Comparison of antagonist potencies at pre- and post-synaptic GABA_B receptors at inhibitory synapses in the CA1 region of the rat hippocampus

Abstract

1. Synaptic activation of γ-aminobutyric acid (GABA)_B receptors at GABA synapses causes (a) postsynaptic hyperpolarization mediating a slow inhibitory postsynaptic potential/current (IPSP/C) and (b) presynaptic inhibition of GABA release which depresses IPSPs and leads to paired-pulse widening of excitatory postsynaptic potentials (EPSPs). To address whether these effects are mediated by pharmacologically identical receptors the effects of six GABA_B receptor antagonists of widely ranging potencies were tested against each response.

2. Monosynaptic IPSP_Bs were recorded in the presence of GABA_A, AMPA/kainate and NMDA receptor antagonists. All GABA_B receptor antagonists tested depressed the IPSP_B with an IC₅₀ based rank order of potency of CGP55679⩾CGP56433=CGP55845A=CGP52432>CGP51176> CGP36742.

3. Paired-pulse EPSP widening was recorded as an index of paired-pulse depression of GABA-mediated IPSP/Cs. A similar rank order of potency of antagonism of paired-pulse widening was observed to that for IPSP_B inhibition.

4. Comparison of the IC₅₀ values for IPSP_B inhibition and paired-pulse EPSP widening revealed a close correlation between the two effects in that their IC₅₀s lay within the 95% confidence limits of a correlation line that described IC₅₀ values for inhibition of paired-pulse EPSP widening that were 7.3 times higher than those for IPSP_B inhibition.

5. Using the compounds tested here it is not possible to assign different subtypes of GABA_B receptor to pre- and post-synaptic loci at GABAergic synapses. However, 5–10 fold higher concentrations of antagonist are required to block presynaptic as opposed to postsynaptic receptors when these are activated by synaptically released GABA.

Introduction

γ-Amino-butyric acid (GABA)_B receptors are present at pre- and post-synaptic loci in highly diverse regions of the vertebrate central nervous system (Bowery, 1993). Postsynaptically, GABA_B receptors activate an inwardly rectifying potassium conductance which hyperpolarizes the neurone (Gähwiler & Brown, 1985). Presynaptically, GABA_B receptors inhibit the release of numerous neurotransmitters (e.g., GABA, glutamate, noradrenaline, 5-HT, substance P, cholecystokinin and somatostatin) through a number of potential mechanisms (e.g., enhancement of certain potassium conductances (Gage, 1992), inhibition of voltage-gated calcium conductances (Campbell et al., 1993; Pfrieger et al., 1994; Wu & Saggau, 1995) and inhibition of release machinery per se (Thompson et al., 1993)). The differential localization of GABA_B receptors raises the possibility that GABA_B receptors at different loci are pharmacologically distinguishable. The importance of this is that it provides the potential to develop drugs that are specifically targeted at regulating the putative inhibitory or disinhibitory roles that these different populations of GABA_B receptors fulfil within the CNS.

The recent development of a series of GABA_B receptor antagonists with widely ranging potencies (Froestl et al., 1995; Froestl & Mickel, 1997) has now enabled this possibility to be examined. To date, this has been performed most extensively using neurochemical methods combined with quantitative pharmacological analysis to examine the presynaptic GABA_B receptors that inhibit glutamate, GABA and somatostatin release. The findings of these studies have been contradictory between groups. Thus, some studies have suggested the existence of multiple GABA_B receptor subtypes, each selectively inhibiting the release of specific neurotransmitters (e.g., Bonanno & Raiteri, 1993b). Other reports have failed to observe such a distinction (e.g., Waldmeier et al., 1994). Likewise, there is controversy as to whether or not postsynaptic GABA_B receptors exist as two distinct GABA_B receptor subtypes (Pham & Lacaille, 1996). However, in both sets of studies GABA_B receptors have been activated predominantly using selective agonists and not by way of endogenously released GABA. As such, the question still remains as to whether pharmacologically distinguishable GABA_B receptors are activated by synaptically released GABA. The answer to this question is important as it will provide a better understanding of the normal physiological and pathological roles that GABA_B receptors play in vivo, most notably in terms of their effects on mnemonic processing (Olpe & Karlsson, 1990; Davies et al., 1991; Mott & Lewis, 1991; Mondadori et al., 1993; Olpe et al., 1993b) and absence epilepsy (Liu et al., 1992; Hosford et al., 1992). Ultimately, this may lead to the development of a new generation of compounds that through targeting of specific GABA_B receptor populations may be more efficacious in treating specific disease states.

The purpose of this study, therefore, was to examine, at GABAergic synapses, the pharmacology of those GABA_B receptors located pre- and post-synaptically that are activated by synaptically released GABA. In this respect, in the CA1 region of the hippocampus, physiological activation of postsynaptic GABA_B receptors results in the late inhibitory postsynaptic potential (IPSP_B) (Dutar & Nicoll, 1998a; Soltesz et al., 1988; Otis et al., 1993; Solís & Nicoll, 1992) whereas activation of presynaptic GABA_B receptors results in paired-pulse depression of synaptic inhibition (i.e. a GABA_B autoreceptor effect: Thompson & Gähwiler, 1989; Deisz & Prince, 1989; Davies et al., 1990; 1991; Olpe et al., 1994) which causes paired-pulse widening of synaptic excitation (Nathan et al., 1990; Nathan & Lambert, 1991; Davies & Collingridge, 1996). By determining the IC₅₀ values for antagonism of the late IPSP, and comparing these with those for antagonism of paired-pulse widening of EPSPs for six structurally different GABA_B receptor antagonists, we have attempted to address whether synaptically activated pre- and post-synaptic GABA_B receptors at GABAergic synapses can be differentiated pharmacologically.

Methods

Biological preparation

Experiments were performed on hippocampal slices obtained from Wistar rats (3–5 weeks old) as described previously (Davies et al., 1990). In brief, animals were cervically dislocated or anaesthetized using halothane (3–5%) and subsequently decapitated in accordance with U.K. Home Office or Swiss Government guidelines. The brain was removed rapidly and the hippocampus left in situ or dissected free. Transverse brain slices (400 μm thick) containing hippocampus, or hippocampal slices per se, were cut using either a Campden virboslicer or Sorval® tissue chopper. Where necessary the hippocampal region was dissected free from other surrounding brain areas. Area CA3 was subsequently removed from all freed hippocampal slices and two of the resultant CA3-ectomized hippocampal slices immediately transferred to an interface recording chamber maintained at 30–32°C. Here slices rested on a nylon mesh at the interface of a warmed perfusing artificial cerebrospinal fluid containing either (mM): NaCl 124; KCl 3.0; NaHCO₃ 26; CaCl₂ 2.0; MgSO₄ 1; D-glucose 10; NaH₂PO₄ 1.25, or NaCl 120; KCl 2.5; NaHCO₃ 30; CaCl₂ 2.5; MgSO₄ 2; D-glucose 10; KH₂PO₄ 1.2, bubbled with a 95% O₂/5% CO₂ mixture. No differences were observed between experiments performed in either solution. Spare slices were stored submerged and oxygenated at room temperature for later use.

Electrophysiological recording

Intracellular recordings were obtained from neurones in stratum pyramidale using glass microelectrodes (60–120 MΩ) filled with potassium methylsulphate (2 M) connected to an Axoclamp-2A amplifier used in discontinuous current-clamp or bridge mode (Axon Instruments, Foster City, CA, U.S.A.). Spike frequency adaptation and input resistance of pyramidal cells were routinely measured throughout each experiment by passing current pulses (amplitude±0.1–0.5 nA, duration 300–700 ms) through the intracellular recording electrode every 30–120 s to depolarize or hyperpolarize the neurone, respectively. In all experiments 6-nitro-7-sulphamoylbenzo-[f]-quinoxaline-2,3-dione (NBQX, 3 μM) or 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 μM), and D-(E)-2-amino-4-methyl-5-phospho-3-pentanoic acid (CGP 40116; 50 μM) or D-2-amino-5-phosphonopentanoate (D-AP5, 50 μM) were present in the perfusing medium to block all ionotropic glutamate receptor-mediated synaptic transmission. In certain experiments picrotoxin was used to block all GABA_A receptor-mediated synaptic inhibition so that pure IPSP_Bs could be isolated. Monosynaptic biphasic IPSPs and pure IPSP_Bs were evoked by delivering a single constant current stimulus (40–140 μA, 0.02–0.2 ms pulse width) using bipolar nickel/chromium or stainless steel stimulating electrodes placed in stratum radiatum close to the recorded neurone, within 500 μm laterally but half to two-thirds the distance down the apical dendritic tree. To quantify the effects of drugs synaptic responses were compared before and after drug treatment at a fixed membrane potential. This was achieved by injecting DC to compensate for any spontaneous membrane potential fluctuations. In all experiments stimulus strengths were set to activate maximal IPSP_Bs at membrane potentials between −62 and −65 mV. The effect of each concentration of the antagonists used was quantified in terms of the percentage reduction of IPSP_B peak amplitude after a 20 min antagonist application, relative to a 20 min baseline period.

Paired-pulse widening of EPSPs was studied using extracellular recording techniques. Extracellular recordings of glutamate-mediated field excitatory postsynaptic potentials (EPSPs) were obtained from stratum radiatum with a NaCl (4 M) filled microelectrode (2–5 MΩ). Synaptic responses were evoked by paired pulse stimulation (2–10 V, 20 μs pulse width) delivered at a fixed interval of 100–200 ms every 30 s to the Schaffer collateral-commissural fibres in the lower third of stratum radiatum using a bipolar stimulating electrode. The magnitude of antagonism of paired-pulse widening of field EPSPs induced by each concentration of the antagonists was calculated using the half widths of the field EPSPs as follows:

The ratio X : Y was then converted to a percentage. Thus, 100% inhibition of paired-pulse widening of EPSPs occurred when the concentration of antagonist was sufficient to cause the second field EPSP half width to be equal to the half width of the first EPSP in the presence of the antagonist (which is equal to the half width of the first EPSP in control conditions).

Drugs

Drugs were administered by addition to the superfusing medium and were applied for a sufficient period (15–20 min) to allow their full equilibration. Picrotoxin, was obtained from Sigma. AP5, CNQX and NBQX were purchased from Tocris-Cookson. D-(E)-2-amino-4-methyl-5-phospho-3-pentanoic acid (CGP 40116), 3-aminopropyl-n-butyl-phosphinic acid (CGP 36742), 3-amino-2-(R)-hydroxypropyl-cyclohexylmethyl-phosphinic acid hydrochloride (CGP 51176A), [3-[[ (3,4-dichlorophenyl) methyl] amino] propyl] -diethoxymethyl-phosphinic acid (CGP 52432), [2-(S)-hydroxy-3-[[1-(S)-(3,4,5-trimethoxyphenyl) - ethyl] amino] propyl] - cyclohexylmethyl-phosphinic acid (CGP 55679), [3-[1-(S)-[[3-(cyclohexylmethyl)hydroxyphosphinyl] - 2 - (S) - hydroxypropyl] amino]ethyl]-benzoic acid (CGP 56433), and [1-(S)-3,4-dichlorophenyl)ethyl] amino-2-(S)-hydroxypropyl-benzyl-phosphinic acid (CGP 55845A) were synthesized de novo by the Chemistry Department at Novartis Pharma AG, Basle, Switzerland. Each drug was dissolved in distilled water or equimolar NaOH at 100–1000 times its final bath applied concentration and was stored frozen until just prior to experimental use. n signifies the number of times each drug was tested, which was the same as the number of slices tested. Each slice was obtained from a separate rat.

Results

Postsynaptic GABA_B receptors

In a first series of experiments postsynaptic GABA_B receptors were activated physiologically. Thus, either a monosynaptic biphasic IPSP comprising a GABA_A receptor-mediated IPSP (IPSP_A) followed by a GABA_B receptor-mediated IPSP (IPSP_B), or an isolated monosynaptic IPSP_B, were evoked in a CA1 pyramidal neurone in the presence of the excitatory amino acid antagonists AP5 or CGP 40116 (50 μM) and CNQX (10 μM) or NBQX (3 μM) to block fast glutamatergic synaptic excitation (Davies et al., 1990). Of the six structurally diverse compounds (Figure 1) tested all abolished IPSP_Bs without substantially affecting IPSP_As. The concentration response relationship for the inhibition of the IPSP_B for each antagonist paralleled those of the other compounds (Figure 2). The respective IC₅₀ values for each compound are provided in Table 1.

Figure 1.

Structures of GABA_B receptor antagonists.

Figure 2.

Comparison of the potency of different GABA_B receptor antagonists to block IPSP_B. Synaptic traces are monosynaptically-activated biphasic IPSPs. Bath application of 0.1 μM CGP 55679 for 15–25 min depressed the IPSP_B without substantially affecting the IPSP_A. Each trace is an average of four consecutive IPSPs and stimulus artefacts have been blanked for clarity. The membrane potential of the neurone was −63 mV. The graph shows plots of the percentage inhibition of IPSP_B versus antagonist concentration for four of the antagonists tested. Data were fitted to the logistic expression Y=M(X^P/[X^P+K^P]) where Y is the percentage inhibition of IPSP_B, X is the antagonist concentration, M is the unconstrained maximum effect, K is the concentration of antagonist producing 50% inhibition (i.e. IC₅₀) and the power P determines the slope of the curve (Barlow & Blake, 1989). Symbols represent mean values and bars standard errors of the mean where these are larger than the symbols.

Table 1.

Comparison of IC⁵⁰ values for antagonism of IPSP_Bs and paired-pulse widening (PPW) of EPSPs

GABA_B autoreceptors

In a second series of experiments the effectiveness of GABA_B receptor antagonists to block GABA_B autoreceptors activated by synaptically released GABA was evaluated. To do this the effects of GABA_B receptor antagonists on paired-pulse widening of AMPA receptor-mediated field EPSPs was tested as this (a) has been reported to be a direct consequence of GABA_B autoreceptor-mediated paired-pulse depression of GABA-mediated synaptic inhibition (Nathan et al., 1990) and (b) provides a potentially more efficient method for quantitative evaluation of GABA_B autoreceptor pharmacology than studying paired-pulse depression of IPSCs. To confirm that this was the case and, further, to establish whether this experimental approach was a fair quantitative representation of the activity of compounds at GABA_B autoreceptors we performed two sets of experiments.

In the first we demonstrated that GABA_B receptor antagonists abolished paired-pulse widening of field EPSPs whether these were recorded in the presence, or absence, of CGP 40116 or D-AP5 to block any activation of NMDA receptor-mediated EPSPs (Figure 3a). Two observations suggested that the effects of the antagonists were due to their block of paired-pulse depression of IPSP_As: Firstly, abolition of IPSP_As using picrotoxin enhanced the duration of the first field EPSP in the pair and thereby occluded paired-pulse widening of field EPSPs since both EPSPs were no longer constrained by this synaptic potential (n=3; Figure 3b). Secondly, GABA_B receptor antagonists had no additional effect on field EPSPs evoked by paired-pulse stimulation in the presence of this GABA_A receptor antagonist (n=3; Figure 3b).

Figure 3.

Paired-pulse widening of field EPSPs is an accurate model for studying the pharmacology of GABA_B autoreceptors. In (a(i)) synaptic traces are extracellularly recorded field EPSPs evoked by a pair of stimuli delivered 100 ms apart in control medium containing 50 μM CGP 40116. Note that the duration of the second field EPSP is longer than that of the first EPSP of the pair. (a(ii)) shows the corresponding responses evoked by the same stimulation protocol in the presence of 1 μM CGP 55845A. Note that under these conditions the durations of the first and second field EPSPs are similar. The far right hand trace is a superimposition of the second field EPSPs illustrated in (i) and (ii) plotted on a faster time base to illustrate the difference in durations of the second field EPSPs evoked in control and in CGP 55845A-containing medium. In (b) superimposed traces are the first (thick line) and second (thin line) field EPSPs of a pair of field EPSPs evoked by a pair of stimuli delivered 200 ms apart in control medium containing 50 μM CGP 40116, in the additional presence of 50 μM picrotoxin and in the additional combined presence of 50 μM picrotoxin and 1 μM CGP 55845A. Note that in control medium the second EPSP is wider than the first whereas in the presence of picrotoxin or the combination of picrotoxin and CGP 55845A the width of each EPSP was identical. Note also that the far right hand responses are pure non-NMDA receptor-mediated field EPSPs and that the increase in amplitude of the second field EPSP in the pair results from paired-pulse facilitation of glutamate release.

In a second series of experiments we used single electrode voltage-clamp recording in the presence of AP5 (40 μM) and CNQX (20 μM) to record monosynaptic biphasic inhibitory postsynaptic currents (IPSCs) (Davies et al., 1990). We compared the concentration response relationship for the antagonism of paired-pulse depression of IPSCs recorded under these conditions with that for antagonism of paired-pulse widening of EPSPs. When two stimuli were delivered 50–1000 ms apart there was a marked reduction in the IPSC evoked by the second stimulus, i.e. paired-pulse depression occurred. This depression was maximal at an interstimulus interval of 100–200 ms and has previously been shown to result from activation of GABA_B autoreceptors (Davies et al., 1990; Nathan & Lambert, 1991; Davies & Collingbridge, 1993). CGP 55845A (0.03–10 μM) inhibited the late component of the IPSC evoked by both stimuli and reversed paired-pulse depression of the early GABA_A receptor-mediated IPSC (n=4). As illustrated in Figure 4 the two relationships closely paralleled each other although that for inhibition of IPSP_B was shifted to the left by a factor of approximately 7. In addition, the concentration response relationship for the antagonism of the IPSC_B by CGP 55845A matched that for its antagonism of IPSP_B, and the concentration response relationship for antagonism of paired-pulse depression of IPSCs mirrored almost exactly that for inhibition of paired-pulse widening of field EPSPs (Figure 4).

Figure 4.

Comparison of the potency of CGP 55845A to inhibit paired-pulse depression of IPSCs with its potency to inhibit paired-pulse widening of field EPSPs. In (a) synaptic traces represent monosynaptic IPSCs evoked by two stimuli delivered 100 ms apart in the presence of D-AP5 (50 μM) and CNQX (10 μM) superimposed on the corresponding responses evoked in the presence of these antagonists plus 1 μM CGP 55845A. Note that in control medium the second IPSC of the pair is reduced compared to the first IPSC and that in the presence of CGP 55845A the peak amplitude of the second IPSC is greatly enhanced such that it now approaches the size of the first IPSC of the pair. In this particular example there is little or no activation of an IPSC_B and the cell was held at a membrane potential of −61 mV. In (b) synaptic responses represent the second field EPSP of a pair of EPSPs evoked by two stimuli delivered 200 ms apart in control and in CGP 55845A-containing medium. Note that in the presence of the GABA_B receptor antagonist the field EPSP is much narrower. The graph in (c) illustrates the concentration response relationships for CGP 55845A-induced antagonism of the IPSP_B, the IPSCB paired-pulse widening of the field EPSP and paired-pulse depression of IPSC_A. Each data plot was fitted to the logistic expression Y=M(X^P/[X^P+K^P]) as described in Figure 2. Note the close correlation between antagonism of paired-pulse widening of EPSP_A and paired-pulse depression of IPSC_A.

Based on these two series of experiments, therefore, it is reasonable to suggest that an analysis of the effects of GABA_B receptor antagonists on paired-pulse widening of field EPSPs provides an accurate measurement of the activity of these compounds at physiologically activated GABA_B autoreceptors. As such, we studied next the ability of a range of antagonists to inhibit this effect. As was the case for IPSP_B, every antagonist tested inhibited paired-pulse widening of EPSPs. Again, the concentration response relationships all paralleled each other and showed similar maximum effects (Figure 5). The rank order of antagonism was the same as that for the IPSP_B although the individual IC₅₀ values were higher. For clarity, a comparison of IC₅₀ values for antagonism of paired-pulse widening of EPSPs and inhibition of IPSP_Bs are given in Table 1.

Figure 5.

Comparison of the potency of different GABA_B receptor antagonists to inhibit paired-pulse widening of field EPSPs. Each of the four superimposed synaptic traces represent the second field EPSP of a pair evoked by two stimuli delivered 200 ms apart in control medium containing 50 μM CGP 40116 superimposed on the corresponding second field EPSP evoked in the presence of the concentration of antagonist indicated. The graph shows plots of the percentage inhibition of paired-pulse widening of field EPSPs versus antagonist concentration for four of the antagonists tested. Data were fitted to the logistic expression Y=M(X^P/[X^P+K^P]) as described in Figure 2.

Comparison of antagonist potency at pre- and post-synaptic GABA_B receptors

Finally, we compared the IC₅₀ values of these six compounds to inhibit the IPSP_B and paired-pulse widening of field EPSPs with those we have previously calculated for phaclofen, 2-hydroxy-saclofen and CGP 35348 (Davies & Collingridge, 1993; Davies et al., 1993). As illustrated in Figure 6 there was a good correlation between IC₅₀ values for each antagonist to inhibit physiologically activated pre- and post-synaptic GABA_B receptors. Thus, the mean IC₅₀ values for the two effects all lay very close to, or within, the 95% confidence limits of a linear regression line that described IC₅₀ values for inhibition of paired-pulse widening of EPSPs that were 7.3 times higher than those for antagonism of IPSP_B.

Figure 6.

Comparison of IC₅₀ values for antagonism of IPSP_B and paired-pulse widening of EPSP_A for different GABA_B receptor antagonists. The graph shows a plot of the IC₅₀ value for antagonism of paired-pulse widening of field EPSPs versus the corresponding IC₅₀ value for antagonism of the IPSP_B for nine different GABA_B receptor antagonists. The line drawn through the data points is a least squares fit regression line and the dashed line bordering it the 95% confidence limits of the fit.

Discussion

The present data demonstrate that, like phaclofen, 2-hydroxy-saclofen and CGP 35348, six additional phosphinic acid derivatives of GABA or corresponding N-substituted analogues (i.e. CGP 36742, CGP 51176A, CGP 55845A, CGP 52432, CGP 56433 and CGP 55679; Froestl et al., 1992; 1995; Froest & Mickel, 1997) are GABA_B receptor antagonists which are capable of antagonizing both pre- and post-synaptic GABA_B receptors at GABA-mediated synapses in the CA1 region of the rat hippocampus. The rank order of potency for this series of antagonists at both populations of GABA_B receptor is identical and fits well with the rank order of potency that can be generated based on the calculated pK_i values for these antagonists obtained from binding studies (Olpe et al., 1993a; Waldmeier et al., 1994; Froestl et al., 1995). Thus, for example, CGP 36742 is approximately 100 fold less potent than CGP 55679, using both electrophysiological and ligand-binding techniques, irrespective of the population of GABA_B receptor tested. Functionally, the only difference between antagonism at pre- and post-synaptic GABA_B receptors is the approximate 7–8 fold higher concentrations that are required to block presynaptic receptors as opposed to postsynaptic receptors, irrespective of the antagonist used.

At first glance, this concentration difference might point to a difference between pre- and post-synaptic GABA_B receptors. Such a situation might be resolved by comparing calculated K_D values for each anatagonist at these different populations. However, this approach may not provide the appropriate information regarding receptors that are activated physiologically because, firstly, this approach will activate both synaptic and extrasynaptic receptors and, secondly, it has been suggested that two pharmacologically distinct GABA_B receptors exist postsynaptically (Pham & Lacaille, 1996), and it is unclear (1) as to which of these are activated by synaptically released GABA, or, indeed (2) whether both are activated simultaneously. Strongest support for heterogeneity of GABA_B receptors, and in particular amongst those which are expressed presynaptically, has come principally from neurochemical release studies. In this respect, Bonanno & Raiteri (1993b) have suggested the existence of at least four separate GABA_B receptor subtypes controlling the release of different neurotransmitters from cortical synaptosomes and K⁺-stimulated brain slices (Bonanno & Raiteri, 1993a; Lanza et al., 1993; Fassio et al., 1994). This subclassification is based on the differential susceptibility of separate GABA_B receptor populations to the agonists baclofen and 3-aminopropylphosphinic acid (3-APPA) and the antagonists phaclofen, CGP 35348 and CGP 52432. Whilst it is difficult to make direct comparisons between the data presented here and that generated using baclofen or 3-APPA to activate GABA_B receptors, it is interesting that our data for both pre- and postsynaptic GABA_B receptors at GABA-mediated synapses fit best with a subtype that is sensitive to all three antagonists mentioned above and, therefore, suggest that these receptors are similar to those that control the cortical release of somatostatin but not GABA or glutamate (Bonanno & Raiteri, 1993b). However, the subclassification of GABA_B receptors suggested by Bonanno & Raiteri (1993b) is not universally accepted, most notably because their observations have not been repeated by others using electrically stimulated release in cortical and dorsal horn slices (Waldmeier et al., 1994; Teoh et al., 1996). In fact, quantitative pharmacological analysis of the antagonism of baclofen-induced inhibition of electrically-induced GABA and glutamate release revealed that K_D values for GABA_B receptor antagonists were similar to those calculated from radioligand binding studies in the same laboratories (Waldmeier et al., 1994), a situation echoed using electrophysiological approaches in the present study. Based on these data and the premise that radioligand binding does not discriminate between pre- and post-synaptic GABA_B receptors it was proposed that either (a) each antagonist does not differentiate between possible GABA_B receptor subtypes or (b) all antagonists are specific for a particular GABA_B receptor subtype and that compounds that activate/antagonize other GABA_B receptor subtypes have yet to be developed. That said, in the dorsal horn CGP 56999A potently antagonized GABA_B receptors controlling the release of GABA and substance P without affecting those which regulate the release of glutamate (Teoh et al., 1996) raising the possibility that pharmacologically distinguishable GABA_B receptors do exist in the CNS. If so, it is unlikely that it will be possible to allocate a particular pharmacological subtype to the GABA_B heteroreceptor on glutamate terminals, another to the heteroreceptor on somatostatin terminals, another to the GABA_B autoreceptor and so on. Indeed, one study using CGP 56999A in the cortex has already failed to demonstrate a differential effect of this antagonist on the GABA_B autoreceptors as opposed to heteroreceptors on glutamate terminals (Waldmeier et al., 1994). In the absence of an exhaustive study to assess the activity of CGP 56999A at all GABA_B receptor populations in the CNS these data raise the intriguing possibility that the pattern of expression of a CGP 56999A-insensitive GABA_B receptor may be regionally restricted to the spinal cord as opposed to other CNS areas.

Previous electrophysiological studies examining the pharmacology of GABA_B receptors have been equally contentious with, to date, different laboratories claiming differences between GABA_B receptors in the neocortex and hippocampus (Deisz et al., 1993; Dutar & Nicoll, 1998b) whilst others reporting no difference in the striatum and hippocampus (Seabrook et al., 1990; Thompson & Gähwiler, 1992). It could be argued on the basis of the present electrophysiological data that different GABA_B receptors exist at pre- and post-synaptic loci at GABAergic synapses in the hippocampal CA1 region. However, it is equally feasible that both populations of GABA_B receptors are pharmacologically identical. In this respect, the rank order of antagonism of pre- and post-synaptic GABA_B receptors is similar and the 7–10 fold concentration difference to antagonize the two populations exists because either (1) each compound tested is a competitive antagonist of GABA_B receptors and GABA_B autoreceptors encounter a higher concentration of synaptically released GABA than do postsynaptic GABA_B receptors even under circumstances where the amount of GABA initially released is the same (e.g., in response to single shock stimulation), or (2) at the presynaptic site GABA_B receptors couple to different effector systems for which there is greater receptor reserve/coupling efficiency. With respect to the former situation there is, at present, no report of the relative concentrations of synaptically released GABA at pre- and post-synaptic GABA_B receptors at GABAergic synapses in the hippocampus. However, it appears that (a) GABA_B autoreceptors may be outside the range of synaptically released GABA since despite their presence in hippocampal cultures they do not appear to account for paired-pulse depression of IPSCs (Yoon & Rothman, 1991; Wilcox & Dichter, 1994) and (b) GABA_B autoreceptors are not saturated in slices since experimental manipulations that increase the concentration and/or availability of GABA enhance paired-pulse depression of IPSCs (Roepstorff & Lambert, 1994). In contrast, numerous studies have suggested differences in the transduction mechanisms coupled to pre- and post-synaptic GABA_B receptors at these synapses (Scherer et al., 1988; Lambert & Wilson, 1993; Pitler & Alger, 1994; Thompson & Gähwiler, 1992). Thus, postsynaptic GABA_B receptors are generally agreed to couple directly to an inwardly rectifying potassium conductance via a pertussis toxin sensitive G-protein (e.g. Gähwiler & Brown, 1985; Dutar & Nicoll, 1998a,1998b) whereas GABA_B autoreceptors are believed to couple to alternative transduction mechanisms. In this respect, it has been suggested that GABA_B autoreceptors may enhance an A-type K⁺ current (Gage, 1992), inhibit an N-type Ca²⁺ current (Doze et al., 1995; Lambert & Wilson, 1996) or inhibit the release machinery per se. Whichever presynaptic mechanism is correct the observation that (−)-baclofen depresses GABA-mediated synaptic responses at lower concentrations than are required to cause postsynaptic hyperpolarization (Davies et al., 1990) points to more effective presynaptic receptor-effector coupling compared to that postsynaptically, assuming identical GABA_B receptors at both loci. The recent cloning of two distinct GABA_B receptor subunits, however, still raises the possibility that pre- and postsynaptic receptors may be pharmacologically distinct (Kaupmann et al., 1997, 1998; White et al., 1998; Jones et al., 1998). That said, the observation that GABA_BR₂ itself exhibits limited binding of existing GABA_B receptor antagonists suggests that both pre- and postsynaptic receptors at GABAergic synapses contain a GABA_BR₁ subunit (i.e. either of the splice variants GABA_BR_1a and GABA_BR_1b). Indeed, pharmacologically, a comparison of the rank order of potency of antagonists to block each of the homomeric GABA_BR₁ receptors, when expressed in COS-7 cells and activated by (−)-baclofen, revealed a similar rank order of potency to that for antagonism of the native receptors studied here (Kaupmann et al., 1997). In addition, quantitative pharmacological evaluation of the cloned GABA_BR_1a and GABA_BR_1b receptors revealed little differences between the two receptors. However, that is not to say that GABA_BR₂ subunits do not exist in pre- and postsynaptic GABA_B receptors at GABAergic synapses. Indeed, it is likely that both receptors are hetero-oligomeric complexes since these receptors when expressed in cell-lines exhibit closer binding affinities to those of native receptors and more effectively activate inwardly rectifying potassium channels which mediate postsynaptic hyperpolarization (Kaupmann et al., 1998; White et al., 1998; Jones et al., 1998). This does not preclude pharmacological differences between pre- and postsynaptic GABA_B receptors since existing antagonists do not bind with high affinity to the GABA_BR₂ subunit and GABA_BR₁ splice variants differ in their extracellular N-terminal region opening up the possibility of differential modulation by specific ligands that, unlike the antagonists developed to date, do not necessarily interact directly with the GABA binding site. In addition, it might be surprising if further subtypes of GABA_B receptor are not discovered in the future since the homologous metabotropic glutamate receptor consists of eight different subtypes, six which couple to the same G proteins (i.e. G_i/o) as GABA_B receptors.

Concluding remarks

Whilst on the basis of the compounds tested in the present study it is not possible to ascribe different GABA_B receptor subtypes to pre- and post-synaptic loci at GABAergic synapses it is clear that substantially higher concentrations of each antagonist are required to block GABA_B autoreceptors than postsynaptic GABA_B receptors. These data when extrapolated to a clinical context and taken in conjunction with the calculated concentrations of antagonist that reach the brain following i.v. administration would suggest that the predominant effect of these drugs would be to block the effects of postsynaptic receptors as opposed to autoreceptors. The exact outcome of such a balance of antagonism is difficult to assess because of the complexity of neuronal circuits and the possibility that in brain regions other than the CA1 area there may be less, or even more, clearcut differential antagonism of separate populations of GABA_B receptors (Deisz et al., 1993). However, on a simplistic level, it might be envisaged that GABA_B receptor antagonists, through their preferential blockade of postsynaptic as opposed to presynaptic GABA_B receptors, might enable increased excitability in the hippocampus particularly during periods of repetitive afferent activity, as for example might occur during learning, and that this might account for their cognitive enhancing properties (Mondadori et al., 1993).

Abbreviations

aCSF

artificial cerebrospinal fluid

EPSP

Excitatory postsynaptic potential

GABA

γ-amino-butyric acid

IPSP/C

Inhibitory postsynaptic potential/current