN VITRO

BY SCOTT M. THOMPSON AND BEAT H. GAHWILER From the Brain Research Institute, University of Zurich, August Forel-Strasse 1, CH-8029 Zurich, Switzerland

(Received 30 July 1991) SUMMARY

1. Intracellular microelectrode recordings were used to study the cellular location, pharmacology, and mechanism of action of y-aminobutyric acidB (GABAB) receptors on pyramidal cells and presynaptic axonal endings in area CA3 of organotypic hippocampal slice cultures. 2. Baclofen (bath applied at 10 /tM) caused a 10-15 mV hyperpolarization of CA3 cells and a 75-100 % decrease in the amplitude of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). Baclofen reduced the amplitude of monosynaptic IPSPs elicited in the presence of excitatory amino acid receptor antagonists, as well as the amplitude of EPSPs elicited after blocking GABAA receptors and reducing subsequent epileptic bursts with excitatory amino acid receptor antagonists. These data indicate that GABAB receptors are located on both excitatory and inhibitory presynaptic elements. 3. The GABAB receptor antagonist CGP 35 348 blocked the postsynaptic action of baclofen, the late IPSP, and the reduction of EPSPs and monosynaptic IPSPs by baclofen. 3-Aminopropylphosphinic acid (3-APA) mimicked all the pre- and postsynaptic actions of baclofen, and its effects were fully antagonized by CGP 35 348. 4. Incubation of cultures with pertussis toxin (500 ng/ml for 48 h) prevented both the postsynaptic hyperpolarization and the block of monosynaptic IPSPs induced by baclofen. The action of baclofen on isolated EPSPs, however, was not affected by pertussis toxin treatment. Stimulation of protein kinase C with phorbol ester (phorbol 12, 13 dibutyrate, 1 /,M for 10 min) reduced all pre- and postsynaptic effects of GABAB receptor activation. 5. Barium (bath applied at 1 mM) prevented both the baclofen-induced hyperpolarization of pyramidal cells and the block of monosynaptic IPSPs by baclofen. In the presence of barium, however, baclofen was fully capable of blocking EPSPs. 6. We conclude that pre- and postsynaptic GABAB receptors are pharmacologically indistinguishable, at present, and that all actions of GABAB receptors are inhibited by stimulation of protein kinase C. Both the postsynaptic action of baclofen and the block of GABA release from interneurons are mediated by pertussis toxin-sensitive G proteins which can be inactivated by stimulation of protein kinase C. Baclofen acts at postsynaptic sites and on the axon terminals of inhibitory MS 9606

S. M. THOMPSON AND B. H. GAHWILER interneurons by activating the same barium-sensitive K+ conductance. GABAB receptors on excitatory axons must, however, work through some other mechanism. 330

INTRODUCTION

GABA (y-aminobutyric acid) is the predominant inhibitory neurotransmitter of the hippocampus. At least two classes of GABA receptor may be distinguished on the basis of their pharmacological properties (Hill & Bowery, 1981). GABAA receptors are selectively activated by muscimol and antagonized by bicuculline. GABAA receptor activation directly opens Cl--permeable ion channels. GABAB receptors are selectively activated by baclofen and are bicuculline insensitive. GABAB receptor activation on the soma and proximal dendrites of hippocampal pyramidal cells leads to an increase in a postsynaptic K+ conductance that can be blocked by barium ions (Newberry & Nicoll, 1984a; Gaihwiler & Brown, 1985). Synaptic release of GABA from inhibitory interneurons mediates a dual component inhibitory postsynaptic potential (IPSP) in the hippocampus: an early, bicuculline-sensitive increase in Clconductance mediated by GABAA receptors, and a late, bicuculline-insensitive increase in K+ conductance mediated by GABAB receptors (Dutar & Nicoll, 1988a). In addition to these postsynaptic actions, application of GABA or baclofen also depresses excitatory postsynaptic potentials (EPSPs) and IPSPs in the hippocampus via presynaptic receptors whose activation decreases transmitter release (Lanthorn & Cotman, 1981; Dutar & Nicoll, 1988b; Thompson & Giihwiler, 1989a; Davies, Davies & Collingridge, 1990; Harrison, 1990; Lambert, Harrison & Teyler, 1991). Pre- and postsynaptic GABAB receptors are reported to have distinct pharmacological and physiological properties (Dutar & Nicoll, 1988b). Phaclofen, a weak GABAB antagonist, has been found by most authors to block the baclofen-activated hyperpolarization and the late IPSP in hippocampal cells, but to leave the presynaptic action unaffected (Dutar & Nicoll, 1988b; Harrison, 1990; cf., however, Davies et al. 1990). Furthermore, the high affinity GABAB agonist 3-aminopropylphosphinic acid (3-APA) is reported to be a potent agonist at presynaptic GABAB receptors (Ong, Harrison, Hall, Barker, Johnston & Kerr, 1990), although its postsynaptic effects are unknown. Pertussis toxin inactivates several types of guanosine triphosphate-binding proteins (G proteins) that are capable of directly activating various K+ channels in hippocampal pyramidal cells (van Dongen, Codina, Olate, Mattera, Joho, Birnbaumer & Brown, 1988). Treatment with pertussis toxin has been shown to block both the baclofen-activated K+ conductance and the late GABAB receptor-mediated IPSP (Andrade, Malenka & Nicoll, 1986; Thalmann, 1987), but not the presynaptic action of baclofen (Colmers & Williams, 1988; Dutar & Nicoll, 1988b; Colmers & Pittman, 1989; cf., however, Scholz, Scholz & Miller, 1990). Activation of protein kinase C with phorbol ester also blocks the postsynaptic action of baclofen and the late IPSP (Baraban, Snyder & Alger, 1985; Andrade et al. 1986), but, unlike pertussis toxin treatment, phorbol esters prevent the inhibition of EPSPs by baclofen as well (Worley, Baraban, McCarren, Snyder & Alger, 1987, Dutar & Nicoll, 1988b). The effect of phorbol esters on the inhibition of IPSPs by baclofen is unknown. Two potential mechanisms exist which could account for the presynaptic action of baclofen. First, an increase in K+ conductance on axon terminals could reduce

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transmitter release by causing a hyperpolarization. Such a mechanism should be blocked by barium, like the postsynaptic potassium conductance is. It is not yet clear, however, whether barium inhibits some or all of the presynaptic actions of baclofen (Allerton, Boden & Hill, 1989; Misgeld, Muller & Brunner, 1989; Lambert et al. 1991). Alternatively the presynaptic action of GABAB receptor activation on dorsal root axons is thought to be mediated by inhibition of axon terminal Ca2+ current (Dunlap & Fischbach, 1981). In summary, it is generally agreed that activation of postsynaptic GABAB receptors leads via a G protein to an increase in a Ba2+-sensitive K+ conductance. The presynaptic mechanism of action remains controversial, and a distinction between effects on excitatory and inhibitory terminals has generally not been made. Portions of these data have been presented in abstract form (Thompson & Gahwiler, 1991; Thompson, Haas & Giihwiler, 1991). METHODS

Preparation of cultures Organotypic slice cultures of hippocampus were prepared using the roller-tube technique as previously described (Gahwiler, 1981). In brief, rat pups (5 to 7 postnatal days old) were decapitated, the hippocampus was dissected free under aseptic conditions, and 400 Fm-thick transverse slices were cut on a tissue chopper. Individual slices were attached to cleaned glass coverslips by embedding them in a thin film of clotted chicken plasma. The coverslip containing the slice was transferred to a sealed culture tube containing 0'75 ml medium, and then placed on a roller drum (10 revolutions/h) in an incubator (36 0C). The culture medium consisted of 25 % heatinactivated horse serum, 50 % Eagle's basal medium and 25 % balanced salt solution with either Hanks' or Earle's salts (GIBCO). Pertussis toxin (List Biological Laboratories) was prepared in Hanks' balanced salt solution at 10 ,ug/ml, and stored for not more than 10 days at 40C. It was applied to some cultures at 500 ng/ml for 24 h, the medium was then replaced and medium containing fresh pertussis toxin added at the same concentration for another 24 h. After 10-14 days in vitro, the slices became thinned to 1-2 cell diameters in thickness, yet retained the cytoarchitectural organization of the intact hippocampus. In addition, during the first two weeks in vitro, pyramidal and granule cells developed their characteristic dendritic morphology, extended axons, and formed functional synapses in an organotypic pattern (Gahwiler, 1984).

Electrophy8iology After 14-30 days in vitro, cultures were transferred to a glass-bottomed recording chamber, fixed to the stage of an inverted microscope (Zeiss Axiovert) equipped with phase and interference contrast optics. The cultures were continuously perfused with warmed (34 0C) saline at a rate of 09 ml/min. The recording solution had the following composition (mM): 137 NaCl, 27 KCl, 28 CaCl2, 3 MgCl2, 8 NaHCO8, 5-6 glucose and 04 NaH2PO4. The pH was adjusted to 7-4 by bubbling with C02, and monitored with Phenol Red (10 mg/l). The chamber had a volume of about 0 5 ml, allowing bath-applied substances to reach equilibrium in about 1 min. Individual CA3 pyramidal cells were visually identified and impaled with microelectrodes. The electrodes were pulled from thin-walled glass capillary tubing (1 mm o.d., 0 75 mm i.d.), filled with 1 M-potassium methylsulphate, and had resistances of 30-80 MCI. Current was injected into the cell via an active bridge circuit whose balance was monitored, and adjusted as necessary, by regular injection of small hyperpolarizing current pulses. Membrane potential was amplified 100 times, filtered at 2 kHz, digitized at 2-10 kHz (Labmaster analog-digital converter, Scientific Solutions, Inc., Solon, OH, USA) and stored on-line on a computer hard disc. Data acquisition and analysis were performed using pClamp software (Axon Instruments) running on a personal computer (COMPAQ Deskpro 386/20). Data were also recorded during the experiment using a chart recorder (Gould Inc., Cleveland, OH, USA). Synaptic responses were evoked with monopolar stimuli (-10 to -100 MA in amplitude, 041 ms in duration) delivered via glass microelectrodes (1-2 ,m tip diameter) filled with 155 mM-NaCl. Stimulating electrodes were placed either in the dentate hilus or granule cell layer, so as to preferentially activate the mossy fibre afferent pathway (see Gahwiler,

32

S. M. THOMPSON AND B. H. GAHWILER

1984), or at the border between stratum pyramidal and stratum radiatum, about 300-400 ,um from the recorded cell, for experiments in which monosynaptic IPSPs were studied. Five IPSPs, or eight EPSPs, were evoked at 0 1 Hz and averaged in all experiments on synaptic potentials. Quantitative data are given as means+ standard deviation. All drugs were applied by bath application. CGP 35 348 (P-[3-aminopropyl]-P-diethoxymethylphosphinic acid), 3-aminopropylphosphinic acid (3-APA), and (-)-baclofen (fi-p-chlorophenylGABA) were provided by Ciba Geigy Ltd, Basle, Switzerland. D-2-Amino-5-phosphonovalerate (APV) and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) were purchased from Tocris Neuramin, Bristol, UK. Bicuculline methochloride (referred to simply as bicuculline in the text) was synthesized from Fluka bicuculline. All substances were prepared as concentrated aqueous solutions and stored at -20 °C until use. Phorbol esters were purchased from Sigma and prepared at i mm in absolute ethanol. RESULTS

GABAB receptors located? As in acutely prepared slices, stimulation of the mossy fibre afferent pathway in hippocampal slice cultures elicits in CA3 pyramidal cells a short latency EPSP, followed by an early GABAA receptor-activated Cl--mediated IPSP and a late GABAB receptor-activated K+-mediated IPSP (Thompson & Gaihwiler, 1989b; Malouf, Robbins & Schwartzkroin, 1990). These IPSPs result from feed-forward and feed-back excitation of GABAergic interneurons and will therefore be referred to as polysynaptic IPSPs. Bath application of baclofen (10 rM) at the resting membrane potential (about -60 mV) caused a rapid hyperpolarization of 10-15 mV, which was accompanied by a substantial decrease in the neuronal input resistance. After injecting depolarizing current to return the membrane potential to the control level, evoked synaptic potentials were found to be greatly reduced (75-100%). Baclofen could be seen to reduce the amplitude of EPSPs as well as both components of the polysynaptic IPSP. To study GABAB receptors on excitatory axon terminals, GABAA receptors were blocked with bicuculline (4 gM) leading to epileptiform discharge. These epileptic bursts result from the uncontrolled spread of excitation between pyramidal cells, and resemble in many respects 'giant EPSPs' (Johnston & Brown, 1981). The amplitude of the epileptic burst was then reduced by fully blocking NMDA receptors with DAPV (20 gm) and incompletely blocking non-NMDA receptors with CNQX (10-20 gM). The EPSPs remaining under these conditions were apparently mediated by non-NMDA receptors because their amplitude was a linear function of membrane potential and they could be reduced by further increasing the concentration of the competitive antagonist CNQX to 40 gM (data not shown). Under these conditions, stimulation in the dentate gyrus gave rise to an EPSP of 3-10 mV in amplitude at membrane potentials of about -70 mV. Application of baclofen (10 4uM) reduced the amplitude of these EPSPs by 87-3 + 7-1 % (n = 3 cells) (Fig. 1A). The effect of baclofen on polysynaptic IPSPs could result from a direct action on inhibitory interneurons or indirectly because of a decreased excitation of interneurons. We therefore examined the effects of baclofen on monosynaptic IPSPs elicited with local stimulation after blocking excitatory amino acid receptors (Davies et al. 1990). In the presence of CNQX and APV (20 gim each), local stimulation elicited a brief IPSP of 10-15 mV in amplitude and 100-200 ms in duration (Fig. 1B). In slice cultures, unlike acute slices (Davies et al. 1990), these monosynaptic IPSPs appeared to be entirely GABAA receptor mediated: they Where are

PRE- AND POSTSYNAPTIC GABAB RECEPTORS

333

exhibited a single exponential decay, they reversed polarity uniformly near the expected Cl- equilibrium potential of about -80 mV, and they were unaffected by GABAB receptor antagonists (Fig. 2). Application of baclofen (10 JM) to monosynaptic IPSPs produced decreases in amplitude of 580 + 17-9 % (n = 6 cells; Fig. A

B

CNQX + APV + bicuculline Baclofen Before

Control Before

Baclofen

Recovery L2 mV 75 ms

Recovery

1.5ms 1~~~~~~~~~~~10 5 mV

CNQX + APV Before

Baclofen

Recovery

L

mV 20 ms

Fig. 1. Effects of baclofen on synaptic transmission. A, in the presence of CNQX (10 /M), APV (20 /M), and bicuculline (4/aM), stimulation elicited a pure EPSP which was decreased 85% by baclofen. Membrane potential = -61 mV. B, upper traces, control polysynaptic responses in another cell are strongly depressed by baclofen. Lower traces, after application of CNQX and APV (20 aM each), local stimulation elicited monosynaptic IPSPs. Baclofen reduced this IPSP by 54%. Membrane potential = -57 mV.

1 B). This effect was slightly smaller than the effect of baclofen on polysynaptic IPSPs in the same cells in control conditions, where decreased excitation of interneurons probably also contributes to the total IPSP depression. Furthermore, unlike the polysynaptic IPSP, complete block of monosynaptic IPSPs was never observed.

Pharmacology of pre- and postsynaptic GABAB receptors CGP 35 348 is a recently developed, high affinity antagonist of GABAB receptors (Olpe, Karlsson, Pozza, Brugger, Steinman, van Riezen, Fagg, Hall, Froestl & Bittiger, 1990). In hippocampal slice cultures, CGP 35 348 (300-500 /tM) completely blocked the hyperpolarization and conductance increase induced by baclofen as well as the late IPSP. The contribution of GABAB receptors to the control IPSP could be isolated by digitally subtracting the IPSP in the presence of CGP 35 348, i.e. the GABAA receptor-mediated component, from the control IPSP containing both GABAA and GABAB components. In slice cultures the isolated GABAB receptor-

S. M. THOMPSON AND B. H. GAHWILER mediated IPSP had an amplitude of 4-10 mV at about -60 mV, and a slow onset, reaching a peak after 220-260 ms (n = 4 cells). Even at high concentrations (1 mM), CGP 35 348 showed no agonist actions such as hyperpolarization or diminution of monosynaptic IPSPs (not shown). CGP 35 348 was equally effective as an antagonist 334

CNQX + APV Before

CGP 35 348 Before

Recovery Before

-

-~Baclofen

After

Baclofen

After

-~Baclofen

After 25 ms

Fig. 2. Effects of CGP 35 348 on presynaptic GABAB receptors on inhibitory interneurons. Monosynaptic IPSPs elicited in the presence of CNQX and APV (20 #m each) are shown before, during and after bath application of 1 4uM-baclofen. In control conditions baclofen caused a 45 % decrease in IPSP amplitude, but after application of 500 ,tM-CGP 35 348 only a 10 % decrease in amplitude. After wash-out of CGP 35 348, baclofen again caused a 45 % decrease in IPSP amplitude. Membrane potential =-58 mV.

of presynaptic baclofen actions. In the presence of 500 /LM-CGP 35 348, baclofen (1 /uM) produced only a 6-6 ± 5-6 % decrease in the amplitude of monosynaptic IPSPs as compared to a 53-1 + 20-2 % decrease in the same cells under control conditions (n = 6 cells; Fig. 2). CGP 35 348 also fully blocked the effects of baclofen on isolated EPSPs (n = 3 cells). 3-Aminopropylphosphinic acid (3-APA) is an high affinity agonist of GABAB receptors, and is reported to be highly selective for presynaptic GABAB receptors (Ong et al. 1990; Pratt, Knott, Davey & Bowery, 1989). Like baclofen, 3-APA (1 gM) elicited a hyperpolarization in CA3 cells of 1541 + 37 mV (n = 18 cells) at about -60 mV, concomitant with an increase in conductance (Fig. 4A). This hyperpolarization, like that elicited by baclofen, was K+ mediated because it was obtained in cells in which the Cl- equilibrium potential was depolarized with respect to the resting membrane potential due to impalement of the cell with KCl-filled recording electrodes. Like baclofen, 3-APA (1 ,tM) also reduced the amplitude of monosynaptic IPSPs by 41-9 + 22-1 % (n = 10 cells; Fig. 4A) and isolated EPSPs by 63-3 + 14-4 % (n = 9 cells; Fig. 4B). All pre- and postsynaptic actions of 3-APA were fully antagonized by 500 ,uM-CGP 35 348 (n = 5 cells). These results, taken together with other recent studies (see Discussion), indicate

PRE- AND POSTSYNAPTIC GABAB RECEPTORS A

335

Pertussis toxin treated Baclofen

b

a

c

C~~~~~~~~~ !/~ ~

CNQX+AP

d

+

I/

/

5mV mv

biuclln

e IrX

f

13 mV

50 Lms

Fig. 3. Effects of baclofen after pertussis toxin treatment. A, upper trace, chart record illustrating the effects of bath-applied baclofen (10 4uM) on a CA3 cell in a culture which was exposed to 500 ng/ml pertussis toxin for 48 h. Five stimuli were delivered in the presence of CNQX and APV (20 /M each) before (a), during (b), and after (c) baclofen. During application of baclofen 02 nA hyperpolarizing current pulses were injected to monitor membrane input resistance. Membrane potential = -55 mV. Lower traces, the monosynaptic IPSPs from the experiment above have been averaged and plotted at an enlarged time scale. B, upper trace, chart recording of CA3 cell after pertussis toxin nA hyperpolarizing current pulses. treatment. Downward deflections result from 0 m2 Eight stimuli were delivered in the presence of CNQX (20SM), APV (20/M), and bicuculline(4oAM) before (d), during (e), and after (f) baclofen. Membrane potential =-70 mV. Lower traces, isolated EPSPs from the experiment above have been averaged and plotted at an enlarged scale. Pertussis toxin treatment thus blocks the postsynaptic action of baclofen and its ability to decrease IPSPs, but not the effects of baclofen on

EPSPs.

that it is not yet possible to distinguish pre- from postsynaptic GABAB receptors

pharmacologically. Mechanism of action of pre- and postsynaptic GABAB receptors Conflicting data exist with regard to the effects of pertussis toxin on the presynaptic actions of baclofen (cf. Dutar & Nicoll, 1988b; Harrison, 1990; Scholz et al. 1990), perhaps due to the difficulties of applying pertussis toxin for experiments

S. M. THOMPSON AND B. H. GAHWILER using acute slices. In hippocampal slice cultures that had been treated with pertussis toxin (500 ng/ml) for 48 h, baclofen did not cause any change in the resting membrane potential greater than + 2 mV, nor any increase in conductance (n = 10 cells in five cultures; Fig. 3). Furthermore, stimulation of mossy fibre afferents 336

A

Control

3-APA

b

a~~~~~~~~~~~~~~ After PDBu 3-APA

C

d

C

5 MV

130o s

5M

10m

B Control

After PDBu Before

Before

mV 12 1ms

3-A

Fig. 4. Effects of stimulation of protein kinase C with phorbol ester on pre- and postsynaptic GABAB receptor-mediated actions. A, to the left, chart record of the response of a CA3 cell to application of the GABAB receptor agonist 3-APA (1 SM) before (upper traces) and after (lower traces) application of 1 ,uM-PDBu for 10 min. The average of five monosynaptic IPSPs in the presence of CNQX and APV (20 /SM each) before (a and c) and in the presence of 3-APA (b and d) are shown superimposed to the right. Membrane potential = -60 mV. B, the effect of 3-APA on isolated EPSPs is shown before (left) and after application of 1 1LM-PDBu for 10 min (right). Stimulation of protein kinase C thus decreases all pre- and postsynaptic effects of GABAB receptor activation. Membrane potential = -70 mV.

elicited EPSPs followed by apparently pure GABAA IPSPs. These polysynaptic IPSPs were shorter in duration than control IPSPs, had a uniform reversal potential around -80 mV, and decayed monoexponentially (n = all 5 cells examined in three cultures; not shown), suggesting that the pertussis toxin treatment had abolished the normal GABAB receptor-mediated late IPSP. In the presence of CNQX and APV, monosynaptic IPSPs were virtually unaffected by application of baclofen (10 /SM; mean decrease = 5-3 + 87 %, n = 7 cells in three cultures; Fig. 3A). This result is thus opposite to that obtained by Scholz et al. (1990) on IPSPs between dissociated embryonic hippocampal neurons. It was also noted that with lower doses of pertussis toxin and/or shorter incubation periods, cultures were occasionally found in which the postsynaptic action of baclofen was fully blocked, but the presynaptic action of

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baclofen on IPSPs remained, probably as a result of incomplete inactivation of G proteins. In contrast, the action of baclofen on isolated EPSPs was never affected by pertussis toxin treatment (Fig. 3B). In five cells (three cultures) in which baclofen exerted no postsynaptic effects, 10 jtM-baclofen nevertheless caused a mean decrease CNQX + APV

Baclofen

C,