Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

A J Irving, Angela Alice Coutts, J Harvey, M G Rae, K Mackie, Guy Smith Bewick, Roger Guy Pertwee

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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 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) 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 CB(1) 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 (100nM) 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 CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) 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.
Original languageEnglish
Pages (from-to)253-262
Number of pages10
JournalNeuroscience
Volume98
Issue number2
DOIs
Publication statusPublished - Jun 2000

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Cannabinoid Receptors
Cannabinoids
Presynaptic Terminals
Neurons
Inhibitory Postsynaptic Potentials
Growth Cones
rimonabant
gamma-Aminobutyric Acid
Cannabinoid Receptor Antagonists
Cannabinoid Receptor Agonists
Glutamate Decarboxylase
Synaptic Vesicles
Optical Imaging
Synaptic Transmission
Neurotransmitter Agents
Axons
Coloring Agents
Central Nervous System
Amino Acids
Antibodies

Keywords

  • Animals
  • Animals, Newborn
  • Axons
  • Cells, Cultured
  • Glutamate Decarboxylase
  • Hippocampus
  • Neural Inhibition
  • Neurons
  • Presynaptic Terminals
  • Rats
  • Receptors, Cannabinoid
  • Receptors, Cell Surface
  • Receptors, Drug
  • gamma-Aminobutyric Acid
  • Cannabinoid
  • GABA
  • Growth cones
  • Hippocampal
  • Immunocytochemistry
  • Synaptic transmission

Cite this

Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons. / Irving, A J; Coutts, Angela Alice; Harvey, J; Rae, M G; Mackie, K; Bewick, Guy Smith; Pertwee, Roger Guy.

In: Neuroscience, Vol. 98, No. 2, 06.2000, p. 253-262.

Research output: Contribution to journalArticle

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T1 - Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

AU - Irving, A J

AU - Coutts, Angela Alice

AU - Harvey, J

AU - Rae, M G

AU - Mackie, K

AU - Bewick, Guy Smith

AU - Pertwee, Roger Guy

PY - 2000/6

Y1 - 2000/6

N2 - 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 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) 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 CB(1) 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 (100nM) 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 CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) 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.

AB - 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 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) 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 CB(1) 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 (100nM) 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 CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) 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.

KW - Animals

KW - Animals, Newborn

KW - Axons

KW - Cells, Cultured

KW - Glutamate Decarboxylase

KW - Hippocampus

KW - Neural Inhibition

KW - Neurons

KW - Presynaptic Terminals

KW - Rats

KW - Receptors, Cannabinoid

KW - Receptors, Cell Surface

KW - Receptors, Drug

KW - gamma-Aminobutyric Acid

KW - Cannabinoid

KW - GABA

KW - Growth cones

KW - Hippocampal

KW - Immunocytochemistry

KW - Synaptic transmission

U2 - 10.1016/S0306-4522(00)00120-2

DO - 10.1016/S0306-4522(00)00120-2

M3 - Article

VL - 98

SP - 253

EP - 262

JO - Neuroscience

JF - Neuroscience

SN - 0306-4522

IS - 2

ER -