PMC

Up-state duration is increased in cultures after withdrawal from chronic treatment with 44 mM EtOH. Example traces: black trace is a representative example of an up-state from a control treated culture and gray trace is an example from a chronic EtOH culture. Graphs show summary data of baseline measures of up-state parameters in control and CET culture (N = 23–26). Horizontal lines represent group means. Asterisks represent significant group differences (p < 0.05).

The effects of WIN are blunted in cultures chronically treated with 44 mM EtOH, without changes in CB1 protein expression. (A) Left: representative traces of up-states recorded from layer V/VI PNs in control and CET cultures under baseline conditions and after application of the CB1 agonist WIN (1 μM). (A) Right: summary data from the experiments comparing the WIN-induced change in up-state parameters between control and CET cultures. (B) Top: summary data for CB1 immunoblots expressed as optical density of 52 kDa band (N = 3–4). Bars represent mean ± s.e.m. Asterisks represent significant group differences: ^*p < 0.05 and ^**p < 0.01.

Model diagram summarizing the effects of CB1 activation on specific synapses under basal conditions and following chronic EtOH treatment. Under baseline conditions, CB1 activation reduces GABAergic and NMDA receptor-mediated glutamate transmission at synapses on layer II/III and layer V/VI PNs. The inhibition of GABAergic transmission onto layer II/III PNs is much greater than at GABA synapses on layer V/VI neurons. There is no difference in the inhibition of glutamate transmission between layers. Following chronic EtOH treatment, the CB1-mediated disinhibition of layer II/III PNs is reduced, and there is a gain of function in CB1 signaling at deep-layer GABA synapses effectively switching the cannabinoid sensitivity between cortical layers. Chronic EtOH treatment does not affect CB1 signaling at glutamate synapses. Abbreviations: CB1, cannabinoid receptor 1; CCK+, cholecystokinin positive; Cl−, chloride ion, ECs, endocannabinoids; EPSP, excitatory post-synaptic potential; GABA, γ-amino-butyric acid; IPSP, inhibitory post-synaptic potential; Na+, sodium ion; WIN, WIN 55,212-2.

CB1-mediated inhibition of glutamatergic transmission is not altered by CET. (A) The effect of WIN on evoked NMDA EPSCs recorded from layer II/III and layer V/VI PNs in control and CET cultures (N = 8–9). White bars represent baseline measures and gray bars represent EPSCs recorded following a 10 min application of WIN (1 μM). The traces to the right are representative examples from each of the four groups. Traces in black are baseline, and the superimposed gray traces are from the same neurons in the presence of WIN. (B) The percent inhibition of EPSCs by WIN in layer II/III and layer V/VI neurons from CET and control cultures. White bars represent control treated cultures and gray bars represent data from CET cultures. All data shown are mean ± s.e.m. Symbols: †, main effect of cortical layer; ^*, significant pair-wise comparison. For all symbols conferring statistical significance: single symbol p < 0.05, double symbol p < 0.01, triple symbol p < 0.001.

CB1-mediated inhibition of GABAergic sIPSCs onto layer V/VI PNs is enhanced following CET. (A) The effect of WIN on spontaneous GABAA IPSCs recorded from layer II/III and layer V/VI PNs in control and CET cultures (N = 7–12). White bars represent baseline measures and gray bars represent IPSCs recorded following a 10 min application of WIN (1 μM). Traces in the middle of (A) are representative examples from each of the four groups. The group of traces to the right is from control cultures, and traces to the left are from CET cultures. The top row of traces is from layer II/III PNs, and the bottom row is from layer V/VI. Black traces represent baseline recordings, and the adjacent gray traces are from the same neuron after a 10 min WIN application. All data shown are mean ± s.e.m. Symbols: †, main effect of EtOH treatment; ^*, significant pair-wise comparison. (B) the percent change of sIPSCs from layer II/III and layer V/VI neurons in control and CET cultures. All data shown are mean ± s.e.m. Symbols: §, interaction; †, main effect of cortical layer; ^*, significant post-hoc comparisons. For all symbols conferring statistical significance: single symbol p < 0.05, double symbol p < 0.01.

CB1-mediated inhibition of GABAergic synapses on layer II/III PNs is blunted following CET. (A) The effect of WIN on evoked GABAA IPSCs recorded from layer II/III and layer V/VI PNs in control and CET cultures (N = 6–13). White bars represent baseline measures and gray bars represent IPSCs recorded following a 10 min application of WIN (1 μM). The traces to the right are representative examples from each of the four groups. Traces in black are baseline, and the superimposed gray traces are from the same neurons in the presence of WIN. (B) The percent inhibition of IPSCs by WIN in layer II/III and layer V/VI neurons from CET and control cultures. White bars represent control treated cultures and gray bars represent data from CET cultures. Right panel shows eIPSC area for individual neurons before (closed circles) and after (open circles) exposure to WIN. All data shown are mean ± s.e.m. Significant interactions are denoted by section signs (§) and significant post-hoc tests are indicated by asterisks (^*). For all symbols conferring statistical significance: single symbol p < 0.05, double symbol p < 0.01, triple symbol p < 0.001.