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. Author manuscript; available in PMC: 2021 Aug 24.
Published in final edited form as: Behav Brain Res. 2020 Nov 6;398:112982. doi: 10.1016/j.bbr.2020.112982

Effects of repeated RU 24969 treatment on the locomotor activity, motoric capacity, and axillary temperatures of male and female preweanling rats

Sanders A McDougall a,*, Jessica L Razo a, Jasmine W Rios a, Jordan A Taylor a
PMCID: PMC8384048  NIHMSID: NIHMS1734615  PMID: 33166571

Abstract

Serotonin (5-HT) 1A and 1B receptors have been implicated in behavioral sensitization, but adult rats appear to develop tolerance to RU 24969 (a 5-HT1A/1B receptor agonist) rather than a sensitized response. The purpose of the present study was to determine whether a one- or four-day pretreatment regimen of RU 24969 would cause sensitization or tolerance in male and female preweanling rats. Depending on experiment, rats were pretreated with RU 24969 (0, 2.5, or 5 mg/kg) for 1 or 4 days (PD 17–20), while testing with lower or higher doses of RU 24969 occurred on PD 22. Locomotor activity, motoric capacity, and axillary temperatures were recorded. The role of Pavlovian contextual conditioning was assessed by administering RU 24969 to rats in either the home cage or a novel environment. On the first pretreatment day, RU 24969 caused both an increase in forward locomotion and motoric impairment, along with a substantial decrease in axillary temperatures. Repeated treatment with the same dose of RU 24969 caused all three dependent measures to show a tolerance response. When given a higher dose of RU 24969 on the test day, the responses lost due to repeated drug treatment were fully (locomotor activity) or partially (motoric capacity and axillary temperatures) reinstated. There was no evidence of behavioral tolerance. Results are consistent with the hypothesis that a subsensitivity of 5-HT1A/1B receptors is at least partially responsible for the tolerance caused by RU 24969, but dispositional tolerance cannot be excluded as a contributing factor.

Keywords: RU 24969, tolerance, behavioral sensitization, locomotor activity, ontogeny

1. Introduction

Serotonin (5-HT) neurotransmission modulates motor system functioning, with most 5-HT receptor subtypes inhibiting locomotor activity [1]. An obvious exception is the 5-HT1 receptor, because stimulating this receptor subtype with the selective 5-HT1A/1B receptor agonist RU 24969 significantly increases locomotor activity in preweanling and adult rats [2–5]. Consistent with this finding, the indirect 5-HT agonist 3,4-methylenedioxymethamphetamine (MDMA) causes a perseverative hyperactivity in adult rats that appears to be mediated by the 5-HT1 receptor [6–8]. When administered repeatedly, MDMA produces a progressively elevated locomotor response that is referred to as behavioral sensitization [9–11]. The sensitized response is context-dependent, meaning that behavioral sensitization is stronger if drug pretreatment and testing occur in the same novel environment [12,13]. Because both RU 24969 and MDMA purportedly induce locomotor activity through actions at the 5-HT1 receptor, it might be expected that repeated treatment with RU 24969 would also produce locomotor sensitization.

Contrary to this reasoning, a number of studies have found that repeated administration of RU 24969 causes tolerance, rather than behavioral sensitization, in adult rats and mice [14,15]. For example, Oberlander et al. [15] reported that four consecutive daily treatments of RU 24969 (5 mg/kg) produced an 80% decline in locomotor activity relative to the first day of testing. Both De Souza et al. [14] and Oberlander et al. [15] concluded that dispositional tolerance, also referred to as pharmacokinetic tolerance (e.g., enhanced metabolism of the drug), was not responsible for the decline in drug action; instead, they hypothesized that repeated RU 24969 treatment caused a down-regulation or subsensitivity of 5-HT1A and/or 5-HT1B receptors. Whether behavioral tolerance contributes to this RU 24969-induced effect has not been investigated. Behavioral tolerance is commonly observed with alcohol and opioid drugs, and is evident when drug pretreatment and testing occur in the same previously novel context [16,17]. Pavlovian processes are often critical for behavioral tolerance, as the drug serves as the unconditioned stimulus (US) and the environmental context serves as the conditioned stimulus (CS) [18–20]. Behavioral tolerance is said to have occurred if repeated CS-US pairings result in a stronger tolerance response.

In sum, depending on the characteristics of the drug and the methodology used, adult rats are capable of exhibiting either behavioral sensitization or tolerance after repeated drug treatment. Immature rats also exhibit these same phenomena [21–24], but some important age-dependent differences are apparent [25]. In terms of behavioral sensitization, sensitized responding produced by dopamine agonists appears to be weaker and less persistent in preweanling rats than adults [26,27]. Moreover, the psychostimulant-induced one-trial behavioral sensitization of adult rats and mice is completely context-dependent [28–31], whereas the one-trial behavioral sensitization of preweanling rats is context-independent [24,32]. Ontogenetic differences involving tolerance are also evident, because rapid and chronic tolerance to ethanol does not emerge until after the preweanling period [33,34], while acute tolerance to ethanol is most pronounced at postnatal day (PD) 16 and declines with increasing age [22]. Therefore, there is abundant evidence that repeated treatment with various classes of drugs causes different patterns of behavioral sensitization and tolerance across ontogeny. Importantly, none of these ontogenetic studies have examined serotonergic compounds.

The methodologies used to assess behavioral sensitization and tolerance often differ in meaningful ways. When studying behavioral sensitization, rats are typically given either a moderate dose (multi-trial procedure) or a high dose (one-trial procedure) of drug during the pretreatment phase, and are often given a lower dose of drug on the test day [35–39]. In contrast, researchers studying drug tolerance frequently administer a moderate dose of the drug during the pretreatment phase, and a higher dose of the drug on the test day. If tolerance has occurred, a high dose should reinstate the responses that were lost due to repeated exposure [15,40,41]. Schuster [41] goes further and recommends administering multiple drug doses during both the treatment and testing phases in order to more fully characterize the pattern of drug tolerance.

The purpose of the present study was to examine the behavioral and physiological effects caused by repeatedly administering RU 24969 to preweanling rats. Young rats were used because this age group has translational relevance to humans [26,42], and ontogenetic comparisons can provide additional levels of analysis when assessing drug action in behavioral or physiological assays [43]. Both one- and multi-trial experimental paradigms were employed (i.e., drug pretreatment occurred on one or four days) and multiple doses of RU 24969 were administered during the pretreatment and test phases. Locomotor activity was the primary dependent variable, although motoric capacity and axillary temperatures were measured in the final experiment. As with adult rats [2], preweanling rats exhibit motor deficits when treated with RU 24969. For this reason, a motoric capacity rating scale was used to quantify the motor movements of saline- and RU 24969-treated preweanling rats. 5-HT1 receptor stimulation causes hypothermia in adult rodents [44,45], thus it was of interest to determine whether this physiological measure would show tolerance after repeated treatment with RU 24969. Lastly, to assess the impact of Pavlovian contextual conditioning factors, rats were pretreated with RU 24969 in either the home cage or a novel environmental context (i.e., the activity chambers).

2. Materials and methods

2.1. Animals

Subjects were 94 male and 94 female preweanling Sprague-Dawley rats (n = 8 subjects per group) that were conceived and born at California State University, San Bernardino (CSUSB). Sires and dams were purchased from Charles River (Hollister, CA). Litters were culled to 10 pups on PD 3. The colony room was maintained at 22–23 °C and kept under a 12:12 light-dark cycle. Food and water were freely available. Testing was done in a separate experimental room and was conducted during the light phase of the cycle. Subjects were cared for according to the “Guide for the Care and Use of Laboratory Animals” [46] under a research protocol approved by the Institutional Animal Care and Use Committee of CSUSB.

2.2. Apparatus

Behavioral testing was done in activity monitoring chambers (26 × 26 × 41 cm) that have a plastic floor, acrylic walls, and an open top (Coulbourn Instruments, Whitehall, PA). Each chamber includes an X–Y photobeam array, with 16 photocells and detectors, that measures distance traveled (cm). Photobeam resolution was 0.76 cm, with the position of each rat being determined every 100 ms. Axillary temperatures were measured using a BAT–12 microprobe thermometer (Physitemp Instruments, Piscataway, NJ).

2.3. Drugs

RU 24969 hemisuccinate (Tocris, Minneapolis, MN) was dissolved in saline and injected intraperitoneally (ip) at a volume of 2.5 ml/kg.

2.4. Procedure

2.4.1. Experiment 1. Effects of repeated RU 24969 treatments on the locomotor activity of male and female preweanling rats: A low test day dose of RU 24969

2.4.1.1. One-trial procedure

On the pretreatment day (PD 20), rats were injected with saline or RU 24969 (2.5 or 5 mg/kg) and immediately placed in activity chambers. On the test day (PD 22), an equal number of rats from each pretreatment group were injected with saline or 1.25 mg/kg RU 24969 and immediately placed in the same activity chambers as before. Distance traveled (DT), which is a measure of locomotor activity, was assessed for 45 min on the pretreatment day and for 120 min on the test day.

2.4.1.2. Multi-trial procedure

During the pretreatment phase (PD 17–20), rats received daily injections of saline or 2.5 mg/kg RU 24969 prior to being placed in activity chambers for 45 min. On the test day (PD 22), rats from each pretreatment group were given a test day injection of saline or 1.25 mg/kg RU 24969 and then immediately placed in activity chambers for 120 min. DT was measured as just described.

2.4.2. Experiments 2. Effects of repeated RU 24969 treatment on the locomotor activity of male and female preweanling rats: A high test day dose of RU 24969

Rats received daily pretreatment injections of saline or 2.5 mg/kg RU 24969 on PD 17–20. Immediately after injections, rats were placed in activity chambers and DT was measured for 45 min. On the test day (PD 22), rats from each pretreatment group were given a test day injection of saline or RU 24969 (5 or 10 mg/kg) and then immediately placed in activity chambers, where DT was measured for 120 min. To assess the longevity of these drug effects, rats pretreated and tested with saline or 5 mg/kg RU 24969 were further subdivided, with rats receiving a subsequent injection of saline or 5 mg/kg RU 24969 on PD 27. DT was measured for 120 min.

2.4.3. Experiment 3. Effects of repeated RU 24969 treatment on the behavior of male and female preweanling rats: Role of environmental context

During the pretreatment phase (PD 17–20), rats received daily injections of saline or 2.5 mg/kg RU 24969. After injections, half of the rats were returned to their home cage (Home Cage condition), while the other half of the rats were immediately placed in activity chambers (Activity Chamber condition), where DT was measured for 45 min. On the test day (PD 22), rats from both the Home Cage and Activity Chamber conditions were injected with saline or 5 mg/kg RU 24969 and immediately placed in activity chambers, where DT was measured for 120 min. Axillary body temperatures were measured 45 min after drug injections on PD 17 and PD 20, and at the completion of behavioral testing on PD 22.

On the same three days (i.e., PD 17, PD 20, and PD 22), the behavior of rats in the activity chambers was coded using a motoric capacity rating scale in which: 0 = asleep or inactive, 1 = normal forward locomotion (no balance disturbances), 2 = forward locomotion with minor balance problems (upright walking, but slightly splayed rear legs), 3 = forward locomotion with moderate balance problems (not dragging, but awkward leg movements), 4 = forward locomotion with major balance problems I (not dragging, but occasional rolling over), 5 = forward locomotion with major balance problems II (minor dragging and rolling over, but focused forward movement), 6 = predominate dragging (prominent dragging and rolling over, with random forward movement), 7 = circular dragging (forward dragging in a circular pattern), and 8 = splayed or “swimming” movements (the scale is adapted from McDougall et al. [47]). Rather than measuring behavior continuously, motoric capacity was quantified using the fixed interval momentary time sampling method described by Cameron et al. [48]. Specifically, the behavior of each rat was assessed for 15 s every 5 min interval by an observer blind to treatment conditions. Data are presented and analyzed across 5 min (pretreatment phase) and 10 min (test day) time blocks.

2.5. Statistics

Litter effects were controlled by assigning no more than one male and one female rat per litter per group [49]. Even using this procedure, statistical analysis of the pretreatment phase necessarily included more than one subject per condition. In this situation, a single litter mean was calculated from multiple littermates assigned to the same pretreatment condition [49,50]. Test day DT and axillary temperature data were analyzed using analysis of variance (ANOVA). Repeated measures ANOVAs were used to analyze day-dependent changes during the pretreatment phase. When the assumption of sphericity was violated, as determined by Mauchly’s test of sphericity, the Greenhouse-Geisser epsilon statistic was used to adjust degrees of freedom [51]. Corrected degrees of freedom were rounded to the nearest whole number and are indicated by a superscripted “a” in the parenthetical statistical reports. Preliminary analyses consistently showed that the DT and axillary temperatures of preweanling rats did not differ according to sex, so the final statistical analyses were collapsed over the sex variable. Tukey tests were used for making post hoc comparisons of significant main effects and interactions.

Unlike for DT and axillary temperature data, the motoric capacity rating scale provided ordinal data. For this reason, the median served as the measure of central tendency and drug effects during the pretreatment phase were analyzed using the Mann-Whitney U test and the Wilcoxon signed-rank test. During the testing phase, data provided by the motoric capacity rating scale were analyzed using both the Mann-Whitney U test and the Kruskal-Wallis H test, with Dunn’s test being used for post hoc comparisons. Regardless of the statistic being used (ANOVA, Tukey, Kruskal-Wallis H tests, Dunn’s test, Wilcoxon signed-rank test, or Mann-Whitney U tests), the significance level was set at P<0.05. Statistical analyses involving ANOVAs and Tukey tests were performed using IBM SPSS Statistics for Windows, Version 24.0 (IBM, Armonk, NY); whereas, nonparametric statistics were performed using GraphPad Prism for Windows, Version 6.07 (GraphPad Software, La Jolla, CA).

3. Results

3.1. Experiment 1. Effects of a low test day dose of RU 24969: tolerance

3.1.1. One-trial procedure

On the single pretreatment day (PD 20), RU 24969 produced a dose-dependent increase in DT, with each successive dose being significantly different from the lower dose (Figure 1) [Pretreatment main effect, F (2, 45) = 54.00, p < 0.001]. On the test day (PD 22), administering a low dose of RU 24969 (1.25 mg/kg) significantly increased the DT scores of male and female preweanling rats (Figure 2) [Test Treatment main effect, F (1, 42) = 10.15, p < 0.005]; however, this effect was less evident in rats pretreated with RU 24969. Specifically, administering 5 mg/kg RU 24969 on PD 20 (i.e., the pretreatment day) significantly decreased DT on the test day [Pretreatment main effect, F (1, 42) = 3.34, p < 0.05]. The various drug effects did not differ according to sex [Pretreatment × Test Treatment × Sex interaction, F (2, 36) = 0.76, p = 0.48].

Fig. 1.

Fig. 1.

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Mean distance traveled (DT) scores (±SD) of male and female preweanling rats (n = 16 rats per condition) on the single pretreatment day. Rats had been injected with saline or RU 24969 (2.5 or 5 mg/kg) immediately before placement in the activity chambers on PD 20. (a) Significantly different from rats pretreated with saline. (b) Significantly different from rats pretreated with 2.5 mg/kg RU 24969.

Fig. 2.

Fig. 2.

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Mean distance traveled (DT) scores (±SD) of male and female preweanling rats (n = 8 rats per group) on the test day. Rats had been pretreated with saline or RU 24969 (2.5 or 5 mg/kg) on PD 20, and injected with saline or 1.25 mg/kg RU 24969 on the test day (PD 22). (a) Significantly different from rats pretreated with saline.

3.1.2. Multi-trial procedure

Overall, RU 24969 increased the DT scores of male and female preweanling rats [Pretreatment main effect, F (1, 30) = 27.77, p < 0.001], but this effect varied across the four-day pretreatment phase (Figure 3). Specifically, the DT of saline-pretreated rats remained stable across the pretreatment phase; whereas, the DT of RU 24969-pretreated rats showed a day-dependent decline [aPretreatment × Day interaction, F (2, 74) = 13.12, p < 0.001]. By PD 20, 2.5 mg/kg RU 24969 no longer caused a significant increase in DT relative to the saline group, and the DT scores of RU 24969-treated rats were significantly reduced on PD 19 and PD 20 when compared to PD 17 (i.e., the day rats were first exposed to RU 24969).

Fig. 3.

Fig. 3.

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Mean distance traveled (DT) scores (±SEM) of male and female preweanling rats (n = 16 rats per condition) across the four-day pretreatment phase. Rats had been injected with saline or 2.5 mg/kg RU 24969 immediately before placement in the activity chambers on PD 17–20. (a) Significantly different from rats pretreated with saline on the same pretreatment day. (b) Significantly different from RU 24969-pretreated rats on PD 17.

Administering 1.25 mg/kg RU 24969 on the test day increased the DT of male and female preweanling rats (Figure 4) [Test Treatment main effect, F (1, 28) = 7.20, p < 0.05]. Even so, a pretreatment regimen of 2.5 mg/kg RU 24969 significantly reduced DT on the test day [Pretreatment main effect, F (1, 28) = 7.69, p < 0.01]. None of these effects differed according to sex [Pretreatment × Sex interaction, F (1, 24) = 1.64, p = 0.21; Test Treatment × Sex interaction, F (1, 24) = 0.10, p = 0.75].

Fig. 4.

Fig. 4.

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Mean distance traveled (DT) scores (±SD) of male and female preweanling rats (n = 8 rats per group) on the test day. Rats had been pretreated with saline or 2.5 mg/kg RU 24969 on PD 17–20, and injected with saline or 1.25 mg/kg RU 24969 on the test day (PD 22). (a) Significantly different from rats pretreated with saline. (b) Significantly different from rats given a test day injection of saline (open bars).

3.2. Experiments 2. Effects of a high test day dose of RU 24969: behavioral reinstatement

Results from the pretreatment phase were essentially identical to those described before (see Figure 3). Briefly, 2.5 mg/kg RU 24969 increased the DT of male and female rats on all days of the pretreatment phase (data not shown) [Pretreatment main effect, F (1, 70) = 124.23, p < 0.001; aPretreatment × Day interaction, F (2, 131) = 10.91, p < 0.001]. RU 24969’s locomotor activating effects exhibited a progressive decline across the pretreatment phase, as RU 24969-induced DT was significantly reduced on PD 19 and PD 20 relative to PD 17 (Tukey tests, p < 0.05).

On the first test day (PD 22), administering 5 or 10 mg/kg RU 24969 increased DT scores relative to rats injected with saline (Figure 5, left graph) [Test Treatment main effect, F (2, 42) = 46.27, p < 0.001]. The pretreatment main effect only approached statistical significance [F (1, 42) = 3.94, p = 0.054]; however, a separate statistical analysis excluding the saline control groups showed that rats pretreated and tested with RU 24969 exhibited more test day DT than rats injected with RU 24969 (5 or 10 mg/kg) for the first time on the test day [Pretreatment main effect, F (1, 28) = 6.40, p < 0.05]. Once again, none of these effects varied according to sex [Pretreatment × Sex interaction, F (1, 24) = 0.02, p = 0.88; Test Treatment × Sex interaction, F (1, 24) = 0.22, p = 0.64].

Fig. 5.

Fig. 5.

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Mean distance traveled (DT) scores (±SD) of male and female preweanling rats (n = 8 rats per group) on the two test days (PD 22 and PD 27). Rats had been pretreated with saline or 2.5 mg/kg RU 24969 on PD 17–20, and injected with saline or RU 24969 (5 or 10 mg/kg) on the first test day (PD 22). Rats pretreated with saline or 2.5 mg/kg RU 24969 on PD 17–20 and PD 22 were further subdivided and injected with saline or 5 mg/kg RU 24969 on the second test day (PD 27). (a) Significantly different from rats injected with saline on the test day (open bars). (b) Significantly different from saline-pretreated rats injected with RU 24969 (5 or 10 mg/kg) on the test day.

Administering 5 mg/kg RU 24969 on the second test day (PD 27; i.e., after 5 drug abstinence days) caused a significant increase in DT relative to the saline groups (Figure 5, right graph) [Test Treatment main effect, F (1, 28) = 57.51, p < 0.001]. Pretreating rats with saline or 2.5 mg/kg RU 24969 on PD 17–22 did not significantly affect performance on PD 27.

3.3. Experiment 3. Effects of repeated RU 24969 treatment: Role of environmental context

3.3.1. Distance traveled.

Once again, male and female preweanling rats given saline exhibited a stable level of responding across the pretreatment phase (an approximate representation of these data can be seen in Figure 3); whereas, 2.5 mg/kg RU 24969 caused a significant enhancement of DT on PD 17 that progressively declined across the pretreatment phase [Pretreatment main effect, F (1, 30) = 61.00, p < 0.001; aPretreatment × Day interaction, F (3, 83) = 7.71, p < 0.001].

Administering a high dose of RU 24969 (5 mg/kg) on the test day increased the DT scores of male and female rats relative to saline controls (Figure 6) [Test Treatment main effect, F (1, 56) = 205.62, p < 0.001]. Notably, neither RU 24969 pretreatment, environmental context, nor sex significantly moderated the effects of RU 24969 on the test day.

Fig. 6.

Fig. 6.

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Mean distance traveled (DT) scores (±SD) of male and female preweanling rats (n = 8 rats per group) on the test day (PD 22). Rats had been pretreated with saline or 2.5 mg/kg RU 24969 in either their home cage or activity chamber on PD 17–20, and injected with saline or 5 mg/kg RU 24969 in the activity chamber on the test day (PD 22). (a) Significantly different from rats given a test day injection of saline (open bars).

3.3.2. Motoric capacity

Analysis of data provided by the motoric capacity scale (Table 1) showed that rats pretreated with 2.5 mg/kg RU 24969 on PD 17 had elevated scores on time blocks 1–12 when compared to saline controls (higher numbers indicate greater motoric disturbances) [Mann-Whitney U tests, p < 0.05]. On all time blocks, RU 24969-treated rats exhibited major balance problems with predominate dragging. On PD 20, after four days of the pretreatment regimen, RU 24969-treated rats no longer exhibited motoric incapacity. Consistent with this finding, the median motoric capacity scores of RU 24969-treated rats were significantly smaller on PD 20 (time blocks 1–9) than PD 17 [Wilcoxon signed-rank tests].

Table 1.

Effects of saline and RU 24969 (2.5 mg/kg) pretreatment on the median motoric capacity scores of preweanling rats (n = 16 rats per condition) across the 45 min sessions on PD 17 and PD 20.

Age-Treatment 5-Min Time Blocks
1 2 3 4 5 6 7 8 9
PD 17
 Saline 0 0 0 0 0 0 0 0 0
 RU 24969 (2.5 mg/kg) 5a 6a 4.8a 6a 6a 6a 6a 6a 6a
PD 20
 Saline 0 0 0 0 0 0 0 0 0
 RU 24969 (2.5 mg/kg) 0b 0b 0b 0b 0b 0b 0b 0b 0b
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Note: Motoric capacity scale: 0 = asleep or inactive, 1 = normal forward locomotion (no balance disturbances), 2 = forward locomotion with minor balance problems (upright walking, but slightly splayed rear legs), 3 = forward locomotion with moderate balance problems (not dragging, but awkward leg movements), 4 = forward locomotion with major balance problems I (not dragging, but occasional rolling over), 5 = forward locomotion with major balance problems II (minor dragging and rolling over, but focused forward movement), 6 = predominate dragging (prominent dragging and rolling over, with random forward movement), 7 = circular dragging (forward dragging in a circular pattern), and 8 = splayed or “swimming” movements.

a

Significantly different from saline-pretreated rats on PD 17 (Mann-Whitney U tests, P<0.05).

b

Significantly different from RU 24969-pretreated rats on PD 17 (Wilcoxon signed-rank tests, P<0.05).

On the test day (PD 22), saline-pretreated rats given RU 24969 (5 mg/kg) for the first time on the test day showed more motoric disturbances than saline controls on all 12 time blocks (Table 2) [Kruskal-Wallis H = 52.62 to 18.07, p < 0.001, and Dunn tests, p < 0.05]. Except for the first time block, these RU 24969-treated rats exhibited moderate balance problems that included awkward movements and occasional rolling over. In comparison, RU 24969-pretreated rats given a high dose of RU 24969 (5 mg/kg) on the test day (i.e., the RU–RU group), exhibited significantly more locomotor activity (along with minor motoric disturbances) than saline controls (i.e., the RU-SAL group) [Dunn tests, p < 0.05], but significantly fewer motoric disturbances than rats treated with RU 24969 for the first time on the test day (i.e., the SAL-RU group) [Mann-Whitney U tests, p < 0.05].

Table 2.

Effects of saline and RU 24969 (2.5 mg/kg) pretreatment, as well as saline and RU 24969 (5 mg/kg) treatment, on the median motoric capacity scores of preweanling rats (n = 16 rats per group) on the test day (PD 22)

Pretreatment-Treatment 10-Min Time Blocks
1 2 3 4 5 6 7 8 9 10 11 12
Saline
 Saline 0 0 0 0 0 0 0 0 0 0 0 0
 RU 24969 (5 mg/kg) 1.5a 4.5a 3.5a 3a 4.5a 3.2a 3.8a 3a 3a 3.6a 3.1a 3.5a
RU 24969 (2.5 mg/kg)
 Saline 0 0 0 0 0 0 0 0 0 0 0 0
 RU 24969 (5 mg/kg) 0.5 1.8a 2ab 1.5a 1ab 1.5ab 1ab 1ab 1ab 1.2ab 1ab 1ab
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Note: Data were collapsed across the home and test chamber conditions. Motoric capacity scale: 0 = asleep or inactive, 1 = normal forward locomotion (no balance disturbances), 2 = forward locomotion with minor balance problems (upright walking, but slightly splayed rear legs), 3 = forward locomotion with moderate balance problems (not dragging, but awkward leg movements), 4 = forward locomotion with major balance problems I (not dragging, but occasional rolling over), 5 = forward locomotion with major balance problems II (minor dragging and rolling over, but focused forward movement), 6 = predominate dragging (prominent dragging and rolling over, with random forward movement), 7 = circular dragging (forward dragging in a circular pattern), and 8 = splayed or “swimming” movements.

a

Significantly different from rats given a test day (treatment) injection of saline (Dunn tests, P<0.05).

b

Significantly different from saline-pretreated rats given a test day injection of 5 mg/kg RU 24969 (Mann-Whitney U tests, P<0.05).

3.3.3. Body temperature

When measured approximately 45 min after injections, 2.5 mg/kg RU 24969 decreased axillary temperatures relative to saline controls (Figure 7) [Pretreatment main effect, F (1, 60) = 389.34, p < 0.001]. On PD 17, the RU 24969-induced reduction in axillary temperatures was significantly more pronounced in rats placed in the test chamber than the home cage [Pretreatment × Environment × Day interaction, F (1, 60) = 10.38, p < 0.005]. RU 24969 (2.5 mg/kg) continued to depress axillary temperatures on PD 20, but the temperature loss was much smaller than on PD 17, and did not differ according to environmental context (Tukey tests, p < 0.05).

Fig. 7.

Fig. 7.

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Mean axillary temperatures (±SEM) of male and female preweanling rats (n = 16 rats per condition) during the pretreatment phase. Rats had been injected with saline or 2.5 mg/kg RU 24969 immediately before placement in the activity chambers or home cage on PD 17–20. (a) Significantly different from rats pretreated with saline on the same pretreatment day. (b) Significantly different from rats pretreated with RU 24969 in the home cage on PD 17. (c) Significantly different from rats pretreated with RU 24969 on PD 17.

On the test day, axillary temperatures did not vary according to environmental context, so data are presented collapsed over this variable (Table 3). Administering RU 24969 (5 mg/kg) on the test day decreased axillary temperatures of male and female preweanling rats [Test Treatment main effect, F (1, 56) = 222.68, p < 0.001]. This RU 24969-induced reduction in axillary temperatures was significantly greater in rats pretreated with saline and not 2.5 mg/kg RU 24969 [Pretreatment × Test Treatment interaction, F (1, 56) = 4.45, p < 0.05].

Table 3.

Mean (SEM) axillary temperatures (°C) of male and female preweanling (n = 16 per group) rats on the test day

Pretreatment: Saline RU 24969 (2.5 mg/kg)
Test Treatment: Saline RU 24969 (5 mg/kg) Saline RU 24969 (5 mg/kg)
36.34 (0.22) 31.73 (0.36)a 36.41 (0.17) 32.95 (0.27)ab
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Note: Rats were pretreated with saline or 2.5 mg/kg RU 24969 on PD 17–20, and were then injected with saline or RU 24969 (5 mg/kg) on the test day (PD 22). Data are collapsed over the Home Cage and Activity Chamber conditions.

a

Significantly different from rats given saline on the test day.

b

Significantly different from saline-pretreated rats given RU 24969 (5 mg/kg) on the test day.

4. Discussion

The present results confirm that a single administration of RU 24969 causes a dose-dependent increase in the locomotor activity of male and female preweanling rats [5]. Tolerance quickly develops to this compound, as a substantial decline in locomotor activity is evident when preweanling rats are given daily injections of RU 24969 (2.5 mg/kg) across a four-day span. When given a lower dose of RU 24969 (1.25 mg/kg) on the test day (PD 22), preweanling rats showed a tolerance response regardless of whether they had received one or four pretreatment injections of RU 24969. Interestingly, RU 24969-pretreated rats given a test day injection of saline also showed a significant reduction in locomotor activity when compared to saline controls. Although a complete time course was not established, weak locomotor tolerance persisted for at least five days after cessation of RU 24969 treatment (i.e., on PD 27).

Tolerance was also apparent when motoric capacity and axillary temperatures were assessed. Specifically, RU 24969 initially caused major motoric impairments (e.g., body dragging, rolling over, etc.), but these effects almost completely disappeared after four daily drug treatments. This day-dependent improvement was not due to 17-day-old rats being more sensitive to the drug than older animals, because rats tested with RU 24969 for the first time on PD 22 also showed major motoric disturbances. In terms of physiological measures, RU 24969 was responsible for a substantial reduction in axillary temperature on the first pretreatment day (activity chamber condition, −6.53 °C relative to saline controls; home cage condition, −3.74 °C). By the end of the pretreatment phase a strong tolerance response was apparent, because RU 24969 only reduced axillary temperatures by −2.28 °C (activity chamber) and −1.22 °C (home cage). RU 24969-induced temperature loss was less pronounced in rats maintained in their home cage, but that result was expected since rats in the home cage could huddle for warmth [52]. Thermoregulatory ability improves across the preweanling period [53,54], thus it is possible that the day-dependent reduction in RU 24969’s effectiveness was due to improved thermoregulation and not tolerance. To answer this question an analysis of test day axillary temperatures is required (see below).

Not only can tolerance be studied by administering the same dose of drug over days, but tolerance can be assessed by injecting rats with a higher dose of drug on the test day. A sufficiently high dose of the target compound should reinstate the behavioral or physiological response that was lost due to repeated exposure [15,40,41]. In the present study, axillary temperatures evidenced an incomplete tolerance, because a high dose of RU 24969 (5 mg/kg) reduced axillary temperatures of RU 24969-pretreated rats (–3.39 °C, relative to saline controls), but not to the same extent as rats given RU 24969 for the first time on the test day (–4.61 °C). It is possible that administering an even higher dose of RU 24969 on the test day would have fully reinstated the reduction in axillary temperatures. On the other hand, administering 5 or 10 mg/kg RU 24969 on the test day not only reinstated a full locomotor response, it caused a sensitization-like potentiation of locomotor activity that was significantly greater than the locomotion shown by rats treated with RU 24969 for the first time on the test day. In this regard, it is important to note that rats given RU 24969 on only the test day showed substantial motoric impairments, including awkward leg movements and occasional rolling over, while rats both pretreated and tested with RU 24969 showed almost complete tolerance to these motoric deficits. Thus, it is likely that the sensitization-like effects just mentioned (i.e., potentiated locomotor activity) were due to an absence of motoric deficits in the RU 24969 pretreatment group rather than a true sensitized response.

There are various types of tolerance (e.g., dispositional, pharmacodynamic, and behavioral tolerance) and they can jointly or singly be responsible for the diminished response to a drug [40,55]. In the present study, there was no evidence that behavioral tolerance was fully or partially responsible for the progressive decline in the behavioral and physiological responses to RU 24969. Behavioral tolerance is not a unitary phenomenon [19,41,42], so this conclusion refers to behavioral tolerance that is a product of Pavlovian contextual conditioning. Whether dispositional tolerance can be excluded as a cause of RU 24969 tolerance is uncertain. De Souza et al. [14] and Oberlander et al. [15] proposed that dispositional tolerance was not responsible for the progressive decline in locomotor responsiveness shown by RU 24969-treated rats and mice, because different behavioral and physiological responses developed tolerance at different rates or not at all. In the present study, locomotor activity, motoric capacity, and axillary temperatures also showed different patterns of RU 24969-induced tolerance, which may rule out dispositional tolerance as the sole cause of the tolerance response. Nonetheless, it is premature to eliminate dispositional tolerance as a possible contributing factor to RU 24969’s actions.

Based on their data, De Souza et al. [14] and Oberlander et al. [15] concluded that pharmacodynamic tolerance was responsible for the progressively diminished response to RU 24969. Specifically, they hypothesized that repeated RU 24969 treatment caused a down-regulation or subsensitivity of 5-HT1A and/or 5-HT1B receptors. Results from the present study are consistent with a pharmacodynamic interpretation involving 5-HT1A/1B receptors. Indeed, the finding that a one- and four-day pretreatment regimen of RU 24969 caused a significant reduction in the test day locomotion of saline-treated preweanling rats can be accounted for by 5-HT1A/1B receptor down-regulation, but not by dispositional tolerance. That locomotor activity, motoric capacity, and axillary temperatures showed different patterns of tolerance may be explained by the relative number of 5-HT1A/1B receptors available for mediating these effects. Specifically, tolerance may develop slowly, or not at all, if there is a large reserve of 5-HT1A/1B receptors in relevant brain regions; whereas, tolerance develops more quickly, and may be longer lasting, in the absence of a receptor reserve [56–58].

An interesting aspect of these findings is that there were sound grounds for hypothesizing that repeated treatment with RU 24969 should cause behavioral sensitization. More specifically, MDMA induces context-dependent behavioral sensitization [12,13], presumably through actions involving the 5-HT1 receptor [7,8]. Although Experiment 2 provided data consistent with a sensitization explanation (i.e., rats pretreated and tested with RU 24969 exhibited more locomotor activity than rats given RU 24969 on only the test day), it was concluded that this sensitization-like effect was a result of motoric impairments in rats receiving RU 24969 for the first time on the test day. Despite this interpretation, it remains possible that repeated treatment with RU 24969 produces the underlying neuroanatomical changes responsible for behavioral sensitization, but that tolerance to the compound masks the expression of a sensitized behavioral response. Although behavioral sensitization was initially referred to as “reverse tolerance” [59,60], it is apparent that the neural mechanisms or metabolic changes responsible for each phenomenon are quite different and are mutually exclusive at a neuronal or receptor level.

In summary, repeated treatment with the 5-HT1A/1B receptor agonist RU 24969 causes a quick developing tolerance in male and female preweanling rats that persists for at least five days. Locomotor activity, motoric capacity, and axillary temperatures all showed a strong tolerance response. There was no evidence that behavioral tolerance was responsible for these effects; however, in agreement with past studies, it is likely that pharmacodynamic tolerance (i.e., a down-regulation or subsensitivity of 5-HT1A/1B receptors) is at least partially responsible for the progressive decline in RU 24969’s effectiveness. Although between-study comparisons can be misleading, there are some dissimilarities in the way preweanling and older animals respond to 5-HT1A/1B receptor agonists. For example, the motoric deficits caused by RU 24969 appear to be more pronounced in preweanling rats than adults, and 5-HT1A/1B receptor stimulation produces a far greater reduction in the body temperatures of preweanling rats when compared to adult rats or guinea pigs [44,45,61].

Acknowledgements

This work was supported by the National Institute of General Medical Sciences [grant number GM083883].

Footnotes

Declarations of interest: none

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