Activation of the cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse

Abstract

The endogenous cannabinoid system has been shown recently to play a crucial role in the extinction of aversive memories. As the amygdala is presumably involved in this process, we investigated the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN-2) on synaptic transmission in the lateral amygdala (LA) of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice. Extracellular field potential recordings and patch-clamp experiments were performed in an in vitro slice preparation. We found that WIN-2 reduces basal synaptic transmission and pharmacologically isolated AMPA receptor- and GABA(A) receptor-mediated postsynaptic currents in wild-type, but not in CB1-deficient mice. These results indicate that, in the LA, cannabinoids modulate both excitatory and inhibitory synaptic transmission via CB1. WIN-2-induced changes of paired-pulse ratio and of spontaneous and miniature postsynaptic currents suggest a presynaptic site of action. Inhibition of G(i/o) proteins and blockade of voltage-dependent and G protein-gated inwardly rectifying K(+) channels inhibited WIN-2 action on basal synaptic transmission. In contrast, modulation of the adenylyl cyclase-protein kinase A pathway, and blockade of presynaptic N- and P/Q- or of postsynaptic L- and R/T-type voltage-gated Ca(2+) channels did not affect WIN-2 effects. Our results indicate that the mechanisms underlying cannabinoid action in the LA partly resemble those observed in the nucleus accumbens and differ from those described for the hippocampus.

Figures

Figure 1

Cannabinoid-induced inhibition of synaptic transmission in the mouse LA. (A) The CB1 agonist WIN-2 (5 μM) reduces FP amplitude to 52 ± 6% of baseline (n = 7; P < 0.05), as measured by extracellular recordings. This effect is completely abolished in the presence of the CB1 antagonist SR (SR 5 μM + WIN-2 5 μM, 94 ± 5%; n = 7; P > 0.05), which itself does not have any effect on FP amplitude (97 ± 2% of baseline; n = 4; data not shown). (B) WIN-2 significantly reduces FP amplitude in CB1+/+ mice to 69 ± 9% of baseline (n = 5; P < 0.05), but not in CB1−/− mice (102 ± 12% of baseline;n = 4; P > 0.05; effect of WIN-2 on CB1−/− vs. CB1+/+; P < 0.05). Representative traces are shown. All data are normalized to the respective baseline values (last 10 min of baseline). Asterisks represent stimulation artifacts. Gray bar shows period of WIN-2 superfusion.

Figure 2

Cannabinoids inhibit glutamatergic synaptic transmission in the LA. (A) Whole-cell patch-clamp recordings show that WIN-2 (5 μM) reduces eEPSC amplitude to 71 ± 10% (n = 6;P < 0.05) and AMPA-eEPSC amplitude to 52 ± 4% (n = 6; P < 0.05) of baseline. WIN-2 enhances PPRs of eEPSCs (from 1.08 ± 0.1 to 1.27 ± 0.13; n = 6;P < 0.05) and AMPA-eEPSCs (from 0.99 ± 0.03 to 1.18 ± 0.04; n = 6; P < 0.05). (B) WIN-2 (5 μM) has no effect on AMPA receptor-mediated synaptic transmission in CB1−/− mice (106 ± 14% of baseline;n = 5; P > 0.05), whereas it significantly reduces AMPA-eEPSC amplitude in the CB1+/+ mice to 59 ± 3% of baseline (n = 4; P < 0.05). Representative traces are shown. All data are normalized to the respective baseline values (last 10 min of baseline). Gray bar shows period of WIN-2 superfusion.

Figure 3

Cannabinoids inhibit GABAergic synaptic transmission in the LA. (A) WIN-2 (5 μM) decreases eIPSC amplitude to 50 ± 10% (n = 8; P < 0.05) of baseline, as measured by whole-cell patch-clamp recording. The agonist also enhances PPR of eIPSCs (control, 1.02 ± 0.01; WIN-2, 1.5 ± 0.06;n = 5; P < 0.05), suggesting a presynaptic inhibition of GABAergic synaptic transmission. (B) WIN-2 (5 μM) significantly reduces eIPSC amplitude in the CB1+/+ mice to 47 ± 6% of baseline (n = 4;P < 0.05) without affecting GABAergic synaptic transmission in CB1−/− mice (105 ± 15% of baseline;n = 4; P > 0.05). Representative traces are shown. All data are normalized to the respective baseline values (last 10 min of baseline). Gray bar shows period of WIN-2 superfusion.

Figure 4

Cannabinoids reduce synaptic transmission through presynaptic mechanisms. (A) The CB1 agonist WIN-2 (5 μM) reduces the frequency of sEPSCs from 4.1 ± 1.1 Hz to 1.4 ± 0.3 Hz (n = 12; P < 0.05). A representative pair of traces and the bar diagram (n = 12) are shown. The amplitudes of the sEPSCs are unaffected by WIN-2, as seen in the bars (control, 8.5 ± 0.12 pA; WIN-2, 8.2 ± 0.04 pA; n = 12;P > 0.05) and one representative example of the cumulative probability of sEPSC amplitudes. (B) Application of WIN-2 (5 μM) also reduces the frequency of mEPSCs recorded in the presence of 1 μM TTX (control, 4.5 ± 0.9 Hz; WIN-2, 2 ± 0.7 Hz;n = 6; P < 0.05). A representative pair of traces and the bar diagram are shown. WIN-2 (5 μM) does not alter mean mEPSC amplitude as seen in the bars (control, 6.6 ± 0.8 pA; WIN-2, 6.6 ± 1.1 pA; n = 6; P > 0.05) and one representative example of the cumulative probability of mEPSC amplitudes.

Figure 5

sIPSCs and mIPSCs recorded in the presence of 3 mM extracellular Ca2+. (A) WIN-2 (5 μM) significantly reduces the frequency of sIPSCs (sIPSCs: control, 8.0 ± 1.6 Hz; WIN-2, 3.6 ± 0.7 Hz; n = 5; P < 0.05). One representative pair of traces and the corresponding bar diagram are depicted. The amplitudes of the sIPSCs are not affected by the agonist, as shown in the bar diagram (control, 13.3 ± 3.4 pA; WIN-2, 12.8 ± 3.5 pA; n = 5; P > 0.05), and one representative example of the cumulative probability of the sIPSC amplitudes. (B) In the presence of 1 μM TTX, WIN-2 (5 μM) had similar effects on mean frequency (control, 3.7 ± 0.6 Hz; WIN-2, 2.0 ± 0.6 Hz; n = 6; P < 0.05) and amplitudes of mIPSCs (control, 5.8 ± 0.8 pA; WIN-2, 5.4 ± 0.8 pA;n = 6; P > 0.05).

Figure 6

Cannabinoid action in the LA involves the activation of Gi/o proteins, but not the inhibition of the AC-PKA pathway. (A) Preincubation of slices with the Gi/o protein inhibitor PTX (5 μg/mL) for 5–7 h abolishes the effects of WIN-2 (5 μM) on extracellularly recorded FP amplitudes (WIN-2, 95 ± 6% of baseline; n = 6; P > 0.05). (B) Application of the AC activator FSK (10 μM), but not of the AC inhibitors MDL-12,330A (10 μM) and SQ 22,536 (50 μM), rapidly increases FP amplitude to 139 ± 12% of baseline (n = 7;P > 0.05). Neither the AC activator (FSK, 100%; FSK + WIN-2, 66 ± 9%; n = 7; P < 0.05), nor the AC inhibitors (MDL/SQ, 100%; MDL/SQ + WIN-2, 63 ± 6%;n = 7; P < 0.05) alter WIN-2-induced reduction of FP amplitude. (C) Inhibition of the PKA by Rp-cAMP (25 μM) does not prevent WIN-2 action on synaptic transmission (Rp-cAMP, 100%; Rp-cAMP + WIN-2, 49 ± 10%; n = 4;P < 0.05). Representative traces are shown. All data are normalized to the respective baseline values (last 10 min of baseline). Asterisks mark stimulation artifacts.

Figure 7

Cannabinoid-induced decrease of basal synaptic transmission involves the modulation of voltage-sensitive and inwardly rectifying K+ channels, but is unaffected by inactivation of pre- and postsynaptic Ca2+ channels. (A) Whole-cell recordings reveal that 4-AP (100 μM; control, 100%; 4-AP, 119 ± 14%; n = 5; P > 0.05) and BaCl2 (control, 100%; BaCl2, 96 ± 6%;n = 6; P > 0.05) slightly affect basal synaptic transmission. Both 4-AP and BaCl2 inhibit the effect of WIN-2 (5 μM) on eEPSC amplitude (4-AP, 100%; 4-AP + WIN-2, 103 ± 10%; n = 5; P > 0.05; BaCl2, 100%; BaCl2 + WIN-2, 108 ± 8%; n = 5;P > 0.05). (B1,B2) Under conditions in which the extracellular Ca2+ concentration is decreased to 0.5 mM, both K+ channel blockers increase eEPSC amplitudes markedly (baseline, 100%; 4-AP, 201 ± 20%; n = 4;P < 0.05; baseline, 100%; BaCl2, 169 ± 26%;n = 4; P < 0.05) and inhibit WIN-2 action on synaptic transmission (4-AP, 100%; 4-AP + WIN-2, 94 ± 15%;n = 4; P > 0.05; BaCl2, 100%; BaCl2 + WIN-2, 78 ± 28%; n = 4;P > 0.05). Similar results are obtained when both 4-AP and BaCl2 are applied together (baseline, 100%; 4-AP + BaCl2, 168 ± 23%; n = 4;P < 0.05; 4-AP + BaCl2, 100%; 4-AP + BaCl2 + WIN-2, 98 ± 22%; n = 4;P > 0.05). (C) Blockade of presynaptic N- and P/Q- type Ca2+ channels with ω-Conotoxin (Cntx; 1 μM) and ω-Agatoxin (Agtx; 200 nM) reduce eEPSC amplitudes to 31 ± 6% (n = 4; P < 0.05) and 66 ± 11% (n = 5; P = 0.05), respectively. Blockade of postsynaptic L- and R/T-type Ca2+ channels with Nifedipine 20 μM (control, 100%; Nifedipine, 94 ± 6%; n = 4;P > 0.05) and Ni2+ 50 μM (control, 100%; Ni2+, 90 ± 5%; n = 5; P > 0.05) does not affect eEPSC amplitude. Neither pre- nor postsynaptic Ca2+ channel blockers affect the WIN-2-induced decrease of the eEPSC amplitude (Cntx, 100%; Cntx + WIN-2, 60 ± 8%;n = 5; P < 0.05; Agtx, 100%; Agtx + WIN-2, 59 ± 11%; n = 5; P < 0.05; Cntx + Agtx, 100%; Cntx + Agtx + WIN-2, 67 ± 7%; n = 4;P < 0.05; Nifedipine, 100%; Nifedipine + WIN-2, 66 ± 10%; n = 5; P < 0.05; Ni2+, 100%; Ni2+ + WIN-2, 63 ± 9%; n = 5;P < 0.05). All bars reflect mean ± SEM.

Figure 7

Cannabinoid-induced decrease of basal synaptic transmission involves the modulation of voltage-sensitive and inwardly rectifying K+ channels, but is unaffected by inactivation of pre- and postsynaptic Ca2+ channels. (A) Whole-cell recordings reveal that 4-AP (100 μM; control, 100%; 4-AP, 119 ± 14%; n = 5; P > 0.05) and BaCl2 (control, 100%; BaCl2, 96 ± 6%;n = 6; P > 0.05) slightly affect basal synaptic transmission. Both 4-AP and BaCl2 inhibit the effect of WIN-2 (5 μM) on eEPSC amplitude (4-AP, 100%; 4-AP + WIN-2, 103 ± 10%; n = 5; P > 0.05; BaCl2, 100%; BaCl2 + WIN-2, 108 ± 8%; n = 5;P > 0.05). (B1,B2) Under conditions in which the extracellular Ca2+ concentration is decreased to 0.5 mM, both K+ channel blockers increase eEPSC amplitudes markedly (baseline, 100%; 4-AP, 201 ± 20%; n = 4;P < 0.05; baseline, 100%; BaCl2, 169 ± 26%;n = 4; P < 0.05) and inhibit WIN-2 action on synaptic transmission (4-AP, 100%; 4-AP + WIN-2, 94 ± 15%;n = 4; P > 0.05; BaCl2, 100%; BaCl2 + WIN-2, 78 ± 28%; n = 4;P > 0.05). Similar results are obtained when both 4-AP and BaCl2 are applied together (baseline, 100%; 4-AP + BaCl2, 168 ± 23%; n = 4;P < 0.05; 4-AP + BaCl2, 100%; 4-AP + BaCl2 + WIN-2, 98 ± 22%; n = 4;P > 0.05). (C) Blockade of presynaptic N- and P/Q- type Ca2+ channels with ω-Conotoxin (Cntx; 1 μM) and ω-Agatoxin (Agtx; 200 nM) reduce eEPSC amplitudes to 31 ± 6% (n = 4; P < 0.05) and 66 ± 11% (n = 5; P = 0.05), respectively. Blockade of postsynaptic L- and R/T-type Ca2+ channels with Nifedipine 20 μM (control, 100%; Nifedipine, 94 ± 6%; n = 4;P > 0.05) and Ni2+ 50 μM (control, 100%; Ni2+, 90 ± 5%; n = 5; P > 0.05) does not affect eEPSC amplitude. Neither pre- nor postsynaptic Ca2+ channel blockers affect the WIN-2-induced decrease of the eEPSC amplitude (Cntx, 100%; Cntx + WIN-2, 60 ± 8%;n = 5; P < 0.05; Agtx, 100%; Agtx + WIN-2, 59 ± 11%; n = 5; P < 0.05; Cntx + Agtx, 100%; Cntx + Agtx + WIN-2, 67 ± 7%; n = 4;P < 0.05; Nifedipine, 100%; Nifedipine + WIN-2, 66 ± 10%; n = 5; P < 0.05; Ni2+, 100%; Ni2+ + WIN-2, 63 ± 9%; n = 5;P < 0.05). All bars reflect mean ± SEM.