Figure 2. Oestrus females exhibit enhanced cocaine actions on the VTA-NAc reward pathway.

(a) Timeline of CPP and paired fibre photometry recordings. (b) Schematic of fibre photometry recording experiments for VTA recordings during conditioning sessions. (c) Representative Ca²⁺ imaging traces from male, oestrus female and dioestrus female mice during saline (left) and cocaine (right) conditioning. (d) Cocaine reduces frequency of VTA Ca²⁺ events (two-way analysis of variance (ANOVA); F_{(2, 8)}=5.792, P<0.05; *P<0.05 as compared with saline). (e) Cocaine-induced frequency reductions in activity were greater during oestrus (one-way ANOVA; F_{(2, 8)}=23.76, P<0.001; ***, P<0.001, ^##P<0.01) when compared with males (#) or dioestrus females (*). (f) Cocaine-induced changes in the amplitude of Ca²⁺ events (two-way ANOVA; F_{(1, 10)}=25.53, P<0.001; ***P<0.001 versus saline). (g) Correlation between percent change in frequency activity of VTA neurons and CPP (r=0.9445; P<0.0001). (h) Schematic of FSCV recordings with bath application of cocaine performed in NAc slices (right) and representative current versus time plots showing cocaine (10 μM) effects on one pulse evoked dopamine release (left). (i) Current versus time plot (left) and colour plots (right) showing the presence of dopamine, as indicated by its oxidation at +0.6 V and reduction at −0.2 V, and the effects of bath application of 10 μM cocaine. (j) Concentration–response curves show that cocaine potency is increased selectively in females during oestrus with no difference in baseline activity (two-way ANOVA; F_{(4, 16)}=15.73, P<0.0001; *P<0.05, ****P<0.0001, ^#P<0.05, ^####P<0.0001). (k) K_i values show that the affinity of cocaine for DAT is increased during oestrus (one-way ANOVA; F_{(2, 6)}=6.564, P<0.05; *P<0.05). (l) Serum estradiol levels were increased during oestrus (one-way ANOVA; F_{(2, 13)}=4.83, P<0.05; *P<0.05). (m) Serum estradiol levels taken immediately before FSCV recordings was positively correlated with cocaine potency (r=0.731; P<0.01). (n) Western blot analysis showing that dopamine transporter levels are not changed over the oestrous cycle (one-way ANOVA F_{(2, 21)}=0.68, P=0.52) (top), yet levels of the phosphorylated Thr53 site on the dopamine transporter were increased during oestrus (one-way ANOVA F_{(2, 21)}=0.37, P<0.05; ^#P<0.05) (bottom). (o) Total ERK levels were not changed between groups (one-way ANOVA F_{(2, 21)}=0.01, P=0.99) (top), whereas phosphorylated ERK levels were increased significantly during oestrus (one-way ANOVA F_{(2, 21)}=3.97, P<0.05; *P<0.05) (bottom). (p) These increased phospho ERK levels were correlated with DAT Thr53 phosphorylation (r=0.47, P<0.05). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 oestrus versus dioestrus (unless otherwise noted); ^#P<0.05, ^##P<0.01, ^###P<0.001, ^####P<0.0001 oestrus versus male. Data represented as mean±s.e.m.