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Published in final edited form as: Nat Neurosci. 2019 Dec 2;23(1):32–46. doi: 10.1038/s41593-019-0537-6

Figure 6. — (a) Diagrams showing the experimental design in which paRac1 is photoactivated 2 hr after cue re-exposure in cocaine-trained rats on withdrawal day 45.

(b) Example images of NAcSh slices (upper) and MSNs (lower) showing HSV-mediated expression of paRac1. All animals used in Fig 6f, Fig 6h-l, Fig 6p, and Fig 6r,s (n = 49 animals) had pa-dnRac1 expression localized within the NAcSh.

(c-e) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example NAcSh MSNs with paRac1 photoactivation after cue re-exposure in saline- (c) and cocaine-trained rats (d), and the effects of naspm (e).

(f) Summary showing that stimulating paRac1 after cue re-exposure did not affect the % silent synapses in saline-trained rats, but locked cocaine-generated silent synapses within their silent state in cocaine-trained rats beyond the presumable 6-hr destabilization window, and CPAMPAR inhibition by naspm did not further increase the % silent synapses (saline = 8.30 ± 2.46, n = 4 animals; cocaine = 32.82 ± 3.48, n = 7 animals; cocaine naspm = 36.32 ± 7.65, n =5 animals, F_2,13=7.64, p=0.0064, one-way ANOVA; *p<0.05, **p<0.01, Bonferroni posttest).

(g) Example NAcSh dendrites of MSNs non-transduced and transduced with paRac1 from saline- and cocaine-trained rats that received photostimulation 2 hr after cue re-exposure. Scale bar, 2.5 μm.

(h) Summary showing the total spine density was increased in cocaine-trained rats 6 hr after cue re-exposure for both non-transduced and transduced MSNs compared to saline-trained rats (saline non-trans = 11.22 ± 0.484, n = 4 animals; saline trans = 11.03 ± 0.199, n = 4 animals; coaine non-trans = 17.88 ± 0.827, n = 3 animals; cocaine trans = 17.67 ± 0.298, n =3 animals, F_1,5=148.0, p<0.0001, RM two-way ANOVA, drug main effect; **p<0.01, Bonferroni posttest).

(i) Summary showing that the density of mushroom-like spines was decreased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the density in non-transduced MSNs remained high (saline non-trans = 3.69 ± 0.130, n = 4 animlas; saline trans = 3.71 ± 0.175, n = 4 animals; cocaine non-trans = 7.96 ± 0.451, n = 3 animals; cocaine trans = 4.69 ± 0.091, n = 3 animals, F_1,5=58.92, p=0.0006, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest).

(j) Summary showing that the density of thin spines was increased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the density in non-transduced MSNs returned to the saline control level (saline non-trans = 6.20 ± 0.280, n =4 animals; saline trans = 5.83 ± 0.128, n = 4 animals; cocaine non-trans = 7.45 ± 0.498, n = 3 animals; cocaine trans = 10.53 ± 0.461, n = 3 animals, F_1,5=55.71, p=0.0007, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest).

(k) Summary showing that the density of stubby spines was increased in cocaine-trained rats 6 hr after cue re-exposure for both non-transduced and transduced MSNs compared to saline controls (saline non-trans = 1.32 ± 0.080, n = 4 animals; saline trans = 1.49 ± 0.135, n = 4 animals; cocaine non-trans = 2.47 ± 0.159, n =3 animals; cocaine trans = 2.45 ± 0.120, n = 3 animals, F_1,5=79.42, p=0.0003, RM two-way ANOVA, drug main effect; **p<0.01, Bonferroni posttest).

(l) Summary showing that the mean spine head diameter was decreased in transduced MSNs from cocaine-trained rats 6 hr after cue re-exposure, while the spine head diameter in non-transduced MSNs remained high (saline non-trans = 0.387 ± 0.005, n = 4 animals; saline trans = 0.402 ± 0.002, n =4 animals; cocaine non-trans = 0.482 ± 0.003, n = 3 animals; cocaine trans = 0.375 ± 0.017, n = 3 animals, F_1,5=65.19, p=0.0005, RM two-way ANOVA, drug x transduction interaction; **p<0.01, Bonferroni posttest).

(m) Diagram showing the experimental timeline for LIMKi experiments.

(n-o) EPSCs evoked at −70mV and +50mV during the minimal stimulation assay (insets) over 100 trials from example paRac1-expressing MSNs from cocaine-trained rats receiving photostimulation 2 hr after cue re-exposure with pretreatment of vehicle (n) or LIMKi (o).

(p) Summary showing that pretreatment with LIMKi prevented the effect of paRac1 stimulation on keeping cocaine-generated synapses in a silent state, such that the % silent synapses were decreased compared to vehicle-treated rats (cocaine vehicle = 32.52 ± 1.45, n = 4 animals; cocaine LIMKi = 7.02 ± 2.11, n = 4 animals, t₆=9.95, p<0.0001, two-sided, unpaired t-test).

(q) Summary showing that cocaine-trained rats with photostimulation of paRac1 during cue re-exposure exhibited comparable levels of cue-induced cocaine seeking as in C450M control rats, measured 6 hr after cue re-exposure (C450 active = 63.89 ± 4.15, n = 9 animals; pa-Rac1 active = 70.38 ± 6.00, n = 8 animals; C450 inactive = 9.33 ± 1.53, n = 9 animals; pa-Rac1 inactive 13.75 ± 2.20, n = 8 animals,F_1,15=0.07, p=0.79, RM two-way ANOVA, withdrawal day 45 lever x virus interaction, n.s. >0.05).

(r) Summary showing that cocaine-trained rats with photostimulation of paRac1 2 hr after cue re-exposure exhibited decreased cue-induced cocaine seeking compared to C450M control rats when measured 6 hr after re-exposure (C450 active = 69.88 ± 4.69, n = 8 animals; pa-Rac1 active = 36.20 ± 2.59, n = 10 animals; C450 inactive = 15.13 ± 2.66, n = 8 animals; pa-Rac1 inactive = 10.10 ± 2.15, n = 10 animals, F_1,16=31.89, p<0.0001, RM two-way ANOVA, withdrawal day 45 lever x virus interaction; **p<0.01, Bonferroni posttest).

(s) Summary showing that cocaine-trained rats with photostimulation of paRac1 2 hr after cue re-exposure exhibited decreased cue-induced cocaine seeking compared to C450M control rats when measured 24 hr after re-exposure (C450 active = 86.13 ± 3.82, n = 8 animals; pa-Rac1 active = 55.88 ± 4.40, n = 8 animals; C450 inactive = 13.00 ± 2.47, n = 8 animals; pa-Rac1 = 9.63 ± 2.61, n = 8 animals, F_1,14=15.55, p=0.0015, RM two-way ANOVA, withdrawal day 45 lever x virus interaction; **p<0.01, Bonferroni posttest). See Supplemental Table 1 for exact p values for all comparisons made during posthoc tests. Data presented as mean±SEM.