High fat diet augments amphetamine sensitization in mice: Role of feeding pattern, obesity, and dopamine terminal changes

Abstract

High fat (HF) diet-induced obesity has been shown to augment behavioral responses to psychostimulants that target the dopamine system. The purpose of this study was to characterize dopamine terminal changes induced by a HF diet that correspond with enhanced locomotor sensitization to amphetamine. C57BL/6J mice had limited (2hr 3 d/week) or extended (24 h 7 d/week) access to a HF diet or standard chow for six weeks. Mice were then repeatedly exposed to amphetamine (AMPH), and their locomotor responses to an amphetamine challenge were measured. Fast scan cyclic voltammetry was used to identify changes in dopamine terminal function after AMPH exposure. Exposure to a HF diet reduced dopamine uptake and increased locomotor responses to acute, high-dose AMPH administration compared to chow fed mice. Microdialysis showed elevated extracellular dopamine in the nucleus accumbens (NAc) coincided with enhanced locomotion after acute AMPH in HF-fed mice. All mice exhibited locomotor sensitization to amphetamine, but both extended and limited access to a HF diet augmented this response. Neither HF-fed group showed the robust amphetamine sensitization-induced increases in dopamine release, reuptake, and amphetamine potency observed in chow fed animals. However, the potency of amphetamine as an uptake inhibitor was significantly elevated after sensitization in mice with extended (but not limited) access to HF. Conversely, after amphetamine sensitization, mice with limited (but not extended) access to HF displayed reduced autoreceptor sensitivity to the D2/D3 agonist quinpirole. Additionally, we observed reduced membrane dopamine transporter (DAT) levels after HF, and a shift in DAT localization to the cytosol was detected with limited access to HF. This study showed that different patterns of HF exposure produced distinct dopamine terminal adaptations to repeated AMPH, which differed from chow fed mice, and enhanced sensitization to AMPH. Locomotor sensitization in chow fed mice coincided with elevated DAT function and increased AMPH potency; however, the enhanced behavioral response to AMPH after HF exposure was unique in that it coincided with reduced DAT function and diet pattern-specific adaptations.

Keywords: Amphetamine; Dopamine; High fat; Microdialysis; Obesity; Voltammetry.

Figure 1. Food Intake and Body Weight

Mice with 24 hr access to a HF diet (n=16) consumed significantly more kcals per pair housed cage over six weeks than mice with access to chow (n=16) and mice with limited access (LimA) to HF (n=13) (A). Binge like intake of the HF diet was observed in LimA mice on days with 2hr access to HF, ~40% of their daily kcals were consumed within the first 30mins of the two hour HF feeding window (B). Increased food intake in the HF group corresponded with progressive weight gain (C) and a significant change in body weight compared to chow and LimA (D). (*p < 0.05, **p <0.01, ***p <0.001)

Figure 2. Plasma insulin, leptin, and triglyceride levels

Circulating insulin (A), leptin (B), and triglycerides (C) were measured to identify possible relationships between metabolic indexes and behavioral or neurochemical changes associated with AMPH exposure. (**p <0.01, ***p <0.001)

Figure 3. Locomotor Habituation

After six weeks of dietary exposure, mice were placed in locomotor chambers, allowed to habituate for 40 min, then administered saline (ip) to identify any injection-stress-induced increases in activity. The patterns of activity during habituation and the saline injection were similar between chow, HF, and LimA mice (A); however, total activity was significantly less in the HF and LimA mice compared to chow (***p < 0.001) (B), indicating less overall activity but similar mobility after HF exposure.

Figure 4. AMPH-Induced Locomotor Activity

Mice with access to a standard lab chow (n=8), a HF diet (n=8), or LimA to a HF diet (n=7) for six weeks were tested for behavioral sensitivity to low-dose AMPH (0.5 mg/kg) after repeated high-dose AMPH (3.0 mg/kg) administration. Mice were given an initial low-dose AMPH injection (A) followed by five high-dose AMPH injections every other day for 10 days (B-F). Mice were then given a low-dose AMPH challenge after a ten day abstinence period (G). A significant increase in locomotor activity was observed after AMPH administration in the HF and LimA groups compared to chow during the first (B), second (C), and fourth (E) dose of 3.0 mg/kg AMPH with percent baseline activity regressing to chow levels by the last high-dose injection (F). The final 0.5 mg/kg AMPH challenge revealed a significant increase AMPH-induced locomotor activity in HF and LimA mice compared to chow. Note the Y-axis for (A) and (G) differ from (B-F) to visually discern activity differences between groups. (*p < 0.05, **p <0.01, ***p <0.001; Blue* denote differences between chow and LimA, Red* denote differences between chow and HF)

Figure 5. Behavioral Sensitization to AMPH

Behavioral sensitization to AMPH was measured by comparing the difference in locomotor activity between the pre- and post- 0.5 mg/kg AMPH injections. Activity patterns from pre and post low-dose AMPH injections for chow (A), HF (B), and LimA (C) groups are overlaid. The post AMPH challenge significantly increased locomotor activity in each group compared to the initial administration of 0.5 mg/kg AMPH; however, the HF and LimA groups displayed greater behavioral sensitization to AMPH (D) compared to Chow, calculated by the percent change in area under the curve following the AMPH injection. (*p < 0.05, **p <0.01, ***p <0.001)

Figure 6. Dopamine Release and Reuptake

Baseline dopamine (DA) function was measured in the NAc using slice voltammetry in mice after six week dietary exposure: chow (n=8), HF (n=8), and LimA (n=6) diets, or 24 hrs following completion of the AMPH sensitization protocol, which was preceded by the six week dietary regimen: Chow AMPH (n=8), HF AMPH (n=8), and LimA AMPH (n=7). (A) Baseline electrically evoked DA release. (B) Baseline maximal rate of dopamine reuptake (Vmax). Representative line traces are shown depicting the difference in baseline DA release and reuptake in slices from AMPH naïve and AMPH exposed chow (C), HF (D), and LimA (E) groups. Peak height represents evoked DA release (μM) and the top third of the curve's downward slope signifies the Vmax plotted over time. (**p < 0.01, ***p < 0.001)

Figure 7. Effect of AMPH Exposure on Vesicular DA Depletion

Dose response curves for AMPH were conducted in brain slices containing the NAc to evaluate the effect of previous AMPH exposure on AMPH-induced DA release. Prior exposure to AMPH did not alter the ability of AMPH to release DA within each dietary group (A-C). In AMPH-naïve mice, AMPH-induced reductions in DA release were greater in the chow versus the HF groups (D); however, no significant differences in the ability of AMPH to release DA was detected between dietary groups in AMPH exposed mice (E). (*p < 0.05)

Figure 8. Effect of AMPH Exposure on Uptake Inhibition

Dose response curves for AMPH were conducted in brain slices containing the NAc to evaluate the potency of AMPH as an uptake inhibitor. Within dietary groups, previous exposure to AMPH-increased AMPH potency in brain slices of chow (A) and HF (B) mice, but not LimA mice (C). Between groups, AMPH was more potent as an uptake inhibitor in the chow versus HF group (D). Moreover, slices from AMPH-exposed mice show a significant increase in AMPH potency as an uptake inhibitor in the chow group compared to the HF and LimA groups (E). (**p <0.01, ***p <0.001)

Figure 9. Effect of AMPH Exposure on DA Autorecpetors

Dose response curves for the D₂/D₃ agonist quinpirole were used to identify changes in DA autoreceptor function in NAc slices. Within dietary groups, AMPH exposure did not alter quinpirole sensitivity in the chow (A) or HF (B) groups, but reduced autoreceptor sensitivity in LimA mice (C). Between groups, LimA showed an increased sensitivity to quinpirole compared to chow in AMPH-naïve mice (D), but no differences in quinpirole sensitivity were detected between groups after exposure to AMPH (E). (*p < 0.05, **p <0.01, ***p <0.001)

Figure 10. HF increases in vivo dopamine in response to AMPH

Microdialysis detection of DA in the NAc. (A) Average basal dopamine levels collected in 20 minute bins for 1 h prior to AMPH. (B) Extracellular dopamine response to AMPH (3.0 mg/kg i.p.) in chow-fed mice and mice with extended (HF) or limited access (LimA) to a HF diet (n=5 for chow and LimA; n=8 for HF). (*p < 0.05, **p <0.01, ***p <0.001)

Figure 11. Effect of HF on the cellular localization of DAT

Western blot analysis for DAT protein levels in membrane (A), cytosolic (B), and tissue lysate (C) from the NAc. Total DAT levels are calculated by adding the density of the 68 and 78 kDa DAT bands. All data is normalized to β-actin and expressed relative to chow DAT levels. (*p < 0.05)