Elsevier

Neuropharmacology

Volume 39, Issue 2, February 2000, Pages 246-253
Neuropharmacology

Impaired inhibition of epileptiform activity by baclofen, but not by adenosine in the weaver hippocampus

https://doi.org/10.1016/S0028-3908(99)00089-1Get rights and content

Abstract

The weaver defect results in a loss of baclofen- and adenosine-gated K+ conductance in the hippocampus of adult homozygous (wv/wv) mice. In addition, suppression of hippocampal epileptiform activity by baclofen is impaired (Jarolimek, W., Bäurle, J., Misgeld, U., 1998. Pore mutation in a G protein-gated inwardly rectifying K+ channel subunit causes loss of K+ dependent inhibition in weaver hippocampus. Journal of Neuroscience 18, 4001–4007). We used wv/wv and wild-type (+/+) mice to determine whether K+ conductance increases are essential for the suppression of epileptiform activity by R-baclofen and adenosine in disinhibited hippocampal slices. In wv/wv mice R-baclofen was less potent by two orders of magnitude in reducing the frequency of spontaneous synchronous burst discharges than in +/+ mice. Endogenous adenosine and adenosine A1 receptor agonists differed only slightly in their efficacy to inhibit spontaneous synchronous burst discharges in wv/wv and +/+ mice. The findings on adenosine A1 receptors suggest that the varied efficacy of R-baclofen in wv/wv and +/+ mice may not be explained solely on the basis of a loss of ligand-gated K+ conductance. Therefore, we investigated the affinity of GABAB receptors for the antagonist CGP55845A in wv/wv and +/+ hippocampi. Schild plot analysis revealed a KD for the GABAB antagonist CGP55845A 10 fold higher in wv/wv than in +/+ mice. The data suggest that an alteration of GABAB receptors could contribute to the reduced efficacy of R-baclofen to suppress hippocampal epileptiform activity in weaver mice, while the suppression by adenosine remains largely unaffected.

Introduction

Baclofen, a selective GABAB agonist, is used in clinical practice for the treatment of spinal spasticity (Penn et al., 1989). The principal action of the drug is the reduction of transmitter release in the mammalian central nervous system. Underlying mechanisms are severalfold. GABAB receptors, like most G protein-coupled receptors, act through various effectors. (1) They inhibit Ca2+ channels, (2) activate a K+ conductance, (3) enhance or reduce adenylyl cyclase activity and (4) reduce constitutive transmitter release by a direct effect on the release machinery (for a review of the literature see Misgeld et al., 1995). Reported EC50 values for the various effects of baclofen in a variety of central neurons and species range from 0.5–5 μM (Wojcik and Holopainen, 1992), and it is common practice to use concentrations of the drug up to 100 μM in experimental studies. Therapeutic concentrations, however, in the cerebrospinal fluid range from 40–100 nM (Knutsson et al., 1974), the latter concentration already producing drowsiness. It is difficult to translate experimental into therapeutic drug concentrations particularly since species differences, pathophysiological changes and different drug kinetics may be involved. Interestingly, spontaneous burst activity in the disinhibited rodent hippocampus provides a paradigm which is effectively inhibited by baclofen in nanomolar concentrations (Swartzwelder et al., 1986). In mice we found an EC50 of R-baclofen of 43 nM under this experimental condition (Jarolimek et al., 1998). It is likely that pre- and postsynaptic actions of baclofen contribute to this effect.

It is unclear to what degree the various known GABAB receptor-coupled effectors are involved in the therapeutic action of baclofen. If the therapeutic action of baclofen rests entirely on a presynaptic inhibition of glutamate release, the K+ conductance increase may not play a major role. There are observations suggesting that the K+ conductance increase is not of major importance in the presynaptic actions of baclofen (Lambert et al., 1991, Lüscher et al., 1997, Wu and Saggau, 1997). Lüscher et al. (1997) reported that baclofen- and adenosine-induced presynaptic inhibition were unchanged in the CA1 and CA3 region of transgenic mice lacking the Kir3.2 gene. They tested the effect of 40 and 100 μM baclofen and adenosine respectively, on evoked field potentials (cf. Fig. 5 in Lüscher et al., 1997). In weaver (wv/wv) mice which carry a point mutation in the pore region of the Kir3.2 subunit (Patil et al., 1995) we found that neither R-baclofen, adenosine nor serotonin induced a significant K+ current in CA3 neurons of wv/wv hippocampal slices (Jarolimek et al., 1998), although the hippocampal morphology (Sekiguchi et al., 1995, Liao et al., 1996) is widely unaltered. We further observed that R-baclofen is rather ineffective in inhibiting the spontaneous synchronous burst discharge (SSBD) of disinhibited wv/wv CA3 neurons. The reduced efficacy of baclofen in wv/wv mice was evident only when using a low range of agonist concentrations because a high concentration (>10 μM) of the agonist would block the SSBD in both wild type (+/+) and wv/wv mice (cf. Fig. 2 in Jarolimek et al., 1998). We assumed that the inefficacy resulted from a loss of K+ conductance activation (Swartzwelder et al., 1986). To test this assumption we investigated the action of adenosine which, via activation of adenosine A1 receptors, has effects on hippocampal neurons similar to the effects of baclofen via GABAB receptors (Greene and Haas, 1991). The results did not support the assumption that a lack of an increase in the K+ conductance is the only reason for the reduced efficacy of R-baclofen in wv/wv mice. Therefore, we also studied the affinity of the potent antagonist CGP55845A to GABAB receptors in wv/wv CA3 neurons.

Section snippets

Methods

Weaver (wv/wv) and wild-type (+/+) (B6CBA) mice were the offspring of parents initially purchased from Jackson Laboratories (Bar Harbor, ME, USA). Weaver mice (wv/wv) were obtained from incross mating of homozygous weaver mice and displayed obvious motor dysfunction. All experiments had been approved by the Animal Care and Use Committees responsible for our institutions and conform to NIH guidelines. Hippocampal slices were prepared from 2- to 7-week-old mice using techniques previously

Effects of R-baclofen on spontaneous and evoked burst discharges

Nanomolar concentrations of R-baclofen reduced the frequency of the SSBD in disinhibited slices from area CA3 of +/+ mice [Fig. 1(A1)], confirming previous results (Jarolimek et al., 1998). The EC50 for the inhibition of the frequency of the SSBD [Fig. 2(B)] was 44±5 nM (n=10 slices). For the inhibition of stimulation evoked burst discharges, however, R-baclofen concentrations in the micromolar range were needed [Fig. 1(A2)]. R-baclofen had no discernible effect on evoked burst discharges at a

Discussion

Inhibition of spontaneous burst activity in disinhibited wv/wv hippocampi by GABAB receptor activation is impaired not only because the K+ conductance is not activated, but also because GABAB receptor properties might have changed. This conclusion is supported by the observation that inhibition of spontaneous burst activity in disinhibited wv/wv hippocampi by adenosine is only slightly reduced, although adenosine A1 receptor activation also does not lead to a K+ conductance increase.

Acknowledgements

The authors thank A. Lewen and C. Heuser for excellent technical assistance. This work was supported by the Sonderforschungsbereich 317/B13 to U.M.

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      Using a high affinity GABAB receptor antagonist, we could block both populations, whereas the weaver mutation in the inward K+ conductance abolished the function of the receptors linked to this conductance. Previously we showed that impaired K+ dependent inhibition mediated by GABAB receptors in the wv/wv CA3 area is associated with drastically reduced suppression of interictal spikes in high [K+]o by the specific GABAB receptor agonist R-baclofen (Jarolimek et al., 1998, 2000). In this study, using not only extracellular, but combined intra- and extracellular recording we show that the occurrence of interictal spikes and their properties are very similar in wv/wv and +/+ CA3 neurons.

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