Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling

Abstract

Endocannabinoids are crucial for the extinction of aversive memories, a process that considerably involves the amygdala. Here, we show that low-frequency stimulation of afferents in the lateral amygdala with 100 pulses at 1 Hz releases endocannabinoids postsynaptically from neurons of the basolateral amygdala of mice in vitro and thereby induces a long-term depression of inhibitory GABAergic synaptic transmission (LTDi) via a presynaptic mechanism. Lowering inhibitory synaptic transmission significantly increases the amplitude of excitatory synaptic currents in principal neurons of the central nucleus, which is the main output site of the amygdala. LTDi involves a selective mGluR1 (metabotropic glutamate receptor 1)-mediated calcium-independent mechanism and the activation of the adenylyl cyclase-protein kinase A pathway. LTDi is abolished by the cannabinoid type 1 (CB1) receptor antagonist SR141716A and cannot be evoked in CB1 receptor-deficient animals. LTDi is significantly enhanced in mice lacking the anandamide-degrading enzyme fatty acid amide hydrolase. The present findings show for the first time that mGluR activation induces a retrograde endocannabinoid signaling via activation of the adenylyl cyclase-protein kinase A pathway and the release of anandamide. Furthermore, the results indicate that anandamide decreases the activity of inhibitory interneurons in the amygdala. This disinhibition increases the activity of common output neurons and could provide a prerequisite for extinction by formation of new memory.

Figures

Figure 1.

LTDi is mediated by endocannabinoids and is independent of postsynaptic Ca2+ influx. A, LFSi of afferents in the LA (100 pulses/1 Hz) leads to a significant long-term depression of IPSCs recorded in principal BLA neurons (LTDi) to 78 ± 5% of control (n = 6; p < 0.05). B, Pretreatment of the slices with the CB1 receptor agonist WIN55,212-2 (5 μm) occludes the induction of LTDi (104 ± 10% of control; n = 5; p > 0.05). LTDi without WIN55,212-2 (data from A) is shown for comparison (squares). C, LTDi is not inhibited when BAPTA (40 mm) is applied to the postsynaptic cell (67 ± 7% of control; n = 7; p < 0.05). Representative traces of IPSCs before (1) and after (2) LFSi are shown. The experiments were performed in C57BL/6JOlaHsd mice.

Figure 2.

Induction of LTDi involves activation of group I mGluRs. A, The unselective group I mGluR agonist DHPG (50 μm) leads to a long-lasting suppression of GABAergic currents similar to LTDi in wild-type mice (CB1+/+; effect of DHPG, 72 ± 5% of control; n = 8; p < 0.05; •) but not in mice lacking CB1 receptors (CB1-/-; effect of DHPG, 101 ± 3%; n = 5; p > 0.05; ○). B, Application of DHPG (50 μm) occludes the induction of LTDi by LFSi (effect of LFSi in the presence of DHPG, 106 ± 13% of control; n = 6; p > 0.05). LTDi without DHPG (data from Fig. 1 A) is shown for comparison (squares). C, The transient application of DHPG (50 μm) also induces a long-lasting decrease of the IPSC amplitude to 64 ± 3% (n = 8; p < 0.05). D, This effect of DHPG is occluded by a preceding induction of LTDi (control, 100%; LFSi, 66 ± 8%; n = 5; p < 0.05; DHPG, 75 ± 4%; n = 5; p > 0.05). Representative traces of IPSCs are shown. The experiments depicted in B-D were performed in C57BL/6JOlaHsd mice.

Figure 3.

Induction of LTDi involves activation of mGluR1, but not of mGluR5. A, LTDi induction is completely inhibited by bath application of the mGluR1 antagonist CPCCOEt (50 μm; effect of LFSi, 105 ± 4% of control; n = 6; p > 0.05). LTDi without CPCCOEt (data from Fig. 1 A) is shown for comparison (squares). B, Preincubation of the slices with the mGluR5 antagonist MPEP (10 μm) has no effect on the induction of LTDi by LFSi (effect of LFSi in the presence of MPEP, 63 ± 10% of control; n = 5; p < 0.05). Representative traces of IPSCs before (1) and after (2) LFSi are shown. The experiments were performed in C57BL/6JOlaHsd mice.

Figure 4.

LTDi is expressed presynaptically and induced postsynaptically via activation of G-proteins. A, Induction of LTDi is accompanied by a reduction of the frequency of sIPSCs from 6.4 ± 0.6 Hz to 2.7 ± 0.5 Hz (n = 5; p < 0.05), whereas the amplitude of sIPSC remains unaltered (control, 7.4 ± 1.1 pA; LFSi, 6.9 ± 0.9 pA; n = 5; p > 0.05). Representative traces of sIPSCs and the cumulative probability of the sIPSC amplitude before and after LFSi are shown. B, Postsynaptic application of the non-hydrolizable GTP analog GTPγ-S (600 μm) via the patch pipette blocks LTDi induction (effect of LFSi in the presence of GTPγ-S, 110 ± 9%; n = 4; p > 0.05). LTDi without GTPγ-S (data from Fig. 1 A) is shown for comparison (squares). Representative traces of IPSCs before (1) and after (2) LFSi are shown. The experiments were performed in C57BL/6JOlaHsd mice.

Figure 5.

LTDi induction does not require activation of PLC and DAG lipase. A, After preincubation of the slices with the specific PLC inhibitor U73122 (5 μm) for at least 60 min, IPSCs were recorded in the presence of 5 μm U73122. Inhibition of PLC does not inhibit the induction of LTDi (effect of LFSi in the presence of U73122, 67 ± 6% of control; n = 7; p < 0.05). B, To block the DAG lipase, slices were preincubated with 100 μm RHC-80267 for at least 60 min. Afterward, control values were recorded in the presence of 50 μm RHC-80267. The DAG lipase inhibitor has no effect on the induction of LTDi (control plus RHC-80267, 100%; LFSi, 68 ± 4%; n = 8; p < 0.05). Representative traces of IPSCs before (1) and after (2) LFSi are shown. The experiments were performed in C57BL/6JOlaHsd mice.

Figure 6.

LTDi involves the activation of the AC-PKA pathway and the release of anandamide. A, LTDi is significantly enhanced in mice lacking the anandamide-degrading enzyme FAAH (FAAH-/- mice, 48 ± 9% of control; n = 6; p < 0.05; ○) compared with the wild-type littermates (FAAH+/+ mice, 78 ± 6% of control; n = 9; p < 0.05; •; LTDi in FAAH+/+ vs LTDi in FAAH-/-, p < 0.05). B, LTDi induction is abolished when AC is blocked postsynaptically by 200 μm SQ22,536 (effect of LFSi in the presence of SQ22,536, 103 ± 15% of control; n = 5; p > 0.05). C, LTDi is also inhibited by the postsynaptic application of the PKA inhibitor Rp-cAMP (25 μm; effect of LFSi in the presence of Rp-cAMP, 101 ± 8% of control; n = 6; p > 0.05). LTDi without SQ22,536 or Rp-cAMP (data from Fig. 1 A) is shown for comparison (squares) in B and C, respectively. Representative traces of IPSCs before (1) and after (2) LFSi are shown. The experiments depicted in B and C were performed in C57BL/6JOlaHsd mice.

Figure 7.

LFSi enhances HFS-induced LTP in the BLA and increases excitatory synaptic transmission in the central nucleus via activation of CB1 receptors. A, HFS of afferents in the LA (2 trains of 100 pulses with 50 Hz and an interstimulus interval of 10 sec) induces an LTP of FPs in the BLA (effect of HFS, 115 ± 3% of control; n = 6; p < 0.05; ○). When LFSi is applied 10 min before HFS, the resulting LTP is significantly enhanced (HFS after LFSi, 143 ± 7%; n = 7; p < 0.05; •; HFS vs HFS plus LFSi, p < 0.05). B, Application of the CB1 antagonist SR141716A (5 μm) for at least 40 min does not affect HFS-induced LTP (control plus SR141716A, 100%; HFS, 118 ± 5%; n = 5; p < 0.05; ○) but abolishes the enhancing effect of LFSi on LTP (LFSi plus HFS with SR141716A, 122 ± 7%; n = 6; p < 0.05; LFSi plus HFS vs LFSi plus HFS plus SR141716A, p < 0.05; •). C, LFSi of afferents in the LA increases the amplitude of EPSCs recorded from principal neurons in the central nucleus (CE; 125 ± 10% of control; n = 6; p < 0.05; •). This effect is abolished by preincubation of the slices with the CB1 receptor antagonist SR141716A (SR; 5 μm; effect of LFSi, 101 ± 5% of control; n = 6; p > 0.05; ○; LFSi vs LFSi plus SR141716A, p < 0.05). Schemes on the left depict the respective positions of stimulation and recording electrodes. Representative traces are shown. The experiments were performed in C57BL/6JOlaHsd mice.