As saccharin was incrementally faded from the administered solutions, mean ± SEM ethanol intakes (g/kg dose; total licks) declined as follows: 0.2S/12E (1.20 ± 0.11; 325 ± 30), 0.05S/12E (0.69 ± 0.08; 173 ± 20), and 10E (0.26 ± 0.05; 72 ± 13). Consumption levels of 10E during FR1 and FR2 reinforcement schedules were comparable to those described below for FR4.
3.1. FR4 schedule of reinforcement
The training history during manipulations in sipper access time following the completion of a FR4 schedule of reinforcement is depicted in sessions 1-25 of . Increasing the sipper access time from 15- to 60-sec during each 10E presentation did not alter the total licks recorded during the 30-min sessions. The mean ± SEM lick values during the last 3 sessions of each access time were 59 ± 14 (15-sec), 70 ± 16 (30-sec) and 61 ± 12 contacts (60-sec). Similarly, increasing sipper access time did not significantly alter the frequency of sipper presentations, with mean ± SEM frequencies of 8.0 ± 0.8 (15-sec), 7.0 ± 0.8 (30-sec) and 8.1 ± 0.6 (60-sec). A significant positive correlation between total session sipper contacts and sipper presentation frequency was found with sipper access periods of 30-sec (r = 0.382; p = 0.001) and 60-sec (r = 0.278; p = 0.022), but not with 15-sec access.
Figure 1 History of reinforcement schedule manipulations during operant conditioning. Depicted are the mean ± SEM for 24 mice per session. FR refers to a ‘fixed ratio’ schedule of reinforcement whereas RR refers to a single ‘response (more ...)
3.2. Comparison of appetitive response measures across reinforcement schedules
In order to estimate the strength of the 10E solution as a behavioral reinforcer across reinforcement schedules, measures corresponding to the initial 4 responses completed during the FR4 schedule contingency were compared with values from the RR4 and RR8 schedules. All mice completed the response requirement under each reinforcement schedule. Manipulation of the reinforcement schedule significantly influenced the response latency [F(2,46) = 4.40; p < 0.05], with the transition from FR4 to RR4 significantly decreasing this latency by 34% (p < 0.05; ). The RR8 schedule was associated with an even more pronounced reduction in the response latency, when compared to the FR4 schedule (p < 0.01). A significant main effect of reinforcement schedule on response rate also was detected [F(2,46) = 4.44; p < 0.05], with a significant decrease in response rate with the RR8 schedule, when compared to the FR4 schedule (p < 0.01). Latency to 1st lick values (time elapsed from the onset of sipper access) did not to follow a normal distribution. Consequently, these values were log10-transformed, at which point a normal distribution was observed. A trend toward significance for reinforcement schedule on the latency to first lick was noted [F(2,46) = 2.81; p = 0.07], with the transition to RR4, and then to RR8 resulting in latency reductions of 54% and 65%, respectively, when compared to the FR schedule. Although alteration of the reinforcement schedule also significantly affected inactive lever responding [F(2,46) = 11.17; p < 0.001], the slight but significant increase was found only with the RR8 schedule.
Table 1 Appetitive measures during FR and RR reinforcement schedules. Values are the mean ± SEM of the last 3 sessions on each reinforcement schedule. Data for the first completed response requirement in mice on the FR4 schedule is depicted (except when (more ...)
3.3. Comparison of ethanol intake and consumption patterns across reinforcement schedules
Transition from a FR to RR schedule of reinforcement favored a significant elevation in 10E self-administration (see ), as evidenced by the significant increase in ethanol dose [F(2,46) = 22.79; p < 0.001], licks [F(2,46) = 28.10; p < 0.001] and 10E volume [F(2,46) = 23.60; p < 0.001]. Ethanol dose, licks and 10E volume were significantly augmented following the transition from FR4 to RR4 by 93% (p < 0.05), 174% (p < 0.001) and 95% (p < 0.05), respectively. Furthermore, the RR8 schedule was associated with significantly greater total licks than those observed with the FR4 (p < 0.001) and RR4 (p < 0.05) schedules. Likewise, the RR8 schedule significantly increased the 10E dose and 10E volume consumed versus the FR4 and RR4 schedules (p < 0.001 for each comparison). Importantly, significant positive correlations between total licks and 10E dose were observed for the FR4 (r = 0.73, p < 0.001, n = 24), RR4 (r = 0.63, p < 0.001, n = 24) and RR8 (r = 0.80, p < 0.001, n = 24) reinforcement schedules, thereby indicating that licks accurately reflected actual ethanol consumption throughout the limited access sessions.
Table 2 Consummatory measures during FR and RR reinforcement schedules. Ethanol intake and lick run dynamics are depicted for mice permitted either intermittent sipper tube access on a FR4 schedule or 30-min continuous access following the completion of a RR4 (more ...)
Consistent with the augmented intakes noted above during the transition from a FR to a RR schedule, the lick run patterns were altered in a manner commensurate with an enhanced onset and maintenance of ethanol drinking. shows that the frequency and size of lick runs were significantly influenced [run frequency: F(2,46) = 46.05; p < 0.001; run size: F(2,46) = 13.29; p < 0.001] by manipulations in reinforcement schedule. Mice responding on a RR4 and RR8 schedule showed elevated lick run frequencies of 168% (p < 0.001) and 195% (p < 0.001), respectively, and increases in lick run sizes of 37% (p < 0.01) and 68% (p < 0.001), respectively, when compared to the FR4 schedule. Notably, the RR8 schedule was affiliated with significantly greater run sizes than those calculated for the RR4 schedule (p < 0.05). also shows that there were significant main effects of reinforcement schedule on the size of the largest lick run [F(2,46) = 20.69; p < 0.001], inter-run interval [F(2,40) = 13.20; p < 0.001], lick run duration [F(2,46) = 21.19; p < 0.001], and lick run rate [F(2,44) = 6.72; p < 0.01].
A separate analysis of the initial lick run of the self-administration session was conducted to more specifically delineate the impact of reinforcement schedule manipulations on drinking onset (). A significant main effect of schedule was determined for the size [F(2,46) = 15.16; p < 0.001], duration [F(2,46) = 7.09; p < 0.01] and lick rate [F(2,43) = 4.35; p < 0.05] of the first run. First lick run durations were significantly increased by approximately 1.9-fold for both the RR4 and RR8 schedules (p< 0.01 for each), whereas first lick run rates fell by 52% (p < 0.01) and 32% (p < 0.05) for the RR4 and RR8 schedules, respectively, when compared to the FR4 schedule. The latency to first lick run also was significantly decreased by the transition from FR4 to RR4 and RR8 schedules [F(2,46) = 14.04; p < 0.001].
3.4. Comparison of cumulative records across reinforcement schedules
Representative cumulative records of responding and sipper contacts for FR4, RR4, and RR8 reinforcement schedules are depicted in . An analysis of the FR4 schedule with 60-sec sipper access revealed that only 53 ± 4% of the sipper presentations were accompanied by a sipper contact (see panel A of ). Furthermore, only 28 ± 4% of the sipper tube presentations yielded a lick run of ≥ 5 licks. Although mice made contact with the sipper tube 66 ± 7% of the time during the first sipper presentation of the session, a point in which the salience of ethanol as a reinforcer would presumably be the greatest, the mean lick run size was only 7 ± 2 contacts (comparable to representative record shown in panel A of ). These observations indicate that when mice were performing on a FR4 schedule, they failed to reliably consume ethanol following each sipper presentation and the initial access to the 10E solution corresponded to negligible levels of drinking.
Figure 2 Representative cumulative records of operant ethanol self-administration during FR4 (panel A), RR4 (panel B) and RR8 (panel C) reinforcement schedules. The top portion of each panel depicts the pattern of cumulative licks. The gray boxes along the abscissa (more ...)
Consistent with the appetitive responding results detailed above (), transition to the RR schedules corresponded to decreases in the response latency but an attenuated response rate, as shown in the representative records for the RR4 (, panel B) and RR8 (, panel C) schedules. Pronounced differences in the onset and maintenance of 10E self-administration between the reinforcement schedules also were readily apparent from the cumulative records, consistent with the first and mean lick run measures reported in , respectively.
3.5. Extinction of ethanol-reinforced responding
Ten mice were selected for an evaluation of extinction. Ethanol dose and BECs were 0.64 ± 0.16 g/kg and 37 ± 11 mg%, respectively, in mice on the RR8 schedule prior to extinction onset. There was a statistically significant positive correlation between 10E dose and BEC for these mice (r = 0.92; p < 0.001; n = 10). A significant influence of extinction session was determined for responding on the active (ethanol-appropriate) lever [F(10,90) = 16.00; p < 0.001]. Mice robustly responded on the active lever during the first day of extinction when compared to their pre-extinction baseline (p < 0.001; ). Responding on the active lever remained significantly elevated over the pre-extinction baseline during sessions 2-6 (p < 0.05 for all), but dropped to levels that were not significantly different from baseline during sessions 7-10. A significant influence of extinction session also was observed for responding on the inactive lever [F(10,90) = 2.03; p < 0.05], with small, but significant increases in responding during extinction sessions 1, 5-6 and 10 when compared to baseline values (p < 0.05 for all).
Lever response frequencies throughout the extinction time course. The mean ± SEM active (filled squares) and inactive (open circles) lever responses recorded during 30-min extinction sessions for 10 mice are depicted.