Cannabinoids decrease the K(+) M-current in hippocampal CA1 neurons

Abstract

Cannabinoid effects on sustained conductances that control neuronal excitability have not been investigated in brain. Here, intracellular voltage-clamp recordings were performed using the rat hippocampal slice preparation to study the postsynaptic effect of cannabinoid agonists on CA1 pyramidal neurons. Superfusion of the cannabimimetics WIN55212-2 or methanandamide onto CA1 neurons elicited an inward steady-state current that reversed near the equilibrium potential for K(+) and voltage-dependently activated from a threshold of approximately -70 mV. The cannabinoid receptor (CB1) antagonist SR141716 did not alter membrane properties but prevented this effect. Further investigation revealed that the inward current elicited by cannabinoids was caused by a decrease of the noninactivating voltage-dependent K(+) M-current (I(M)). Cannabinoids had no effect in slices pretreated with the M-channel blocker linopirdine. Assessment of the I(M) relaxation indicated that cannabinoids decreased I(M) in a concentration-dependent manner, with a maximum inhibition of 45 +/- 3% with WIN55212-2 (EC(50) of 0. 6 microM) and 41 +/- 5% with methanandamide (EC(50) of 1 microM). Cannabinoids did not affect the inwardly rectifying cationic h-current (I(h)). The cannabinoid-induced I(M) decrease was prevented by SR141716 but remained unaffected by the muscarinic receptor antagonist atropine. Conversely, the cholinergic agonist carbamylcholine decreased I(M) in the presence of SR141716, indicating that cannabinoid and muscarinic receptor activation independently diminish I(M). It is concluded that cannabinoids may postsynaptically augment the excitability of CA1 pyramidal neurons by specifically decreasing the persistent voltage-dependent I(M).

Fig. 1.

Cannabinoids elicit an inward steady-state current. A, Selected current traces obtained with anI–V protocol. This representative CA1 pyramidal neuron held at −56 mV was subjected to three different voltage steps sequentially applied and superimposed at each condition (voltage protocol at bottom left). Superfusion of 5 μm mAEA induced an inward steady-state current at depolarized potentials (170 pA at −42 mV) but had no effect in the hyperpolarized range. RMP was −69 mV. B, Net currents averaged from five neurons exposed to 5 μm mAEA. The cannabinoid elicited a voltage-dependent inward current that reversed at −87 mV, with recovery to control values on washout of the drug.C, Plot of the mAEA-induced conductance derived fromB. G_mAEA was calculated asI_mAEA/(V − V_rev), whereI_mAEA is the mAEA-induced current,V is the command potential, andV_rev is the reversal potential. The conductance was voltage-dependent and activated at approximately −75 mV.

Fig. 2.

The cannabinoid inward current is concentration-dependent. Averaged steady-state currents elicited with different concentrations of mAEA: 0.1 μm(n = 3), 0.25 μm(n = 4), 1 μm (n= 4), 5 μm (n = 5), and 10 μm (n = 4). The amplitude of the inward current increased with the concentration of mAEA. The threshold response was 0.25 μm, and the maximum effect was reached with 5 μm.

Fig. 3.

The cannabinoid inward current is elicited via activation of CB1 receptors. A, Selected current traces from a neuron exposed to the CB1 antagonist SR1 (1 μm) and mAEA (5 μm) in the presence of SR1. SR1 alone had no effect but completely prevented the mAEA response. RMP was −67 mV, andV_H was −59 mV. B, Net currents averaged from seven neurons exposed to 1 μm SR1 alone and three neurons exposed to 5–10 μm mAEA in the presence of SR1. The antagonist completely prevented the mAEA effect.C, Net currents elicited by WIN-2 in the absence (2 μm; n = 6) or presence (2–5 μm; n = 5) of 1 μm SR1. WIN-2 elicited a voltage-dependent inward current that was completely prevented by SR1.

Fig. 4.

Cannabinoids decreaseI_M. A, Current recordings showing I_M relaxations from a neuron held at −44 mV. Hyperpolarizing voltage commands (3 steps superimposed, protocol at bottom left) were applied to deactivateI_M (slow relaxation at command onset). WIN-2 elicited an I_M decrease associated with an inward holding current (dotted line is control holding current). The I_M relaxations identified withletters are magnified and superimposed on thefarright for comparison. RMP was −67 mV. B, Average of I_Mamplitude in nine cells tested with 2–5 μm WIN-2. The cannabinoid decreased I_M by 44% with recovery to 85% of control upon washout. C, Net steady-state currents from five neurons exposed to the selectiveI_M inhibitor linopirdine, followed by WIN-2. Linopirdine (10 μm) elicited a voltage-dependent inward current because of blockade of M-channels. Further addition of 2 μm WIN-2 had no effect, indicating that cannabinoids affected only I_M. D, Recordings showing I_h relaxations observed with hyperpolarizing voltage commands to −103 and −119 mV (V_H of −58 mV). Superfusion of 5 μm mAEA did not alter I_hamplitude. RMP was −68 mV.

Fig. 5.

The cannabinoid-inducedI_M decrease is concentration-dependent.A, I_M recordings from a neuron exposed to 1 μm mAEA. Superfusion of mAEA decreased I_M by 27% (I_M relaxations magnified on the far right) and elicited a limited inward holding current. RMP was −68 mV, and V_H was −47 mV.B, Superfusion of 5 μm mAEA produced a larger I_M decrease (by 58% on this cell; relaxations magnified on far right) associated with a pronounced inward holding current. RMP was −71 mV, andV_H was −43 mV. C, Dose–response curve of I_M inhibition by WIN-2 (filled circles) or mAEA (open squares). The threshold response was below 0.2 μm, and maximal effects were obtained with 3 μm WIN-2 (EC₅₀ of 0.6 μm;dashed line) to inhibit I_M by 45% and 6 μm mAEA (EC₅₀ of 1 μm;dotted line) to inhibitI_M by 41%.

Fig. 6.

The cannabinoid-inducedI_M decrease is mediated via CB1 receptors.A, I_M relaxation elicited with a 10 mV hyperpolarizing step (V_H of −42 mV). A first application of 1 μm WIN-2 decreasedI_M by 47%. After washout of WIN-2 coincident with superfusion of 1 μm SR1, a second application of WIN-2 in the continued presence of SR1 had no effect onI_M. The bottom panel shows the magnified I_M relaxations. RMP was −71 mV. B, Average of I_Mamplitude on five neurons exposed to 1–5 μm WIN-2 in slices treated with 1 μm SR1, showing the lack of effect of the cannabinoid in presence of the CB1 antagonist. C, SR1 also prevented the I_M decrease expected with superfusion of 5 μm mAEA. RMP was −67 mV, andV_H was −48 mV.

Fig. 7.

Cannabinoid and muscarinic receptor agonists independently decrease I_M. A,I_M relaxation elicited with a 10 mV hyperpolarizing step (V_H of −47 mV) in the presence of the muscarinic receptor antagonist atropine (1 μm). Carbachol (CCh, 5 μm) had no effect on I_M because of blockade of muscarinic receptors, but addition of 2 μm WIN-2 in the continued presence of atropine decreased I_M. RMP was −67 mV. B, AverageI_M amplitude on five cells exposed to 1 μm atropine, followed by 2 μm WIN-2. The cannabinoid-induced I_M decrease was unaffected by the muscarinic receptor antagonist. C,I_M relaxation elicited with a 10 mV hyperpolarizing step (V_H of −44 mV) in the presence of SR1. WIN-2 had no effect on I_Mbecause of blockade of CB1 receptors, but 5 μm CCh decreased I_M (washout performed in atropine). RMP was −69 mV.

Fig. 8.

Summary chart of I_Minhibition by cannabinoids. Superfusion of SR1 alone did not affectI_M amplitude (2% augmentation). WIN-2 decreased I_M by 45%, an effect prevented in the presence of SR1 (3% decrease) but unaltered by atropine (44% decrease). Comparable results were obtained with mAEA that decreasedI_M by 41% in absence of SR1 and by 6% in presence of the CB1 antagonist.