Biperiden reverses the increase in 50-kHz ultrasonic vocalizations but not the increase in locomotor activity induced by cocaine

Abstract

Cocaine use disorder (CUD) is a worldwide public health problem, associated with severe psychosocial and economic impacts. Currently, no FDA-approved treatment is available for CUD. However, an emerging body of evidence from clinical and preclinical studies suggests that biperiden, an M1 muscarinic receptor antagonist, presents potential therapeutic use for CUD. These studies have suggested that biperiden may reduce the reinforcing effects of cocaine. It is well established that rodents emit 50-kHz ultrasonic vocalizations (USV) in response to natural rewards and stimulant drugs, including cocaine. Nonetheless, the effects of biperiden on the cocaine-induced increase of 50-kHz USV remains unknown. Here, we hypothesized that biperiden could antagonize the acute effects of cocaine administration on rat 50-kHz USV. To test this hypothesis, adult male Wistar rats were divided into four experimental groups: saline, 5 mg/kg biperiden, 10 mg/kg cocaine, and biperiden/cocaine (5 and 10 mg/kg, i.p., respectively). USV and locomotor activity were recorded in baseline and test sessions. As expected, cocaine administration significantly increased the number of 50-kHz USV. Biperiden administration effectively antagonized the increase in 50-kHz USV induced by cocaine. Cocaine administration also increased the emission of trill and mixed 50 kHz USV subtypes and this effect was antagonized by biperiden. Additionally, we showed that biperiden did not affect the cocaine-induced increase in locomotor activity, although biperiden administration per se increased locomotor activity. In conclusion, our findings indicate that administering biperiden acutely reduces the positive affective effects of cocaine, as demonstrated by its ability to inhibit the increase in 50-kHz USV.

Drug addiction is a worldwide public health problem associated with severe psychosocial, physiological, and economic impacts ^1,2. Recent reports have highlighted the prevalence of cocaine use disorder (CUD) affecting approximately 21 million people worldwide ². Cocaine administration is associated with positive behavioral effects including euphoria, increased arousal, and alertness ¹. These effects primarily occur due to cocaine’s ability to increase dopamine levels in the ventral and dorsal striatum by inhibiting dopamine transporter (DAT) activity ³. Therefore, cocaine can alter the neural mechanisms that mediate motivated behavior and reward-seeking by elevating extracellular dopamine levels in the nucleus accumbens (NAc) ^1,3.

Acetylcholine is an important modulator of striatal dopamine activity and involves a complex interaction among different families of muscarinic and nicotinic receptors ^4,5. Cholinergic activity within the NAc denotes a critical role in the stimulus-reward association and on the expression of motivated behaviors ⁵. Previous studies have shown that acetylcholine potentiates the seeking and rewarding effects of cocaine ⁶. Microdialysis studies have shown increased acetylcholine levels in the NAc and ventral tegmental area during cocaine self-administration ^6,7. Pharmacological inhibition of muscarinic receptors has also been shown to reduce the drug-seeking and reinforcing effects of cocaine ^8,9. In this regard, M1 muscarinic receptor antagonists have emerged as a potential treatment for CUD, as they could decrease the cocaine-induced craving and seeking ¹⁰, cocaine self-administration ⁹ as well as cocaine-induced conditioned place preference (CPP) ^8,11 and ethanol-induced CPP ¹². Biperiden is a muscarinic receptor antagonist previously employed for treating Parkinson’s disease. It exhibits a high affinity for M1 receptors, although effects on M4 receptors are also observed ¹³. Dieckmann et al. ¹⁰ showed that biperiden decreased cocaine-seeking and relapse in CUD patients. Preclinical studies also revealed that biperiden decreased cocaine-induced CPP ^8,11. Given the lack of effective FDA-approved treatments for CUD, additional studies are required to further evaluate the potential therapeutic use of biperiden for CUD treatment. Fifty-kHz ultrasonic vocalizations (USV) are described as pro-social signals emitted by rodents, potentially reflecting a positive emotional state ^14,15. The increase in the emission of 50-kHz calls is a well-described effect of stimulant drugs, reflecting a potential euphoric effect ^14,16,17. To our knowledge, no prior study has investigated the effects of biperiden on the cocaine-induced increase in 50-kHz USV. Therefore, based on previous reports that biperiden decreases cocaine seeking, craving, and reinforcing effects ^8,10,11 our study aimed to test whether acute biperiden administration could mitigate the acute effects of cocaine on 50-kHz USV and locomotor activity.

Forty-seven male Wistar rats (3 months old) weighing 300–350 g were used in this study. Rats were provided by the animal core facility at the University Federal do Parana and were housed in polypropylene cages (41×34×16 cm) with 3 or 4 rats per cage. Food and water were provided ad libitum and the housing room temperature was maintained at 22 ± 2°C with a 12h/12h light/dark cycle, light phase starting at 7:00 am. Experiments were conducted during the light phase. At the end of the experiments, rats were euthanized with an overdose of pentobarbital. The study was approved by the Universidade Federal do Parana Ethics Committee for Animal Care (protocol 932) and complied with both Brazilian (11.794/8 October 2008) and International Laws (EC Council Directive, 24 November 1986; 86/ 609/EEC).

The cocaine-HCl used in this study was a donation from the Brazilian Federal Police (inquiry #5016610–91.2019.4.04.7000/PR). Cocaine was 98% pure, as determined by nuclear magnetic resonance. Cocaine-HCl and biperiden (Cinetol, Cristalia, Brazil) were dissolved in saline (0.9% w/v) to achieve concentrations of 10 mg/ml and 5 mg/ml, respectively.

Rats were randomly assigned to the following groups: saline/saline (n = 11), biperiden/saline (n = 12), cocaine/saline (n = 12), and cocaine/biperiden (n = 12). During the 3 days before the USV recording, rats were handled for 5 min once a day. Next, rats were submitted to a baseline session in which USV and locomotor activity were recorded for 40 minutes. No administration was performed during this session. The next day, rats received intraperitoneal (i.p.) administrations of 10 mg/kg cocaine ¹¹, 5 mg/kg biperiden ¹², or vehicle (saline) immediately before a 40-minute test session. All rats received two i.p. injections according to their experimental group, with the second injection given immediately after the first injection.

In the baseline and test sessions, rats were gently placed into a 40×40×40 cm acrylic box, with the floor covered with fresh bedding. Room luminosity was maintained at 4 lux and room temperature was 20 ± 2 °C. Data acquisition started immediately after the rats were placed into the acrylic box. USV emissions were recorded using a CM16 UltraSoundGate Condenser microphone controlled by the Avisoft Recorder 2.7 software (Avisoft Bioacoustics, Germany). The microphone was positioned 25 cm above the center of the acrylic box. The recorded USV files were analyzed using the DeepSqueak 2.6.1 software, followed by manual acceptance or rejection of the 50-kHz calls. Previous studies have shown that besides increasing the total number of 50-kHz, cocaine differentially altered the emission of certain USV subtypes (flat, step, and mixed)¹⁸. In order to evaluate if biperiden could decrease the effect of cocaine on these 50-kHz USV subtypes, 50-kHz USV were further classified into four subtypes: flat, trill, step, and mixed, according to predefined criteria ¹⁹. Video recordings of locomotor activity were obtained using a camera positioned above the acrylic box. Analysis of the locomotor activity was performed automatically using the Ethovision XT software (Noldus, The Netherlands).

For statistical analysis, normal distribution and equal variances among groups were tested by the D′Agostino & Pearson and Bartlett tests, respectively. For the data that did not exhibit a normal distribution, a Y=log(Y+2) transformation was applied. Data that met normal distribution and equal variance were analyzed by using one-way ANOVA or two-way repeated measures (RM) ANOVA, followed by Tukey’s post hoc test. The Welch ANOVA test was conducted for the data that presented a normal distribution but not an equal variance, followed by Tamhane’s post hoc test. All statistical analyses were performed using the GraphPad Prism software (San Diego, USA). Differences were considered statistically significant if p < 0.05.

During the baseline session, no significant differences among groups were observed in the total number of 50-kHz USV (Fig. 1A; F_(3,43) = 1.81; p = 0.15, one-way ANOVA). On the other hand, during the test session, a significant difference in the total number of 50-kHz USV was observed (Fig. 1B; W_(3,22.2) = 12.64; p < 0.001, Welch ANOVA). Tamhane’s post hoc test indicated that the cocaine/saline-treated group exhibited significantly more 50-kHz calls compared to saline/saline (p < 0.001) and biperiden/saline groups (p = 0.004). Interestingly, rats treated with cocaine/biperiden showed a significant decrease in the number of 50-kHz USV compared to the cocaine-saline group (p = 0.03; Tamhane’s test), indicating that biperiden antagonized the cocaine-induced increase in 50-kHz USV. No significant differences were observed between the biperiden/saline and cocaine/biperiden groups compared to the saline/saline group (p = 0.85).

Figure 1. Biperiden antagonized the increase of 50-kHz USV induced by cocaine.

A. Total number of 50-kHz USV during the baseline session. B. Total number of 50-kHz USV during the test session. C. Fifty-kHz USV across the test session. Bars represent the mean ± SEM. Open circles represent individual data from A and B. * p < 0.05 comparing cocaine/saline and saline/saline, # p < 0.05 comparing cocaine/saline and biperiden/saline, & p < 0.05 comparing cocaine/saline and cocaine/biperiden and ^ p < 0.05 comparing each group to the respective first bin of time (5 min). Saline/saline (SAL), biperiden/saline (BIP), cocaine/saline (COC), cocaine/biperiden (COC/BIP).

Analyses of 50-kHz calls across the test session using a two-way RM ANOVA showed significant main effects of time (Fig. 1C, F_(1.9,81.8) = 156.4; p < 0.001), and treatment (F_(3,43) = 9.37; p < 0.001). However, the interaction between these factors was not significant (F_(21,301) = 0.71; p = 0.82). Tukey’s post hoc test showed that starting from the fifteenth minute after the beginning of the session all groups presented a significantly lower number of USV when compared to the first 5 minutes (Fig. 1C; p’s < 0.05). Tukey’s post hoc test also showed that compared to the saline/saline group, cocaine/saline administration significantly increased the number of 50-kHz calls during all the time points on the test session (Fig. 1C; p’s < 0.01). This cocaine-induced effect was antagonized in rats that received cocaine/biperiden administration. Rats that received cocaine/biperiden administration had a significantly lower number of USV when compared to the cocaine/saline group (p’s < 0.01) and were not different from the saline/saline and saline/biperiden groups across the session (p’s > 0.05).

Next, we investigated whether the biperiden effect was specific to certain subtypes of 50-kHz USV. No group differences were observed in the total number of flat (Fig. 2A, W_(3,22) = 1.13; p = 0.35, Welch ANOVA) and step calls (Fig. 2B, F_(3,43) = 1.79; p = 0.16, one-way ANOVA). However, significant differences were observed in the total number of trill (Fig. 2C; W_(3,21.6) = 15.63; p < 0.001; Welch ANOVA) and mixed calls (Fig. 2D, W_(3,23.2) = 24.99, p < 0.001; Welch ANOVA). Tamhane’s post hoc test showed that the numbers of trill and mixed calls in the cocaine/saline group were significantly higher, compared to saline/saline (p’s < 0.001) biperiden/saline (p’s < 0.001) and cocaine/biperiden groups (p < 0.001 and p = 0.02, respectively for trill and mixed calls). No significant differences between cocaine/biperiden and the saline/saline groups on trill and mixed calls were observed (p = 0.97 and p = 0.99, respectively) and between cocaine/biperiden and biperiden/saline groups (p = 0.70 and p = 0.16). Also, the total number of mixed calls in the biperiden/saline group was significantly lower compared to the saline/saline group (Fig. 2D; p = 0.005, Tamhane’s test).

Figure 2. Biperiden administration antagonized cocaine’s effect on trill and mixed 50-kHz calls.

Bars represent means ± SEM of flat (A) step (B), trill (C), and mixed calls (D) calls. Open circles represent individual data. * p < 0.05 comparing cocaine/saline and saline/saline, # p < 0.05 comparing cocaine/saline and biperiden/saline, & p < 0.05 comparing cocaine/saline and cocaine/biperiden and + p < 0.05 comparing biperiden/saline and saline/saline. Saline/saline (SAL), biperiden/saline (BIP), cocaine/saline (COC), cocaine/biperiden (COC/BIP).

Analyses of locomotor activity during the test session showed a significant difference in the total distance traveled among the groups (Fig. 3A, W_(3,22.1) = 7.87, p < 0.01; Welch ANOVA). Tamhane’s test showed that cocaine/saline administration significantly increased locomotor activity compared to the saline/saline group (p = 0.04). Rats treated with biperiden/saline or cocaine/biperiden also exhibited increased locomotor activity compared to the saline/saline group (p = 0.02 and p = 0.02, respectively; Tamhane’s test). No significant differences were observed among the biperiden/saline, cocaine/biperiden, and cocaine/saline groups (p = 0.99).

Figure 3. Effects of biperiden administration on locomotor activity during the test session.

A. Bars represent the mean ± SEM total distance traveled during the test session. Open circles represent individual data. B. Representative examples of individual rat tracks during the test session. + p < 0.05 comparing biperiden/saline and saline/saline, * p < 0.05 comparing cocaine/saline and saline/saline. $ p < 0.05 comparing cocaine/biperiden and saline/saline. Saline/saline (SAL), biperiden/saline (BIP), cocaine/saline (COC), cocaine/biperiden (COC/BIP).

Previous studies have shown that biperiden reduced cocaine and ethanol-induced CPP in rodents ^8,11,12 and also cocaine-seeking and relapse in CUD patients ¹⁰. In the present study, we tested the hypothesis that biperiden can antagonize the cocaine-induced increase on 50-kHz USV. We confirmed our initial hypothesis by showing that the acute administration of biperiden effectively antagonized the cocaine-induced increase in the total number of 50-kHz USV. However, no effect of biperiden was observed on cocaine-induced increase in locomotor activity. Our results are in agreement with previous reports that biperiden can mitigate some, but not all effects of cocaine, i.e. cocaine sensitization as demonstrated by Ramos et al. ¹¹.

Fifty-kHz USV are considered a pro-social behavior exhibited by rats, usually elicited in response to appetitive stimuli, novelty, or social interaction ¹⁴. The analysis of such call emission is a well-established model of the positive affective state induced by cocaine and other stimulant drugs and natural rewards ^14,15. Previous studies have demonstrated that systemic administration of cocaine can increase the emission of 50-kHz USV and this effect is proposed to be associated with the increase in dopamine extracellular levels in NAc ^15–18. Cocaine self-administration can also increase acetylcholine levels in NAc ⁶. Therefore, our results showing that biperiden antagonized the cocaine-evoked 50-kHz calls suggest that biperiden can decrease the rewarding effect of cocaine. In our study we also showed that cocaine specifically increased the number of trill and mixed subtypes of USV and this effect was antagonized by the biperiden administration. Biperiden also had a per se effect by decreasing the number of mixed calls. To our knowledge, no previous study evaluated the effect of biperiden on the emission of 50-kHz USV. The increase in trill and other frequency modulated USV subtypes is a well described effect of cocaine ^14,15,18. In rats, the emission of trills, and other frequency-modulated calls are described to potentially reflect different hedonic states, but the precise biological meaning of the trill, and other USV subtypes is still not completely understood ^14,15,20.

Our findings of biperiden antagonizing the cocaine-induced increase in USV contrast with the lack of effect of biperiden on the cocaine-induced increase in locomotor activity. In agreement with previous reports ²¹ we showed that biperiden administration increased locomotor activity per se. Also, the increase in locomotor activity is a well-described stimulant effect of cocaine ^16,18. As previously described ¹¹, we showed that biperiden did not mitigate the cocaine effects on locomotor activity. These contrasting results on USV and locomotor activity may be attributed to the differential effects of biperiden as a M1 and M4 receptor antagonist. Rojas-Carvajal et al. ²² showed that scopolamine, a non-specific muscarinic receptor antagonist, decreased the spontaneous emission of 50-kHz USV. Additionally, Wang et al. ²³ showed that selective knockout of the M4 receptors had no effect on USV elicited during social interaction in mice. Therefore, it is possible that biperiden mitigated the effect of cocaine on 50-kHz USV because the affinity of biperiden for M1 receptors is higher than the affinity for M4 receptors ¹³. This interpretation is supported by previous studies reporting that M1 receptor antagonists decreased dopamine release and neuronal excitability in the NAc ^24,25.

The cholinergic system plays an important and complex role in the neuronal mechanisms underlying cocaine addiction, involving different receptor subtypes, and direct and indirect modulatory activities ^4,7,25. Cholinergic activity is particularly associated with learning and memory mechanisms that contribute to the persistence of cocaine use. Previous studies demonstrated that biperiden administration could alter memory consolidation during cocaine-CPP test, thereby attenuating cocaine-seeking ^8,11. However, in the context of our study, using an acute administration protocol, we suggest that rather than affecting memory mechanisms, biperiden might have induced a decrease in euphoria and the positive affective state induced by cocaine. However, further investigations are required to expand the comprehension of biperiden’s effect over the broader spectrum of the cocaine effects. Future studies should consider evaluating biperiden’s effects on chronic cocaine treatment using different animal models of addiction. Also, these studies should consider using female subjects, as sex differences are reported after cocaine administration ³.

In conclusion, our study contributed to the relevant literature by showing for the first time that the biperiden administration antagonized the effect of cocaine on rats’ 50-kHz USV, which suggests that biperiden can attenuate the rewarding effects of cocaine. Further investigation on the potential therapeutic application of biperiden for the treatment of CUD is encouraged given the clinical and preclinical evidence that biperiden could mitigate certain cocaine-induced effects ^8,10,11.