A recreational dose of methylphenidate, but not methamphetamine, decreases anxiety-like behavior in female rats

Accepted Manuscript Title: A recreational dose of methylphenidate, but not methamphetamine, decreases anxiety-like behavior in female rats Authors: Jessica A. Boyette-Davis, Heather R. Rice, Roanne I. Shoubaki, Chantal M.F. Gonzalez, Marcela N. Kunkel, Devon A. Lucero, Paige D. Womble, Fay A. Guarraci PII: DOI: Reference:

S0304-3940(18)30408-7 https://doi.org/10.1016/j.neulet.2018.06.005 NSL 33636

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

7-4-2018 1-6-2018 5-6-2018

Please cite this article as: Boyette-Davis JA, Rice HR, Shoubaki RI, Gonzalez CMF, Kunkel MN, Lucero DA, Womble PD, Guarraci FA, A recreational dose of methylphenidate, but not methamphetamine, decreases anxiety-like behavior in female rats, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

RUNNING HEAD: STIMULANTS AND ANXIETY

A recreational dose of methylphenidate, but not methamphetamine, decreases anxiety-like

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behavior in female rats Jessica A. Boyette-Davis*a,, Heather R. Riceb, Roanne I. Shoubakib, Chantal M. F. Gonzalezb , Marcela N. Kunkela, Devon A. Lucerob, Paige D. Wombleb, Fay A. Guarracib

of Psychology and Behavioral Neuroscience, St. Edward’s University, Austin,

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a Department

of Psychology, Southwestern University, Georgetown, TX 78626 USA

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b Department

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TX 78704 USA

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* Corresponding Author: [email protected]; 3001 S Congress, Austin TX 78704 USA

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MPH and METH effects on anxiety in females have not been previously studied. Acute and chronic dosing was used to determine changes in anxiety during the EPM. MPH, given acute or chronic, decreased anxiety in adult female rats. METH and MPH altered locomotion during an open field test, but not during the EPM. Adult female rats given MPH show evidence of an anxiolytic effect.

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    

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Highlights

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ABSTRACT

Methylphenidate (MPH) and methamphetamine (METH) are two commonly abused psychomotor stimulants that impact anxiety, but in a manner that is currently unclear. This study adds to the literature by testing the effects of MPH and METH on anxiety in adult

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female rats, which has not previously been studied. In Experiment1, changes in anxiety-like behavior were determined using the Elevated Plus Maze (EPM) following either an acute injection of saline, METH (1mg/kg), or MPH (10mg/kg). Changes in general locomotion

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were measured using the open field test. MPH, but not METH, significantly decreased anxiety; MPH and METH were associated with increased activity in the open field. In

Experiment2, we compared the effects of three once daily injections of saline to MPH

(10mg/kg) or METH (1mg/kg). As with the acute dosing, repeated exposure to MPH, but not METH, decreased anxiety, and both drugs increased locomotion. Neither acute nor

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chronic dosing produced a change in locomotion during the EPM, indicating that the

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anxiolytic effects of MPH are independent of changes in locomotor behavior. These findings

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add further clarification to the literature investigating the psychoactive properties of MPH,

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with a special and needed emphasis on female behavior.

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locomotion

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Keywords: methylphenidate; Ritalin; methamphetamine; elevated plus maze; anxiety;

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1. Introduction MPH is a psychostimulant drug commonly prescribed for the treatment of attention

deficit hyperactivity disorder (ADHD) that can produce feelings of alertness, energy, and enhanced concentration [1,2,3]. Estimates indicate that up to 8% of the population abuses MPH [3]. Side effects of MPH include headache, decreased appetite, and insomnia [4]; abuse can enhance these side-effects, and add confusion, psychosis, and irritability to the long list

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of psychiatric effects [1]. An additional behavioral effect is that of anxiety. Anxiety levels have been shown to be lessened following acute MPH exposure [5]; however, increased anxiety is a common consequence of MPH abuse [1]. Animal studies confirmed that MPH

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exposure can alter anxiety. Acute [6] and chronic [7] MPH (5mg/kg) administration to male and female rat pups produced increased anxiety-like behavior in the EPM in adulthood, especially for the male rats [7]. A lower acute dose (3mg/kg) given to male and female

juvenile rats produced anxiolytic responses in females only [8]. In contrast, adult male rats chronically exposed to a high dose (10mg/kg) exhibited increased anxiety in the EPM

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[9,10]. The effects of MPH on anxiety are therefore mixed, and may be related to multiple

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factors, including the age and sex of the subjects, as well as the amount of exposure to the

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drug (i.e. acute/repeated).

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METH, another commonly abused psychomotor stimulant functions similarly to

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MPH [11]; however, instead of inhibiting dopamine transporters as MPH does, METH acts

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as a substrate for monoamine transporters, promoting the release of dopamine [11]. METH has been recognized as one of the most abused drugs in the United States, especially among

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women [12,13]. Psychiatric manifestations of METH use include euphoria, hallucinations, and increased sexual desire [14,15].

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Like MPH, METH also seems to impact anxiety. In fact, increased anxiety is one of

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the most common consequences of METH use [16], and this change can impair recovery for those who seek abstinence [17]. In animal studies, acute exposure may be anxiolytic, whereas repeated exposure may be anxiogenic. For example, one report found that a single injection of METH (5mg/kg) in male rats decreased anxiety in the EPM [18]. Others have reported that acute exposure to METH (1mg/kg) produced an anxiolytic effect in female

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rats, but not in males [19]. However, as with humans, repeated use of METH seems to be anxiety-inducing in rats. A recent study found that administration of 2mg/kg of METH for 14 days resulted in an increase in anxiety-like behaviors in male rats [20].

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Therefore, acute administration of both MPH and METH seems to produce anxiolytic behaviors, whereas repeated administration appears to be anxiogenic; however, as is clear from the literature, more work is needed to better determine the parameters of MPH and METH effects on anxiety. The current study was designed to assess the effects of MPH or METH on anxiety-like behavior in adult female rats using dosing regimens previously

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shown by our lab to affect emotional behaviors, such as sexual motivation.

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2. Materials and Methods

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2.1 Subjects

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Forty-nine adult female Long-Evans rats (200-300g) were purchased from Envigo

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(Indianapolis, IN) (Experiment1: n=26; Experiment2: n=23). Rats were group housed in

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hanging polycarbonate cages. Aspen wood shavings were used for bedding and a red plastic tube was provided for enrichment. Food and water were available ad libitum.

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Temperature and humidity were monitored daily. Lighting was maintained on a reversed 12:12 hour light-dark cycle (lights off at 10:00a.m.). Behavioral testing occurred during the

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dark phase under dim red light. To control for hormone levels, one week prior to

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behavioral testing, the experimentally naïve animals were bilaterally ovariectomized under a ketamine-xylazine cocktail (50mg/kg KCl + 50mg/kg Xylazine; Sigma-Aldrich, St. Louis, MO). Animals were monitored daily for 7 days after the surgery for any signs of distress or illness. Rats were given 10g of estradiol benzoate (EB) 48 hours and 1mg of progesterone (P) (Sigma-Aldrich) 4 hours prior to behavioral tests. Hormones were dissolved in sesame

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oil and injected subcutaneously in the flank. . All subjects received minimal handling, except that necessary for weekly weighing, cage cleaning, and injections (e.g., anesthesia, hormones, drug treatment). The experimental procedures were approved by the

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Institutional Animal Care and Use Committee where the research took place and were in accordance with the current Guide for the Care and Use of Laboratory Animals. 2.2 Drug Treatment

For Experiment1, subjects were randomly assigned to receive a single i.p. injection of either MPH (10mg/kg, n=10), METH (1mg/kg, n=7), or sterile physiological saline (0.9%

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NaCl; 1ml/kg, n=9). For Experiment2, subjects were randomly assigned to receive one i.p.

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injection per day for 3 consecutive days of either MPH (10mg/kg, n=8), METH (1mg/kg,

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n=7), or saline (0.9% NaCl; 1ml/kg, n=8). Behavioral testing was conducted 20 minutes

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after the only (Experiment1) or the last (Experiment2) injection by experimenters who

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were blind to the treatment condition. These doses and regimens were used because they

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have been shown to be similar to doses taken recreationally by humans [1], as well as have been shown to alter other emotional behavior, such sexual motivation in female rats

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[21,22,23,24]. Furthermore, we found in pilot studies that lower doses of MPH (2.5 and 5mg/kg) administered acutely or repeatedly failed to produce any effects on anxiety-like

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behavior or locomotion.

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2.3 Elevated Plus Maze The EPM is a plus-shaped apparatus commonly employed to test for anxiety-like

behaviors in rodents. It consists of four arms arranged in a plus shape, elevated 50cm off the floor. Two arms are 10cm wide platforms that extend away from the maze (open arms), whereas the other two arms are 10cm platforms that contain walls that are 50cm high

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(closed arms). A computer program (EPMscore.exe) [25] was used to capture the amount of time spent in the open and closed arms. Each trial lasted 5 minutes. 2.4 Open-Field Test

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The open field test is a standard test used for quantifying locomotion in rodents. Testing began immediately after the EPM by placing each subject in the middle of a clear Plexiglas enclosure (101cm long x 32cm high x 37cm wide). Line markings were placed

every 5cm on the floor of the enclosure. Line crossings (scored as all 4 paws crossing a line) were recorded for 5 minutes.

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All behavioral tests were recorded with digital, low-light video cameras (Sony

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behaviors were scored by trained observers.

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DCRHC65) and used to measure inter-rater reliability between multiple observers. All

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2.5 Data Analysis

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All statistical analyses were calculated using IBM SPSS Statistics 25.0. For each

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experiment, a one-way analysis of variance (ANOVA) was used to examine the effect of drug treatment (MPH, METH, Saline) on each of the dependent measures recorded during

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the EPM test: the percentage of time spent in the open arms, the percentage of time spent in the closed arms, number of entries into the open arms, number of entries into the closed

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arms. Tukey’s HSD post-hoc comparisons were calculated for each dependent measure (α

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set at .05). Paired t-tests were calculated within each drug treatment comparing behavior in the open arms to behavior in the closed arms (i.e., open vs. closed arm entries, open vs. closed arm percentage time). For the open field, the total number of line crossings recorded was analyzed with a one-way ANOVA. To assess differences between drug treatments as the test progressed,

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one-minute bins were analyzed. Tukey’s HSD comparisons were calculated for each dependent measure (e.g., total number of line crossings, number of line crossing per onemin bin; α set at .05).

3.1 Experiment 1: Acute Administration of MPH or METH

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3. Results

A one-way ANOVA was calculated on each of the four dependent measures of anxiety (i.e., percentage of time spent in open arms, number of open arm entries,

percentage of time spent in closed arms, and number of closed arm entries), with drug

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treatment as the independent variable. We found a significant main effect of drug

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treatment on percentage of time spent in the open arms (F2, 23 = 3.82, p = .037). Post-hoc

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comparisons revealed rats treated with MPH spent significantly more time in the open

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arms than those treated with saline (p = .029) (Fig. 1A). No other post-hoc comparisons

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reached statistical significance (p’s > .05). Although there was only a trend for an effect of

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drug treatment on percentage of time spent in the closed arms (F2, 23 = 3.23, p = .056), posthoc comparisons revealed rats treated with MPH spent significantly less time in the closed

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arms than rats treated with saline (p = .048) (Fig. 1A). No other post-hoc comparisons reached statistical significance (p’s > .05). There was no significant main effect of drug

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treatment on number of entries into the closed arms or number of entries into the open

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arms (Fig. 1B) (p’s > .05). Saline-treated rats spent significantly less time in open arms than in closed arms (t8 = 3.82, p = .003), but entered into the open and closed arms at the same rate (p > .05), validating percentage of open arm time as an indication of anxiety in this study.

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A one-way ANOVA was used to analyze general locomotor behavior during the entire open field test, with drug treatment as the independent variable. There was a significant main effect of drug treatment on the total number of line crossings (F2, 23 = 7.85,

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p = .003). Post-hoc comparisons revealed subjects treated with MPH (p = .003) or METH (p = .023) made significantly more line crossings than saline-treated rats. No other comparisons reached statistical significance (p’s > .05) (Fig. 2A).

One-way ANOVAs were calculated on line crossings made during one-minute bins. There was a significant main effect of drug treatment on the number of line crossings

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during the 2nd (F2, 23 = 6.01, p = .008), 3rd (F2, 23 = 8.96, p = .001), 4th (F2, 23 = 8.54, p = .002),

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and 5th bin (F2, 23 = 7.49, p = .003). Post-hoc comparisons revealed subjects treated with

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MPH made significantly more line crossings than saline-treated rats during the second bin

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(p = .007). However, when compared to saline-treated subjects, females treated with either

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MPH or METH made significantly more line crossings during the third, fourth, and fifth bin

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of the open field (range of p’s = .002-.034) (Fig. 2B). No other comparisons reached statistical significance (p’s > .05).

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3.2 Experiment 2: Repeated Administration of MPH or METH We found a significant main effect of drug treatment on percentage of time spent in

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the open arms in the EPM (F2, 20 = 7.13, p = .005). Post-hoc comparisons revealed rats

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treated with MPH spent significantly more time in the open arms than saline-treated rats (p = .003) (Fig. 3A). No other comparisons reached statistical significance (p’s > .05). We also found a significant main effect of drug treatment on percentage of time spent in the closed arms (F2, 20 = 3.67, p = .044). Post-hoc comparisons revealed subjects treated with MPH spent significantly less time in the closed arms than rats treated with saline (p = .035) (Fig.

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3A). No other post-hoc comparisons were significance (p’s > .05). Furthermore, there was no significant main effect of drug treatment on closed arm entries or on open arm entries (p’s > .05) (Fig. 3B). Saline-treated rats spent significantly less time in open arms than in

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closed arms (t7 = 4.11, p = .005), but entered the open and closed arms at the same rate (p > .05).

We found a significant main effect of drug treatment on the total number of line crossings made during the open field (F,2 20 = 11.03, p = .001) following a 3-day dosing

regimen, with Tukey’s HSD showing MPH (p = .001) and METH (p = .018) significantly

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increased line crossings compared to saline (Fig. 4A). No other comparisons reached

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statistical significance (p’s > .05).

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A significant main effect of drug treatment on the number of line crossings made

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during the 2nd (F2, 20 = 12.24, p = .0001), 3rd (F2, 20 = 7.89, p = .003), 4th (F2, 20 = 14.06, p =

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.0001), and 5th bin (F2, 20 = 13.48, p = .0001) was found. Subsequent post-hoc comparisons

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revealed subjects treated with MPH or METH made significantly more line crossings than saline-treated rats during the second, third, fourth, and fifth bin (range of p’s = .0001 to

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.040) (Fig. 4B). No other post-hoc comparisons were significant (p’s > .05). 3. Discussion

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The results of the present study indicate that a high, non-therapeutic dose of MPH

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(10mg/kg) [26] administered to female rats produced decreased anxiety, as evidenced by significantly more time spent in the open arms of the EPM than those treated with saline. This effect was found for both acute and repeated exposure to this drug. In contrast, female rats treated with METH did not significantly differ from saline-treated rats in anxiety-like behavior, after acute or repeated dosing.

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In contrast to what others have reported (i.e., an increase in anxiety-like behavior) [9,10], we observed evidence of anxiolytic behaviors during the EPM test following both acute and repeated administration of MPH (10mg/kg). One plausible explanation for this

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seeming contradiction is the sex of the subjects being tested. An abundance of literature indicates that females are more sensitive to the effects of psychostimulants, and some have concluded that this effect is so established as to state that “the response to

psychostimulants is sex-dependent” (pg. 398) [27]. Examples of enhanced behavioral

responses to psychostimulants in females include faster onset of addictive patterns of use

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and increased subjective reports of desired drug effects, like euphoria [28]. Hormonal

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influence is the most obvious explanation for these effects. For example, pretreating

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women with estradiol just prior to d-amphetamine exposure produced feelings of wanting

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more drug and experiencing more drug-induced pleasant sensations [29]. Becker [30]

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hypothesized that estrogen enhances dopaminergic signaling in reward pathways

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following psychostimulant exposure, increasing behavioral responses to these drugs in females. Given that our rats were hormonally primed to mimic the estrus phase of the

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reproductive cycle when estrogen levels peak, our observed effects of MPH were a function of an interaction with circulating estradiol levels. Therefore, it may be the case that a dose

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of 10mg/kg of MPH in males produces anxiety-like behaviors, but the same dose in females

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produces decreases in these behaviors due to estrogen-mediated mechanisms. Motaghinejad and colleagues [9,10] also exposed their rats repeatedly to MPH prior to testing anxiety, whereas the female rats in Experiment1 were tested 20 minutes following a single injection. Nevertheless, in Experiment2 we found that daily administration of the same dose of MPH for three consecutive days robustly decreased anxiety-like behavior

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during the EPM. Specifically, female rats treated daily with MPH for three days spent more time in the open arms and less time in the closed arms when compared to female rats treated with saline. Therefore, when these findings are viewed collectively, it seems likely

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that sex differences are the critical factor differentiating these studies and not the number of injections.

Although it is also possible that the results are related to the dose of MPH used, we propose this is unlikely. Pilot data using lower doses (2.5 and 5mg/kg; data not shown)

failed to produce any change in behavior, suggesting that a high dose, similar to doses used

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recreationally, is driving anxiolytic effects in female rats. This finding could have important

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implications for those focusing on the addictive aspects of MPH. The lower doses tested in

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pilot studies have been previously shown to alter anxiety, but only in pups exposed to MPH

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[6, 7, 8]. The dose used here (10mg/kg) has been shown to effectively alter behavior (i.e.

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sexual behavior) in female rats of this strain [24], and this dose was used by others

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investigating MPH and anxiety in male rats, allowing for a comparison between sexes [9,10]. Although these previous findings support the use of the dose given in the present

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study and provide a comparison, it is important to note that the election of a single-dose approach presents a limitation to the conclusions that can be drawn from our findings.

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Based on the findings of Macúchová [19], we expected acute METH to decrease

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anxiety, whereas repeated administration would increase anxiety, but we found no evidence of such effects of METH. There are, however, many methodological reasons that could explain why our findings were dissimilar to others, including the time at which testing occurred following drug injection (i.e., 45 minutes later in their study versus 20 minutes post-injection here), the strain of the rat used (i.e., Wistar versus Long Evans), and

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lighting (i.e., the present study used a reverse light/dark cycle and tested under dim red light). Because the dose of METH used in the present study has been used in our lab to demonstrate altered behavior under the same hormonal and testing parameters [22], the

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lack of change in anxiety following METH is not simply due to the inability of this dose to alter female rat behavior. Instead, methodological differences and sex/hormonal

differences offer a more plausible explanation for the discrepancy between studies.

Interestingly, Macúchová [19] also reported that METH increased entries into the open arms of the EPM compared to saline-treated animals, which we did not observe.

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However, female rats treated repeatedly with either MPH or METH in the present study did

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display increased locomotion, as evidenced by increased line crosses during the open field

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test. These results are consistent with other studies investigating the stimulant properties

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of MPH and METH [31]. Although locomotion could confound measures of anxiety in the

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EPM, we propose that the effects of MPH on anxiety are not an artifact of changes in

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locomotor behavior. Only MPH increased time spent in the open arms, whereas both MPH and METH administration increased locomotion in Experiments1 and 2. Importantly, there

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were no significant differences between drug treatment conditions on measures of activity during the EPM (e.g., open arm entries, closed arm entries). Therefore, the increase in time

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spent in the open arms for female rats treated with this high dose of MPH (10mg/kg)

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observed in Experiments1 and 2 can best be explained as actual changes in anxiety-like behavior. In contrast to what has been found in male rats, repeated exposure to MPH robustly decreased anxiety. Repeated METH exposure did not alter anxiety in female rats in Experiment2. Like the acute effects of MPH or METH, sex differences could explain the

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discrepancy between our results from repeated exposure in Experiment2 and previous studies with male subjects. 5. Conclusions

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In conclusion, either acute or repeated exposure to a high dose of MPH produced decreases in anxiety-like behavior that were independent of locomotor stimulation. The

effect of MPH on anxiety in the present study is consistent with reports from people who

use this drug. When viewed in light of other studies, the findings we present here suggest that female Long-Evans rats tested during behavioral estrus, following either acute or

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repeated exposure to a high dose of MPH, display behaviors evident of an anxiolytic effect.

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Such effects were not found for another, similar psychostimulant: METH. Although it is

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possible that drug effects on general locomotion can confound behavior observed during

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the EPM, the effects of MPH on anxiety in the present study appear to be independent of

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changes in locomotion when tested in female rats during behavioral estrus. The use of MPH

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in the present study clarifies some of the specific parameters under which MPH is anxiolytic. Future studies should be conducted to build upon these findings, including

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incorporation of known anxiolytic drugs such as diazepam, addition of higher doses of

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MPH, and inclusion of behavioral testing across the phases of the estrous cycle.

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Declarations of interest: None Funding: The work was funded by the Howard Hughes Medical Institute, through the Undergraduate Science Education Program (Grant #52007558), which provided summer research stipends to some of the authors (F.A.G., P.W., C.G., and D.L.) and the supplies to conduct the research described in the present manuscript.

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Author contributions: All authors contributed to study design, data collection and

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rat open-field behaviors, Physiol. Behav. 55 (1994) 441-446.

Figure Captions

Fig. 1 Acute treatment with MPH (10mg/kg) decreased anxiety-like behavior as measured

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by percentage of time spent in the open and closed arms during the EPM (A: Acute saline:

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n=9; Acute METH 1 mg/kg: n=7; Acute MPH 10 mg/kg: n=10). Neither MPH nor METH

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affected entries into the open or closed arms during the EPM test (B). Data are presented as

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means (± SEM). An asterisk (*) indicates a significant difference between MPH-treated

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subjects and saline-treated subjects, p<.05. A pound symbol (#) indicates a significant

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difference between percentage of time spent in the open arms and percentage of time spent in the closed arms within the saline treatment group, p<.05.

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Fig. 2 Acute treatment with MPH (10mg/kg) or METH (1mg/kg) increased line crossing

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during the entire open field test (A: Acute saline: n=9; Acute METH 1 mg/kg: n=7; Acute MPH 10 mg/kg: n=10). MPH significantly increased line crossings during the 2nd, 3rd, 4th

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and 5th minute of the open field test when compared to saline treatment; METH increased line crossings during the 3rd, 4th and 5th minute of the open field test when compared to saline treatment (B). Data are presented as means (± SEM) during the entire 5-min test or during each 1-min bin of the test. An asterisk (*) indicates a significant difference between drug-treated subjects (MPH or METH) and saline-treated subjects, p<.05.

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Fig. 3 Repeated treatment with MPH (10mg/kg per day for 3 days) decreased anxiety-like behavior as measured by percentage of time spent in the open and closed arms during the EPM (A: Repeated saline: n=8; Repeated METH 1mg/kg: n=7; Repeated MPH 10mg/kg:

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n=8). Neither MPH nor METH affected entries into the open or closed arms during the EPM test (B). Data are presented as means (± SEM). An asterisk (*) indicates a significant

difference between MPH-treated subjects and saline-treated subjects, p<.05. A pound

symbol (#) indicates a significant difference between percentage of time spent in the open arms and percentage of time spent in the closed arms within the saline treatment group,

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p<.05.

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Fig. 4 Repeated treatment with MPH (10mg/kg per day for 3 days), as well as repeated

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treatment with METH (1mg/kg per day for 3 days) increased line crossing during the entire open field test (A: Repeated saline: n=8; Repeated METH 1mg/kg: n=7; Repeated

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MPH 10mg/kg: n=8). MPH or METH treatment significantly increased line crossings during

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the 2nd, 3rd, 4th and 5th minute of the open field test compared to saline treatment (B). Data are presented as means (± SEM) during the entire 5-min test or during each 1-min bin of

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the test. An asterisk (*) indicates a significant difference between drug-treated subjects

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(MPH or METH) and saline-treated subjects, p<.05.

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