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NEUROPHARMACOLOGY
School of Biomedical and Molecular Sciences, University of Surrey, Guildford, United Kingdom (Y.C.); and Eli Lilly & Co. Ltd., Erl Wood Manor, Windlesham, United Kingdom (N.M.-R., E.S.)
Received December 12, 2005; accepted February 27, 2006.
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Abstract |
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, 1998; White et al., 1998). The agonist binding site in the GABABRs is limited to the GABAB(1) subunit (Kniazeff et al., 2002), whereas the GABAB(2) subunit links to G-proteins (Galvez et al., 2001; Uezono et al., 2006) of the pertussis toxin-sensitive family, i.e., Gi/Go (Odagaki and Koyama, 2001). The recently discovered GABABR-positive allosteric modulators (Urwyler et al., 2001, 2003), exemplified by the compound CGP7930, have been shown to act on the GABAB(2) subunit (Binet et al., 2004) to enhance the binding and the function of GABABR agonists, GABA or baclofen, without showing any intrinsic agonistic efficacy.
The GABABRs exert their inhibitory functions in the central nervous system by increasing potassium conductance postsynaptically to reduce neuronal excitability and by inhibiting calcium conductance presynaptically to reduce neurotransmitter release (for review, see Bowery et al., 2002; Bettler et al., 2004). The GABABR agonist baclofen has been used in clinical studies to treat spasticity, pain, and addiction, but the muscle relaxant, hypothermic, and sedative side effects of baclofen, together with patients' increasing tolerance, limit its potential therapeutic utility. Recently, the therapeutic targeting of GABABRs has been further exploited by testing the GABABR-positive allosteric modulators in behavioral models. CGP7930 and another GABABR-positive allosteric modulator GS39783 have been shown to reduce cocaine self-administration (Smith et al., 2004), the cocaine-induced lowered intracranial self-stimulation threshold (Slattery et al., 2005), and anxiety (Cryan et al., 2004). In these behavioral experiments, the GABABR potentiators were also shown to be devoid of the typical side effects associated with GABABR agonists.
To elucidate the mechanisms underlying the anti-addictive actions of the GABABR potentiators, we have recently shown that CGP7930 can indeed enhance the GABABR-mediated inhibition of dopamine neuronal activity in the ventral tegmental area (Chen et al., 2005b). In the present study, we examined the mode of action by the GABAB potentiator CGP7930 on GABABR-mediated modulations of synaptic transmission in the hippocampal CA1 area.
GABABRs are expressed in abundance in the hippocampus, and their modulatory roles in the CA1 synaptic transmission are well described (Nicoll, 2004). GABABR activation causes a slow postsynaptic hyperpolarization and the presynaptic inhibition of neurotransmitter release, including auto-inhibition of GABA release and heterosynaptic inhibition of Glu release (Thompson and Gahwiler, 1992; Wu and Saggau, 1995). Using the rat hippocampal CA1 neuronal network, we have dissected out the measures for GABABR-mediated modulations of both the excitatory and inhibitory synaptic transmission and examined the effects of CGP7930 on these GABABR-mediated modulations of synaptic transmission. Our results revealed a selective potentiation by CGP7930 of baclofen-induced modulation of synaptic inhibition. Some of these results have previously been published as an abstract (Chen et al., 2005a).
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Materials and Methods |
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One slice was transferred to an MED64 probe (Panasonic, Osaka, Japan), with an interelectrode distance of 150 µm. The slice was carefully positioned to have the CA1 area over the 64-electrode array as shown in Fig. 1A. The slice was weighed down with a nylon wire mesh and a tungsten ring, submerged in ACSF saturated by 95% O2/5% CO2 at 31–32°C at a perfusion rate of 1 to 2 ml/min. Of the 64 planar surface electrodes, two positioned in the stratum radiatum were selected for stimulation to activate two Schaffer collateral pathways. A negative electrical pulse of 10 to 70 µA with 0.2-ms duration was applied once every 30 s to the two pathways alternately. Recordings of field excitatory postsynaptic potentials (fEPSPs) or the population spikes (PSs) were obtained simultaneously from a number of electrodes positioned in the stratum radiatum and stratum pyramidale, respectively (Fig. 1), using Performer 2 (Panasonic). Experiments were only conducted when signals recorded adjacent to the stimulation electrodes were comparable with microelectrode recordings, i.e., the amplitude of fEPSPs reached 1 mV and the PS reached 2 mV. The 20 to 80% initial slope and the half-width (HW) of the fEPSP were measured by Performer 2. The amplitude of the PS (the vertical distance from the negative peak to a line drawn between the two positive peaks) was measured offline using pClamp 9 (Axon Instruments, Union City, CA). Recordings that met the criteria of the fEPSP amplitude >0.2 mV (n = 6–14 for each experiments) or the PS amplitudes >0.5 mV (n = 3–6 for each experiment) were selected for analysis, and the averaged results was used as n = 1 experiment (see Fig. 2B). In averaging multiple recordings from one slice we took into account the variability between recording sites and hence generated highly repeatable results between experiments.
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(RS)-Baclofen and CGP55845 were purchased from Tocris Cookson Inc. (Bristol, UK). CGP7930 was synthesized at the Lilly chemistry laboratories (Lilly UK). CGP7930 was dissolved in dimethyl sulfoxide, and the final dimethyl sulfoxide in ACSF is <0.1%. Numerical data in the text and error bars in figures are expressed as the means ± S.E.M. Statistical comparisons were made with one-way analysis of variance or two-tailed Student's t test, unpaired, or paired when stated.
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Results |
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The GABABR antagonist CGP55845 at 1 or 10 µM(n = 11) or the GABABR potentiator CGP7930 at 30 µM (n = 6) caused no significant changes to the fEPSP slope or the PS amplitude, indicating a low tonic level of GABAB receptor-mediated modulation of synaptic excitation. The GABABR agonist baclofen reduced the GluR-mediated excitatory synaptic transmission and produced concentration-dependent inhibition of the fEPSP slope and the PS amplitude (Fig. 2) (n = 6, P < 0.001, one-way analysis of variance), in agreement with previous findings that GABABR activation can inhibit excitatory synaptic transmission by presynaptic inhibition of glutamate release (Davies et al., 1993; Isaacson et al., 1993; Ziakopoulos et al., 2000), as well as postsynaptic hyperpolarization of CA1 cells (Newberry and Nicoll, 1984). Baclofen was applied cumulatively with escalating concentrations at 0.1, 1, 3, and 10 µM, followed by full reversal with the antagonist CGP55845 at 1 µM (Fig. 2A). At the low concentrations of 0.1 and 1 µM, baclofen had no effects on the fEPSP slope or the PS amplitude. At the high concentrations of 3 and 10 µM, a reduction of up to 70% was achieved by baclofen. The percentage reduction for both the fEPSP and the PS are in good agreement (Fig. 2, B and C, 
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The GABAB-positive allosteric modulator CGP7930 has been reported to enhance the potency and efficacy of GABABR agonists (Urwyler et al., 2001). CGP7930 has also been shown previously by us to potentiate baclofen-induced inhibition of dopamine neuron firing in rat ventral tegmental slices (Chen et al., 2005b). Here we wanted to see whether CGP7930 is able to enhance the effects of baclofen on CA1 excitatory transmission. The effects of 30 µM CGP7930 (Chen et al., 2005b) on baclofen-induced modulation of the fEPSP and the PS were examined in a set of six experiments. CGP7930 was preapplied for 20 min and then coapplied with escalating concentrations of baclofen in the same paradigm as the experiments above. The inhibition caused by baclofen was thus compared between control experiments and experiments with CGP7930 coapplied (Fig. 2). The baclofen concentration-response curves for the fEPSP slope and the PS amplitude were plotted and fitted with sigmoidal curves. The curves with CGP7930 coapplied were found to be shifted slightly to the left, for both the fEPSP slope (IC50 of baclofen: 5.7 µM for control versus 4.2 µM for +CGP7930) and the PS amplitude (IC50 of baclofen: 6.8 µM for control versus 3.3 µM for +CGP7930), but no statistical differences were found (P > 0.05, n = 6) (Fig. 2, B and C). A larger shift was observed with the PS amplitude, perhaps due to the fact that more factors can influence the excitability of the cells, including inhibition in the slice. The inability of CGP7930 to shift the baclofen concentration-response curve of the fEPSP slope (Fig. 2B) showed that CGP7930 was not able to potentiate the inhibitory effects of baclofen on synaptic excitation in CA1 cells.
CGP7930 Enhanced the Inhibitory Effects of Baclofen on Synaptic Inhibition. On close examination of the shape of the fEPSP, it was noted that baclofen caused significant widening of the fEPSP by slowing down the returning phase, even at the low concentrations of 0.1 and 1 µM baclofen, which did not affect the fEPSP initial slope (Fig. 3A). It was also noted that the fEPSPs recorded in the stratum radiatum were of a range of HWs (mean = 5.0 ms and S.D. = 1.6 ms, n = 72), with narrow ones recorded proximal to the cell body and wider ones in distal dendritic field. The effects of baclofen on fEPSP HW were analyzed with all the fEPSP recordings (amplitude >0.2 mV) in a slice, and an averaged effects of baclofen was used as n = 1. Comparing the results from the two sets of experiments of baclofen control versus baclofen + CGP7930, it was found that the widening effect of baclofen on the fEPSP was significantly potentiated by CGP7930, especially at low concentrations of baclofen (Fig. 3B). On average, in baclofen control experiments, 0.1 µM baclofen caused a small widening of the HW of the fEPSP (106.3 ± 1.1% of control, P < 0.05, n = 8), without affecting the slope of the fEPSP (102.5 ± 2.5% of control, P > 0.05) (Fig. 2). With the coapplication of 30 µM CGP7930, baclofen at 0.1 µM increased the HW by 20.6 ± 3.0% (P < 0.01, Student's t test) (Fig. 3C) more than the control. At 1 µM baclofen, the HW of the fEPSP was widened by 36.6 ± 2.5% (P < 0.001, n = 8) in control experiments; but in the presence of CGP7930, the increase was 61.8 ± 7.3%, which was significantly (P < 0.01) enhanced from the control. CGP7930 applied alone in the pretreatment period did not cause a significant change (103.0 ± 1.2% of control, P > 0.05) to the HW. At higher concentrations of baclofen (3 and 10 µM), no further increases in HW were recorded, indicating a saturation of the effect. However, the potentiation by CGP7930 was also found to be significant (P < 0.05) at 3 µM baclofen (Fig. 3C).
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Changes in the excitatory drive by varying the stimulus intensity or during the application of 6-cyano-7-nitroquinoxaline-2,3-dione did not cause the widening of the fEPSP, but a reduction of GABAAR activation caused the slowing down of the returning phase of the fEPSP and hence the widening of the HW (data not shown). It is also known that the activation of N-methyl-D-aspartate receptors by the removal of extracellular magnesium ions produced a slow and protracted excitatory potential in CA1 that will cause widening of the fEPSP (Chen et al., 1999). However, baclofen is not known to cause the removal of magnesium block of the N-methyl-D-aspartate receptor channels, but baclofen is well demonstrated to reduce GABA release to inhibit synaptic inhibition (Bowery et al., 2002). To confirm that the fEPSP HW widening by baclofen is due to reduced GABAAR-mediated synaptic inhibition, we tested the effects of baclofen when GABAAR-mediated synaptic inhibition was fully blocked. In the presence of 10 µM bicuculline, baclofen at 1, 3, and 10 µM was apparently prevented from causing the widening of the fEPSP, and CGP7930 failed to potentiate the effects (n = 2, data not shown). However, in these experiments, the epileptiform activity present in the evoked responses caused "ripples" in the returning phase of the fEPSP, hindering accurate measurement of the fEPSP HW from a majority of the recordings, especially the ones from the proximal regions of the dendritic field. An alternative and more robust measurement is needed to examine the direct modulations of synaptic inhibition.
In the hippocampal CA1 pyramidal cells, stimulation of the Schaffer collateral-commissural fibers causes the monosynaptic GluR-mediated excitation, which is followed immediately by the delayed disynaptic GABAAR-mediated feed-forward and feedback synaptic inhibition. The reduction of the delayed inhibition can affect the returning phase of the fEPSP and cause the widening. This synaptic inhibition can also manifest by inhibiting the second PS evoked by a paired stimuli with a short interval (interpulse interval = 15 ms), known as the paired-pulse inhibition of the PS (Cornish and Wheal, 1989). Paired-pulse recordings of the PS were therefore conducted to measure the strength of the delayed synaptic inhibition, and the effects of baclofen were compared with or without CGP7930.
In control slices with inhibition intact, the second pulse generates an inhibited PS, resulting in a paired-pulse ratio (PPR = PS2/PS1) <1 (0.53 ± 0.06, n = 12) (Fig. 4A). In these experiments, stimulus intensities were adjusted to evoke the PS of 
90% of the maximal amplitude. Although the PSs were recorded by electrodes positioned in the CA1 pyramidal cell layer, the fEPSP slopes were also monitored simultaneously by electrodes in the stratum radiatum (see Fig. 1). When synaptic excitation is not altered, changes in synaptic inhibition can be demonstrated by changes in the PPR of the PS.
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The effects of the GABAB potentiator CGP7930 were then assessed on the PPR of the PS. After the coapplication of baclofen with 30 µM CGP7930, it was found that larger increases in the PPR were observed at both 0.1 and 1 µM baclofen (Fig. 4B). A 9-fold (P < 0.05) and a 3-fold (P < 0.01) enhancement of the PPR were found at 0.1 and 1 µM baclofen, respectively (Fig. 4C). The GABABR antagonist CGP55845 reversed the effects of baclofen but had no effects on the PPR on its own (data not shown). The GABABR allosteric modulator CGP7930 alone also had no significant effects on PPR (Fig. 4C). These results show that CGP7930 significantly potentiated the baclofen-induced suppression of the synaptic inhibition, despite the lack of effects by CGP7930 on baclofen-induced modulation of the synaptic excitation.
Apart from the selective modulation by CGP7930 on the synaptic inhibition, we also found that baclofen showed enhanced sensitivity for modulating the inhibitory event. As shown in Fig. 2, baclofen at 0.1 and 1 µM had no significant effects on the fEPSP slope, which measures synaptic excitation. However, both 0.1 and 1 µM baclofen showed significant effects on the modulation of synaptic inhibition, measured by the increase in PPR of the PS (Fig. 4). Taken together, it appears that the GABABRs that modulate the synaptic inhibition were more sensitive to baclofen, and their functions were selectively potentiated by CGP7930.
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Discussion |
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). In our experiments, we confirmed the inhibitory effects of baclofen on synaptic excitation, measured mainly as the fEPSP slope, and on delayed synaptic inhibition, measured by the paired-pulse inhibition of the PS. We have also found that baclofen showed higher potency for modulating the synaptic inhibition than the synaptic excitation in CA1, similar to an observation in CA3 (Lei and McBain, 2003). More interestingly, when testing the effects of the GABAB allosteric modulator CGP7930, we observed potentiation of baclofen's effects on synaptic inhibition only. Baclofen's inhibitory effects on synaptic excitation were not enhanced by CGP7930. We have, therefore, observed in the hippocampal CA1 area a differential modulation of synaptic transmission by the GABAB allosteric modulator CGP7930, which also correlates with a differential sensitivity to baclofen of the two synaptic pathways.
The differential modulation by CGP7930 on synaptic transmission reported here is probably due to a different effect by this compound on the presynaptic autoreceptors located on the inhibitory GABA terminals compared with that on the presynaptic heterosynaptic receptors on the excitatory Glu terminals. Effects on postsynaptic GABABRs on CA1 cells would equally affect the measurements of both the excitatory and the inhibitory components so that a selective effect on the autoreceptors versus the heteroreceptors could explain the experimental results. Numerous electrophysiological and biochemical studies indicate pharmacological differences between presynaptic auto- and heteroreceptors, and between pre- and postsynaptic receptors in various brain tissues (Bowery et al., 2002; Bettler et al., 2004). Differences between presynaptic auto- and heteroreceptors in the CA3 area of the hippocampus included the finding that GABAA receptor-mediated synaptic inhibition was more sensitive to baclofen (Lei and McBain, 2003) and to pertussis toxin and barium treatments (Thompson and Gahwiler, 1992). In measuring GABA, Glu, and other neurotransmitter release, several agonists and antagonists displayed pharmacologically distinct potencies between the presynaptic GABAB auto- and heterosynaptic receptors (Bonanno and Raiteri, 1993; Teoh et al., 1996; Bonanno et al., 1997). Now, for the first time, in the hippocampal CA1 area, we have demonstrated that the novel GABAB receptor-positive allosteric modulator, CGP7930, selectively potentiated baclofen-induced activation of presynaptic autoreceptors.
GABABRs are involved in many neuronal functions and diseases, and the GABABR agonist baclofen has been used clinically to treat spasticity, pain, and addiction. However, agonist-induced side effects and tolerance have limited the therapeutic potential of baclofen. Based on considerable evidence pointing to the existence of pharmacologically distinct native GABABR subtypes (Bowery et al., 2002; Bettler et al., 2004), it was hoped that subtype-selective agonists and antagonists could dissociate their therapeutic effects from the side effects. However, to date, no subtype-selective agonists or antagonists were found. In particular, expression cloning of GABABRs failed to reveal pharmacologically distinct receptor subtypes (Kaupmann et al., 1998), even though two major GABAB(1) isoforms were identified as 1a and 1b. Several intracellular mechanisms have also been shown to influence the GABABR function, which include receptor G-protein coupling, type of G-proteins, G-protein effecter coupling, and subunit phosphorylation states (Couve et al., 2002; Bettler et al., 2004). However, the existence of any subtypes of GABABRs is still uncertain.
The therapeutic utility of GABABR modulation was further exploited recently by the discovery of GABABR-positive allosteric modulators (Urwyler et al., 2001, 2003) and their in vivo potencies in behavioral experiments of reward (Smith et al., 2004; Slattery et al., 2005) and anxiety (Cryan et al., 2004). In addition to the efficacy of the GABABR-positive allosteric modulators in disease models, they were also found to be devoid of agonistic side effects. This profile enhanced the therapeutic potential of the GABABR-positive allosteric modulators, but the underlying mechanisms remain largely unexplained, although the use of an allosteric modulator may reduce drug tolerance developed in patients using baclofen for a prolonged period (Abel and Smith, 1994). Few studies have been conducted to examine the effects of the potentiators at the synaptic level and to elucidate their modulation of synaptic transmission. Our previous work (Chen et al., 2005b) showed that CGP7930 potentiated the baclofen-induced reduction in firing of ventral tegmental dopamine neurons and provided a mode of action for GABABR potentiators in reducing the rewarding effects of cocaine. Now in the hippocampus, we have demonstrated a novel mechanism of the preferential modulation by CGP7930 on the synaptic inhibition in the CA1 area. Discussions on the relevance of in vitro concentrations and in vivo doses were attempted in our previous article (Chen et al., 2005b) and by Smith et al. (2004). Our previous findings showed that 10 µM CGP7930 was ineffective in experiments using brain slices and yet a 100 µM concentration was insoluble in ACSF, so the optimal concentration of 30 µM was used in the present experiments. Other studies using native brain tissues also indicated that the effective range of CGP7930 was between 10 and 100 µM (Onali et al., 2003; Urwyler et al., 2005). Tissue penetration and compound solubility may have affected the apparent potency of the compound compared with that in in vitro experiments using membrane preparations (EC50 of 1–5 µM) (Urwyler et al., 2001). After in vivo administration of an effective dose (30 µmol/kg) of CGP7930, brain concentrations of the compound were estimated to be approximately 300 nmol/kg 1 h later (Smith et al., 2004). A higher brain concentration may be expected 10 to 15 min after compound administration, when the behavioral experimental procedures were carried out (Carai et al., 2004; Smith et al., 2004).
GABABR potentiators have no intrinsic agonistic effects and hence require the presence of an agonist to exert their actions. Because of the extrasynaptic location of the GABABRs (Scanziani, 2000), endogenously released GABA may need to escape the synaptic cleft to reach the presynaptic GABABRs. In our experiments, we used the exogenous ligand, baclofen. As a result, both the presynaptic GABAB auto- and heterosynaptic receptors were exposed to the same concentrations of the agonist, and the effects on both pathways were recorded simultaneously using a multielectrode system. The effects of low concentrations of baclofen probably represent more closely the physiological effects of GABABR activation. In our experiments, the significant effects of CGP7930 were indeed found when it was coapplied with low concentrations of baclofen. Accordingly, the differential modulation by CGP7930 on synaptic inhibition versus synaptic excitation is not due to the accessibility of the endogeneously released GABA to the presynaptic GABAB auto- versus heterosynaptic receptors but to the selective sensitivity of the presynaptic GABAB autoreceptors to the positive allosteric modulator.
Our work has thus revealed a novel pattern of allosteric modulation on native GABABRs in their modulation of synaptic transmission. The differential potentiation of GABABR-mediated functions by CGP7930 provides a synaptic mechanism by which the GABAB potentiators may exert their in vivo efficacies without the untoward side effects of the GABABR agonists, in support of GABAB potentiators as an exciting new and better therapeutic alternative to GABAB agonists.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS:GABAAR, GABAA receptor; GABABR, GABAB receptor; CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)-phenol; GS39783, N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine; ACSF, artificial cerebrospinal fluid; fEPSP, field excitatory postsynaptic potential; PS, population spike; HW, half-width; CGP55845, (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl)phosphinic acid; GluR, glutamate receptor; PPR, paired-pulse ratio.
Address correspondence to: Dr. Ying Chen, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK GU2 7XH. E-mail: ying.chen{at}surrey.ac.uk
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) were constructed by sigmoidal fitting to the averaged percentage control values of the fEPSP slope (B) or the PS amplitude (C) at the corresponding baclofen concentrations (n = 6). No significant differences were found between the curves with or without CGP7930.
