Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission

Abstract

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 mum) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1(f/f;CaMKIIalphaCre)), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncaged l-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1(f/f;Dlx5/6-Cre)), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.

Figure 1.

In the BLA, activation of CB1 does not affect FP and EPSC amplitudes of mice lacking CB1 expression in principal neurons. A, WIN (5 μm) reduces FP amplitudes in wild-type CB1^f/f mice to 69.5 ± 5.2% of baseline (n = 9). This effect is completely abolished in CB1^{f/f;CaMKIIαCre} mice (5 μm WIN; 107.4 ± 5.0%; n = 9). B, C, WIN significantly reduces isolated glutamatergic FPs (B) and AMPA-EPSCs (C) in CB1^f/f mice to 73.1 ± 8.4% (n = 9) and to 55.3 ± 6.4% (n = 8) of baseline, respectively, but not in the CB1^{f/f;CaMKIIαCre} mice. Representative traces are shown. Data are normalized to the respective baseline values (10 min of baseline). Asterisks represent stimulation artifacts. Picro, Picrotoxin; CGP, CGP 35348.

Figure 2.

In the BLA, CB1 activation reduces FP and EPSC amplitudes of mice lacking CB1 expression in GABAergic interneurons. A, In CB1^f/f and CB1^{f/f;Dlx5/6-Cre}, WIN (5 μm) depresses FP amplitudes significantly (CB1^f/f: 60.2 ± 10.3%, n = 7; CB1^{f/f;Dlx5/6-Cre}: 44.0 ± 10.4%, n = 7). B, WIN reduces the amplitude of isolated AMPA-EPSCs to 47.1 ± 10.0% (n = 8) and to 44.2 ± 6.5% of baseline in CB1^f/f and in CB1^{f/f;Dlx5/6-Cre}, respectively (n = 9). Picro, Picrotoxin; CGP, CGP 35348.

Figure 3.

In the hippocampus and somatosensory cortex, CB1 on glutamatergic neurons is necessary for the WIN-induced inhibition of glutamatergic transmission. A, In the CA1 region of the hippocampus, EPSPs are reduced to 49.6 ± 4.3% (n = 12) after application of WIN (5 μm) in CB1^f/f mice but not in slices of CB1^{f/f;CaMKIIαCre} mice (109.9 ± 4.2%; n = 12). B, AMPA-EPSC amplitudes are decreased by WIN only in CB1^f/f mice (EPSCs of CB1^f/f: 52.7 ± 11.1%, n = 6; EPSCs of CB1^{f/f;CaMKIIαCre}: 96.9 ± 8.2%, n = 6). C, In the somatosensory cortex of layer II neurons, WIN reduces the AMPA-EPSC amplitude in CB1^f/f mice to 49.7 ± 5.0% (n = 8), whereas in CB1^{f/f;CaMKIIαCre} mice, WIN has no effect (105.4 ± 4.3%; n = 8). Representative traces are shown. Picro, Picrotoxin; CGP, CGP 35348.

Figure 4.

In neurons of limbic structures, cannabinoids reduce glutamatergic synaptic transmission exclusively through presynaptic mechanisms. A, Although the CB1 agonist WIN (5 μm) reduces evoked AMPA-EPSCs in the BLA (to 69.9 ± 6.0%; n = 7; filled circles), inward currents induced by the uncaging of glutamate (3–5 ms, 1 mW, 1 μm laser spot diameter; size of uncaging area, ∼10 μm) are not affected by WIN (105.1 ± 4.6%; n = 7; open circles). B, In the CA1 region of the hippocampus, only synaptic responses are decreased by WIN (AMPA-EPSC: 67.8 ± 6.0, n = 7, filled circles; laser-induced responses: 105.1 ± 4.6%, n = 7, open circles).