Forty eight young adult, postnatal day 56 (P-56) to P-58 female Sprague-Dawley rats, weighing 170 - 180 g upon arrival, were acquired from Harlan, Indianapolis, IN, USA. Rats arrived in multiple shipments and were housed individually in a cage that became their test cage in the sound proof experiment room on a 12:12 night/ dark cycle (lights on at 06:00) at an ambient temperature of 21˚C ± 2˚C and relative humidity of 37% - 42%. Food pellets and water were provided ad libitum to the animals. After 5 to 7 days of acclimation, animals were weighed and randomly divided into four groups, control (n = 12), 3.0 mg/kg i.p. cocaine (n = 12), 7.5 mg/kg i.p. cocaine (n = 12), and 15 mg/kg i.p. cocaine (n = 12) groups, and placed back in their open field testing/home cages (40.5 × 40.5 × 31.5 cm) in the same room. An additional acclimation period of 24 to 36 hours was followed by 11 consecutive recording days (Table 1). All effort was made to minimize the number of animals used and their suffering. The experimental protocol was approved by our animal welfare committee.

Locomotor activity was recorded using the computerized animal activity monitoring (CAAM; AccuScan In struments, Inc., Columbus, OH) system. During eleven non-stop recording days, the recordings were stopped

ED: experimental day; All injections were i.p. and equalized to volume of 0.8 cc/animal and were given at 07:00.

only for animal handling between 06:30 and 07:00. The sound-proof experimental room was closed during all other hours and the animals were not disturbed. The 12 open field cages each had two levels of 16 infrared beams and their sensors consisted of the CAAM system. The interruptions of these beams were counted and compiled by AccuScan Analyzer and downloaded to PC every 10 minutes. Then OASIS software organized these data into several different locomotor movement indices, such as total distance traveled and horizontal and stereotypic activities [7] [20] [23] - [27] .

Cocaine was dissolved in 0.9% isotonic saline solution (0.9% NaCl). All the injections were of the same volume (0.8 cc) to insure that different responses were not due to differences in injection volumes. Injections were made intra-peritoneal (i.p.) between 06:30 and 07:00.

On experimental day 1 (ED 1) (baseline control data), all groups were given a single injection of 0.8 ml of 0.9% NaCl solution, and the recording started immediately after injection, approximately at 07:00. On ED 2 to 7, animals received a single injection daily of either; saline, 3.0 mg/kg cocaine, 7.5 mg/kg cocaine, or 15.0 mg/kg cocaine depending on the group assignments (Table 1). EDs 8 to 10 were the washout period, and the locomotor activity was recorded without any injection. A single re-challenge injection, of identical dose as given in ED 2 through 7, followed on ED 11 (Table 1). Locomotor activity was recorded nonstop for 11 days with animal handling occuring each day. The experiment procedures conformed to the guidelines of NIH and the declaration of Helsinki and approved by our local Animal Welfare Committee. In preliminary experiments four female rats were kept together in one cage from P40 to P80 with the intention that all the female rats would cycle on the same day. Vaginal smears [28] were taken daily prior to daily psychostimulant injections. It was found that the drug treatment did not alter the estrous cycle. Thus, all the experimental groups started in the proestrous day and lasted for 11 days, i.e., about three estrous cycles.

Two data analyses were used. 1) Six 10 min recording bins were summed and averaged to produce hourly histograms with standard errors. They showed 24-hour activity for each locomotor index, horizontal activity (HA), total distance (TD) traveled, and number of stereotypy (NOS); 2) The same 10 min counts/bins were used as plotting points for statistical calculation using the cosine curve statistical analysis (CCSA) test [29] , which is a test that examines whether circadian activity pattern is significantly altered between the two experimental days (EDs) being compared. The CCSA test fit the data points of each recording point into a cosine curve and estimated three parameters, mesor (average activity level), amplitude (distance from mesor to highest point), and acrophase (time at which the maximum amplitude occurs) in order to more accurately model the cyclic nature of the activity over a 24-hour cycle. These three components were used to determine whether locomotor activity diurnal rhythm pattern has been significantly altered [29] . There was a dose dependent increase in locomotor activity immediately after the cocaine injection. This effect was profound and lasted as long as three hours. This immediate increase in locomotion as a response to the injection skewed the statistical parameterization of the CCSA test, as the data points were poorly fit using a cosine curve. Therefore, in order to account for the immediate effect of the drug and to create a statistically appropriate cosine curve, the initial three hours of the locomotor activity after the injection were eliminated. To maintain consistent parameterization and analysis of the protocol all subsequent calculation were based on the removal of the first three hours after cocaine exposure. Since the focus of this study is on the activity pattern of the whole day and not the immediate effect post-inject- tion, the first three hours of the data after the cocaine injections were excluded in the CCSA analysis for all groups.

To analyze the data, four comparisons were made: 1) Acute effect―compared the activity of ED 1 (control recording with saline) with activity of ED 2 (the first day of cocaine injection) to observe whether a single (acute) injection is able to change the locomotor diurnal rhythm activity pattern; 2) Induction phase―comparison of the activity of EDs 1 against the activity obtained on ED 7 (the last day of the drug maintenance treatments; Table 1) to observe whether the six days of treatments induce changes in activity pattern; 3) Washout phase―comparison between activity of ED 1 to activity of EDs 8, 9, and 10 to observe whether the activity alterations induced during the maintenance period persists in absence of daily cocaine injections (Table 1); and 4) Expression phase― comparison of the activity obtained on EDs 1 with the activity of ED 11 (i.p. cocaine re-challenge) to observe whether the chronic treatment elicits a long lasting effect; only the HA index was discussed in this paper, because all the other locomotor indices exhibited similar findings.

Figure 1 shows the control group’s (rats treated with saline on ED 1 to ED 7 and ED 11) hourly histogram and the superimposed graphs of the temporal 10 minutes counts/bin of horizontal activity (HA) for experimental days (EDs) 1, 2, 7, 8, and 11. As reported in previous studies, rats treated with saline only showed increases in activity during first few minutes post injection and then the activity levels returned to baseline [7] [20] . The diurnal HA rhythm pattern stayed similar with minor non significant fluctuations throughout the 11 experimental days period. The CCSA test comparing the activity pattern of ED 1 with ED 2 to ED 11 indicated no significant changes in diurnal HA patterns between these EDs. Figure 1 and Table 2 show the histogram and the CCSA test data for ED’s 1, 2, 7, 8, and 11. Other locomotor indices also yielded similar results (not all graphs are shown). Since every new shipment of rats exhibit different baseline activity, and based on the saline control observation that all the day exhibit similar activity pattern as ED1, any significant differences using the CCSA test between the data of saline ED 1 and other EDs of cocaine treated days can and was considered as the drug effects.

Figure 2 shows the hourly histogram of ED 1 and ED 2 and the CCSA test graph for HA index of 7.5 mg/kg cocaine group. There were increases in the first three hours post injection, but the activity levels were decreased throughout the rest of ED 2. The CCSA test indicated that acute cocaine injection induced significant changesin the HA patterns between ED 2 and ED 1 (Table 3). Asimilar observation was obtained from the 15.0 mg/kg i.p. cocaine group. However, the 3.0 mg/kg i.p. cocaine group, in the CCSA test, did not show any significant changes in locomotor circadian rhythms between ED 1 and ED 2 for any of the indices compared (Table 3).

The hourly HA histograms and the corresponding temporal graph of the CCSA test of the 7.5 mg/kg cocaine

Significance was set at P < 0.05. LI: locomotor index; Mesor: mean level of cosine curve; Amp: amplitude— height of the peak above the mean level; Acrotime: time at which peak occurs; Comparing all the experimental days of control group shows no significant change in circadian rhythm throughout the experiment, i.e. activity obtained on ED 1 following saline was similar to that obtained on ED 2, 7, 8, and 11.

Significance was set at P < 0.05. Gray highlighted field indicates the significant changes between the two compared experimental days. Only the results with significant differences were shown. LI: locomotor indicess; Mesor: mean level of cosine curve; Amp: amplitude―height of the peak above the mean level; Acrotime: time at which peak occurs.

group comparing ED 7 to ED 1 are displayed in Figure 3. As with the comparison between EDs 1 and ED 2, there were increases in locomotor activity in the first two to three hours post cocaine injection. The HA was increased in some hours of the dark phase and decreased during some hours of light and dark phase. The CCSA test comparing the activity levels post cocaine injection on ED 7 with ED 1 indicated significant differences between the diurnal rhythm patterns in the two EDs (Table 3). Similar findings were recorded for the other two cocainedos age groups as well (Table 3).

Figure 4 is similar to Figure 3 but describes the comparison between the data of ED 1 and ED 8, of these groups (3.0; 7.5; and 15.0 mg/kg cocaine) the first washout day (Table 1). There were some increases in locomotor activity during dark phase on ED 8 compared to ED 1. These fluctuations in activities resulted in significant changes between the diurnal rhythms of ED 1 and 8, according to the CCSA test (Table 3). Similar findings were recorded for other dosage groups as well (Table 3) and at ED 9 and 10 (data not shown).

Figure 5 compares the data of ED 1 to that of ED 11 for 7.5 mg/kg cocaine group. The figure shows that following cocaine injection on ED 11, there are increases in locomotor activity in the first two to three hours of light phase and during some hours of the dark phase. These changes resulted in significantly altered diurnal rhythm on ED 11 compared to ED 1 according to the CCSA test (Table 3). Similar findings were recorded for other dosage groups as well (Table 3).