Article Figures & Data
Figures
- Figure 1.
Single in vivo administration of cocaine abolishes eCB-LTD and increases CC-Homer protein levels. a, Top, Sample fEPSPs before (control) or after induction of eCB-LTD in mice injected in vivo with saline (sham) or cocaine (20 mg/kg) taken at the time indicated on graph below. Bottom, Average time courses of eCB-LTD. Single cocaine injection (n = 27) abolished eCB-LTD (compared with sham; n = 17; 50 min after tetanus; ***p < 0.05). The dashed line represents 100%. b, Representative Western blots of animals administered saline (sham) or cocaine in vivo. The blots show protein levels for CC-Homer using a pan-Homer antibody in the NAc 1 d after injection. Densitometry measurements are expressed as percentage of control ± SEM. Single cocaine exposure (n = 7) markedly increased CC-Homer protein levels in the NAc, compared with saline (n = 7; ***p < 0.05). Immunoblots were quantified by scanning densitometry. c, Top, Densitometry measurements expressed as percentage of control ± SEM and representative Western blots of animals administered saline (sham) or cocaine in vivo. The blots show protein levels for CC-Homer using a pan-Homer antibody in the NAc 1 week after single cocaine injection. One week after a single exposure to cocaine, CC-Homer protein levels were back to normal (n = 5) compared with saline (n = 5; p > 0.05). Bottom, eCB-LTD expressed as percentage of baseline 50 min after LTD induction: eCB-LTD was identical 1 week after a single exposure to cocaine (n = 7) and in sham mice (n = 17; p > 0.05). The dashed line represents 100%.
- Figure 2.
Role of D1R and NMDAR in the cocaine-induced blockade of eCB-LTD. a, Cocaine-induced inhibition of eCB-LTD was prevented by a D1R antagonist. Animals that had received SCH23390 (0.5 mg/kg) displayed eCB-LTD when coinjected with cocaine (n = 9; ***p < 0.05 compared with cocaine alone). D1R -/- mice display normal eCB-LTD (n = 7) but were insensitive to cocaine administration compared with wild type (n = 9; ***p < 0.05). The dashed line represents 100%. b, Representative Western blots of animals administered saline (sham), SCH23390 (SCH; 0.5 mg/kg) alone, or SCH23390 plus cocaine (SCH + Coc) in vivo. The blots show protein levels for CC-Homer using a pan-Homer antibody in the NAc 1 d after injection. Densitometry measurements are expressed as percentage of control ± SEM. In SCH23390-injected animals, single cocaine exposure (n = 6) did not alter CC-Homer protein levels in the NAc, compared with saline (n = 5; p > 0.05) and SCH23390 alone (n = 6; p > 0.05). c, Cocaine-induced inhibition of eCB-LTD is prevented by an NMDAR antagonist. Animals receiving MK801 display normal eCB-LTD when coinjected with cocaine (n = 13; ***p < 0.05 compared with cocaine alone). The dashed line represents 100%. d, Representative Western blots of animals administered saline (sham), MK801 (1 mg/kg) alone, or MK801 plus cocaine (MK801 + Coc) in vivo. The blots show protein levels for CC-Homer using a pan-Homer antibody in the NAc 1 d after injection. Densitometry measurements are expressed as percentage of control ± SEM. In MK801-injected animals, single cocaine exposure (n = 8) did not alter CC-Homer protein levels in the NAc, compared with saline (n = 8; p > 0.05) and MK801 alone (n = 8; p > 0.05).
- Figure 3.
CB1R presynaptic functions and protein levels are not altered by single in vivo cocaine exposure. a, CB1R-mediated fEPSP inhibition. Single cocaine injection (n = 5) did not alter the CB1R-mediated fEPSP inhibition [compared with sham; n = 4; 20 min after application of the CB1R agonist CP55,940 (10 μm); p > 0.05]. b, Densitometry measurements expressed as percentage of control ± SEM. Single cocaine exposure (n = 6) did not alter CB1R protein levels in the NAc, compared with saline (n = 5; p > 0.05).
- Figure 4.
A single in vivo cocaine exposure does not induce a generalized alteration of prefrontal cortex-NAc synapses. a, Single cocaine injection (n = 7) did not alter mGluR2/3-LTD [compared with sham; n = 10; 60 min after application of the selective mGluR2/3 agonist LY354740 (200 nm) for 10 min; p > 0.05]. The dashed line represents 100%. b, Single cocaine exposure did not change NAc protein levels of mGluR2/3. Densitometry measurements are expressed as percentage of control ± SEM. Single cocaine exposure (n = 4) did not alter mGluR2/3 protein levels in the NAc, compared with saline (n = 3; p > 0.05). c, Input-output curves for sham-treated mice and mice treated with a single injection of cocaine were similar. d, Left, Paired-pulse ratios were augmented after eCB-LTD induction in sham animals (1.21 ± 0.0910 min before tetanus compared with 1.36 ± 0.09 50 min after tetanus; n = 12; ***p < 0.05 but not in cocaine-treated animals (1.17 ± 0.08 10 min before tetanus compared with 1.12 ± 0.09 50 min after tetanus; n = 20; p > 0.05). Right, Baseline paired-pulse ratios were identical in vehicle- and cocaine-treated animals [1.21 ± 0.09 (n = 12) in sham animals compared with 1.17 ± 0.08 (n = 20) in cocaine-treated animals; p > 0.05], suggesting that basal probability of transmitter release was unchanged after cocaine injection. tet, Tetanus.
- Figure 5.
Cocaine administration causes a loss of surface mGluR5. a, Single cocaine exposure did not change total NAc protein levels of mGluR5. Representative Western blots of animals administered saline (sham) or cocaine in vivo are shown. The blots demonstrate protein levels for mGluR5 using an mGluR5 antibody in the NAc 1 d after injection. Densitometry measurements are expressed as percentage of control ± SEM. Single cocaine exposure (n = 6) did not alter total mGluR5 protein levels in the NAc, compared with saline (n = 5; p > 0.05). b, Representative Western blots of animals administered in vivo with saline (sham) or cocaine showing biotinylated mGluR5 in the NAc 1 d after injection. Densitometry measurements are expressed as percentage of control ± SEM. Single cocaine exposure (n = 9) decreased biotinylated mGluR5 protein levels in the NAc, compared with saline (n = 8; ***p < 0.05). c, Average time courses of mGluR5-mediated LTD. Single cocaine injection (n = 11) abolished mGluR5-LTD [compared with sham; n = 13; 40 min after application of the selective mGluR5 agonist (S)-DHPG (100 μm) for 10 min; p < 0.05]. Coc, Cocaine.
- Figure 6.
Schematic illustrating the interactions between the eCB-mediated retrograde signaling and the CC-Homer family of scaffolding proteins at excitatory NAc synapses. a, In naive or sham animals, retrograde eCB-LTD is normally induced after activation of postsynaptic mGluR5 and production of eCB. b, One day after cocaine exposure, heightened protein levels of CC-Homer caused the intracellular retention of postsynaptic mGluR5. The associated loss of postsynaptic mGluR5 surface expression prevents the induction of eCB-dependent retrograde signaling. The effects of cocaine on eCB-dependent LTD require D1R and NMDAR. RyR, Ryanodine receptor; G, G-protein; iGluRs, ionotropic GluRs.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental Fig. 1 - Supplementary figure 1: Typical western blot obtained using the Pan-Homer antibody. To control specificity of the antibody the first lane was loaded with lysate of COS-7 cells transfected with Homer1a-myc and the second lane with lysate of COS-7 cells transfected with Homer1b-myc (please note the upward shift due to the myc tag).The other lanes correspond to NAc lysate from sham or cocaine treated mice. In our extracts, the pan-Homer antibody recognizes only one band at around 45KDa.
