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. Author manuscript; available in PMC: 2021 Nov 15.
Published in final edited form as: Neuropharmacology. 2020 Aug 16;179:108276. doi: 10.1016/j.neuropharm.2020.108276

Chronic risperidone administration leads to greater amphetamine-induced conditioned place preference.

Mark E Bardgett 1, Tyler Downnen 2, Casey Crane 3, Emily C Baltes Thompson 4, Brittany Muncie 5, Sara A Steffen 6, Justin R Yates 7, James R Pauly 8
PMCID: PMC7572744  NIHMSID: NIHMS1626511  PMID: 32814089

Abstract

Risperidone is an atypical antipsychotic drug used increasingly in children to manage symptoms of ADHD and conduct disorder. In rats, developmental risperidone administration is accompanied by increased locomotor activity during adulthood, as well as heightened sensitivity to the locomotor stimulating effects of amphetamine. This study compared sensitivity to the rewarding effects of amphetamine, as measured by conditioned place preference (CPP), between groups of rats administered chronic risperidone (3.0 mg/kg, s.c.) during development (postnatal days 14-42) or adulthood (postnatal days 77-105). Locomotor activity in a novel test cage and amphetamine-induced CPP were measured beginning three and four weeks, respectively, after the final risperidone injection. Female rats administered risperidone early in life were more active than any other group tested. Previous risperidone administration enhanced amphetamine CPP regardless of sex, and this effect appeared more prominent in the developmentally treated group. The density of forebrain dopamine transporters, a primary target of amphetamine, was also quantified in rats administered risperidone early in life and found to be reduced in the medial anterior, posterior, and ventral caudate nucleus. These results suggest that chronic risperidone treatment modifies later locomotor activity and sensitivity to the reinforcing effects of amphetamine, perhaps via a mechanism related to decreased forebrain dopamine transporter density.

Keywords: antipsychotic, dopamine, dopamine transporter, adolescence, development, reinforcement, psychostimulant

1. Introduction

Risperidone is second-generation, atypical antipsychotic drug used mainly in adults as an effective treatment for schizophrenia. Its mechanism of action is likely related to its antagonism of dopamine D2 and serotonin 5HT2A receptors in midbrain, striatal, and frontal cortical regions (see Bardgett, 2004 for review). In adult rats, long-term antagonism of these receptors can lead to a compensatory state of dopamine supersensitivity, including elevated forebrain D2 receptor densities and increased locomotor sensitivity to dopamine agonists (Samaha et al., 2007; Tadokoro et al., 2012). It has been suggested that these compensatory changes linger after cessation of antipsychotic administration for an amount of time equal to or less than the duration of drug administration (Muller and Seeman, 1978; Stevens et al., 2016). The presence of a dopaminergic supersensitivity state, even if temporary, is of concern since it may enhance the reinforcing effects of many drugs of abuse that are elicited by increased dopaminergic activity (Samaha, 2014).

Over the past two decades, there has been a surge in antipsychotic prescriptions, primarily risperidone, to children with psychiatric disorders (Kalverdijk et al., 2017; Olfson et al., 2012). They are mainly used to manage the behavioral symptoms of attention deficit hyperactivity disorder (ADHD) and other impulse control disorders, and boys are more likely to receive them than girls (Olfson et al., 2012, Sultan et al., 2019). Until recently, data characterizing the effects of risperidone on neural and behavioral development were lacking. However, over the past several years, we have used rats as a model system to study the long-term outcomes of early-life risperidone administration. Briefly, adult rats administered risperidone from postnatal days 14–42 are hyperactive (Bardgett et al., 2013), an effect not always seen after adult risperidone administration (Stevens et al., 2016), and exhibit deficits in pre-weaning and juvenile social behavior (Gannon et al., 2015). Adult rats administered risperidone early in life also display impairments in spatial working memory (Bardgett et al., 2019) and a heightened sensitivity to the locomotor-stimulating effects of amphetamine (Stubbeman et al., 2017). Many of these effects are consistent with the state of dopamine supersensitivity observed after adult antipsychotic administration (Samaha et al., 2007; Tadokoro et al., 2012), but notably they persist well into young adulthood (Bardgett et al., 2013; Stevens et al., 2016), suggesting a greater degree of permanence in comparison to the effects of chronic risperidone administration during adulthood.

Given that rats administered risperidone early in life are more sensitive to the locomotor activating effects of amphetamine, it is important to determine if such treatment alters other behavioral effects of amphetamine. At a cellular level, amphetamine elevates synaptic dopamine and other monoamine neurotransmitters by “competing” with them for uptake by monoamine transporters located on presynaptic and vesicular membranes, thus preventing their removal of from the synapse (Heal et al., 2013; Zahniser and Sorkin, 2009). Among its many behavioral effects, amphetamine possesses reinforcing properties, such as inducing preferences for specific spatial locations based on multiple pairings of the drug with the location, i.e., conditioned place preference (CPP) (Carr and White, 1986; Spyraki et al., 1982; Yates et al., 2013). Previous work has shown that acute risperidone administration during adulthood prevents the acquisition of a CPP reinforced by 3,4-methylenedioxymethylamphetamine (“Ecstasy”) or even non-stimulant drugs, such as morphine (Manzanedo et al., 2001; Roger-Sanchez et al., 2013). The purpose of this research was to compare the effects of chronic risperidone administered during development or adulthood on later amphetamine-induced CPP. The density of dopamine transporters (DAT) in the forebrain was also measured in a subgroup of rats that received chronic risperidone or vehicle administration during development. Our hypothesis was that adult rats administered risperidone early in life would exhibit a more pronounced amphetamine CPP since we previously reported that early-life risperidone led to greater locomotor responses to amphetamine during adulthood (Stubbeman et al., 2017). Moreover, it was predicted that this change would be more marked after chronic risperidone administration during development versus adulthood since the latter treatment, unlike the former, has not been associated with later hyperactivity (Stevens et al., 2016). Finally, forebrain DAT density was expected to be lower in adult rats administered risperidone early in life, since fewer DATs might lead to more excessive levels of synaptic dopamine and contribute to the heightened locomotor activity previously noted in these rats (Bardgett et al., 2013; Stevens et al., 2016; Stubbeman et al., 2017).

2. Materials and methods

2.1. Experiment 1. Effects of risperidone on locomotor activity and conditioned place preference

2.1.1. Animals and housing

Long-Evans female and male rats (n = 57 & 49, respectively) were used and derived from pregnant dams (Envigo, Indianapolis, IN) that arrived at the animal facility on gestational day 14. Litters were culled on postnatal day 8 to eight total pups comprised of three-four pups of each sex (based on the availability of female and male pups from each litter). Rats were weaned on postnatal day 21. Upon weaning, rats were housed two per cage with continuous access to food and water. Within each cage, there was one rat from the risperidone group and one rat from the vehicle group. The lights in the housing room were on between 6:30 and 18:30. All experimental procedures were carried out according to the Current Guide for the Care and Use of Laboratory Animals (USPHS) under a protocol approved by the Northern Kentucky University Institutional Animal Care and Use Committee. A timeline of experimental events for this experiment and the second experiment is depicted in Figure 1.

Figure 1.

Figure 1.

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Timeline of treatment and testing in the cohorts of rats used in the conditioned place preference and dopamine transporter experiments. PND = postnatal day, day of birth considered PND 0.

2.1.2. Drugs

The dose of risperidone used (3.0 mg/kg, sc) was based on our previous behavioral work (Bardgett et al., 2013; 2019; Gannon et al., 2015, Stevens et al., 2016; Stubbeman et al., 2017) and reports demonstrating the effects of early-life risperidone on neurotransmitter receptor levels (Choi et al., 2009; 2010; Moran-Gates et al., 2007). Risperidone was dissolved in a small volume of 10% glacial acetic acid, brought to volume with 0.9% saline, and adjusted to a pH ~6.2 with 1M sodium hydroxide. Control rats were administered the vehicle solution, which was prepared in an identical manner to that of the risperidone solution except that risperidone was not added. Subcutaneous injections were administered at a volume of 2.0 ml/kg of body weight for the developing rats and 1.0 mg/ml for the adult rats – the larger volumes in the pups allowed for more precise delivery of the risperidone dose. The National Institute of Mental Health’s Chemical Synthesis and Drug Supply program kindly provided the risperidone.

Two cohorts of rats were studied – one that received drug injections during development and another that received injections during adulthood. Within each cohort, there were four groups of rats (12-16 per group): male and female rats injected with vehicle or risperidone. Rats were weighed and administered risperidone or vehicle daily between 10:00 and 14:00 during the light cycle from either postnatal day 14 through 42 or postnatal day 77 through 105. The former period was chosen for study since it is considered analogous to the time between early childhood and late adolescence in humans (Andersen, 2005; Spear, 2000). Given that many young children receive antipsychotic drugs continuously over long periods of time across this age period (Constantine et al., 2012; Kalverdijk et al., 2008), the timing of the injections was meant to mimic prolonged antipsychotic drug exposure during childhood and early adolescence in humans.

2.1.3. Locomotor activity

Eighteen days after the last risperidone injection, locomotor activity was recorded for 20 minutes on five consecutive days. Testing occurred between 9:00 a.m. and 4:00 p.m. each day. These tests were intended to determine if previous risperidone administration affected locomotor activity long after cessation drug injections. Locomotor activity was measured in clear polypropylene cages (51 cm long x 26.5 cm wide x 32 cm high) covered with wire tops and inserted into SmartFrame Cage Racks (Kinder Scientific, Poway, CA). Locomotor activity was defined by the number of photobeam breaks generated within a given time period. All locomotor testing occurred in a dark room.

2.1.4. Amphetamine-induced Conditioned Place Preference

Conditioned place preference testing began 25 days after the final risperidone injection. Testing and conditioning occurred in three-compartment place preference chambers (MED-CPP-013AT, Med Associates, St. Albans, VT) enclosed within sound-attenuating cubicles (ENV-020M, Med Associates, St. Albans, VT). On the first day of the procedure, rats were allowed to explore all three compartments of the CPP chamber for 15 minutes (pretest), and the time spent in each compartment was recorded. On the 2nd, 4th, 6th, and 8th day of the procedure, rats received subcutaneous injections of either 0.5 or 1.5 mg/kg of D-amphetamine (Sigma, dissolved in 0.9% saline, 1.0 ml/kg) and were immediately confined for 30 minutes to the initially non-preferred compartment. Yates and colleagues (2012; 2013) had previously reported that these amphetamine doses produced significant conditioned place preference. All rats received a subcutaneous injection of 0.9% saline (1.0 ml/kg) on the 3rd, 5th, 7th, and 9th days of the procedure, and were confined for 30 minutes to the initially preferred compartment. Activity was measured by photobeams located within the conditioning chambers on days 2-9. On day 10, rats were allowed to explore all three compartments for 15 minutes (posttest) and the time spent in each compartment was recorded.

2.2. Experiment 2. Effects of risperidone on locomotor activity and dopamine transporters

2.2.1. Animals, drug administration, and locomotor activity

The density of dopamine transporters within the striatum and frontal cortex was measured in a separate group of adult rats administered risperidone from postnatal days 14-42. In this study, six Long-Evans dams with litters (Envigo, Indianapolis, IN) arrived at postnatal day 7. A total of 20 pups from these litters were used for this experiment. Ten rats (6 females, 4 males) received daily injections of 3.0 mg/kg of risperidone and 10 rats (6 females, 4 males) received vehicle. On postnatal days 49 and 50, all rats were tested for locomotor activity as described above for one hour each day.

2.2.2. Autoradiography

On postnatal day 62, brains from all rats were collected via rapid decapitation. Once removed from the skull, the brain was hemidissected, and the left hemisphere was frozen in powdered dry ice and stored at −80° C until further processing. The left hemisphere was sliced on a Leica cryostat into a series of 16 μm thick sagittal sections. [125I]-RTI-55 (Perkin-Elmer Life Sciences, Boston, MA, USA) was used to detect dopamine transporter sites and remaining autoradiography was performed as described by van Bregt et al., (2012). The optical density of regions within the medial striatum and frontal cortex, as shown in Figure 2, and roughly analogous to plate # 81 of Paxinos and Watson (1986), was quantified using ImageJ software (v. 2.0). Raters were blind to the treatment status of each brain. Mean uncalibrated optical density was calculated for each individual region from a series of nine contiguous sections. Non-specific binding was determined by measuring the optical density of a similarly sized area located within the corpus callosum just posterior to the sampled striatal sites (Figure 2). This value was subtracted from all density measures, and the product was used for statistical analyses.

Figure 2.

Figure 2.

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Areas included in autoradiographic analysis. In A., 1. Anterior caudate, 2. Posterior caudate, 3. Dorsal caudate, 4. Ventral caudate, and 5. Nucleus accumbens. “N” indicates the area within the corpus callosum that was sampled for a measurement of non-specific binding. In B., 1. Superficial prefrontal cortex, and 2. Deep layers of prefrontal cortex. Each circle represents the actual size of the area sampled for the optical density measures.

2.3. Statistical analysis

In experiment 1, locomotor activity data collected prior to the CPP experiments were averaged for each cohort across the five days of testing. These averages were compared between groups using a three-way analysis of variance (ANOVA) with drug (risperidone vs. vehicle), sex, and age of administration as between-groups factors. For the locomotor data gathered during the CPP experiment, the effects of amphetamine and saline on locomotor activity during the respective conditioning days were averaged across days for each drug. Since there were no significant differences in activity observed between the two amphetamine doses during the conditioning days, the data from the amphetamine conditioning sessions, as well as the saline conditioning days, were analyzed using a three-way analysis of variance with drug (risperidone vs. vehicle), sex, and age of administration as between-groups factors. For each age cohort in the CPP experiment, a two-way ANOVA was used to compare difference in the time spent in the amphetamine-paired compartment at pretest vs. posttest with drug (risperidone vs. control groups) serving as a between-groups factor and time (5 min intervals) as a within-subjects factor. These analyses did not include an assessment of the effects of the different amphetamine doses or sex since neither exerted a significant main effect or interactive effect with any other variable. The same analysis was conducted on the time spent in the saline-paired compartment at pretest vs. posttest.

In the second experiment, locomotor data were averaged across the two days of testing, and a two-way ANOVA was used to compare these averages using drug administration (risperidone vs. vehicle) and sex as between-group factors. For the dopamine transporter data, the same analysis was used to compare the relative optical density measured in each brain region.

All post-hoc analyses of main effects or interactions were performed using two tailed t-tests or Fishers Protected Least Significant Difference tests. In all analyses, significant differences were accepted if p < .05.

3. Results

3.1. Locomotor activity

Four weeks after the last injection of risperidone or vehicle, locomotor activity was tested for 20 minutes a day over five consecutive days. While activity decreased significantly across the testing days F(4, 240) = 38.55, p < .0001, there was no statistical interaction between time and risperidone. Therefore, the data were averaged across the five days into a single score. Using this average score, a significant interaction was observed between age of administration, sex, and risperidone, F(1, 98) = 4.686, p = .033 (Figure 3). Female rats administered risperidone early in life were significantly more active in comparison to female rats administered vehicle early in life (p = .013, two-tailed t test) or any other group. This same effect was not observed in male rats administered risperidone early in life, nor in rats of either sex administered risperidone later in life.

Figure 3.

Figure 3.

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Average locomotor activity recorded for 20 minutes a day across five days at 18 days after the final risperidone injection. Females administered risperidone early in life (YOUNG) were more active than all groups, * p = .013 vs. young female vehicle rats. Each bar represents mean ± S.E.M. Number of rats per group listed in each bar.

3.2. Conditioned place preference

One week after locomotor testing, conditioned place preference was measured in all rats. The conditioning effects of two doses of amphetamine, 0.5 and 1.5 mg/kg, were compared between female and male rats. Because there were no interactive effects of amphetamine dose or sex with other independent variables, the data from each amphetamine dose group and sex were combined for subsequent analysis. Two rats from the developmental cohort were excluded from the analyses because they received injections of amphetamine while being placed on the incorrect side of the apparatus. Place preference was defined as the time spent in the amphetamine conditioned compartment on the test day minus the time spent in the same compartment during the pretest.

There were significant effects of risperidone, F(1,100) = 4.169, p = .044, and age, F(1,100) = 5.516, p = .021 on place preference (Figure 4a). Rats that were administered risperidone, and rats that were older demonstrated greater place preference in comparison to rats that were administered vehicle or were younger, respectively. However, these differences appeared to be mainly driven by a contrast between rats administered risperidone or vehicle early in life. A post-hoc analysis revealed that rats administered risperidone early in life exhibited a significantly greater place preference than rats administered vehicle early in life (p = .034, two-tailed t test). A similar significant difference was not observed between rats administered risperidone or vehicle during adulthood.

Figure 4.

Figure 4.

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Conditioned place preference scores. The time spent at posttest in the compartment paired with amphetamine is depicted in A. and in the compartment paired with saline in B. Each bar represents mean difference score ± S.E.M. between time spent in specified compartment at posttest minus pretest. In A., rats that received risperidone early in life (YOUNG) spent more time in the amphetamine conditioned compartment than rats that received vehicle early in life, * p = .034. Number of rats per group listed in each bar.

The same comparisons were made between age and risperidone administration on the amount of time spent in the non-conditioned (saline) compartment on the test day minus the time spent in the same compartment on the pretest day. These analyses showed that while overall rats spent less time in the non-conditioned chamber at post-test relative to pretest, there was no main effect of age or risperidone, and no statistical interaction between these factors on this measure (Figure 4b).

Locomotor activity was recorded during each 30 minute conditioning trial in the place preference chambers after saline and amphetamine administration. In separate analyses of the activity seen after saline or amphetamine administration, there was a significant effect of time across test days, F(3, 288) = 26.021 & 26.827, p < .0001, respectively. Activity was significantly decreased and increased across days after saline and amphetamine administration, respectively, but since there were no time x treatment interactions, the data were averaged across days for all subsequent analyses. After saline administration, there were significant main effects of age, sex, and risperidone, F(1, 96) = 26.726, 4.338, & 6.490 p ≤ .0001, .040, & .012 respectively, on activity (Figure 5a), but no interactions observed between these effects. Younger rats were found to be more active than older ones, and females were more active than males. Rats that received chronic risperidone administration were more active than those that received vehicle. Post-hoc analyses indicated females administered risperidone early in life were more active than females administered vehicle early in life (p = .02, two-tailed t test) – none of the other individual group comparisons were significant.

Figure 5.

Figure 5.

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Locomotor activity during conditioning sessions in the CPP chambers. Activity recorded after saline administration depicted in A. and after amphetamine administration in B. In A., younger rats were more active than older ones, female rats were more active than males, and risperidone rats were more active than vehicle. Female rats administered risperidone early in life (YOUNG) were more active than females administered vehicle early in life, * p = .02. In B., female rats were more active than males, and risperidone rats were more active than vehicle. Male rats administered risperidone during adulthood (ADULT) were more active than males administered vehicle during adulthood, * p = .02. Each bar represents mean ± S.E.M. Number of rats per group listed in each bar.

When the activity data generated after amphetamine injection were analyzed, there was no significant difference observed as a function of the amphetamine dose used. Therefore, all data were collapsed across the two amphetamine doses. In the subsequent analyses, no significant interactions were discovered, but there were significant main effects of sex and risperidone, F(1, 96) = 3.929 & 6.727 p = .050 & .011 respectively (Figure 5b). As seen after saline injections, females were more active than males, and rats that were previously administered chronic risperidone were more active than rats administered vehicle. Post-hoc analysis indicated that males administered risperidone during adulthood were more active than those that were administered vehicle (p = .02, two-tailed t test) – no other individual group comparisons were significant.

To assess whether rats that received risperidone were more sensitive to the locomotor activating effects of amphetamine, the activity score for each rat generated after saline injection was subtracted from the same score generated after amphetamine injection. Analysis of this difference score using a three-way ANOVA with age, sex, and risperidone as between-groups factors did not reveal any significant effects or interactions between these variables.

3.3. Dopamine transporter density

Given the effects of chronic risperidone, especially when administered early in life, on amphetamine conditioned place preference, we quantified and compared the relative density of forebrain dopamine transporters – a chief target of amphetamine in the brain – between rats that were administered risperidone or vehicle early in life. In this group of rats, early-life risperidone led to increased locomotor activity when measured one week after the cessation of treatment, F(1, 16) = 6.706, p = .02 (Figure 6). Like the data reported in the CPP experiment, females administered risperidone early in life demonstrated significantly greater locomotor activity relative to females administered vehicle (p = .004, two-tailed t test) – the same comparison between males in each treatment group was not significant. In several regions of the caudate nucleus (Figure 2), the relative density of dopamine transporters was decreased in rats that received risperidone early in life (Figure 7). Significant decreases were observed in the anterior, posterior, and ventral regions of the medial caudate, F(1, 16) = 6.437, 5.341, & 8.861, p = .02, .03 & .009 for each respective region, but not in the dorsal medial caudate, the medial nucleus accumbens, or the frontal cortex. Males possessed significantly higher DAT density in the anterior, posterior, and dorsal caudate when compared to females, F(1, 16) = 5.266, 5.896, & 4.55, p = .04, .03, & .05, respectively. There were no sex differences observed elsewhere, and no interactions occurred between treatment and sex.

Figure 6.

Figure 6.

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Locomotor activity recorded for 60 minutes a day across two days at one week after the final risperidone injection. Females administered risperidone were more active than female vehicle rats, * = p < .004. Each bar represents mean ± S.E.M. Number of rats per group listed in each bar.

Figure 7.

Figure 7.

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Dopamine transporter density in the medial striatum and frontal cortex. Autoradiographic images of [125I]-RTI-55 binding in adult rats administered vehicle or risperidone during development are depicted on the left. The arrows in the top image indicate subregions of the caudate in which DAT density differed between vehicle and risperidone groups. The graph on the right shows the mean optical density ± S.E.M. for each region, and the asterisks denote subregional differences between groups, p < .03 - .009. There were 10 rats in each group. AC, PC, DC, & VC – anterior, posterior, dorsal, and ventral caudate, NA – nucleus accumbens, SFC & DFC – superficial and deep layers of frontal cortex.

4. Discussion

One of the primary findings of this study was that chronic risperidone administration led to later enhancement of amphetamine CPP. The idea that chronic antipsychotic drug administration alters later sensitivity to reward has been considered previously in adult rats. Bédard et al. (2011; 2013) have shown that, after cessation of chronic haloperidol administration in adult rats, responding on a lever previously paired with reward increases – an effect further enhanced by acute amphetamine injection. In terms of early-life antipsychotic drug administration and CPP, Vinish et al. (2013) discovered that adult rats administered the atypical antipsychotic drug, olanzapine, during development were more sensitive to the rewarding effects of amphetamine in a CPP test. Our work extends these findings by focusing on the effects of the most widely used antipsychotic drug in children, risperidone (Kalverdijk et al., 2017; Olfson et al., 2012). While acute risperidone administration has been shown interfere with stimulant- and opioid-induced CPP in adult animals (Manzanedo et al., 2001; Roger-Sanchez et al., 2013), the effects of chronic risperidone, administered either before or after adolescence, on later CPP have not been reported. In the present study, there was a main effect of risperidone on amphetamine CPP, although the difference between the risperidone and vehicle groups appeared more pronounced in the rats that received risperidone during development as opposed to those that received it during adulthood. The lack of a marked effect produced by adult risperidone administration, despite the reports (Bédard et al., 2011; 2013) of the heightened reward preference produced by adult antipsychotic administration, could be related to differences in antipsychotic drugs (risperidone vs. haloperidol) or, more likely, long-term persistence in the effects of developmental antipsychotic administration as opposed to the more transient effects of adult antipsychotic administration (Stevens et al., 2016).

In our CPP study, the rats treated with vehicle during development demonstrated preference scores during adulthood that were not only lower compared to rats administered risperidone during development but lower than any other group tested. As such, the apparent difference in amphetamine CPP seen between the adult rats treated with risperidone or vehicle early in life could be attributed to either a heightened sensitivity in the former group or a uniquely low preference in the latter group. It is doubtful that any procedural differences or anomalies led to this outcome since the amphetamine CPP procedure was similar to the one reported by Yates et al. (2012; 2013), although they used preference ratios in their analyses. The developmental group was tested at approximately 67 days of age and the adult group at 130 days of age, and a main effect of age was revealed in the study. Most previous work assessing age effects on CPP has compared conditioning between periadolescent rats (28-45 days of age) and young adult rats (65-80 days of age). Among these studies, Mathews et al. (2010) found no difference in amphetamine CPP between male Long-Evans rats at 45 and 69 days of age, whereas methamphetamine and nicotine CPP appear to weaken with age (Lenoir et al., 2015; Zakharova et al., 2009). Perhaps work is merited to compare CPP across a broader range of adult ages in order to determine whether the age effect observed in the present study is typical, and to more confidently assess the impact of chronic risperidone administration on later CPP.

As previously reported (Bardgett et al., 2013; 2019), animals that received risperidone early in life demonstrated greater levels of locomotor activity in an open field weeks after the cessation of treatment. This same effect was not observed in rats that received risperidone as adults, consistent with previous work (Stevens et al., 2016). Also, as reported before (Bardgett et al., 2019), the elevating effect of early-life risperidone on locomotor activity during adulthood was primarily confined to female rats in both cohorts tested in the present study. The enhanced effect of developmental risperidone in females is consistent with the observation that adult female rats are more sensitive to long-term behavioral effects of prolonged aripiprazole administration during late adolescence (Freeman et al., 2017). On the other hand, risperidone administration administered via food intake from postnatal day 22-50 increases locomotion to a greater extent in male relative to female Sprague-Dawley rats when activity is tested three weeks after treatment cessation (De Santis et al., 2016). Despite these conflicting data, the interaction between risperidone and sex deserve greater scrutiny, since chronic risperidone administration has been shown to increase estradiol levels in rats (Baptista et al., 2002) and exert neurochemical effects that are sex-specific (Lian et al., 2016), while estrogen appears to augment some of the behavioral effects of antipsychotic drugs (Madularu et al., 2014), which is consistent with the present study.

Amphetamine increased activity during the CPP conditioning days. There was no evidence of amphetamine-induced sensitization across days and the level of activity produced by each amphetamine dose was similar. The lack of a dose effect on activity may owe to the limited (30 minute) session since we have reported dose-dependent differences in activity with doses in the same range when using longer testing periods in an open field (Stubbeman et al., 2017). Rats previously administered risperidone demonstrated elevated activity in the CPP chamber during both the saline and amphetamine conditioning sessions in a manner that was independent of sex or age of administration. Previous risperidone administration did not enhance the effects of amphetamine on locomotor activity. This finding is in contrast to a recent report from our lab that early-life risperidone increases the effect of amphetamine on locomotor activity (Stubbeman et al., 2017). Again, the differences in the length of activity recording and the testing environment could account for the distinct outcomes between the studies. Collectively, the open field and CPP locomotor data indicate that the effects of long-term antipsychotic drug administration on later ambulatory activity depend on a myriad of development, situational, temporal, and sex-related factors. But the interaction of these factors should not take away from the general idea that early-life antipsychotic drug treatment alters locomotor activity later in life.

Because amphetamine targets the dopamine transporter, we compared the density of forebrain dopamine transporters between adult rats administered risperidone and vehicle early in life. In examining sagittal sections from the medial striatum, adult rats that received developmental risperidone administration possessed significantly fewer dopamine transporters in the anterior, posterior, and ventral caudate relative to vehicle-administered controls. This finding is consistent with a recent study (De Santis et al., 2016) that also found fewer striatal dopamine transporters in male, but not female, adult rats administered risperidone orally during development. A decrease in striatal DATs could reflect a loss of transporters or dopaminergic terminals. Although the first explanation appears the simplest, studies of chronic haloperidol administration in adults have shown a loss of tyrosine-hydroxylase positive neurons in the substantia nigra (Reynolds et al., 2011), raising the possibility that chronic risperidone treatment may likewise decrease dopamine-producing neurons in the midbrain and their terminal fields in the striatum. In either case, reduced DAT activity could conceivably lead to larger tonic levels of synaptic dopamine, and fewer transporters would be expected to enable lower doses of amphetamine sufficiently elevate dopamine and more easily elicit CPP. While we did not observe a loss of DATs in the nucleus accumbens, an area most associated with amphetamine CPP (Carr and White, 1986), the other striatal regions where DAT reductions were seen in risperidone-treated rats that have been implicated in reward-guided behavior (Yin and Knowlton, 2006) as well as in human studies of reward gains and losses related to psychostimulant use (Bischoff-Grethe et al., 2017). It is therefore plausible that decreases in striatal DATs contribute to the enhanced amphetamine CPP seen in adult rats administered risperidone early in life.

5. Conclusions

The work presented here may have implications for substance abuse and use disorders. Samaha (2014) previously proposed that chronic antipsychotic drug treatment during adulthood creates a transient syndrome of dopamine supersensitivity that enhances the locomotor and rewarding effects of drugs. Our findings here, along with our previous work (Stubbeman et al., 2017), extend this idea by showing that chronic antipsychotic drug administration during early postnatal development yields a similar syndrome that may be more permanent. These preclinical data have implications for the use of antipsychotic drugs in the management of conduct disorder and ADHD - disorders already associated with a greater risk for substance abuse later in life (Harstad and Levy, 2014, Levy et al., 2014; Salvo et al., 2012). Perhaps limitations on the use of antipsychotics in children, as has already taken place in the United States (Schmid et al., 2015), and the dose and length of treatment when necessary will ensure that any adverse impact of treatment on brain development is minimized.

Highlights.

  • Female rats administered risperidone early in life are more active as adults.

  • Chronic risperidone increases later sensitivity to reinforcing effects of amphetamine.

  • Early-life risperidone leads to fewer striatal dopamine transporters in adulthood.

Acknowledgements

This work was supported by the National Institute of Health (grant numbers P20GM103436, R15DA041708). This funding source had no other role in the research other than funding. All authors declare no conflict of interest.

Footnotes

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Contributor Information

Mark E. Bardgett, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076

Tyler Downnen, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076.

Casey Crane, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076.

Emily C. Baltes Thompson, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076

Brittany Muncie, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076.

Sara A. Steffen, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076

Justin R. Yates, Department of Psychological Science, Northern Kentucky University, Highland Heights, KY 41076

James R. Pauly, Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40504

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