2.1. Behavioral Studies
After 14 self-administration sessions, the animals showed stable lever-pressing rates during the last 3 self-administration days with less than a 10% difference in their daily intake of cocaine (
Figure 1). The mean number of cocaine infusions per day during the last 3 self-administration days varied from 26 to 29 while during 14 experimental sessions the animals received 164.9 ± 9.2 mg/rat of cocaine.
Figure 1 shows the behavioral responses of the rats that underwent cocaine self-administration, extinction training, and cocaine-induced reinstatement of seeking behavior. A two-way ANOVA for repeated measures for animals previously self-administered cocaine for tissue level of endocannabinoids and NAEs and expression of cannabinoid receptor experiments showing the significant effect of lever × session interaction (F(24,336) = 10.72;
p < 0.001 and F(24,336) = 9.47;
p < 0.001, respectively). The post hoc analyses revealed a greater frequency of presses on the “active” lever than on the “inactive” lever from the 1st or 2nd cocaine self-administration session till the 1st or 2nd extinction day. During extinction training, neither drug nor drug-paired stimuli were given in response to lever pressing, resulting in a gradual decrease in “active” lever presses (
p < 0.001). Moreover, an intraperitoneal injection of cocaine induced a significant increase of “active” lever presses during the reinstatement of drug seeking in rats that previously self-administered cocaine (
p < 0.01). There was no alteration in inactive lever presses after exposure to priming.
In the yoked cocaine and yoked saline groups, the difference between pressing the “active” versus the “inactive” lever failed to reach significance both in the experiment used to evaluate the endocannabinoid and NAEs tissue level and in the expression of cannabinoid receptors (a two-way ANOVA for repeated measures: Yoked cocaine + i.p. cocaine: F(24,336) = 0.83; p = 0.69 and F(24,288) = 1.27; p = 0.18; Yoked saline + i.p. cocaine: F(24,336) = 0.59; p = 0.79 and F(24,288) = 0.31; p = 0.99; Yoked saline + i.p. saline: F(24,336) = 0.45; p = 0.83 and F(24,288) = 0.31; p = 0.99, respectively).
2.2. Tissue Level of Endocannabinoid and NAEs in Rat Brain Structures
In the yoked saline + i.p. saline group, the AEA levels ranged from 11.37 to 16.33 ng/g, with the highest concentration observed in the nucleus accumbens and the lowest in the frontal cortex. As shown in
Figure 2, reinstatement of seeking behavior in the animals which had undergone a prior procedure of cocaine self-administration resulted in a changes in the AEA levels in all of the analyzed brain structures: the cerebellum (F(3,30) = 12.52;
p < 0.001), nucleus accumbens (F(3,28) = 23.41;
p < 0.001), hippocampus (F(3,30) = 6.43;
p < 0.01), dorsal striatum (F(3,29) = 4.88;
p < 0.05), frontal cortex (F(3,28) = 6.70;
p < 0.05), and prefrontal cortex (F(3,28) = 12.46;
p < 0.001). A significant decrease in the AEA level was observed in the cerebellum and nucleus accumbens in the animals which self-administered (at least
p < 0.01) or passively received (
p < 0.001) cocaine in comparison to the yoked saline animals. Instead, in the prefrontal cortex (
p < 0.01) and hippocampus (
p < 0.05) an increase was reported only in cocaine self-administered rats. Moreover, the animals that had previously self-administered cocaine showed a reduction of the AEA level in the dorsal striatum and frontal cortex when compared to the yoked saline + i.p. cocaine and yoked cocaine + i.p. cocaine rats which, during restatement of seeking-behavior, received an intraperitoneal injection of cocaine (10 mg/kg).
The concentration of 2-AG in the control (yoked saline + i.p. saline) group ranged from 3.21 to 5.12 μg/g, with the highest concentration in the dorsal striatum and the lowest in the nucleus accumbens. Cocaine-induced reinstatement influenced the 2-AG levels in the following structures: the frontal cortex (F(3,28) = 11.27;
p < 0.001), hippocampus (F(3,29) = 33.38;
p < 0.001), dorsal striatum (F(3,30) = 26.22;
p < 0.001), nucleus accumbens (F(3,29) = 3.65;
p < 0.05), and cerebellum (F(3,28) = 6.23;
p < 0.01), but not in the prefrontal cortex (F(3,29) = 2.74). The post hoc analyses revealed a significant decrease in the 2-AG level in the frontal cortex (
p < 0.001) and cerebellum (
p < 0.05), whilst in the hippocampus (
p < 0.001) and in the nucleus accumbens (
p < 0.05), an increase was reported in rats that had self-administered cocaine in comparison to the corresponding yoked saline + i.p. saline group. In the animals passively administered cocaine, a significant enhancement in the 2-AG level in the dorsal striatum (
p < 0.001) and a significant reduction in the cerebellum (
p < 0.05) was observed (
Figure 3).
In the yoked saline + i.p. saline group, the OEA levels ranged from 21.78 to 58.78 ng/g, with the highest concentration in the nucleus accumbens and the lowest in the prefrontal cortex. Cocaine treatment during reinstatement of seeking behavior resulted in a change in the OEA level in the all studied structures: the prefrontal cortex (F(3,30) = 24.36;
p < 0.001), frontal cortex (F(3,30) = 3.82;
p < 0.05), hippocampus (F(3,30) = 29.15;
p < 0.001), dorsal striatum (F(3,29) = 53.84;
p < 0.001), nucleus accumbens (F(3,30) = 69.02;
p < 0.001), and cerebellum (F(3,29) = 20.80;
p < 0.001). In the rats that had self-administered cocaine and those given yoked cocaine injections, a significant increase in the OEA levels was observed in the prefrontal cortex (
p < 0.001) and dorsal striatum (
p < 0.001) when compared to the yoked saline + i.p. saline rats. On the other hand, both groups previously administered cocaine, actively or passively, demonstrated a significant reduction of the level of OEA in the hippocampus (
p < 0.001) and cerebellum (
p < 0.001). Moreover, the animals previously self-administered cocaine displayed a reduction of the OEA level in the nucleus accumbens (
p < 0.001) in comparison to all of the yoked groups. The Dunnett’s Multiple Comparison test also revealed a significant decrease of the OEA level in the hippocampus of the rats which had previously received saline and were exposed to noncontingent reinforcements (
p < 0.001) when compared to the corresponding yoked saline + i.p. saline group (
Figure 4).
The concentration of PEA ranged from 30.90 to 83.60 ng/g in the control (yoked saline + i.p. saline) group, with the highest concentration in the nucleus accumbens and the lowest in the prefrontal cortex. Cocaine administration induced changes in the PEA levels in all of the structures (the prefrontal cortex (F(3,30) = 231.20;
p < 0.001), frontal cortex (F(3,30) = 59.06;
p < 0.001), hippocampus (F(3,29) = 6.59;
p < 0.001), dorsal striatum (F(3,29) = 68.15;
p < 0.001), nucleus accumbens (F(3,30) = 75.41;
p < 0.001), and cerebellum (F(3,29) = 81.28;
p < 0.001)). During cocaine-induced reinstatement in rats previously self-administered cocaine, an increase in the PEA levels was observed in the prefrontal cortex (
p < 0.001), the hippocampus (
p < 0.05), and dorsal striatum (
p < 0.001), while a decrease was noted in the nucleus accumbens (
p < 0.001) and cerebellum (
p < 0.001). In rats passively administered cocaine, increases in the PEA levels in the frontal cortex (
p < 0.001) and dorsal striatum (
p < 0.001) and decreases in the cerebellum (
p < 0.001) were reported (
Figure 5).
2.3. Expression of the CB1 and CB2 Receptor Protein
In the cocaine-induced reinstatement phase, we observed a decrease in the expression of CB1 receptor protein in the ventral tegmental area (F(3,24) = 4.95;
p < 0.01) in both of the studied groups and in the control group with a cocaine i.p. dose. Furthermore, significant changes were observed in the lateral septal nuclei (F(3,24) = 8.54;
p < 0.001) and prefrontal cortex (F(2,14) = 8.46;
p < 0.01), and an increase of the expression of CB1 receptors was observed in the group of rats that had actively self-administered cocaine (at least
p < 0.05). Interestingly, significant changes were demonstrated in the dorsal striatum (F(3,24) = 4.93;
p < 0.05), and a decrease in the CB1 receptors level was observed in the yoked cocaine + i.p. cocaine group. A one-way ANOVA did not show changes in CB1 receptor expression in the basolateral and basomedial amygdala (F(3,24) = 1.06), nucleus accumbens core (F(3,24) = 2.43) and shell (F(3,24) = 0.46), medial globus pallidus (F(3,24) = 1.69), hippocampus (F(3,24) = 1.23), and substantia nigra (F(3,24) = 2.72). The results of the analysis are shown in
Figure 6 (top panel).
In the CB2 receptor expression, we noticed statistically significant changes in the lateral septal nuclei (F(3,24) = 9.85;
p < 0.001), and the post hoc tests demonstrated an increase in the receptor level in chronic and acute cocaine-treated rats (
p < 0.001). Moreover, significant changes were noted in the prefrontal cortex (F(3,24) = 11.39;
p < 0.001), and an increase in the receptors was noticed in active cocaine self-administered (
p < 0.001) and yoked saline + i.p. cocaine (
p < 0.05) animals exposed to cocaine intraperitoneal injection during reinstatement. Furthermore, significant changes were demonstrated in medial globus pallidus (F(3,24) = 6.53;
p < 0.01), while an increase was observed only in yoked cocaine + i.p. cocaine rats (
p < 0.01). Also in the dorsal striatum a significant changes in the CB2 receptor expression were indicated (one-way ANOVA (F(3,24) = 3.57;
p < 0.05); a decrease in the receptors level in the passive cocaine group was noted in comparison to the other experimental groups. In the nucleus accumbens core (F(3,24) = 1.14), nucleus accumbens shell (F(3,24) = 0.29), basolateral and basomedial amygdala (F(3,24) = 0.80), the substantia nigra (F(3,24) = 2.58), hippocampus (F(3,24) = 1.60), and ventral tegmental area (F(3,24) = 1.77), the level of CB2 receptors was unchanged. The results of analysis are shown in
Figure 6 (bottom panel).
Table 1 shows the observed changes in tissue levels of eCS and NAEs and changes in the expression of cannabinoid receptors (CB1 and CB2) in the rat brain structures.