Elsevier

Neuropharmacology

Volume 43, Issue 4, September 2002, Pages 503-510
Neuropharmacology

Pharmacological separation of cannabinoid sensitive receptors on hippocampal excitatory and inhibitory fibers

https://doi.org/10.1016/S0028-3908(02)00157-0Get rights and content

Abstract

Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB1 cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona et al., 1999, Hájos et al., 2000, Hájos et al., 2001). The novel receptor does not correspond to CB2, since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB1 receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC50 values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC50 value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 μM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 μM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB1. We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 μM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 μM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs.

These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB1 than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB1 (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.

Introduction

A well-known effect of a synthetic cannabinoid WIN 55,212-2 is the inhibition of both glutamatergic and GABAergic neurotransmission in several brain regions, including the hippocampus, by acting on presynaptic terminals (Misner and Sullivan, 1999, Hájos et al., 2000, Hoffman and Lupica, 2000). To date, only one cloned cannabinoid receptor (CB1) has been identified in the CNS (Matsuda et al., 1990). Using light and electron microscopic techniques, immunocytochemical analyses revealed that CB1 receptors are present on a subset of GABAergic axon terminals in the hippocampus (Katona et al., 1999, Hájos et al., 2000) and in the basolateral amygdala (Katona et al., 2001). Glutamatergic terminals, however, were found to be immunonegative for CB1. Combined physiological and pharmacological studies in CB1 receptor knockout mice verified that cannabinoids suppress hippocampal inhibition via CB1 receptors (Hájos et al., 2000, Hájos et al., 2001, Wilson et al., 2001). In contrast, the reduction of glutamatergic transmission by WIN 55,212-2 was unchanged in mice lacking CB1 receptors compared to wild types (Hájos et al., 2001). In agreement with these findings, binding studies in CB1 receptor knockout mice have demonstrated that WIN 55,212-2 could still stimulate GTPγS binding in brain membranes, although with reduced efficacy (Breivogel et al., 2001). Taken together, in the hippocampus, CB1 receptors mediate inhibition of GABA release, while a different, so far unidentified cannabinoid sensitive receptor should be responsible for the modulation of glutamatergic synaptic transmission. The routinely used cannabinoid antagonist SR 141716A does not distinguish between CB1 and the new CB receptor, as it was shown to antagonize WIN 55,212-2-induced inhibition of both eIPSCs and EPCSs (Misner and Sullivan, 1999, Hájos et al., 2000, Hoffman and Lupica, 2000, Hájos et al., 2001).

One of the putative endogenous ligands of cannabinoid receptors is anandamide (Devane et al., 1992). Recent data, however, indicate that anandamide can also act as a full agonist on VR1 vanilloid receptors (Zygmunt et al., 1999, Smart et al., 2000), and has a substantial structural similarity to vanilloid ligands such as capsaicin (Szallasi and Di Marzo, 2000). The presence of VR1 receptors in the brain, including the hippocampus, has been shown by both binding studies and immunostaining (Acs et al., 1996, Mezey et al., 2000, Sanchez et al., 2001), although other studies have found no VR1 expression in the CNS (e.g. Caterina et al., 1997). In addition, recently published data indicated that anandamide and vanilloid ligands might act at the same site in the hippocampus (Al-Hayani et al., 2001). Thus, it seems possible that vanilloid ligands can differentially modulate the effects of cannabinoids on hippocampal excitation and inhibition. To elucidate whether these differences underlie a possible pharmacological separation between two distinct types of cannabinoid receptor present on excitatory and inhibitory axon terminals, we first compared the potency of WIN 55,212-2 to reduce excitatory and inhibitory postsynaptic currents (EPSC, IPSC) and demonstrated that—unlike SR 141716A—the cannabinoid antagonist AM251 is selective for CB1, and does not affect cannabinoid-induced reduction of EPSCs. The effect of vanilloid ligands was then tested on both types of neurotransmission, which turned out to be another tool to distinguish CB1 and the new CB receptor.

Section snippets

Materials and methods

Experiments were carried out according to the guidelines of the institutional ethical codex and the Hungarian Act of Animal Care and Experimentation (1998. XXVIII. section 243/1998), which is in full agreement with the regulation of animal experiments in the European Union. All efforts were made to minimize pain and suffering and to reduce the number of animals used.

Male Wistar rats (15–22 days old) were deeply anaesthetized with sodium pentobarbital (70 mg/kg, i.p.) or with isoflurane followed

The synthetic cannabinoid WIN 55,212-2 reduces monosynaptically evoked EPSCs and IPCSs with different affinity

Binding studies obtained in CB1 receptor knockout mice (Breivogel et al., 2001) suggest that the cannabinoid ligand WIN 55,212-2 may have a lower affinity to cannabinoid sensitive receptors present on glutamatergic axon terminals compared to CB1 receptors located on GABAergic axon endings. Therefore we performed concentration response analyses for the inhibitory effect of WIN 55,212-2 on both EPSCs and IPSCs evoked by focal electrical stimulation in CA1 pyramidal cells. As shown in Fig. 1, WIN

Discussion

The ten-fold difference in EC50 values of WIN 55,212-2 mediated inhibition of evoked EPSCs and IPSCs strongly support our previous conclusion that distinct receptors regulate cannabinoid inhibition of hippocampal glutamatergic and GABAergic synaptic transmission. An EC50 value for inhibition of IPSCs (0.138 μM) comparable to that obtained in our present study (0.24 μM) was estimated in hippocampal slice preparation by Hoffman and Lupica (2000). Furthermore, [35S]GTPγS binding stimulated by WIN

Acknowledgements

This work was supported by OTKA (No: T32251), the Howard Hughes Medical Institute, and NIH (NS30549). N.H. was supported by Bolyai Scholarship. We thank Drs I. Mody and I. Katona for comments on the manuscript.

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