Experiment 1a and b: Food restriction modestly potentiates VS-reinforced responding over repeated sessions

We examined effects of food restriction in rats tested on a PR schedule (n = 24). One of the AL rats in the PR group was eliminated from all analyses, since it did not respond on the levers (only two leverpresses during the entire 14 daily sessions). Before implementing the food restriction procedure (i.e., sessions 1–2), we confirmed that food restriction groups did not differ from AL groups on any of the measures: active and inactive leverpressing, crossing or body weights (sessions 1 and 2 in Fig. 1a). During the first two sessions, rats responded on the active lever more than the inactive lever [lever: F(1,21) = 26.18, p < 0.0001], and leverpress rates decreased between sessions 1 and 2 [session: F(1,21) = 5.83, p < 0.05]. Weights increased over the 2 days [F(1,21) = 33.73, p < 0.0001]. Following these sessions, the rats in the food restriction groups received limited daily rations, the weights of the food-restricted rats gradually decreased to 89 % of those of session 2 over 12 days, while the AL rats gradually gained weights to 113 % (PR) [Tukey's test following a feeding × session interaction: F(11,231) = 64.84, p < 0.0001]. Feeding condition played an important role in leverpressing. Active, but not inactive, leverpressing of food-restricted rats was significantly potentiated more than the active or inactive leverpressing of AL rats [lever × feeding: F(1,21) = 4.71, p < 0.05, followed by Tukey's test]. Although the means shown in Fig. 1a suggest a gradual increase in active leverpressing in food-restricted, but not AL, rats, the effect was not detected by a feeding × lever × session interaction. While lever discrimination was robust throughout the experiment [lever: F(1,21) = 61.21, p < 0.0001], the rats further increased active, but not inactive, leverpressing between sessions 3 and 14 [lever × session: F(11,231) = 3.91, p < 0.005]. Based on the significant feeding condition × lever interaction and the significant lever × session interaction, food restriction appears to have moderately potentiated VS-reinforced responding, and the effect emerged over repeated testing.

To confirm that food restriction effects detected with the PR schedule are not schedule-specific, we examined effects of food restriction with another behavioral schedule, a VR2, which we thought to induce similar levels of leverpressing as the PR. Again, we confirmed that food restriction groups did not differ from AL groups on any of the measures during sessions 1–2 (Fig. 1b). Rats responded on the active lever more than the inactive lever [lever: F(1,14) = 28.80, p < 0.0001] and gained weight [F(1,14) = 13.63, p < 0.005]. The weights of the food-restricted rats gradually decreased by 12 % of those of session 2 over the next 12 days, while the AL rats gradually gained weights by 8 % [feeding × session: F(7,98) = 243.22, p < 0.0001]. The rats on the VR2 schedule also responded on active lever more than inactive lever [lever: F(1,14) = 44.43, p < 0.0001]. Mean leverpresses of VR2 rats shown in Fig. 1b also suggests a gradual increase in active leverpressing in food-restricted rats. However, effects of food restriction were not statistically significant [feeding × lever × session: ns; feeding × session: F(11,154) = 2.41, p = 0.060; feeding × lever: ns; feeding: ns].

Food restriction had no significant effect on locomotor activity [feeding: F(1,21) = 0.83, p = 0.37 and F(1,14) = 0.29, p = 0.60 for PR and VR2 schedules, respectively]. However, AL rats tended to have greater locomotor activity in early sessions and decrease it thereafter, while food-restricted rats tended to maintain it at a similar level between sessions 2 and 14 in PR rats [feeding × session: F(11,231) = 1.74, p = 0.065 and F(7,98) = 1.38, p = 0.22 for PR and VR2 schedules, respectively].

Experiment 1c: SCH similarly decreases VS-reinforced responding and locomotor activity

Previous studies showed that injections of dopamine receptor antagonists including SCH readily decrease spontaneous locomotor activity or exploration (Hoffman and Beninger 1985; Bardo et al. 1993; Bevins et al. 2002). We examined whether SCH similarly decreases VS-reinforced responding and locomotor activity. IP injections of 25 μg/kg, but not 12.5 μg/kg, SCH significantly decreased crossing (Fig. 1c) [F(2,28) = 18.91, p < 0.0001]. Similarly, the 25 μg/kg, but not 12.5 μg/kg, dose significantly decreased leverpressing [dose: F(2,28) = 12.96, p < 0.0005]. Food restriction did not significantly interact with SCH for either leverpressing or crossing [feeding: ns; feeding × dose: ns; feeding × dose × lever: ns].

Experiment 1d: AMPH potentiates VS-reinforced responding in food-restricted rats

Our previous study found that systemic injections of AMPH moderately potentiate VS-reinforced responding (Shin et al. 2010). Here, we examined whether food restriction further augments AMPH-potentiated VS-reinforced responding. While food restriction or AMPH alone potentiated leverpressing [feeding: F(1,14) = 8.19, p < 0.05; dose: F(3,42) = 16.61, p < 0.0001], these factors differentially altered active and inactive leverpressing (Fig. 1d). The injections of the 0.3 and 1, but not 3, mg/kg doses significantly increased active leverpressing in the food-restricted group (n = 8), while only the 1 mg/kg dose significantly increased active leverpressing in AL group (n = 8) [feeding × lever × dose interaction: F(3,42) = 8.35, p < 0.005, followed by Tukey's test]. Active leverpressing of food-restricted rats with the 1 mg/kg dose was greater than that of AL rats with the same dose (p < 0.05; Fig. 1d). The 1 mg/kg dose slightly increased inactive leverpressing of food-restricted rats compared to its vehicle value. All doses of AMPH significantly potentiated locomotor activity [dose: F(3,42) = 41.92, p < 0.0001; feeding × dose: ns]: the 1 mg/kg dose was more effective than the 0.3 or 3 mg/kg dose in stimulating locomotor activity. Thus, while AMPH injections similarly increased locomotor activity between food-restricted and AL rats, AMPH injections increased active leverpressing much more potently in food-restricted rats than AL rats. It should be noted that prior experience with SCH injections did not seem to have influenced these effects of AMPH, since we obtained similar AMPH's effects in subsequent experiments (2b, c), involving rats without SCH experience.

Experiment 2a: Food restriction alone is not sufficient to potentiate VS-reinforced responding

The data shown in Fig. 1a suggest that food restriction moderately potentiates VS-reinforced responding. However, it is unclear whether food restriction alone is sufficient in causing it because the food-restricted rats received repeated behavioral tests during which they gradually reduced weights. Thus, it may be repeated tests, instead of food restriction. Here, we examined with larger numbers of rats (food restriction: n = 16; AL: n = 16) whether a mere 2-week period of food restriction without behavioral test is sufficient for such potentiation. Figure 2a shows the data. First of all, the weights of food-restricted rats were significantly different from those of AL rats starting in session 1 [feeding: F(1,30) = 563.46, p < 0.0001; feeding × session: F(3,90) = 81.66, p < 0.0001, followed by Tukey's test]. The rats clearly discriminated active lever from inactive lever [lever: F(1,30) = 57.12, p < 0.0001], and the discrimination improved over sessions with decreasing inactive leverpressing [lever × session: F(3,90) = 9.38, p < 0.0001]. Food restriction did not alter overall leverpressing [feeding: F(1,30) = 0.27, p = 0.61]; moreover, a feeding × lever × session interaction was not present [feeding: F(3,90) = 0.26, p = 0.81]. However, the food-restricted rats tended to increase leverpressing, while the AL rats decreased it over sessions [feeding × session: F(3,90) = 3.36, p < 0.05]. Therefore, a mere 2-week period of food restriction is not sufficient in potentiating VS-reinforced responding, although food restriction can affect VS-reinforced leverpressing over repeated behavioral sessions. The 2-week food restriction only had a tendency to decrease crossing [feeding: F(1,30) = 3.77, p = 0.062]. The next experiment examined whether additional repeated testing along with repeated injections of AMPH increase VS-reinforced responding.

Experiment 2b and c: Repeated injections of AMPH sensitize locomotor activity, but not VS-reinforced responding

Repeated injections of AMPH are known to sensitize locomotor activity (Robinson and Becker 1986). However, previous studies reported that repeated injections of AMPH do not sensitize responding reinforced by LHS or intracranial self-stimulation (Wise and Munn 1993; Cabeza de Vaca et al. 2004). We examined whether repeated injections of AMPH sensitize VS-reinforced responding. The 1 mg/kg dose was chosen because it was the most effective dose in potentiating VS-reinforced responding (Fig. 1d), and therefore, it gives enough room for detecting decrease in the behavior as a result of repeated AMPH injections. In addition, injections were repeated eight times to see whether increase in VS-reinforced responding in food-restricted rats (Fig. 1a) can be replicated here in saline-injected food-restricted rats.

The food-restricted (n = 16) and AL (n = 16) rats used in the above experiment were divided into AMPH and saline injection groups while maintaining the same feeding conditions. Consistent with the finding shown in Fig. 1d, we observed a significant feeding × drug × lever interaction [F(1,28) = 4.53, p < 0.05 with a 2 feeding × 2 drug × 8 session × 2 lever ANOVA] (Fig. 2b). Active lever counts of food restriction/AMPH rats were greater than any other lever counts of the rats on food restriction, AL, AMPH or saline (p values < 0.05) except active lever counts of food restriction/saline rats (p = 0.21; Tukey's test). This is explained by food restriction/AMPH rats tended to decrease active leverpressing (p < 0.05, the session 10 value compared to its vehicle value) and the food restriction/saline rats tended to increase active leverpressing (p < 0.05, the session 11 value compared to its vehicle value) while AL/saline or AL/AMPH rats did not significantly change active leverpressing over the course of the sessions [feeding × drug × lever × session interaction: F(7,196) = 3.40, p < 0.005]. The latter result is consistent with the effect of food restriction on VS-reinforced responding shown in Fig. 1a. Inactive leverpress levels did not significantly change in all four groups (Tukey's test).

Following a 3-week injection-free period, we examined effects of different AMPH doses on leverpressing. A 2 feeding × 2 drug experience × 5 dose × 2 lever ANOVA indicates that none of the effects involving prior drug experience was statistically significant. We then conducted 2 feeding × 2 drug experience × 5 dose ANOVAs separately between active and inactive lever responses, to clearly determine effects of the factors on each lever. Again, prior AMPH experience had no significant effect on active or inactive leverpressing. A feeding × dose interaction is significant for active lever [F(4,112) = 8.99, p < 0.0005]. The 0.3 and 1 mg/kg doses significantly increased active leverpressing in food-restricted rats, but not in AL rats, while the 3 mg/kg dose significantly decreased active leverpressing in both groups (Tukey's test). Although the mean of saline-experienced food-restricted rats at the 3 mg/kg dose does not appear to be lower than its 0 mg/kg dose, the mean is distorted by one rat pressing 796 times, while others pressing less than 20. A feeding × dose interaction is also significant for inactive lever [F(4,112) = 5.86, p < 0.05]. The 3 mg/kg dose significantly decreased inactive leverpressing in both food-restricted and AL rats, while other doses had no significant effect in either group. The interaction appears to be explained differential responding between the two groups at the 1 mg/kg dose: this dose tended to decrease inactive leverpressing in AL rats, while tending to increase it in food-restricted rats. The mean response of saline-experienced food-restricted rats at the 1 mg/kg dose is relatively high; however, because this is largely caused by one rat pressing over 400 times, it was not significantly greater than the 0 mg/kg dose.

Repeated AMPH injections increased crossing counts over sessions, while saline injections did not [session × drug: F(7,196) = 3.60, p < 0.01 with a 2 feeding × 2 drug experience × 5 dose ANOVA]. While AMPH group had greater crossing than saline group's in any of sessions 5–12, AMPH in sessions 6–12 had greater crossing than that in session 5 (Tukey's test). Although food-restricted rats receiving AMPH tended to have greater crossing counts than AL rats receiving AMPH, effects of food restriction are not significant [feeding: ns; feeding × session: ns; feeding × session × drug: ns]. However, when effects of different AMPH doses were examined 3 weeks later, food restriction significantly potentiated crossing [feeding: F(1,28) = 12.34, p < 0.005] and a significant feeding × dose interaction was found [F(4,112) = 7.14, p < 0.001]. Both food-restricted and AL groups increased crossing at the 0.3 and 1 mg/kg doses, and AL, but not food-restricted, group showed significant decrease in crossing at the 3 mg/kg dose. Also, a significant drug experience × dose interaction was detected [F(4,112) = 10.63, p < 0.0001]. While both AMPH- and saline-experienced groups increased crossing at the 0.3 and 1 mg/kg doses when compared to respective saline values, AMPH-experienced group significantly increased crossing at the 0.1 mg/kg dose as well when compared to the saline value of saline-experienced group; moreover, the 3 mg/kg dose significantly decreased crossing in AMPH-, but not saline-, experienced group (Tukey's test). Therefore, these results suggest that prior AMPH-exposure shifted the dose response curve to the left and are consistent with the notion that prior AMPH-exposure sensitized rats with respect to locomotor activity.

It should be noted that repeated AMPH injections affected body weight gain in AL rats. Although injections were separated by 48 h, the AL rats that received repeated injections of AMPH gained weights at a lower rate than the AL rats receiving saline injections [drug × session: F(7,98) = 4.59, p < 0.05]. Interestingly, after 3 weeks of no injection, the difference in weight between the AMPH- and saline-treated AL rats was significant [drug experience: F(1,14) = 6.17, p < 0.05; drug experience × session: ns]. The same analyses do not apply to food-restricted rats, because experimenters controlled their weights.

Experiment 3a: Acute food restriction does not augment AMPH-potentiated VS-reinforced responding

Experiments 1d and 2b,c showed that food restriction readily augments AMPH-potentiated VS-reinforced responding. The food-restricted rats used in these experiments had been on the food restriction condition for, at least, 2 weeks when they received the first AMPH injection. Thus, it is not clear whether chronic food restriction is necessary in inducing the potentiating effect. Previous studies suggest that the potentiating effect of food restriction on LHS-reinforced responding depends on continuous food restriction over several days or weeks (Carr and Wolinsky 1993; Carr 2002). Although LHS is distinct from VS, common mechanisms may be responsible for augmenting effects of food restriction on AMPH-potentiated responding reinforced by LHS and VS. If this is so, augmenting effect of food restriction is absent after a 24-h food deprivation, and food restriction's effects on AMPH-potentiated VS-reinforced responding grow larger and larger over the course of 2 weeks. To test this hypothesis, we compared AMPH-potentiated responding of AL rats with that of food-restricted rats on food restriction days 0, 1, 3, 7, and 14.

When food-restricted rats received AMPH for the first time after a 24 h food deprivation (day 1), they did not display marked increase in VS-reinforced responding (Fig. 3a). Their active leverpressing did not significantly increase from that of no-injection day (day 0) or from that of AL rats. However, the food-restricted rats increased VS-reinforced responding more and more over the course of the 2 weeks as the food restriction condition continued. Similar to our observations shown in Figs. 1d and 2b, after 2 weeks of continuous food restriction, the food-restricted rats displayed marked increase in VS-reinforced responding compared to AL control rats. These observations are supported by a significant feeding × lever × day (0, 1, 3, 7, and 14) interaction [F(4,72) = 6.04, p < 0.005, followed by Tukey's test with a 2 feeding × 2 lever × 5 day ANOVA]. In contrast to active leverpressing, inactive leverpressing was not significantly altered by AMPH in either AL or food-restricted rats over the course of 14 days (Tukey's test). These results support the hypothesis stated above.

Crossing counts of days 1, 3, 7, and 14 were greater than that of day 0, and crossing counts of day 3, 7, and 14 were greater than that of day 1 (Fig. 4c) [length: F(4,72) = 164.14, p < 0.0001, followed by Tukey's test]. Moreover, the food-restricted group displayed more pronounced increase in crossing than the AL group [feeding × length: F(4,72) = 3.49, p < 0.05]. Food-restricted group's crossing counts of days 1, 3, 7, and 14 were greater than that of day 0, and those of days 3, 7 and 14 were greater than that of day 1, while AL group's crossing counts of days 1, 3, 7, and 14 were greater than that of day 0, but crossing counts of days 3, 7, and 14 were not greater than that of day 1 (Tukey's test). These data suggest that crossing was sensitized by repeated AMPH injections and continuing food restriction. The data on body weight are also shown in the figure. Over the 14-day period, the weights of food-restricted rats gradually decreased to 85 % of the day 0, while those of AL rats gradually increased to 107 % during the same period (Fig. 3) [feeding × length: F(4,72) = 98.57, p < 0.0001].

Experiment 3b: SCH similarly decreases food restriction/AMPH-potentiated VS-reinforced responding and locomotor activity

To determine the role of dopamine in food restriction/AMPH-potentiated VS-reinforced responding and locomotion, effects of SCH were examined. Both doses of SCH (12.5 and 25 μg/kg, SC) significantly decreased AMPH-potentiated active leverpressing, but not inactive leverpressing (Fig. 3b) [dose × lever: F(2,18) = 11.31, p < 0.001, followed by Tukey's test]. Similar to active leverpressing, both SCH doses also decreased locomotor activity [dose: F(2,18) = 209.45, p < 0.0001]. Thus, these results suggest that dopamine transmission via D1 receptors is similarly important between food restriction/AMPH-potentiated VS-reinforced responding and locomotor activity.

Experiment 4a and b: Lithium treatments reduce VS-reinforced responding, but augment it when combined with AMPH

Lithium has been the most effective treatment in the manic phase of bipolar disorder (Jefferson 1990; Bowden et al. 1994). Symptoms of mood disorders include changes in motivation and reward seeking (Hasler et al. 2006); therefore, it is interesting to determine how lithium alters VS-reinforced responding under food restriction. Rats were fed with either lithium or control diet for more than 3 weeks before testing. In a separate group of rats (n = 10), we confirmed that this lithium diet regimen achieved a serum concentration of lithium equaling 0.61 mM ± 0.05 (mean±SEM). This level is comparable to the therapeutic levels of human bipolar patients, which ranges from 0.5 to 1.2 mM (Gould et al. 2007).

The lithium treatment decreased leverpressing (Fig. 4a) [lithium: F(1,20) = 5.51, p < 0.05]: it selectively decreased active leverpressing, but not inactive leverpressing [lithium × lever: F(1,20) = 8.14, p < 0.01, followed by Tukey's test]. When the rats were injected with AMPH, however, the lithium treatment further augmented VS-reinforced responding (Fig. 4b) [lithium × lever × dose: F(4,80) = 4.69, p < 0.05]. AMPH doses of 0.5, 1 and 2, but not 4, mg/kg increased active leverpressing in the lithium diet group, while doses of 0.5 and 1, but not 2 or 4, mg/kg increased it in the control diet group; inactive leverpressing was not significantly altered by AMPH doses in either group (Tukey's test). Thus, we found a novel effect of lithium on AMPH-potentiated behavior.

Consistent with previous studies, the lithium treatment decreased locomotor activity (Fig. 4a) [lithium: F(1,20) = 7.84, p < 0.05]. Locomotor activity decreased over the repeated sessions [session: F(7,140) = 10.11, p < 0.0001, followed by Tukey's test; diet × session: F(7,140) = 2.00, p = 0.11]. The lithium treatment did not selectively alter AMPH-potentiated locomotor activity between the groups (Fig. 4b) [lithium × dose: ns: diet: ns]: all doses of AMPH (0.5–4 mg/kg) potentiated crossing [dose: F(4,80) = 141.14, p < 0.0001, followed by Tukey's test].