Er:ropean Journal of Pharmacology, 212 (1992) 291-294

291

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

lL1P 21015 Short communication

y-Hydro:~butyrate hyperpolarizes hippocampal neurones by activating GABA~ receptors X i n m i n Xie a n d T r e v o r G. S m a r t Department of Pharmacology, 7"he School of Pharmacy, London, U.K.

Received 7 November 1991, revised MS received 31 December 1991, accepted 7 January 1992

y-llydroxybutyrate, a naturally occurring substance present in lhc mammalian central nervous system caused a dosc-dependent (0.25-10 mM) hyperpolarization and small membrane conductance increase when applied to hippocampal CA1 pyramidal neurones in vitro. This action was reversibly inhibited by the GABA~ antagonist, CGP 35348 (20-100 p.M) and divalent cations, Zn -~+ and Ba~ ~, but not by the GABA A antagonist bicuculline (50 /~M). These results suggest that GABAn receptors may mediate the actions of y-hydroxybutyrate. y-Hydroxybutyrate; GABA (y-aminobutyric acid); GABA ~ recepturs; CGP35348; Hippocampus; Zn 2~

1, Introduction 7-Hydroxybutyrate is a naturally occurring analogue of G A B A in the mammalian brain, and is derived primarily following y-aminobutyric acid ( G A B A ) metabolism via GABA-transaminase and succinic semialdehyde reductase. Recently ncurochemical and pharmacological studies have suggested that y-hydroxybutyrate may play a neurotransmitter or neuromodulator role in the central nervous system (Vaycr ct al., 1987; Vaycr and Maitre, 1989). Indeed, y-hydroxybutyrate does fulfill some of the criteria for a putative transmitter. Endogenous levels of y-hydroxybutyrate in the rat brain are estimated at approximately 2.5 /xM and appear heterogeneously distributed in brain tissue (Snead, 1991). The hippocampus contains a very high level and is also one of the richest regions of specific y-hydroxybutyrate binding sites. Moreover, voltage and C.a2 - d e p e n d e n t release of -/-hydroxybutyrate has been reported and an active uptake mechanism has also been observed (Vayer et al., 1987; Vayer and Maitre, 19891. Functionally, y-hydroxybutyrate inhibits the discharge rate of nigral and neocortical cells in vivo (Olpe and Koella, 1979), and produces a membrane potential hyperpolarization with a small conductance increase in nigrostriatal neurones in vitro (Harris et al., 1989).

Correspondence Io: X. Xie, Deparlment of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WC1N lAX, U.K. Tel. 44.71.753 5896, fax 44.71.278 0622.

Both actions in vivo and in vitro arc resistant to bicuculline, but further definition of the receptors mediating these responses is not yet available. Interestingly, y-hydroxybutyrate is also used as an experimental tool to induce absence-like seizures in rats, which is considered to b c a reasonable model for petit real epilepsy (Snead, 1991). Rather high doses (3.5-5 m m o l / k g ) are required for induction of epilepsy, but thc receptors responsible for this effect have also not been identified. The present study concentrates on the action of y-hydroxybutyrate on hippocampal pyramidal neurones in vitro and attempts to define the receptors most likely mediating the actions of this substance.

2. Materials and methods Experiments were pcrfl)rmed on 400 /xm thick transverse hippocampal slices obtaincd from adult Wistar rats as described previously (Xie and Smart, 1991). Slices were submerged and superfused with a Krebs solution containing (mM): NaC1 118; KCI 4.7; CaCI 2 2; MgCl 2 2; N a H C O 3 25; glucose 11, bubbled with 95% 0 2 - 5 % CO2, pH 7.4 at 30°C. lntracellular recordings were made from CA1 pyramidal cells using 3 M KCIor 4 M K acetate-filled microelectrodes. Drugs were dissolved in Krebs and bath-applied at known concentrations. The cells had membrane potentials of - 64 + 7 mV (mean _+ S.D., n = 48) and action potential amplitudes of 85-100 mV. CGP 35348 (p-[3-aminopropyl]p-diethoxymethyl-phosphinic acid) was provided by

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3. Results

Bath application of 7-hydroxybutyrate (0.25-10 mM) caused dose-dependent hyperpolarizations in all hippocampal CA1 neurones studied (n = 28). At high concentrations (5-10 raM), y-hydroxybutyratc induced a clear, but slow onset and long-lasting hypcrpolarization (4-8 mV) from the resting membrane potential coupled with a small incrcasc in the input conductance (fig. 1A) y-Hydroxybutyratc also depressed cell ex-

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Fig. 1. Effects of CGP 35348 and zinc on the 7-hydroxybutyrate (GHB)-induccd hypcrpolarization in hippocampal CA1 n e u m n c s . (A) Upper traces are chart records of 10 mM y-hydroxybutyrate-induced responses before and during 20 /~M-CGP 35348 application. Upward deflections arc action potentials evoked by depolarizing current injection ( + 0 . 4 hA, 300 ms). l)ownward deflections arc hyperpolarizing electrotonic potentials following current pulse injection ( - 0 . 4 hA, 300 ms, 0.2 ltz) to monitor the input conductance. Membrane potential --60 mV adjusted with DC current injection. I,ower traces from the samc cell were obtained after washing out CGP 35348 for 10 rain. The y - h y d m ~ b u t y r a t e responses were inhibited by 300 p~M zinc. The upward dcflections are spontaneous large depolarizing potentials indnced by zinc. (13) In a different cell, superimposed clcctrotonic potentials (lower traces) produccd by depolarizing or hyperpolarizing current pulses (lower traces) wcrc recordcd under coutrol conditions, during the application and subsequent recovery from y-hydrox3,'butyrate (10 raM) and during co-application of y-hydroxybutyratc (10 m M ) + C(}P 35348 (20/x M). Resting membrane potential - 6 2 inV. Note that in both A and B, at the peak of the y-hydroxybutyrate-induced hyperpolarizing responses, the membrane potential was adjusted to the original resting lcvcl with I).C. positive current injection. Both cells were recorded using 3 M KCl-fillcd microclcctrodcs.

Fig. 2. ~ffec~ of CGP 35348 and bicuculline on ~-hydro~'butyrate-induced responses. (A) In a CAI neurone voltage-clamped a~ .-60 mV holding potential (V~). bath-applied ~-hydroxybutyratc (10 raM. horizontal bars) induced an outward membrane current and a conductance increase (up~cr trace) in control ~ c b s . Brict hyperpolarizing voltage s~cps ( - 2 0 mV, 300 ms, 0.3 Hz; lower ~race) were a~plicd ~o monitor lhc m e m b r a n e conduclancc. In the presence of 50 NM CGP 35348. ~-hydro~'butyrate-induced rcsNmscs were inhibited. Te~rodotoxin (1 ~ M ) was applied throughoul the experiment. (B) The ~-hydro~'hutyrate-induced hyperpolarization was unaffected by bicucullinc. Chart records from a different CA1 cell wi~h a resting mc~nhrane ~ t e n t i a l of ---65 inV. ~-Ilydroxy~u~yratc (10 raM) indnced a hyperpolarization in fl~c absence (uppcr trace) and presence (lower ~race) of 50 ~ M bicucullinc. Upward deflections are spontaneous action potentials and downward deflections represent hyperpolarizing electrotonic potentials ( - 0 . 4 nA, 3 ~ ms, 0.2 Hz). During lhc peak of the response DC currem was used to repolarize the membrane potential.

citability, manifest by a reduction in action potential firing evoked by the direct injection of depolarizing current pulses (fig. IB). It is possible that the y-hydroxybutyrate-induced hyperpolarization resulted from changes in the tonic release of certain neurotransmitters or modulators onto CA1 neurones (cf. Vaycr et al., 1987; Vayer and Maitre, 1989). However, in the presence of tetrodotoxin (1 /x M)which prcvcntcd synaptic transmission, y-hydroxybutyrate still produced a hyperpolarizing effect on the postsynaptic neuronal membrane. Pyramidal ncurones voltage-clamped near their resting potentials ( - 6 0 mV), responded to y-hydroxybutyrate application (10 mM) with a small outward membrane current and an associated increasc in the input conductance (fig. 2A). The current-voltage rcla-

293

tionship revealed that the reversal potcntial for the y-hydroxybutyrate-induced current was approximately - 9 0 inV. Repeated application of y-hydroxybutyrate at intervals of approximately 10 min, produced no apparent desensitization in the responses. The characteristics of y-hydroxybutyratc responses werc quite si:nilar to those of the established G A B A ~ receptor agonist baclofen on these cells (cf. Dutar and Nicoll, 1988). Indeed, the hypcrpolarization or outward currc.nt induced by y-hydroxybutyrate was also inhibited by the GABA n antagonist, CGP 35348 (Olpc et al., 1990) in a dose-rclated manner (20-100 /zM; n = 12; figs. 1 and 2A). The -y-hydroxybutyrate response recovered within 10 min following removal of CGP 35348 (fig. 1A). CGP 35348 (20-100 p.M) had no direct effect on the membrane potential, input resistance or cell excitability. When the recording microelectrodcs were filled with 3 M KCI to incrcase the intracellular CI concentration, y-hydroxybutyrate still produced a hypcrpolarizing responsc which was not affected by thc GABA,x antagonists bicuculline (50 /xM; fig. 2B) or picrotoxin (50 ~M) In contrast, GABA (1 mM) always evoked a depolarizing rcsponse in CAI neuroncs when recording with KCl-fillcd microclectrodes and these responses werc also blocked by bicuculline (20-50 /zM). However, bath-application of GABA at higher concentrations (4 mM) in the prcscnce of bicuculline and picrotoxin, produced a small hyperpolarization (3-5 mV) which was blocked by CGP 35348 (50-100 /xM). Furthermore, R-baclofcn (10-20 p.M), a potent and specific GABA ~ agonist, also induced a slow hyperpolarization with a small increase in the membrane conductance which was reversibly inhibited by CGP 35348 (50-300 p.M) (data not shown). The divalent cations, Ba 2+ and Zn 2+ have been reported to inhibit baclofen-induced responses in hippocampal neurones (G~ihwiler and Brown, 1985; Xie and Smart, 1991). It was of interest, therefore, to examine whether these cations can affect the y-hydroxybutyratc-induced response. Bath application of zinc (300/zM) for 5 min induced periodic spontaneous depolarizing potentials previously shown to be mediated by G A B A A receptors (Xie and Smart, 1991) and also inhibited the 7-hydroxybutyratc response (fig 1B, n = 5). Ba 2- (500-1000/xM) inhibited the hyperpolarization induced by either R-baclofen (10 ~ M ) or by y-hydroxybutyratc (data not shown; cf. Harris et al., 1989). To further identify the receptor activated by y-hyCroxybutyrate, the antagonists CGP 35348 and zinc were used in conjunction with 5-hydroxytryptamine (5HT). 5-HT was used since 5-HT~, receptors are suggested to share the same potassium conductance mechanism with G A B A n receptors in hippocampal neurones (Dutar and Nicoll, 1988). Hippocampal CAI

neurones responded to bath-applied 5-HT (10-20 p.M) with a membrane hyperpolarization (5-8 mV) and a small increase in conductance. Neither CGP 35348 (20-100 /xM) nor zinc (300 p.M) inhibited this response to 5-HT (data not shown).

4. Discussion

The present study demonstrated that y-hydroxybutyrate has a very weak but consistent hypcrpolarizing action on hippocampal CAI pyramidal cells. This action was sensitive to the selective GABA u antagonist CGP 35348, where micromolar concentrations were able to completely inhibit the response induced by millimolar concentrations of y-hydroxybutyrate. These results suggest that the y-hydroxybutyrate action is mediated by G A B A ~ receptors. Although the y-hydroxybutyratc-evoked conductance changes in hippocampal neurones were small, preliminary observations suggest that the responses appear to be mediated by an increase in potassium conductance rather than by increases in chloride conductance The hyperpolarizing responses induced by y-hydroxybutyrate were unaffected by using either K acetate- or KCl-filled microelcctrodes. Moreover, the reversal potential of approximately - 9 0 mV for y-hydroxybutyrate-induced current was close to the calculated potassium equilibrium potential under our recording conditions. The y-hydroxybutyratc responses were also resistant to the traditional GABA~x antagonists but sensitive to Ba 2" and Zn 2+. Thcsc pharmacological properties would be expected as a result of activating GABA~ receptors with the agonists, baclofen or GABA (G~ihwilcr and Brown, 1985; Harris et al., 1989; Xie and Smart, 1991) which can lead to an increased potassium conductance (Dutar and Nicoll, 1988). y-Hydroxybutyratc, at 300-600 /xM, has been rcported to act on specific binding sites and produce increases in cyclic guanosine monophosphate and inositol phosphate levels in rat hippocampal slices. These effects were antagonized by opiate antagonists, such as naloxone (Vayer and Maitre, 1989). Howcvcr, at 250500 /zM, y-hydroxybutyrate only produced very small hyperpolarizing responses in hippocampal neuroncs, and the cffccts observed at highcr concentrations (5-10 mM) were not blockcd by 20 /xM naloxonc (unpublishcd observation). The concentrations of y-hydroxybutyratc required to produce a significant response in hippocampal ncuroncs, as well as in nigrostriatal neurones (Harris ct al., 1989) are clearly rather high and much higher than wc would normally employ to resolve an agonist effect. However, -,/-hydroxybutyratc is used as an expcrimcntal tool in inducing absence seizures and this usually rcquircs a dose of 3.5-5 m m o l / k g

294

(Sncad, 1991) The resultant extracellular concentrations of y-hydroxybutyrate achieved in the brain following in vivo application are predicted to attain millimolar concentrations and as such, on the basis of our study, are sufficiently high to activate GABA B receptors. It is unlikely that endogenous y-hydroxybutyratc will naturally attain such concentrations in the brain following neuronal activity (Snead, 1991; Bernasconi et al, in press), and thus this action is unlikely to be of any physiological consequence. However, a role for GABA B receptors in the induction of absence seizures has recently been suggested (Bernasconi ct al., in press). Our study may therefore provide a basis for the mechanism underlying the effect of y-hydroxybutyratc in vivo and its role in the generation of the absence-like seizures. Acknowledgement Wc thank Prof N.G. Bowe~, for advice and Dr. H.-R. Olpc for kindly providing CGP 35348.

References Bcrnasconi, g., J. Lauber, C. Marescaux, M. Vergncs, P. Martin, V. Rubio, T. l,eonhardt, N. gcymann and II. Bittiger, Experimental

absence seizures: potential role of y-hydrox'ybutyric acid and GABA~ receptors, J. Neural. Transm. (in press). Dutar, P. and R.A. Nicolk 1988, A physiological role for GABA~ receptors in the central nervous system, Nature 332, 156. Gfihwiler, B.H. and D.A. Brown, 1985, GABAn-rCceptor-activatcd K- current in voltage-clamped CA3 pyramidal cells in hippocampal cultures, Proc. Natl. Acad. Sci. U.S.A. 82, 1558. llarris, N.C., C. Wcbb and S.A. Greenfield, 1989, The effects of y-hydroxybutyrate on the membrane properties of guinea-pig pars compacta neuroncs in the substantia nigra in vitro, Neuroscience 31,363. Olpe, H.-R. and W.P. Koella. 1979. Inhibition of nigral and neocortical cells by y-hydroxybutyrate: a microiontophoretic investigation, Eur. J. Pharmacol. 53. 359. Olpe, H.-R., G. Karlsson, M.F. Pozza, F. Brngger, M. Steinmann, H.V. 1,',iczen, G. Fagg, R.G. t Iall, W. FroestI and H. Bittigcr, 1990, CGP35348: a centrally active blocker of GABA n receptors, Eur. J. PharmacoI. 187, 27. Snead, O.C., 1991, The y-hydroxybutyrate model of absence seizures; correlation of regional brain levels eft y-hydroxybutyric acid and y-butyrolactone with spike wave discharges, Neuropharmacology 33, 161. Vayer, P. and M. Maitre. 1989, y-IIydroxybutyrate stimulation of the formation of cyclic GMP and inositol phosphates in rat hippocampal slices, J. Neurochem. 52, 1382. Vayer, P., P. Mandel and M. Maitre, 1987, Gamma-kydroxybntyrate, a possible neurotransmitter, Life Sci. 41, 1547. Xie, X.M. and T.G. Smart, 1991, A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission, Nature 349, 521.