Activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the lateral amygdala

Abstract

Previously, we found that in the lateral amygdala (LA) of the mouse, WIN55,212-2 decreases both glutamatergic and GABAergic synaptic transmission via activation of the cannabinoid receptor type 1 (CB1), yet produces an overall reduction of neuronal excitability. This suggests that the effects on excitatory transmission override those on inhibitory transmission. Here we show that CB1 activation by WIN55,212-2 and Delta(9)-THC inhibits long-term depression (LTD) of basal synaptic transmission in the LA, induced by low-frequency stimulation (LFS; 900 pulses/1 Hz). The CB1 agonist WIN55,212-2 blocked LTD via G(i/o) proteins, activation of inwardly rectifying K+ channels (K(ir)s), inhibition of the adenylate cyclase-protein kinase A (PKA) pathway, and PKA-dependent inhibition of voltage-gated N-type Ca2+ channels (N-type VGCCs). Interestingly, WIN55,212-2 effects on LTD were abolished in CB1 knock-out mice (CB1-KO), and in conditional mutants lacking CB1 expression only in GABAergic interneurons, but were still present in mutants lacking CB1 in principal forebrain neurons. LTD induction per se was unaffected by the CB1 antagonist SR141716A and was normally expressed in CB1-KO as well as in both conditional CB1 mutants. Our data demonstrate that activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the LA. These findings suggest that CB1 expressed on either glutamatergic or GABAergic neurons play a differential role in the control of synaptic transmission and plasticity.

Figure 1.

Cannabinoids block the induction of LTD of synaptic transmission in the LA. (A) Low frequency stimulation (LFS; 900 pulses/1 Hz) of afferents induces a LTD of extracellularly recorded excitatory postsynaptic field potentials (FPs) in the LA (n = 7; P < 0.05; filled circles). The CB1 receptor agonist WIN55,212-2 blocks the induction of LTD at the concentration of 2.5 μM (WIN 2.5 μM: n = 7; P > 0.05; open circles), while the lower concentration of 1.0 μM has no effect on LTD induction (n = 6; P < 0.05; open squares). (B) The same stimulation paradigm depresses intracellularly recorded EPSCs in the LA (n = 10; P < 0.05; filled triangles), without changing the paired-pulse ratio (interstimulus interval [ISI] 50 msec; n = 10; P > 0.05; filled squares). LTD of EPSCs is also blocked by WIN55,212-2 (2.5 μM; n = 7; P > 0.05; open triangles). Representative traces are shown. All data are normalized to the respective control values (last 10 min before LTD induction). Asterisks mark the stimulation artifacts.

Figure 2.

The effect of WIN55,212-2 on LTD is mediated by CB1 receptors. (A) Pretreatment of the slices with the CB1 receptor antagonist SR141716A (SR; 2.5 μM) prior to WIN55,212-2 application prevents the effect of WIN55,212-2 and restores LTD induction (n = 6; P < 0.05; open circles). Pretreatment of the slices with the antagonist SR141716A per se does not affect LTD induction (n = 5; P < 0.05; filled circles). (B) WIN55,212-2 (2.5 μM) does not inhibit LTD of EPSCs in mice lacking CB1 receptors (CB1-KO; n = 5; effect of LFS: P < 0.05; open circles) but in slices from their wild-type littermates (n = 4; effect of LFS: P > 0.05; filled circles). (C) In the presence of Δ⁹-THC (1 μM), LFS only induced weak LTD (n = 6; P > 0.05; open circles). In control slices, LFS induced LTD (n = 6; P < 0.05; filled circles). Representative traces are shown. All data are normalized to the respective control values (last 10 min before LTD induction). Asterisks mark the stimulation artifacts.

Figure 3.

Activation of CB1 receptors located on GABAergic interneurons are necessary for the WIN55,212-2-induced inhibition of LTD. (A) In slices of wild-type (CB1^f/f) and CB1^{f/f;CaMKIIaCre} mice, low frequency stimulation (LFS; 900 pulses/1 Hz) induces a LTD of EPSCs in the LA (n = 6; P < 0.05; filled circles). The CB1 receptor agonist WIN55,212-2 (WIN; 2.5 μM) blocks the induction of LTD (n = 6; P > 0.05; open circles) in both genotypes. (B) In slices of CB1^f/f and CB1^{f/f;Dlx5/6Cre} mice, LFS (900 pulses/1 Hz) of afferents induces a LTD of EPSCs in the LA (n = 6; P < 0.05; filled squares). WIN55,212-2 (WIN; 2.5 μM) blocks the induction of LTD (n = 6; P > 0.05; open squares) only in slices of CB1^f/f mice but not in slices of CB1^{f/f;Dlx5/6Cre} mice. All data are normalized to the respective control values (last 10 min before LTD induction).

Figure 4.

CB1 receptor–mediated inhibition of LTD in the LA involves activation of G_i/o proteins and inhibition of the adenylate cyclase-protein kinase A pathway. (A) Preincubation of the slices with pertussis toxin (PTX; 5 μg/mL) for 5–7 h at 37°C prevents the inhibiting effect of WIN55,212-2 (WIN; 2.5 μM) on LTD induction (n = 7; P < 0.05). (B) The selective activation of the adenylate cyclase with 10 μM forskolin (FSK) abolishes the effect of WIN55,212-2 (WIN; 2.5 μM), and restores LTD (n = 5; P < 0.05). (C) In accordance with this result, WIN55,212-2 (WIN; 2.5 μM) also fails to block LTD when the protein kinase A is activated by 25 μM Sp-cAMPs (n = 5; P < 0.05). Representative traces are shown. All data are normalized to the respective control values (last 10 min before LTD induction). Asterisks mark the stimulation artifacts.

Figure 5.

CB1 receptor–mediated inhibition of LTD in the LA involves the activation of K_irs. (A) When voltage-dependent K⁺-channels are blocked with 100–300 μM 4-AP, WIN55,212-2 (2.5 μM) still inhibits LTD induction under the condition of standard extracellular Ca²⁺ concentration of 2 mM (n = 7; P > 0.05; open circles). Similar results are obtained when the extracellular Ca²⁺ concentration is decreased to 1 mM (n = 7; P > 0.05; filled circles) to eliminate the problem of a possible interaction of K⁺-channel blockade with presynaptic Ca²⁺ influx. (B) However, blockade of K_irs with BaCl₂ (300 μM) prevents the effects of WIN55,212-2 (WIN; 2.5 μM) and restores LTD induction. This is the case under the conditions of both standard (2 mM; n = 5; P < 0.05; open circles) and decreased (1 mM; n = 4; P < 0.05; filled circles) extracellular Ca²⁺ concentration. In slices of CB1^{f/f;CaMKIIαCre} mice, where CB1 is expressed exclusively on interneurons, but not on glutamatergic principal neurons, BaCl₂ inhibits the effect of WIN55,212-2 on LTD under the conditions of both 1 and 2 mM Ca²⁺ concentration (1 mM; n = 6; P < 0.05; open squares; 2 mM; n = 6; P < 0.05; filled squares). (C) Control experiments show that an extracellular Ca²⁺ concentration of 1 mM is not sufficient to induce LTD (n = 5; P > 0.05; filled circles). However, in the presence of both 100–300 μM 4-AP (n = 9; P < 0.05; open circles) and 300 μM BaCl₂ (n = 4; P < 0.05; filled triangles), LFS again induces LTD similar to control values. Representative traces are shown. All data are normalized to the respective control values (last 10 min before LTD induction). Asterisks mark the stimulation artifacts.

Figure 6.

CB1 receptor activation increases K_ir currents in interneurons of the LA. (A) Current-voltage (I–V) curves of membrane currents elicited by depolarization ramps at time points of control (a), WIN55,212-2 (WIN; 2.5 μM) (b), BaCl₂ (300 μM) (c), BaCl₂ plus WIN55,212-2 (WIN; 2.5 μM) (d), and using slices of CB1-KO mice (WIN; 2,5 μM) (e) are shown. At a membrane potential of –100 mV, WIN55,212-2 (WIN; 2.5 μM) increases the membrane current (a) to 148 ± 15% (b; n = 4; P < 0.05). In the presence of the K_ir-channel blocker BaCl₂ (300 μM; c) and in CB1-KO (e), WIN55,212-2 does not affect the membrane current anymore (n = 4; P > 0.05). (B) The I-V curve of the WIN55,212-2–induced current is obtained by subtracting the current in the control from that during exposure to the CB1 receptor agonist WIN55,212-2 (b − a).

Figure 7.

CB1 receptor activation decreases Ca²⁺ influx through presynaptic N-type VGCCs of interneurons via inhibition of the PKA. (A) Using Ba²⁺ as the charge carrier, voltage-gated calcium channel (VGCC) currents are evoked by step depolarization from a holding potential of –70 mV to 0 mV for 300 msec. WIN55,212-2 (WIN; 2.5 μM) reduces the VGCC-mediated currents significantly (n = 4; P < 0.05; left). In the presence of cadmium (Cd²⁺; 10 μM), step depolarization from –70 mV to 0 mV does not evoke any currents anymore, proving that the measured currents are mediated by VGCCs (n = 3; P > 0.05; right and Aa). The WIN55,212-2-mediated effect on VGCC was voltage-independent (n = 5; Ab). (B) N-type VGCCs are isolated by application of the T/R-type VGCC blocker nickel (Ni²⁺, 50 μM), the L-type VGCC blocker nifedipine (NIF; 20 μM), and the P/Q-type VGCC blocker ω-agatoxin IVA (AgTx; 200 nM). WIN55,212-2 (WIN; 2.5 μM) clearly reduces the remaining current (n = 5; P < 0.05). This effect of WIN55,212-2 is abolished in the presence of the PKA blocker Rp-cAMPs (25 μM; n = 3; P > 0.05), which itself already reduces N-type VGCC currents (n = 3; P < 0.05). (C,D) WIN55,212-2 (2.5 μM) does not reduce N-type VGCC currents in slices pretreated with the CB1 antagonist SR141716A (2.5 μM; n = 5; P > 0.05) or in slices of CB1-KO mice (n = 4; P > 0.05).