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