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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

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