Intra-accumbal administration of shRNAs against CART peptides cause increases in body weight and cocaine-induced locomotor activity in rats

Abstract

In order to examine the effect of cocaine and amphetamine regulated transcript (CART) peptide depletion in adult rats, CART shRNAs or scrambled control shRNAs were administered bilaterally into the nucleus accumbens (NAc). There was an increase in body weight of the shRNA injected rats compared with the rats injected with the scrambled RNA. This is compatible with the data showing a role for the peptide in body weight and food intake. Also at this time, there was about a two-and-a-half fold increase in cocaine-mediated locomotion in the shRNA injected rats compared to the control rats. This finding is critical support for the hypothesis that endogenous CART peptides in the NAc inhibit the actions of cocaine and other psychostimulants. In immunohistochemical experiments on these same animals, there was a decrease in the staining density of CART peptide in the NAc of the shRNA injected rats. These data show that shRNA can reduce CART peptides in the NAc and that endogenous CART peptides influence body weight and cocaine-induced locomotor activity (LMA).

Figure 1

Sequences of shRNAs and Scrambled control RNA.

Figure 2

Percent change in body weights of rats over time after intra-NAc injections of either CART shRNA or scrambled RNA (control). A 2-way ANOVA shows that there is a significant difference between rats injected with CART shRNA (n = 3) and controls (n = 3) (F 1, 92 = 50.09, *** p < 0.0001). Bonferroni post tests showed significant differences between treatments at 21–23 weeks (*p < 0.05). At 23 weeks, rats treated with CART shRNA or control RNA had gained 208 ± 9 g or 170 ± 22 g respectively over their weight at entry into the vivarium. CART shRNA-injected rats show a significant increase in percent weight gain compared to controls. The arrows show the time points when the rats were administered saline and cocaine (i.e. at 8, 14 and 23 weeks).

Figure 3

Time course of the effect of saline or cocaine on locomotor activity in rats 23 weeks after intra-NAc injections of either CART shRNA or scrambled RNA control. The y-axis represents the total distance (LMA) covered by the rat, while the x-axis represents the time points (10 min intervals). The arrows show the times of injections and the thin vertical lines separate the different components (basal, saline, cocaine) of the time course. For analysis, A 2-way ANOVA for repeated measures was used to analyze the different components of the time course. To determine if there was any difference between CART shRNA and controls in basal locomotion, the LMA at times 10–30 min was analyzed. To determine the effect of saline, the LMA (time = 40–60 min) after saline injection was analyzed. To determine the effect of cocaine, the LMA (time = 70–90 min) after cocaine injection was analyzed. There was no significant difference between CART shRNA and control for basal LMA (F 1, 4 = 0.001111, p = 0.9750) or after saline injection (F 1, 4 = 0.004408, p = 0.9503), but there was a significant difference after cocaine injection (F 1, 4 = 11.62, *p = 0.0270). The Bonferroni post hoc tests differences between CART shRNA and controls are shown in the figure at 70 and 80 min: *p < 0.05.

Figure 4

Basal, saline- or cocaine-mediated LMA in rats 8, 14 and 23 weeks after intra-NAc injections of either CART shRNA or control RNA. The y-axis represents the total LMA by the rat 30 minutes after no injection (basal) or after injections of saline or cocaine (10 mg/kg ip). These data were calculated from time course data (see time course data for week 23 in Figure 3). Basal values are the sum of the activity at 10, 20 and 30 minutes. Similarly, saline values are the sum of activity at 40, 50 and 60 minutes. Also, cocaine values are the sum of the activity at 70, 80 and 90 minutes for all animals. There is no difference in basal, saline-, and cocaine-induced locomotion between CART shRNA and control (n = 3 in each group) at 8 (F 1, 12 = 1.339, p = 0.2697) or 14 weeks (F 1, 12 = 0.07599, p = 0.7875). There is a difference in cocaine-induced locomotion between CART shRNA and control (n = 3 in each group) at 23 weeks (F 1, 12 = 7.576, **p = 0.0175). The differences determined by Bonferroni post hoc tests are shown in the figure: *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5

Correlation analysis between % weight change and locomotor activity in rats at 8, 14 and 23 weeks after intra-NAc injections of either CART shRNA or scrambled RNA control. The y-axis represents the % weight change compared to weight at pre-treatment, while the x-axis represents the total locomotor activity in 30 minutes after cocaine injection. The analysis shows that there is a correlation between % change in weight (8, 14, 23 weeks) and LMA (8, 14, 23 weeks) for the shRNA treated rats (r = 0.8576, R² = 0.7354, **p = 0.0031), while there is no correlation for the controls (r = −0.4222, R² = 0.1783, p = 0.2576).

Figure 6

Immunohistochemical localization of CART Peptides in the NAc. The representative section from an animal on the left was injected with the scrambled control RNA, while the representative animal on the right was injected with the shRNA against CART peptide. CART peptide levels are reduced in the animal on the right.

Figure 7

The effect of intra-accumbal administration of anti-CART shRNA and scrambled shRNA on CART peptide density in the NAc. Immunohistochemical analysis was done 23 weeks after the microinjections of CART shRNA and scrambled controls (Figure 6). Statistical analysis shows that there is a significant difference (* p < 0.05) in CART peptide levels in the nucleus accumbens between CART shRNA and control (n = 6 measurements in each group: each animal had a total of 2 microinjections, 1 microinjection into each hemisphere. therefore 2 measurements corresponding to each hemisphere of the brain were taken/rat). The measurement of the NAc CART peptide density was calculated relative to the dorsal striatum which acts as a background (the dorsal striatum contains no CART peptide.

Figure 8

Correlation analysis between CART density and cocaine-induced LMA in rats at 23 weeks after intra-NAc injections. The data represents individual values for all rats in both the shRNA and scrambled RNA groups. Each animal had a total of 2 microinjections (bilateral), one into each hemisphere and therefore 2 measurements corresponding to each brain were taken/rat for the CART density in the NAc (see Figure 7 legend). The difference between cocaine and saline total LMA (cocaine LMA 30 min – saline LMA 30 min) for each rat was plotted on the x-axis and the CART peptide density for the rat was plotted on the y-axis. The difference between cocaine and saline LMA represents the actual cocaine effect. The analysis shows that there is a correlation between CART density and cocaine-saline LMA (r = −0.5992, R² = 0.3591, *p = 0.0395).