Novel approach to data analysis in cocaine-conditioned place preference

Abstract

Only a subgroup of human drug users progress from initial drug taking to drug addiction. The learned associations between the effects of the drug and the environment in which it is experienced is an important aspect of the progression to continued drug taking and drug seeking. These associations can be modeled using the conditioned place preference (CPP) paradigm, although no current method of CPP analysis allows for the identification of within-group variability among subjects. In this study, we adapted a 'criterion' method of analysis to separate 'CPP expressing' from 'non-CPP expressing' rats to study more directly within-group variability in the CPP paradigm. Male Sprague-Dawley rats were conditioned with cocaine (5, 10, 20 mg/kg) or saline in an unbiased three-chamber CPP apparatus in either a single-trial or four-trial CPP procedure. A classification and regression tree analysis of time spent in the cocaine-paired chamber established a time of 324 s spent in the cocaine-paired chamber as the criterion for cocaine CPP expression. This criterion effectively discriminated control from cocaine-conditioned rats and was reliable for rats trained in both single trial and four-trial CPP procedures. The criterion method showed an enhanced ability to detect effective doses of cocaine in the single-trial CPP procedure and a blockade of CPP expression by MK 212 (0.125 mg/kg) treatment in a subgroup of rats. These data support the utility of the criterion analysis as an adjunct to traditional methods that compare group averages in CPP.

Figure 1. Distribution of time spent in the initially least-preferred chamber demonstrates a significant difference between saline and cocaine conditioning

Fig. 1A, Rats in control (n=121, open circles) and cocaine (n=158, black circles) groups were tested for the expression of CPP in a 15 min session. The amount of time spent by each rat during the test session in the initially least-preferred chamber was categorized according to 25 sec bins. χ² analysis revealed a significant difference between the two distributions (p<0.001). Fig. 1B, Rats in control (open box) and cocaine groups (grey box) were tested for the expression of CPP in a 15 min session. The line in the center of the box represents the median time, and the top and bottom of the box represent the 75^th and 25^th percentiles, respectively. The black circles represent the 95^th and 5^th percentiles. The solid line (“criterion”) represents the 324 s criterion for the expression of cocaine CPP.

Figure 2. The distribution of time spent in the initially least-preferred chamber does not depend on number of conditioning sessions

Fig. 2A, Following conditioning with either a single pairing with the environment of 20 mg/kg of cocaine (open square, n=71) or four pairings of 10 mg/kg of cocaine (open triangle, n=87), animals were tested for the expression of CPP in a 15 min session. The amount of time spent by each rat in the initially least-preferred chamber during the test session was categorized according to 25 sec bins. No differences in the percentage of animals meeting the CPP expression criterion were observed between the two conditioning procedures. Fig. 2B, Rats in single- (open box) and four-trial groups (grey box) were tested for the expression of CPP in a 15 min session. The line in the center of the box represents the median, and the top and bottom of the box represent the 75^th and 25^th percentiles, respectively. The filled circles represent the 95^th and 5^th percentiles. The solid line represents the 324 s criterion for the expression of cocaine CPP.

Figure 3. Cocaine produces a CPP following a single or four pairings of cocaine and environment

Following either a single (Fig. 3A) or four (Fig. 3B) conditioning sessions in which the environment was paired with cocaine (0, 5, 10, 20 mg/kg), the amount of time rats (n=11–12/group) spent during a 15 minute drug-free test session in the initially least-preferred chamber was observed. Fig. 3A, left, Criterion analysis comparing the proportion of animals spending at least 324 s during the test session in the initially least-preferred chamber revealed a significant effect of cocaine (10 or 20 mg/kg) conditioning. Fig. 3A, right, Traditional analysis comparing the mean time spent in the initially least-preferred chamber during the test session by each conditioning group revealed a significant effect of cocaine (20 mg/kg) conditioning. Fig. 3B, left, Criterion analysis demonstrated a significant effect of cocaine (5, 10, 20 mg/kg) conditioning following four pairings. Fig 3B, right, Traditional analysis also demonstrated a significant effect of cocaine (5, 10, 20 mg/kg) after four pairings. * p< 0.05 vs 0 mg/kg cocaine (control)

Figure 4. MK 212 treatment blocks expression of a single trial cocaine (20 mg/kg) CPP in the criterion but not traditional analysis

Following preconditioning, animals were conditioned with saline (open bars) or cocaine (20 mg/kg, grey bars) to the initially least-preferred side of the chamber. Ten minutes before the test session, animals were treated with MK 212 (0, 0.125, 0.25 mg/kg). Fig. 4A, Criterion analysis of the percentage of animals meeting or exceeding the 324 s criterion for CPP expression revealed a blockade of expression of a single trial cocaine CPP by MK 212 (0.125 mg/kg). Fig. 4B, Traditional analysis comparing the mean group time during the test session in the initially least-preferred chamber demonstrated that MK 212 treatment did not alter the expression of a single trial cocaine CPP. Fig. 4C, Composite analysis demonstrated overlap of criterion and traditional analyses; the amount of time spent during the test session in the initially least-preferred chamber by individual animals (open circles); proportion at top gives the number of animals meeting the criterion/number in treatment group. Dashed line (- - -) represents 324 s criterion. Black boxes represent group mean ± SEM. *p<0.05 vs vehicle-saline; ^ p<0.05 vs vehicle-cocaine; cond=conditioning drug, test=injection before test session.