Methamphetamine has been shown to effect locomotor activity and anxiety-like behavior in mice during methamphetamine withdrawal (Rauhut & Curran-Rauhut, 2018). Research suggests that locomotor activity increases immediately after methamphetamine exposure in mice (Rud, Do, & Siegel, 2016). Locomotor activity decreases over time in mice receiving methamphetamine injections after 90 minutes (Zombeck, Gupta, and Rhodes, 2009). This time period may not be adequate to properly determine behavior during withdrawal as previous studies have analyzed behavior up to 5 hours after methamphetamine injections and observed significant differences in locomotor activity (Rauhut & Curran-Rauhut, 2018). Previous research analyzing pharmacokinetic properties of methamphetamine, which showed the half-life of methamphetamine 56.6 minutes for doses of 0.64 mg/kg, were used to calculate the necessary dose and time period to induce methamphetamine withdrawal during testing for the current study (Brien, Kitney, & Peachey, 1978).
The use of methamphetamine may also influence anxiety levels. Methamphetamine withdrawal in rats effects anxiety-like behavior, with rats experiencing withdrawal exhibiting greater anxiety-like behavior (Hajheidari, Miladi-Gorji, & Bigdelli, 2015). Data has also shown that mice receiving methamphetamine doses of 1mg/kg exhibit greater anxiety-like behavior up to five hours after methamphetamine administration (Rauhut & Curran-Rauhut, 2018). An animal models was used in the current study to control external factors that humans experience, which is difficult to recreate in a laboratory setting. Anxiety-like behaviors may influence drug seeking behavior during periods of withdrawal and is an important factor when analyzing drug use.
Previous research analyzing the stress response has shown that mice receiving methamphetamine treatment had elevated corticosterone levels up to two hours after injection (Zuloaga, Johnson, Agam, & Raber, 2014). Methamphetamine exposure has also been shown to increase corticosterone levels in animal models during withdrawal periods (Jacobskind, Rosinger, Gonzalez, Zuloaga, & Zuloaga, 2018). This suggests that the Hypothalamic Pituitary (HPA) Axis is activated in response to methamphetamine treatment and methamphetamine use may be perceived as a physiological stressful event. Human methamphetamine users have also shown an increase in cortisol levels in response to a stressful test, suggesting that methamphetamine use causes an increase in the stress response (King, Alicata, Cloak, & Chang, 2010). Methamphetamine may be causing an increased stress response which could influence anxiety and depression behavior.
Additionally, the effect of housing conditions is important to analyze in respect to anxiety behavior. Hajheidari, Miladi-Gorji, and Bigdeli (2015) found that rats who received methamphetamine treatment spent more time in closed arms of a maze and avoided the open space, exhibiting greater anxiety-like behavior. Rats housed in an enriched environment spent less time immobile during a forced swim task, and the rats who received methamphetamine spent the most time immobile (Hajheidari, Miladi-Gorji, & Bigdeli, 2015). This suggests that the interaction of housing and drug treatment influence anxiety and depressive-like behavior in rodents. In humans, methamphetamine withdrawal has been significantly associated with depression symptoms, with 64.6% of participants showing symptoms of depression (Zhang et al., 2014). Using an animal model is useful to understand the impact methamphetamine withdrawal may have on depressive-like behavior when an association exists in humans between methamphetamine use and depression.
The current study seeks to further understand the relationship between methamphetamine withdrawal and housing conditions on behavioral responses. There is a lack of research on the effects housing conditions have on methamphetamine withdrawal symptoms and plasma corticosterone levels. The responses to methamphetamine withdrawal may be important in understanding drug administering behavior. We predicted that mice experiencing methamphetamine withdrawal will have greater anxiety-like and depressive-like behavior than controls. We predicted that mice exposed to methamphetamine will have increased levels of plasma corticosterone. Additionally, we predicted that mice housed in the enriched environment would exhibit less anxiety-like and depressive-like behaviors, along with decreased plasma corticosterone levels.
Method
Subjects
Female mice of strain C57BL/6J (N=20) were obtained for testing from The Jackson Laboratory (Bar Harbor, ME, USA). At the beginning of testing, the mice were three months old. After arrival, the mice were housed in four groups of five. Two groups were housed in standard conditions consisting of a food and water dish and tissue scraps. The other two groups were housed in an enriched environment, which consisted of a running wheel and tunnels along with mesh wrapping around the cage which the mice could climb. The mice were given food and water ad libitum. The mice were housed in a room that maintained 12 hour light and dark cycles, with lights turned on at 8:00 am. Testing took place during the light phase. When tested, noise levels were kept at 55 decibels and the light level was kept at 551 lux during testing. Approval was obtained from the University of St. Thomas’s Institutional Animal Care and Use Committee (IACUC) prior to testing.
Injections
Mice were given three intraperitoneal injections total. Half of the mice received methamphetamine (0.1mL, 1mg/kg) and the other half received saline (0.1mL). Injections were given one week apart. The first injection took place one week before the open field test. All mice received an injection of either methamphetamine (1mg/kg) or saline four hours before testing began to induce withdrawal when testing began (Brien, Kitney, & Peachey, 1978), while the other half received a saline injection. The groups were counterbalanced within housing conditions.
Open-Field Task
The open-field task was used as a measure of anxiety-like behavior and locomotor activity (Struntz & Siegel, 2018). The walls of the chambers were made of Plexiglas. The percentage of time in the center, distance travelled, and the number of center entries were measured using Anymaze software. The first week of testing, the mice were placed in open field chambers for a duration of five minutes. Prior to the beginning of testing, all mice were handled for a minute to acclimate them to being transported to the testing area. The open field chamber had dimensions of 40 cm x 40 cm and the center was defined as the inner 20 cm x 20 cm. Between trials, the open field chambers were cleaned with 70% isopropanol.
Porsolt Forced-Swim Task
The Porsolt forced-swim task utilized the same open field chambers with a water filled beaker one week after the open-field task and was used to measure depressive-like behavior (Hajheidari, Miladi-Gorji, & Bigdeli, 2015). Anymaze software was used to measure the percentage of time immobile. The second week, the mice were again injected with either a methamphetamine solution (0.1mL, 1mg/kg) or saline solution (0.1mL) intraperitoneally The mice were injected four hours prior to the beginning of testing in order to induce withdrawal. A Porsolt forced swim test was performed the second week. Mice were placed in a 1 liter beaker filled with 700 mL of lukewarm water for three minutes. Anymaze software quantified the time the mice were mobile, based upon a mobility threshold. Between trials, the beakers were washed with water.
Corticosterone ELISA
At the end of the experiment, 12 of the mice were euthanized via cervical dislocation and decapitation, with three mice from each experimental condition. Blood was collected and stored at -80ºC. A competitive ELISA kit from Abcam (Cambridge, MA, USA) was used to measure plasma corticosterone levels. All kit instructions were followed. A microplate reader was used for the ELISA kit which measured wavelengths of 450 nm.
Data Analysis
To perform statistical analyses, IBM SPSS Statistics software was utilized. A 2-way ANOVA was run to determine the effects of drug treatment and housing on the time spent in the center, number of entries into the center, distance travelled, percent time spent immobile during the Porsolt forced-swim task, and plasma corticosterone levels.
Results
No main effect of treatment on the percentage of time spent in the center during the open field test was observed, F(1, 16)=3.115, p=0.097. No main effect of housing on percentage of time spent in the center was observed, F(1,16)=0.307, p=0.587. No significant interaction was observed between the housing condition and drug treatment on the percentage of time spent in the center, F(1,16)=0.127, p=0.726 (Figure 1).
A main effect of treatment on the number of center entries was observed, F(1,16)=6.755, p=0.019. No main effect of housing on the number of center entries was observed, F(1,16)=3.066, p=0.099. Additionally, no significant interaction was seen between housing and drug treatment on number of entries, F(1,16)=0.809, p=0.382 (Figure 2).
No main effect of treatment on the distance travelled was observed, F(1,16)=0.244, p=0.628. No main effect of housing on distance travelled was observed, F(1,16)=1.113, p=0.307. No significant interaction was seen between the drug treatment and housing conditions on the distance travelled, F(1,16)=0.826, p=0.377 (Figure 3).
No main effect of treatment on percentage of time spent immobile was observed, F(1,16)=0.091, p=0.767. No main effect of housing on percentage of time spent immobile was observed, F(1,16)=0.157, p=0.697. No significant interaction was seen between the drug treatment and housing condition on percentage of time immobile, F(1,16)=0.242, p=0.630 (Figure 4).
No main effect of treatment on corticosterone levels was observed, F(1,8)=0.027, p=0.874. No main effect of housing on corticosterone levels was observed, F(1,8)=0.001, p=0.977. No significant interaction was observed between the treatments and corticosterone levels, F(1,8)=0.04, p=0.847 (Figure 5).
Discussion
The purpose of the current study was to analyze the effect of housing conditions and methamphetamine withdrawal on anxiety-like and depressive-like behavior, along with locomotor activity and plasma corticosterone levels. We found that methamphetamine withdrawal and housing conditions did not affect locomotor activity in the open-field, plasma corticosterone levels, or depressive-like behavior in the Porsolt forced-swim task. We found that mice experiencing methamphetamine withdrawal experienced decreased anxiety-like behavior as measured by the number of center entries in the open-field, but not when analyzing the percent time in the center.
Methamphetamine exposure did not affect the locomotor activity of mice. This does not correspond to previous research which has shown that mice exhibit reduced locomotor activity up to five hours after methamphetamine exposure (Rauhut & Curran-Rauhut, 2018). This may have been due to the time interval of each test. Mice were only tested over a period of five minutes, while previous studies have analyzed locomotor activity over periods of twenty minutes (Struntz & Siegel, 2018). The current study had limited time to test the animals, which is why open-field tests were performed over five minutes. Housing condition did not affect locomotor activity either. Enriched housing has been shown to reduce depressive-like behavior in mice (Hajheidari, Miladi-Gorji, & Bigdelil, 2015), but little research has been done to see how show the effect of housing conditions on locomotor activity during methamphetamine withdrawal.
Plasma corticosterone levels were not affected by housing condition or methamphetamine exposure. This does not fit with previous studies that have shown an increase in plasma corticosterone levels in mice during withdrawal (Zuloaga, Johnson, Agam, & Raber, 2014). Interestingly, methamphetamine exposure has been shown to significantly increase plasma corticosterone levels in females (Jacobskind, Rosinger, Gonzalez, Zuloaga, & Zuloaga, 2018). This may be because the current study collected blood samples more than five hours after methamphetamine exposure as opposed to two hours that was seen in previous studies (Zuloaga, Johnson, Agam, & Raber, 2014). Additionally, housing condition did not affect corticosterone levels. Research has shown that an enriched environment decreases anxiety levels in rodents (Hajheidari, Miladi-Gorji, & Bigdeli, 2015) which may be related to plasma corticosterone levels. However, the current study failed to observe any significant effect of housing on plasma corticosterone. Additionally, when pipetting the ELISA samples, the pipette volume changed in between loading, so some wells may have received more blood than others. The difference in blood volumes may have changed the concentrations of plasma corticosterone levels in some wells and not others.
Methamphetamine withdrawal did not affect depressive-like behavior either. In humans, methamphetamine withdrawal has been significantly associated with depressive behavior (Zhang et al., 2014). Our animal model did not follow the same pattern. Rodent models have found that methamphetamine withdrawal is associated with greater depressive-like behavior (Hajheidari, Miladi-Gorji, & Bigdeli, 2015). The previous study was using 4 mg/kg subcutaneous doses of methamphetamine, while the current dose used was 1 mg/kg. This may have contributed to the lack of an observed effect. Future studies may wish to examine a range of doses to determine the effect of methamphetamine on depressive-like behavior in rodent models. Additionally, the previous study (Hajheidari, Miladi-Gorji, & Bigdeli, 2015) used rats instead of mice. The difference in species may have contributed to the difference in depressive-like behavior. Housing condition also did not affect depressive-like behavior. This also did not follow previous studies that have shown a decrease in depressive-like behavior in rodents housing in enriched conditions (Hajheidari, Miladi-Gorji, & Bigdeli, 2015). The previous study’s enriched environment contained elements that the current study lacked, such as rope, balls, and a swing. The lack of an observable effect may have been due to a difference in the type and extent of environmental enrichment. Future studies may want to further analyze this relationship and examine if the extent and type of enrichment is a factor in depressive-like behavior.
Methamphetamine affected one aspect of the anxiety-like behavior tested. Mice experiencing methamphetamine withdrawal entered the center of the open-field more times than saline controls, which corresponds to less anxiety-like behavior. This is not seen in previous studies which have shown that mice experiencing methamphetamine withdrawal have greater anxiety-like behavior (Hajheidari, Miladi-Gorji, & Bigdelli, 2015; Rauhut & Curran-Rauhut, 2018). However, previous studies exposed the rodents to methamphetamine over four weeks (Rauhut & Curran-Rauhut, 2018) while the current study had only exposed mice to methamphetamine twice when the open-field task was performed. Additionally, mice were exposed to estradiol (Rauhut & Curran-Rauhut, 2018) while the current study did not expose the rodents to any hormones. Housing conditions did not affect the number of center entries in the current study. Previous studies have shown a decrease in anxiety-like behavior in rodents who are exposed to environmental enrichment (Hajheidari, Miladi-Gorji, & Bigdelli, 2015). As stated above, the previous study’s enriched environment contained components not available to the rodents in the current study and the previous study analyzed rats, which may affect the effect of housing on anxiety-like behavior.
Methamphetamine and housing did not affect the other aspect of anxiety-like behavior tests, the percent time in the center of the open-field. However, mice experiencing methamphetamine withdrawal after four hours have been observed to spend less percent time in the center (Rauhut & Curran-Rauhut, 2018). The current study only analyzed behavior over five minute trials while other studies have performed trials over twenty minutes periods (Struntz & Siegel, 2018). Housing condition did not affect this behavior either. Again, previous studies have shown that environmental enrichment decreases anxiety-like behavior in rodents (Hajheidari, Miladi-Gorji, & Bigdelli, 2015), but the environmental enrichment contained different apparatus. Additionally, the rodents were housed in the enriched environment up to 45 days before behavioral testing (Hajheidari, Miladi-Gorji, & Bigdelli, 2015) while the current study’s mice were housed in the environmentally enriched setting for two weeks prior to the open-field test. The duration of which the mice are housed in their environment may affect anxiety-like behavior as it pertains to the open-field task.
The current study found no conclusive evidence that methamphetamine withdrawal depressive-like behavior, locomotor activity, or plasma corticosterone levels in mice. This may show that the HPA axis is not activated response to methamphetamine during withdrawal as has been postulated in other studies. This is important when understanding human methamphetamine withdrawal and what factors may contribute to humans using methamphetamine recurrently. This study suggests that depressive-like behavior and plasma corticosterone does not significantly affect this process. The current study also analyzed the effect of environmental enrichment to attempt to better model the environments that humans experience. However, housing condition did not significantly affect the above behaviors or plasma corticosterone levels. This suggests that one’s environment does not significantly impact the behaviors associated with methamphetamine withdrawal regarding locomotor activity, depressive-like behavior, and plasma corticosterone.
The effects of methamphetamine on anxiety-like behavior were contradictory. The results showed that methamphetamine withdrawal decreased anxiety-like behavior as it pertains to the number of center entries in the open-field. This does not correspond to previous studies and suggests that methamphetamine does not induce anxiety-like behavior. However, no significant effect of methamphetamine was observed on the other aspect of anxiety-like behavior, the percent time in the center of the open-field. This suggests that perhaps a different test should be used in the future to analyze anxiety-like behavior since this task gave contradictory results. Housing did not affect either anxiety-like behavior tested, suggesting that the environment does not significantly affect anxiety-like behavior in rodents. However, because previous studies have shown an affect (Hajheidari, Miladi-Gorji, & Bigdelli, 2015), future studies may want to investigate further the relationship between housing and anxiety-like behavior as it pertains to methamphetamine withdrawal since the type of environmental enrichment can vary widely. Additionally, the current study used C57BL/6J mice, which may have affected behavior based on genetic differences. Future studies may want to analyze the same measurements as the current study with other strains of mice to fully determine the effects of methamphetamine withdrawal and housing condition.