Chronically dysregulated corticosterone impairs dopaminergic transmission in the dorsomedial striatum by sex-divergent mechanisms

Chronic corticosterone (CORT) treatment increases total plasma CORT in male mice and decreases plasma corticosteroid binding globulin (CBG) levels in both sexes

To chronically elevate plasma CORT levels during the rest period, we implanted male and female mice with subcutaneous slow-release CORT pellets (35 mg, 60-day release); control groups received Placebo pellets of the same size. Slow-release pellets were used to increase circulating CORT levels during the rest period (the light phase for mice), thereby disrupting circadian rhythms of CORT [35, 36] as observed in some individuals with MDD [5, 6]. This approach differs from another commonly used approach, CORT administration via drinking water, which preferentially increases circulating CORT levels during the active phase, when mice drink more often [37]. To test if slow-release CORT pellet treatment chronically elevated plasma CORT levels during the rest period, we collected blood from Placebo- and CORT-treated mice at zeitgeber time 4-6 (ZT4-6, 4–6 h after lights on) four weeks after implantation and used an enzyme-linked immunosorbent assay (ELISA) to quantify total plasma CORT (Fig. 1A). There was a significant effect of treatment (Two-way ANOVA, F(1, 37)=41.18, p < 0.0001), a significant effect of sex (F(1,37) = 11.78, p < 0.01), and a significant interaction between treatment and sex (F(1,37) = 17.25, p < 0.001). Notably, we found that CORT pellet implant increased total plasma CORT in male mice only and resulted in higher levels of resting CORT in male vs female mice (Placebo Male vs CORT Male, Tukey’s multiple comparisons p < 0.0001; CORT Male vs CORT Female, p < 0.0001; Fig. 1B). This sex difference in total plasma CORT four weeks after pellet implantation is consistent with previous studies in rats [38], and likely occurs due to sex differences in hypothalamic-pituitary-adrenal (HPA) axis responsivity [39]. However, a limitation of measuring total plasma CORT is that it includes both free and protein-bound CORT. Free CORT can cross the blood-brain barrier, while protein-bound CORT cannot [40,41,42]. Corticosteroid binding globulin (CBG) is the primary blood protein that binds CORT. Thus, we questioned if chronic CORT treatment decreased CBG, which could augment circulating levels of free CORT, even in the absence of changes in total levels. Chronic CORT treatment decreased CBG levels with no evidence of sex difference (Fig. 1C; Two-way ANOVA, significant effect of treatment, F(1,30)=16.30, p < 0.001; no sex x treatment interaction). Treatment did not affect estrous cyclicity of females (Fig. S1). We concluded that circulating levels of free CORT are likely elevated in both male and female mice after treatment with subcutaneous slow-release CORT pellets but to differing degrees of severity. Due to the significant sex difference in plasma CORT levels after CORT treatment, we separated the sexes for analysis in all following experiments.

Fig. 1: Chronic corticosterone treatment increases plasma CORT levels in males, decreases plasma CBG levels in both sexes, and impairs motivation.figure 1

A Experimental timeline for pellet implantation and plasma CORT and CBG measurements. B Plasma corticosterone (ng/mL) in male and female mice implanted with a placebo or corticosterone (35 mg; CORT) pellet. Two-way ANOVA, main effect of treatment ****p < 0.0001, main effect of sex p < 0.01, main effect of treatment x sex interaction p < 0.001, multiple comparisons ####p < 0.0001. C Plasma CBG (ng/mL) in male and female mice implanted with a placebo or corticosterone (35 mg; CORT) pellet. Two-way ANOVA, main effect of treatment ***p < 0.001. D Experimental timeline for pellet implantation and operant behavior paradigms with schematic of fixed ratio (FR) paradigms. E Days to reach criterion for FR1 in male mice. Unpaired two-tailed t-test *p < 0.05. Each point represents an individual. F Days to reach criterion for FR1 in female mice. Unpaired two-tailed t-test ****p < 0.0001. Each point represents an individual. G Percent active nosepokes over days of FR1 until criterion was met in Placebo- (grey) and CORT-treated (pink) male mice. Each point represents mean ± SEM percent active nosepokes for a given day of FR1 training. H Percent active nosepokes over days of FR1 until criterion was met in Placebo- (black) and CORT-treated (purple) female mice. Each point represents mean ± SEM percent active nosepokes for a given day of FR1 training. I Active nosepoking rates of Placebo- (N = 8) and CORT- (N = 9) treated male mice across operant behavior paradigms. Two-way ANOVA, main effect of treatment *p < 0.05, main effect of treatment x paradigm interaction p < 0.001. J Active nosepoking rates of Placebo- (N = 7) and CORT- (N = 7) treated female mice across operant behavior paradigms. Two-way ANOVA, main effect of treatment *p < 0.05, main effect of treatment x paradigm interaction p < 0.001. K Inactive nosepoking rates of Placebo- and CORT-treated male mice across operant behavior paradigms. Two-way ANOVA, main effect of treatment *p < 0.05. L Inactive nosepoking rates of Placebo- and CORT-treated female mice across operant behavior paradigms. M Reward rates of Placebo- and CORT-treated male mice across operant behavior paradigms. N Reward rates of Placebo- and CORT-treated female mice across operant behavior paradigms Two-way ANOVA, main effect of treatment **p < 0.01, main effect of treatment x paradigm interaction p < 0.01, multiple comparisons #p < 0.05. O Time to completion of operant session (in minutes) for Placebo- and CORT-treated male mice across operant behavior paradigms. Two-way ANOVA, main effect of treatment x paradigm interaction p < 0.05. P Time to completion of operant sessions (in minutes) for Placebo- and CORT-treated female mice across operant behavior paradigms. Two-way ANOVA, main effect of treatment ***p < 0.001, main effect of treatment x paradigm interaction p < 0.05. Data presented as mean ± SEM.

Chronic CORT treatment impairs motivated reward-seeking in male and female mice

Chronic CORT treatment has previously been shown to impair reward-seeking behaviors in male mice [14, 15]. However, it was unclear what effect chronic CORT treatment would have on female mice. To assess reward-seeking behaviors in both sexes, we used operant training. Four weeks after Placebo or CORT pellet implantation, mice began training on a fixed ratio-1 (FR-1) schedule, then advanced to FR-3 and FR-5 (Fig. 1D). We found that CORT treatment significantly increased the number of days it took both sexes to reach criterion on the FR-1 task (Fig. 1E, F; Unpaired two-tailed t-test, p < 0.05 male, p < 0.0001 female). However, CORT-treated mice readily learned the association between the active nosepoke and reward. CORT- and Placebo-treated mice similarly discriminated between the active and inactive nosepokes during the initial days of FR-1 training (Fig. 1G, H), but CORT-treated mice were slower to use this associative knowledge to reach the criterion of obtaining 30 rewards per session. This finding suggests that CORT-treated mice have intact reward learning but are less motivated to attain rewards than Placebo-treated mice. After FR-1 criterion was met, CORT-treated mice exhibited decreased rates of nosepoking across FR-3 and FR-5 sessions (Fig. 1I, J; males: significant effects of treatment [Two-way ANOVA F(1,15) = 5.554, p < 0.05], day of training [F(2.513,37.70) = 37.70, p < 0.0001], and an interaction between treatment and day of training [F(5,75) = 4.749, p < 0.001]; females: significant effects of treatment [Two-way ANOVA F(1,12) = 5.098, p < 0.05], day of training [F(3.267,39.20) = 87.47, p < 0.0001], and an interaction between treatment and day of training [Two-way ANOVA F(5,60) = 7.294, p < 0.0001]). Again, no deficit in active nosepoke discrimination was observed (in fact, CORT-treated males made significantly fewer inactive nosepokes than Placebo-treated males, Fig. 1K, L; males: Two-way ANOVA, significant effect of treatment [F(1,15)=6.123, p < 0.05]; females: no significant effect of treatment). Therefore, as for initial FR1 training, decreased rates of active nosepoking in CORT-treated mice do not stem from impaired learning, but likely arise due to decreased motivation. Motivational deficits in CORT-treated mice are further supported by their impaired rate of rewards earned, particularly on the final days of FR-3 and FR-5 (Fig. 1M, N; males: Two-way ANOVA, significant effects of day of training [F(3.422,51.34) = 7.968, p < 0.0001], and an interaction between day of training and treatment [F(5,75)=3.320, p < 0.01); females: Two-way ANOVA, significant effects of treatment [F(1,12)=16.15, p < 0.01], day of training [F(3.275,39.30)=5.835, p < 0.01], and an interaction between day of training and treatment [F(5,60) = 4.427, p < 0.01]). While CORT-treated males earned the same number of rewards as Placebo-treated males across operant training (Fig. S2A), they took longer to earn those rewards, especially on the final days of FR-3 and FR-5 (Fig. 1O; Two-way ANOVA, significant effect of an interaction between treatment and day of training, p < 0.01). CORT-treated females also took longer to earn rewards (Fig. 1P; Two-way ANOVA, significant effects of treatment [F(1,12) = 19.20, p < 0.001], day of training [F(3.360,40.32) = 3.978, p < 0.05], and an interaction between treatment and day of training [F(5,60) = 2.648, p < 0.05]) and earned significantly fewer total rewards (Fig. S2B) than Placebo-treated females. These results suggest continued motivational impairments throughout training. In female mice only, CORT treatment significantly decreased reward port entry rates (i.e., actions to retrieve earned rewards, Fig. S2C, D), suggesting that CORT treatment may also induce anhedonia in females.

Chronic CORT treatment does not impair phasic dopamine transmission during reward-seeking

After observing an effect of CORT treatment on reward-seeking in both sexes, we questioned if CORT treatment was impairing phasic dopamine transmission in the striatum. To examine phasic dopamine transients in Placebo- and CORT-treated mice, we injected a virus encoding the fluorescent dopamine sensor, dLight1.3b (AAV9-CAG-dLight1.3b), into the NAcc and DMS. We implanted a fiber optic over each injection site and recorded dLight1.3b transients during operant training using fiber photometry. We found that CORT treatment did not affect phasic dLight1.3b transients in the NAcc or DMS (Fig. S3), thus we pursued measures of tonic dopamine activity.

Chronic CORT treatment decreases tissue dopamine content of the dorsomedial striatum (DMS) in female mice

To investigate whether chronic CORT treatment influenced dopamine content of the striatum, we analyzed tissue samples from the NAcc and DMS of Placebo- and CORT-treated mice using high-performance liquid chromatography and electrochemical detection (HPLC-ECD) of dopamine. CORT treatment did not affect NAcc dopamine content in either sex (Fig. 2B), but significantly decreased DMS dopamine content in female mice (Fig. 2C; Unpaired two-tailed t-test, p < 0.01). Therefore, although acute CORT has effects on NAcc dopamine [43], DMS dopamine is more sensitive to chronic CORT treatment in females.

Fig. 2: Chronic corticosterone treatment significantly decreases tissue dopamine content of the dorsomedial striatum (DMS) in female mice.figure 2

A Experimental timeline for pellet implantation, tissue punches, and HPLC-ECD of dopamine. B Tissue dopamine content of the nucleus accumbens core (NAcc) of placebo- and CORT-treated male and female mice. C Tissue dopamine content of the DMS of placebo- and CORT-treated male and female mice. Unpaired two-tailed t-test, **p < 0.01. Each point represents an individual. Data presented as mean ± SEM.

Chronic CORT treatment impairs dopamine transporter (DAT) function in the DMS of male mice

One mechanism that regulates levels of tonic dopamine in the striatum is modulation of dopamine transporter (DAT) function [24]. By altering decay rates of phasic dopamine transients, changes in DAT function can alter the timescale for integration of phasic dopamine signals, allowing or disallowing the buildup of tonic levels when dopamine neurons are active. Chronic DAT impairment can also cause compensation in the dopamine system, altering the rate of synthesis of new dopamine [24]. To investigate DAT function in mice chronically treated with CORT, we assayed dopamine dynamics in an ex vivo slice preparation. We injected a virus encoding the fluorescent dopamine sensor, dLight1.3b (AAV9-CAG-dLight1.3b), into the NAcc and DMS, and implanted Placebo or CORT pellets during the same surgery. Four weeks later, we prepared striatal tissue sections and electrically evoked dopamine release while imaging dLight1.3b fluorescence (Fig. 3A, Fig. S4). To mimic tonic and phasic dopamine neuron firing, we used a single stimulation pulse or a burst of 5 pulses at 20 Hz, respectively. We quantified the decay of evoked dLight1.3b transients by calculating a ‘tau-off’ value and used it as a measure of the speed of extracellular dopamine clearance [31, 44]. CORT treatment did not significantly increase tau-off in male (Fig. 3B, L) or female (Fig. 3G, Q) mice in response to either one or five pulses at baseline in the DMS or NAcc (Fig. S5). However, the lack of change could be due to compensation for chronic DAT impairment. To elucidate how DAT activity was contributing to tau-off, we washed a DAT inhibitor, GBR12909 (1 µM, ‘DATi’), onto the slice. We also tested the contribution of another monoamine transporter, Organic Cation Transporter 3 (OCT3) [45], to dopamine clearance by washing on an OCT3 inhibitor, normetanephrine (50 µM; ‘OCTi’). OCT3 is a low-affinity, high-capacity non-specific monoamine transporter [46]. Although OCT3 does not regulate synaptic dopamine levels as effectively as DAT, CORT binds OCT3 directly and inhibits reuptake, making it important to examine in our studies [45, 47,48,49]. In the NAcc, we did not observe any significant effect of CORT treatment on tau-off after DAT and OCT3 inhibition in either sex (Fig. S5). Using one stimulation pulse in the DMS, we did not observe significant differences in tau-off between Placebo- and CORT-treated mice after DAT and OCT3 inhibition (Fig. 3D–F, I–K), although there was a trending effect of CORT treatment in male mice (Fig. 3D, Two-way ANOVA, F(1,16)=3.636, p = 0.07). In response to five pulses in the DMS, DAT inhibition slowed dopamine clearance in Placebo-treated males, but had no effect in CORT-treated males, indicating DAT function is impaired in the DMS of CORT-treated males (Fig. 3N–P; Two-way ANOVA, significant effect of treatment, F(1,14) = 8.566, p < 0.05). In females, CORT treatment did not impair DAT or OCT3 function in the DMS (Fig. 3S–U). CORT treatment did not affect the amplitude of dLight1.3b fluorescence elicited by one or five stimulation pulses in the DMS of either sex (Fig. S6).

Fig. 3: Chronic corticosterone treatment impairs ex vivo dopamine transporter (DAT) function in the dorsomedial striatum (DMS) of male mice.figure 3

A Experimental timeline for viral injection, pellet implantation, and slice imaging experiments. B dLight1.3b fluorescence tau-off values after a single electrical stimulation of the DMS in acute tissue slices from male mice. C Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence. after a single electrical stimulation of the DMS in acute tissue slices from male mice. D Fold change of tau-off values of dLight1.3b fluorescence in the presence of inhibitors for the dopamine transporter (DATi) and organic cation transporter 3 (OCTi), normalized to tau-off values of dLight1.3b fluorescence in the absence of any transporter inhibitors, after a single electrical stimulation of the DMS in acute tissue slices from male mice. Two-Way ANOVA, trending effect of treatment p = 0.07. E, F Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a single electrical stimulation of the DMS in acute slices from Placebo- (E) and CORT- (F) treated male mice, in the presence and absence of DATi and OCTi. G dLight1.3b fluorescence tau-off values after a single electrical stimulation of the DMS in acute tissue slices from female mice. H Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a single electrical stimulation of the DMS in acute tissue slices from female mice. I Fold change of tau-off values of dLight1.3b fluorescence in the presence of DATi and OCTi, normalized to tau-off values of dLight1.3b fluorescence in the absence of any transporter inhibitors, after a single electrical stimulation of the DMS in acute tissue slices from female mice. J, K Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a single electrical stimulation of the DMS in acute tissue slices from Placebo- (J) and CORT- (K) treated female mice, in the presence and absence of DATi and OCTi. L dLight1.3b fluorescence tau-off values after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from male mice. M Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from male mice. N Fold change of tau-off values of dLight1.3b fluorescence in the presence of DATi and OCTi, normalized to tau-off values of dLight1.3b fluorescence in the absence of any transporter inhibitors, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from male mice. Two-Way ANOVA, main effect of treatment p < 0.05, multiple comparisons *p < 0.05. O, P Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from Placebo- (O) and CORT- (P) treated male mice, in the presence and absence of DATi and OCTi. Q dLight1.3b fluorescence tau-off values after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from female mice. R Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from female mice. S Fold change of tau-off values of dLight1.3b fluorescence in the presence of DATi and OCTi, normalized to tau-off values of dLight1.3b fluorescence in the absence of any transporter inhibitors, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from female mice. T, U Average dLight1.3b fluorescence traces, normalized to the peak of dLight1.3b fluorescence, after a 20 Hz, 5 pulse electrical stimulation of the DMS in acute tissue slices from Placebo- (T) and CORT- (U) treated female mice, in the presence and absence of DATi and OCTi. Points represent the average of 2–3 sweeps from a single individual. Data presented as mean ± SEM.

To verify our ex vivo results, we designed an experiment to examine DAT function in vivo using fiber photometry. We injected a virus encoding dLight1.3b (AAV9-CAG-dLight1.3b) into the DMS of male and female mice and implanted a fiber optic in DMS for in vivo recording during behavior (Fig. S7). Four weeks later, mice were placed in an open field to collect baseline locomotor and dLight1.3b fluorescence data (Fig. 4A). After ten minutes of baseline data collection, mice were injected with the DAT inhibitor, GBR12909 (20 mg/kg, i.p.), and returned to the open field for forty minutes. In mice with high DAT activity, injection of a DAT inhibitor should increase locomotion, with a concordant increase in extracellular dopamine in DMS (measured as a change in the dLight1.3b fluorescence area-under-the-curve (AUC)). After DAT inhibition, locomotion of CORT-treated male mice was blunted relative to Placebo-treated male mice (Fig. 4D; Two-way ANOVA, significant effect of treatment [F(1,17) = 6.776, p < 0.05], trending effect of time [F(2.344,39.86) = 2.835, p = 0.06]). Furthermore, CORT-treated male mice exhibited significant blunting of dLight1.3b AUC after DAT inhibition compared to Placebo-treated male mice (Fig. 4E; Two-way ANOVA, significant effects of treatment [F(1,9) = 5.418, p < 0.05], time [F(2.439,21.95) = 7.083, p < 0.01], and the interaction between treatment and time [F(49,441) = 2.979, p < 0.0001]).

Fig. 4: Chronic corticosterone treatment impairs in vivo dopamine transporter (DAT) function in the dorsomedial striatum (DMS) of male mice.figure 4

A Experimental timeline for viral injection, fiber optic implant, pellet implantation, and open field behavior. B Representative image of dLight1.3b viral spread and fiber optic implantation site (outlined in dashed white line). Scale bar equals 500 micrometers. C Representative activity traces of male mice (Placebo, top; CORT, bottom) during the ten-minute baseline period (‘Baseline’) and the last ten minutes of recorded activity after injection of the DAT inhibitor GBR12909 (20 mg/kg, i.p., ‘DATi’). D Velocity of Placebo- (N = 10) and CORT- (N = 9) treated male mice, in averaged five-minute bins, before and after injection with the DAT inhibitor, GBR12909 (20 mg/kg, i.p.; injection time indicated by vertical dashed line). Two-Way ANOVA, main effect of treatment *p < 0.05, trending effect of time p = 0.06. E Change in dLight1.3b area-under-the-curve (AUC) relative to the minute average of the ten-minute baseline period prior to injection with the DAT inhibitor, GBR12909 (20 mg/kg, i.p.; injection time indicated by vertical dashed line) in male mice. Two-Way ANOVA, main effect of treatment *p < 0.05, main effect of time p < 0.01, main effect of treatment x time interaction p < 0.0001. Placebo N = 5, CORT N = 6. F Representative activity traces of female mice (Placebo, top; CORT, bottom) during the ten-minute baseline period (‘Baseline’) and the last ten minutes of recorded activity after injection of the DAT inhibitor GBR12909 (20 mg/kg, i.p., ‘DATi’). G Velocity of Placebo- (N = 6) and CORT- (N = 8) treated female mice, in averaged five-minute bins, before and after injection with the DAT inhibitor, GBR12909 (20 mg/kg, i.p.; injection time indicated by vertical dashed line). Two-Way ANOVA, main effect of time p < 0.001. H Change in dLight1.3b AUC relative to the average of the ten-minute baseline period prior to injection with the DAT inhibitor, GBR12909 (20 mg/kg, i.p.; injection time indicated by vertical dashed line) in female mice. Two-Way ANOVA, main effect of time p < 0.01. Placebo N = 5, CORT N = 6. Data presented as mean ± SEM.

DAT inhibition could increase dLight1.3b AUC by increasing the decay constant of dLight1.3b transients, leading to a larger AUC per transient. Longer dopamine clearance times (indicated by higher decay constants) could then slowly increase baseline dLight1.3b fluorescence, reflecting a slow buildup of tonic dopamine. Such an increase in baseline fluorescence would also contribute to an increase in dLight1.3b AUC following DAT inhibition. To differentiate between these two effects, we analyzed the decay constants of dLight1.3b transients recorded during open field behavior before and after DAT inhibition. We found that DAT inhibition increased both the decay time constants of in vivo dLight1.3b transients and led to a buildup of baseline dLight1.3b fluorescence (Fig. S8). We observed non-significant trends in which both of these effects were greater in Placebo-treated males than CORT-treated males (Fig. S8; Two-way ANOVA, effect of treatment p = 0.06 for decay constants, p = 0.08 for baseline fluorescence). Thus, we concluded that the significant difference in DMS dLight1.3b AUC between Placebo- and CORT-treated males after DAT inhibition (Fig. 4E) is the result of a combined effect on the decay of individual dLight1.3b transients and an increase in the baseline dLight1.3b fluorescence due to integration of slowly decaying transients.

In female mice, we did not observe differences between treatment conditions (Fig. 4F–H). We observed a significant effect of time on locomotion (Fig. 4G; Two-way ANOVA, F(2.062,24.75) = 9.222, p < 0.001) and on dLight1.3b AUC (Fig. 4H; Two-way ANOVA, F(2.642,23.78) = 7.124, p < 0.01) after DAT inhibition in both Placebo- and CORT-treated groups. We concluded that CORT treatment impairs DAT function in the DMS of male, but not female, mice. However, from these data it was unclear how CORT treatment impaired DAT function.

Chronic CORT treatment decreases phosphorylation of DAT at threonine-53

To assess how CORT treatment impaired DAT function, we examined DAT expression and post-translational modifications of DAT, which regulate reuptake activity [24, 50, 51]. Specifically, we examined phosphorylation at threonine-53, a known regulatory site [51,52,53,54]. We collected DMS tissue punches from Placebo- and CORT-treated male and female mice and fractionated the tissue homogenate to isolate membrane-bound proteins. We then performed western blots probing for DAT and Thr53 phospho-DAT (pDAT). We found that CORT treatment had no effect on total levels of membrane-bound DAT in males or females (Fig. 5C, D). However, CORT treatment significantly decreased pDAT in male mice (Unpaired two-tailed t-test, p < 0.05), but not female mice (Fig. 5E, F). These results suggest that CORT treatment impairs DMS DAT function in male mice by decreasing phosphorylation of DAT at threonine-53, and further supports the conclusion that DMS DAT is unaffected by CORT treatment in female mice.

Fig. 5: Chronic corticosterone treatment decreases phosphorylation of the dopamine transporter (DAT) at threonine-53 in the dorsomedial striatum (DMS) of male mice.figure 5

A Experimental timeline for pellet implantation, tissue punches, and western blot experiments. B Representative western blots for phosphorylated DAT at threonine-53 (‘pDAT’), DAT, and beta-actin (‘Actin’) from DMS tissue samples of male mice (top) and female mice (bottom). C Membrane-bound DAT expression, normalized to Actin and plotted as a percent of Placebo expression, from DMS tissue samples of male mice. D Membrane-bound DAT expression, normalized to Actin and plotted as a percent of Placebo expression, from DMS tissue samples of female mice. E pDAT expression, normalized to DAT and plotted as a percent of Placebo expression, from DMS tissue samples of male mice. Unpaired two-tailed t-test *p < 0.05. F pDAT expression, normalized to DAT and plotted as a percent of Placebo expression, from DMS tissue samples of female mice. Each point represents a single individual. Data presented as mean ± SEM.

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