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, 59 (1-2), 121-7

Delta9-tetrahydrocannabinol Is a Full Agonist at CB1 Receptors on GABA Neuron Axon Terminals in the Hippocampus

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Delta9-tetrahydrocannabinol Is a Full Agonist at CB1 Receptors on GABA Neuron Axon Terminals in the Hippocampus

Nora Laaris et al. Neuropharmacology.

Abstract

Marijuana impairs learning and memory through actions of its psychoactive constituent, delta-9-tetrahydrocannabinol (Delta(9)-THC), in the hippocampus, through activation of cannabinoid CB1 receptors (CB1R). CB1Rs are found on glutamate and GABA neuron axon terminals in the hippocampus where they control neurotransmitter release. Previous studies suggest that Delta(9)-THC is a partial agonist of CB1Rs on glutamate axon terminals in the hippocampus, whereas its effects on GABA terminals have not been described. Using whole-cell electrophysiology in brain slices from C57BL6/J mice, we examined Delta(9)-THC effects on synaptic GABA IPSCs and postsynaptic GABA currents elicited by laser-induced photo-uncaging (photolysis) of alpha-carboxy-2-nitrobenzyl (CNB) caged GABA. Despite robust inhibition of synaptic IPSCs in wildtype mice by the full synthetic agonist WIN55,212-2, using a Tween-80 and DMSO vehicle, Delta(9)-THC had no effects on IPSCs in this, or in a low concentration of another vehicle, randomly-methylated beta-cyclodextrin (RAMEB, 0.023%). However, IPSCs were inhibited by Delta(9)-THC in 0.1% RAMEB, but not in neurons from CB1R knockout mice. Whereas Delta(9)-THC did not affect photolysis-evoked GABA currents, these responses were prolonged by a GABA uptake inhibitor. Concentration-response curves revealed that the maximal effects of Delta(9)-THC and WIN55,212-2 were similar, indicating that Delta(9)-THC is a full agonist at CB1Rs on GABA axon terminals. These results suggest that Delta(9)-THC inhibits GABA release, but does not directly alter GABA(A) receptors or GABA uptake in the hippocampus. Furthermore, full agonist effects of Delta(9)-THC on IPSCs likely result from a much higher expression of CB1Rs on GABA versus glutamate axon terminals in the hippocampus.

Figures

Figure 1
Figure 1
Effects of cannabinoid agonists on GABAA receptor-mediated currents evoked synaptically, or through uv laser photolysis of CNB-caged GABA (50 μM) in WT mouse hippocampal CA1 pyramidal neurons. All recordings were performed during blockade of AMPA/kainate receptors with DNQX (10 μM) and NMDA receptors with APV (40 μM). A. Representative signal averages of outward GABAA receptor currents activated through either electrical stimulation (synaptic) or local photolysis of caged GABA by uv light (λ, applied at arrow) focused through the microscope objective. Note that both the synaptic and the photolysis GABA responses were completely blocked by the GABAA channel blocker picrotoxin (100 μM). B. Effects of the cannabinoid agonist WIN55,212-2 (dissolved in Tween-80/DMSO) on the mean time course of GABAA currents elicited through electrical stimulation or CNB-GABA photolysis in the same CA1 pyramidal neurons (n = 6 neurons). C. Mean time course of the effect Δ9-THC (dissolved in Tween-80/DMSO) on photolysis- and synaptically-evoked GABAA currents (n = 5 pyramidal neurons). Note the absence of an effect of Δ9-THC on IPSCs or photolysis-evoked currents in these experiments. A gluconic acid-based intracellular solution was used in these experiments.
Figure 2
Figure 2
Effects of different concentrations of RAMEB on Δ9-THC inhibition of IPSCs, and concentration response comparison with WIN55,212-2. A. Mean time course of the effect of Δ9-THC (10 μM) dissolved in 0.023% RAMEB solution on synaptic- and photolysis-evoked GABAA-mediated currents recorded in the same CA1 pyramidal neurons (n = 9 neurons). Note the absence of a significant effect of Δ9-THC on GABA currents in this concentration of the vehicle. B. Mean time course of the effect of Δ9-THC (10 μM) on photolysis- and synaptically-evoked GABAA currents (n = 11 neurons), using a higher concentration of the RAMEB vehicle (0.1%). Note the significant inhibition of the synaptically-evoked IPSCs by Δ9-THC using the 0.1% RAMEB solution, and the absence of effects on the photolysis-evoked currents. C. Concentration-dependent effect of Δ9-THC, suspended in 0.1% RAMEB, and WIN55,212-2 in Tween-80-DMSO, on synaptically evoked IPSCs. The EC50 for Δ9-THC was 1.22 μM (95% C.I. = 0.86 μM to 1.73 μM). Note that the maximal effects of Δ9-THC and WIN55,212-2 on IPSCs are comparable (p > 0.05, t-test). The number of neurons used for each point on the concentration-response curve was 5-7. The data shown in all subsequent figures used Δ9-THC in the 0.1 % concentration of RAMEB.
Figure 3
Figure 3
Effects of Δ9-THC on photolysis-evoked and synaptic GABAA currents in WT (CB1+/+) and KO(CB1-/-) mice. The GABAA receptor-mediated currents were evoked by alternating electrical stimulation of the hippocampal slice (synaptic) with uv laser photolysis of caged GABA in the same pyramidal neurons. A1. Signal averages of synaptic and photolysis-evoked GABA currents obtained prior to (black lines), and during Δ9-THC (10 μM) application (gray lines) in a CA1 pyramidal neuron from a CB1+/+ mouse. A2. The mean time course of the effect of Δ9-THC on photolysis-evoked and synaptic GABAA currents (n = 11 CA1 pyramidal neurons). Note the significant effect of Δ9-THC on synaptic IPSCs and the absence of an effect on photolysis-evoked currents. B1. Representative signal averages demonstrating the lack of Δ9-THC (gray lines) effect on synaptic and photolysis-evoked responses in CA1 pyramidal neuron obtained from CB1-/- mice (n = 6). B2. Mean time course demonstrating the lack of Δ9-THC effect on the GABAA currents. A gluconic acid-based intracellular solution was used in these experiments.
Figure 4
Figure 4
Δ9-THC acts presynaptically to reduce GABAergic neurotransmission in the hippocampus. A. Effect of the competitive GABA uptake inhibitor nipecotic acid (10 μM, gray line) on averaged (n = 6-9 individual sweeps) inward currents evoked by CNB-GABA photolysis (uv laser flash applied at λ) in a CA1 pyramidal neuron. The control sweep is shown in black. Also shown (dashed lines) are single exponential curves fitted to the decay phase of the currents that were used to calculate the decay time constants (tau). Note that nipecotic acid significantly increased the tau value in this cell (control tau =100.8 ms, 95% C.I. = 99.0-102.7 ms; nipecotic acid tau =120.9 ms, 95% C.I. = 118.3-123.5 ms). Currents shown here and in B are scaled to the same amplitude. B. Lack of an effect of Δ9-THC (10 μM, gray line) on photolysis-evoked GABA current decay in a CA1 pyramidal neuron (control tau = 116.8 ms, 95% C.I. = 115.0-118.7 ms; Δ9-THC tau = 116.9 ms, 95% C.I. = 114.4-119.5 ms). C. Summary of the effect of nipecotic acid and Δ9-THC on decay tau values for GABA currents evoked by synaptic stimulation and photolysis of CNB caged GABA. Values represent ratios of the tau value acquired after drug application to that obtained during the control period. Note that Δ9-THC did not significantly affect synaptic or photolysis-evoked GABA current decay, whereas nipecotic acid prolonged the tau values for each of these currents. *, p < 0.01; **, p < 0.001, paired t-test. These data indicate that GABA uptake was unaffected by Δ9-THC under the present recording conditions.

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