Alaproclate, a Selective Serotonin Reuptake Inhibitor, Reduces Marble Burying Behavior in C57BL/6J Mice Kazuyuki Yamada *

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Alaproclate, a selective serotonin reuptake inhibitor, reduces marble burying behavior in C57BL/6J mice Kazuyuki Yamada *

Abstract Defensive burying is a widely observed behavior in rodents in response to noxious items, such as foul food, shock prods, and unusual objects such as glass marbles, upon encountering them. Marble burying behavior (MBB) has often been analyzed for the assessment of anxiety in animals and screening of drugs. In the MBB protocol, rodents are introduced into a cage with glass marbles laid with equidistant on thick flooring material. Following this, rodents will seek to immediately and eagerly bury the marbles. This behavior is analogous to the repetitive behaviors of patients with obsessive compulsive disorder (OCD). Therefore, MBB is a standard animal model of OCD that is used to assess therapeutic agents and evaluate drug efficacy. Many drugs have been used to examine their effect on MBB and OCD, and most research has focused on selective serotonin reuptake inhibitors (SSRIs). Although previous studies demonstrated that classic SSRIs such as fluoxetine and fluvoxamine reduced MBB, newly developed SSRIs have not been fully examined. Therefore, in the present study, we studied another SSRI, alaproclate, in the ordinal MBB test procedure in C57BL/6J mice model. The results show that alaproclate also reduced MBB without affecting locomotor activity. The present findings provide new evidence that SSRIs reduce MBB.

Key Word: anxiety, marble burying behavior, selective serotonin reuptake inhibitor, mouse, obsessive- compulsive disorder

Ⅰ. Introduction Ⅱ. Method Ⅲ. Results Ⅳ. Discussion Ⅴ. Conclusion

Ⅰ. Introduction contact. In response to the electric shock, the Defensive burying is an avoidance behavior rodents push, spray, and dig the flooring material, widely exhibited by rodents in response to noxious directing it towards the prod [4]. In the MBB stimuli, such as foul food [1-3], shock prods protocol, rodents are introduced into a cage with [4], anesthetized immobile conspecifics [5], and glass marbles laid out equidistantly on the thick unusual objects such as glass marbles [6]. Two flooring material. In response to the presence of protocols describe the assessment of defensive marbles, the rodents immediately resort to burying burying behavior: the “shock prod method” and them [6]. The rodents’ eagerness and compulsion the “marble burying behavior (MBB).” In the first to bury the marbles is analogous to the repetitive protocol, a prod is placed in the rodent’s cage, behaviors described in patients with obsessive- and an electric shock is delivered upon physical compulsive disorder (OCD); therefore, MBB is

* Professor, School of Management, Shizuoka Sangyo University

─ 135 ─ 環境と経営第25巻第１号（2019年） an important animal model of OCD that can be as an animal model of OCD [14]. Burying is a used to develop therapeutic agents and evaluate its normal rodent behavior [15], which is expressed efficacy. in the presence of non-anxiogenic or anxiogenic OCD is defined by the NIH Health Information stimuli, and harmful items. Therefore, if MBB as follows: “OCD is a common, chronic, and is induced by anxiety, repeated exposure should long-lasting disorder in which a person has lead to habituation and a reduction in anxiety, uncontrollable, reoccurring thoughts (obsessions) which would eventually lead to a reduction in and behaviors (compulsions) that he/she feels the number of buried marbles. Previous studies the urge to repeat over and over [7].” Selective have not reported any habituation effect using serotonin reuptake inhibitors (SSRIs) and other the MBB protocol [12, 13]. This suggests that antidepressants are effective in the treatment of MBB is not associated with anxiety and/or OCD [8]; however, the detailed pathology of OCD neophobia to novel/unfamiliar items. However, and the underlying mechanisms of action of these benzodiazepine anxiolytics, such as diazepam drugs remain unclear. Thus, several animal models and chlordiazepoxide, reduce MBB [12]. This have been developed to elucidate the neural discrepancy may show that MBB is a partial, mechanisms underlying OCD. Food-deprived not complete, model of OCD. Taken together, rats show exaggerated drinking behavior when these results indicate that burying harmless glass provided with small food pellets periodically, as marbles is comparable with unreasonable repetitive defined by a fixed time (FT) or a variable time (VT) behaviors in patients with OCD. schedule. This exaggerated drinking behavior is Many studies have shown that different defined as schedule-induced polydipsia (SIP) [9, neurotransmitters, such as monoamines 10]. Lesions in the orbitofrontal cortex also lead to (dopamine [16], norepinephrine [17], serotonin, compulsive lever-pressing in rats during operant and histamine [18]), amino acids (GABA [19], lever-pressing training to earn food pellets [11]. glutamic acid [20]), cannabinoids [21], and their These behaviors are robust and retain positive associated pathways contribute to MBB and reactivity to clinically effective drugs in the OCD. Serotonergic neurotransmission plays a treatment of OCD; however, the development of key role in MBB and OCD. Selective serotonin rodent models remain time-intensive and difficult, reuptake inhibitors (SSRIs), the first-line drugs since, they require extensive food restriction for the treatment of OCD [8], are also strong training, surgery, and recovery. Therefore, these inhibitors of MBB. As shown in Table 1, a lot animal models may not be suitable for use in of previous studies show the MBB-decreasing drug screening involving considerable number effect of classical SSRIs such as fluoxetine and of animals. Conversely, rodents spontaneously fluvoxamine, and other SSRIs. Given the strong bury marbles in the absence of any experimental correlation of SSRIs with MBB inhibition, there maneuver to enhance motivation, such as food/ exists the possibility of the involvement of the water restriction, and/or extensive training. serotonergic pathway in obsessive rodent behavior. Additionally, MBB does not show habituation after Therefore, we tested the effects of alaproclate, an repeated trials [12, 13]. Therefore, this is a useful SSRI, on MBB in mice. method for measuring changes in within-subject design experiments. MBB is stably observed in Ⅱ. Methods mice, which are much smaller than rats, and easily Animals: amenable to gene-modifying technology. Female mice were used on account of the Some studies address the concerns and previous studies indicating an ease of observation controversies regarding the suitability of MBB of MBB in them [13]. Forty female mice were

─ 136 ─ Alaproclate, a selective serotonin reuptake inhibitor, reduces marble burying behavior in C57BL/6J mice Table 1 Effect of SSRIs on MBB and spontaneous activity Agents MBB Activity reference [reference No.] citalopram reduced no effect Njung'e K, Handley SL. 1991 [13] Takeuchi H, et al. 2002 [23] Li X, Morrow D, Witkin JM. 2006 [25] Dankoski EC, et al. 2014 [33] escitalopram reduced no effect Taylor GT, et al.2017 [37] fluoxetine reduced partially decreased Abe M, et al. 1998 [22] no effect Takeuchi H, et al. 2002 [23] Hirano K, et al. 2005 [24] Li X, Morrow D, Witkin JM. 2006 [25] Umathe SN, et al. 2011 [29] Mosienko V, et al. 2012 [30] Arora T, et al. 2013 [31] Nardo M, et al. 2014 [32] Chanchal R, et al. 2015 [34] decreased Grassi G, et al. 2016 [35] no effects Rodriguez MM, et al. 2017 [36] fluvoxamine reduced no effect Njung'e K, Handley SL. 1991 [13] Ichimaru Y, et al. 1995 [21] Hirano K, et. al. 2005 [24] Shinomiya K, et. al. 2005 [26] Harasawa T, et al. 2006 [27] Sugimoto Y, et al. 2007 [28] indalpine reduced no effect Njung'e K, Handley SL. 1991 [13] sertraline reduced no effect Hirano K, et. al. 2005 [24] paroxetine reduced no effect Hirano K, et. al. 2005 [24] Shinomiya K, et. al. 2005 [26] zimeldine reduced no effect Njung'e K, Handley SL. 1991 [13] commercially procured (JCL Inc., Tokyo, Japan) at was implemented (lights were switched on at 0800 7 weeks of age (four mice were excluded because hour). Food and water were freely available in the of health problems, such as skin injury). Since, a breeding cages. All behavioral experiments were stronger MBB was observed on isolation housing conducted during the light cycle between 1300 and when compared with group housing [38], the mice 1700. All experimental procedures were performed were individually housed for 4 weeks, after 1 week with strict accordance to the guidelines of the of acclimation to the breeding environment in Institute of Physical and Chemical Research group housing (n=4 per cage). The breeding and (RIKEN), and were approved by the institute’s experimental rooms were air-conditioned (22±1°C, Animal Investigation Committee. 50–60% humidity), and a 12-h light-dark cycle

─ 137 ─ 環境と経営第25巻第１号（2019年） Drug treatment: plastic cages with sawdust for 30 min without Alaproclate hydrochloride (CAS: 60719-82-6; presenting glass marbles (habituation trial), and SIGMA-RBI, USA), was diluted in physiological then returned to their home cages. Following saline (Kobayashi, Japan), and administered by flattening of the sawdust layer in the habituated intraperitoneal (i.p.) infusion. The drug dose was cages, 20 glass marbles were introduced; evenly determined based on the previous study [39] spaced, 5 cm apart, on the sawdust. Mice were (20 mg/kg body weight). The drug solution was then reintroduced into the same cage in which prepared immediately before the experimentation. they had been habituated. After 30 min, the test was terminated by removing the mice from the Locomotor activity measurement: cage, and the number of marbles, for which more A clear acrylic enclosure (30× 30× 30 cm), than two-thirds of the surface area was covered with the side-walls and floor covered externally with sawdust when viewed from directly above with white paper to block external visual stimuli, the field, was counted. Image J (https://imagej.nih. was used as the experimental field for measuring gov/ij/) was used to determine whether the marbles the locomotor activity of mice. Luminance at the were covered with sawdust or not. After each trial, center of the field was 100 lux, and background the plastic cages, plastic ceilings, and the glass noise was approximately 50 dB. Each mouse was marbles were washed with tap water and cleaned introduced into the open field, facing a corner with 70% alcohol, and the used sawdust was of the field, and allowed to explore freely for 5 replaced with a fresh stock. min. Behavior of the mouse was recorded by a charge coupled device (CCD) camera attached Statistical analysis: to a computer, and analyzed using ANY-maze Statistical analyses were conducted using (Stoelting, USA); except for vertical movements, SPSSTM version 25 statistical software (Japan which were manually analyzed. The total distance IBM, Tokyo, Japan). A Student’s t-test was used traveled (cm), the total time mobile (sec), the total to compare the continuous data of the two groups, resting time (sec), and the total number of vertical and Mann-Whitney U-test was used to analyze the movements were used as indices of spontaneous ordinal scale data. activity. After each trial, the open field was washed with tap water, cleaned using 70% alcohol, and Ⅲ. Results dried with a paper towel. Figure 1 shows the results of the locomotor activity measurement: the saline control (n=11), Marble burying test: and alaproclate-treated (n=8) mice did not show In the marble burying test, independent animal statistically significant differences in the total groups from the locomotor activity measurement distance traveled (Figure 1A: t=-0.144, p=0.887, were used in order to exclude the effects of prior n.s.), the total time mobile (Figure 1B: t=-0.924, drug exposure and/or infusion-induced stress. The p=0.368, n.s.), and the total number of vertical protocol of the marble burying test was identical movements (Figure 1D: t=-0.665, p=0.515, n.s.). to the previous study [40]. Briefly, 20 clear glass On the other hand, the alaproclate-treated mice marbles (light blue-colored, 20 mm diameter) were exhibited a statistically significant decrease in the used for individual test. Opaque cages (30 × 36 resting time (Figure 1C: t=2.462, p<0.05). Figure 2 × 13 cm) were constructed using smooth, opaque shows the results of the marble burying test. Mice plastic; with a vinyl ceiling containing air holes, treated with alaproclate (n=7) buried significantly and a layer of sawdust (JCL Inc., Tokyo, Japan), fewer marbles compared to the mice treated with 5 cm deep. Mice were placed individually in the saline (n= 10) (U=2.5, p<0.05).

─ 138 ─ Alaproclate, a selective serotonin reuptake inhibitor, reduces marble burying behavior in C57BL/6J mice

Figure 1 Results of the activity measurement. A: Mean distance traveled (cm), B: Mean time mobile (sec), C: Mean time rest (sec), D) Mean number of vertical movements. Data represent mean + SEM.

Ⅳ. Discussion In the present study, we demonstrated that alaproclate, an SSRI, reduced MBB in female mice without affecting spontaneous locomotor activity. These results add new evidence on the effect of SSRIs and the contribution of serotonergic neural transmission to MBB. In many studies, spontaneous and/or locomotor activity were measured together with the MBB in order to negate the sedative or stimulatory effects of drugs from the decreasing number of marbles in MBB (see Table 1). In the present study, we confirmed the locomotor activity of mice using an open field method, aiding in the detailed measurement of locomotor activity. Both, saline- and alaproclate-treated mice exhibited Figure 2 Results of the marble burying test. Circles near-identical measurements for the total distance represent the number of buried marbles for each traveled, the total time mobile, and the total mouse. Bars represent median of the groups.

─ 139 ─ 環境と経営第25巻第１号（2019年） number of vertical movements (Figure 1A, B, the differential effects of the environment on MBB D). However, alaproclate-treated mice showed and OCD. Presently, many serotonergic agents, a statistically significant decrease in the resting including SSRIs, are available. Although not all time (Figure 1C). The resting time was defined agents are approved for therapeutic use (only four as the time taken by the mouse to relocate from SSRIs- escitalopram, fluvoxamine, paroxetine, its current occupied area (100 ms in the present and sertraline have been approved, but fluoxetine study), and it was measured when the mouse did and zimeldine have not been approved in Japan) not show apparent locomotion. The sum of the because of their efficacy and side effects [22, 42], total time mobile and resting time did not equate unapproved drugs and agents whose development to the total experimental time (300 sec). The for therapeutic use has been discontinued, decreased resting time observed in the alaproclate- including alaproclate, may be useful to delineate treated mice suggested that they had increased the neural mechanisms underlying the MBB stereotypic movement/behavior during the activity reducing effect of SSRIs, and in the pathogenesis measurement. As the activation syndrome was a and pathophysiology of OCD. representative side effect of SSRIs [41], decreased resting time may be attributable to the activation Ⅴ. Conclusion effects of alaproclate. Therefore, a detailed In the present study, we have demonstrated that analysis of the stereotypic movement/behavior in alaproclate, whose effect on defensive burying mice treated with alaproclate and other SSRIs is had not been examined till now, decreased MBB required. Although this study was preliminary and without affecting the locomotor activity, and conducted on a small scale, it strongly indicates increased the resting time. The latter suggests that that the detailed analysis of the spontaneous/ alaproclate may induce stereotypic movements locomotor activity is significant determinant of in open field experiments, and indicates the drug-induced effects on MBB. requirement for a detailed analysis of the As highlighted previously, many studies have spontaneous/locomotor activity to assess the clearly indicated that SSRIs reduce MBB in mice efficacy of drugs. Despite the preliminary nature (Table 1). As SSRIs inhibit 5-hydroxytryptamine and small size of the study, it provides new (5-HT) reuptake by binding to serotonin evidence that treatment with SSRI decreases MBB transporter (5-HTT), a reduction in MBB in in female mice. response to SSRIs is most likely related to increased 5-HT concentration in the synaptic Acknowledgements cleft. Drugs such as fenfluramine, that increase I would like to thank Ms. Homma C. for her the brain 5-HT concentration, also reduce MBB technical assistance, and Editage (www. Editage. [13]. Thus, the 5-HT concentration in the CNS jp) for English language editing. should play a key role in MBB. SSRIs are the first- line drugs for the treatment of OCD [8]; however, References there is a 3-6 week interval between treatment [1] Wilkie DM, MacLennan AJ, Pinel JP. Rat onset and therapeutic response [33]. Contrary to defensive behavior: burying noxious food. J OCD, treatment with SSRIs immediately reduce Exp Anal Behav. 1979;31(3):299-306. doi: the number of marbles buried in MBB cases. 10.1901/jeab.1979.31-299 Therefore, it is rather important to ensure if the [2] Poling A, Cleary J, Monaghan M. Burying by discrepancy between the effect of SSRIs on MBB rats in response to aversive and nonaversive and OCD, can be attributed to the differences in stimuli. J Exp Anal Behav. 1981;35(1):31-44. the neural mechanisms of MBB and OCD, or to doi: 10.1901/jeab.1981.35-31

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