Functional expression of cell surface cannabinoid CB1 receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

doi:10.1016/S0306-4522(00)00120-2

Neuroscience

Volume 98, Issue 2, June 2000, Pages 253-262

https://doi.org/10.1016/S0306-4522(00)00120-2 Get rights and content

Abstract

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB₁) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB₁ receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB₁ receptor. Highly punctate CB₁ receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB₁ receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100 nM) markedly inhibited (by 77±6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB₁ receptor antagonist SR141716A (100 nM).

In conclusion, the present data show that cell surface CB₁ receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.

Section snippets

Materials

Cytosine β-d-arabinofuranoside, dialysed fetal bovine serum, paraformaldehyde, picrotoxin, protease type XIV, protease type X, tetrodotoxin, Triton X-100 and monoclonal antibodies against GABA, GAP-43 and MAP2 were purchased from Sigma Chemical Company, Dorset, U.K. Minimal essential medium (MEM) was purchased from Gibco, Paisley, U.K. The monoclonal antibody to synapsin I was purchased from Chemicon International, Temecula, CA, U.S.A. Monoclonal anti-GAD (clone GAD-6) was purchased from

Visualization of cell surface CB₁ receptor immunoreactivity with an antibody raised against an N-terminal epitope

CB₁ receptor immunoreactivity on the cell surface of living hippocampal neurons was detected using the N-terminal antibody. From approximately three days in culture, labelling was present on a network of fine neurites, with little or no somatic signal (Fig. 1). In young cultures, some of these fibres expressed contiguous, beaded labelling, whereas on others the immunoreactivity was localized to discrete puncta. Interestingly, putative growth cones exhibited intense CB₁ receptor staining (Fig. 1

Discussion

Using a combination of immunohistochemical and electrophysiological techniques we have demonstrated that CB₁ cannabinoid receptors in cultured hippocampal neurons are expressed at inhibitory terminals, where their activation results in the inhibition of GABA release.

Conclusion

This present study has enabled the visualization of synaptic, plasma membrane clusters of CB₁ receptors in cultured hippocampal neurons. These receptors are present at the majority of inhibitory GABAergic terminals where their activation leads to the presynaptic inhibition of transmitter release. CB₁ receptors are also widely expressed at GABAergic growth cones, suggesting that they also play an important role in neuronal development.

Acknowledgements

The authors wish to thank the Wellcome Trust, grants 047368 and 055291, and NIDA, grants DA00286 and DA11322, for financial support. AJI and JH are Wellcome Trust Research Career Development Fellows.

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Functional expression of cell surface cannabinoid CB1 receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

Abstract

Section snippets

Materials

Visualization of cell surface CB1 receptor immunoreactivity with an antibody raised against an N-terminal epitope

Discussion

Conclusion

Acknowledgements

Neuropharmacology

Neuroscience

Eur. J. Pharmac.

Eur. J. Pharmac.

Neuropharmacology

Pharmac. Ther.

Neuropharmacology

Fedn Eur. biochem. Socs Lett.

Neuroscience

Neuron

Neuroscience

Neuroscience

Neuroscience

Effects of the endogeneous cannabinoid, anandamide, on neuronal activity in rat hippocampal slices

Br. J. Pharmac.

Characterization of GABAergic neurons in hippocampal cell cultures

J. Neurocytol.

Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction

Science

Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture

J. Neurosci.

Presynaptic inhibition of GABAergic inputs to rat substantia nigra pars reticulata neurones by a cannabinoid agonist

NeuroReport

Functional expression of cell surface cannabinoid CB₁ receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

Visualization of cell surface CB₁ receptor immunoreactivity with an antibody raised against an N-terminal epitope