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Distinct behavioral traits and associated brain regions in mouse models for obsessive–compulsive disorder

Distinct behavioral traits and associated brain regions in mouse models for obsessive–compulsive... Background: Obsessive–compulsive disorder (OCD) is a mental disease with heterogeneous behavioral pheno- types, including repetitive behaviors, anxiety, and impairments in cognitive functions. The brain regions related to the behavioral heterogeneity, however, are unknown. Methods: We systematically examined the behavioral phenotypes of three OCD mouse models induced by pharma- cological reagents [RU24969, 8-hydroxy-DPAT hydrobromide (8-OH-DPAT ), and 1-(3-chlorophenyl) piperazine hydro- chloride-99% (MCPP)], and compared the activated brain regions in each model, respectively. Results: We found that the mouse models presented distinct OCD-like behavioral traits. RU24969-treated mice exhibited repetitive circling, anxiety, and impairments in recognition memory. 8-OH-DPAT-treated mice exhibited excessive spray-induced grooming as well as impairments in recognition memory. MCPP-treated mice showed only excessive self-grooming. To determine the brain regions related to these distinct behavioral traits, we examined c-fos expression to indicate the neuronal activation in the brain. Our results showed that RU24969-treated mice exhibited increased c-fos expression in the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), prelimbic cortex (PrL), infralimbic cortex (IL), nucleus accumbens (NAc), hypothalamus, bed nucleus of the stria terminalis, lateral division, intermediate part (BSTLD), and interstitial nucleus of the posterior limb of the anterior commissure, lateral part (IPACL), whereas in 8-OH-DPAT-treated mice showed increased c-fos expression in the ACC, PrL, IL, OFC, NAc shell, and hypo- thalamus. By contrast, MCPP did not induce higher c-fos expression in the cortex than control groups. Conclusion: Our results indicate that different OCD mouse models exhibited distinct behavioral traits, which may be mediated by the activation of different brain regions. Keywords: Obsessive–compulsive disorder, Behavior, Brain area, Neuronal activity Highlights • Three mouse models of obsessive–compulsive disor - *Correspondence: huangly55@mail.sysu.edu.cn; liboxing@mail.sysu.edu.cn; wenshl@mail.sysu.edu.cn der (OCD) were established. Department of Psychology, The Fifth Affiliated Hospital, Sun Yat-Sen • RU24969, 8-OH-DPAT, or MCPP administration University, No.52 Meihua West Road, Zhuhai 519000, Guangdong caused specific OCD-like behaviors. Province, China Neuroscience Program, Department of Physiology, Guangdong • Regional neuronal activity was assessed using c-fos Provincial Key Laboratory of Brain Function and Disease, Zhongshan expression levels. School of Medicine, Sun Yat-Sen University, Guangzhou 510810, China 3 • Expression of c-fos differed among the three newly Neuroscience Program, Department of Pathophysiology, Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan established OCD mouse models. School of Medicine, Sun Yat-Sen University, Guangzhou 510810, China © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Behav Brain Funct (2021) 17:4 Page 2 of 14 • Distinct OCD symptoms may be treated by region- In this study, we systematically examined the behav- specific targeted therapy. ioral phenotypes of three OCD mouse models induced by pharmacological reagents (RU24969, 8-OH-DPAT, and MCPP), and compared the activated brain regions in each model, respectively. Our results showed that dif- Introduction ferent OCD mouse models exhibited distinct behavioral Obsessive–compulsive disorder (OCD) is a mental dis- traits, which may be mediated by the activation of differ - order mainly characterized by obsessive and compul- ent brain regions. sive behaviors. The prevalence of OCD is ~ 2.3% in the population [1], and most of the patients develop symp- toms before 35 years old [2]. Selective serotonin reup- Materials and methods take inhibitors comprise the first-line treatment of OCD Animals patients, but approximately half of the OCD patients All experiments were performed in accordance with fail to fully respond to this treatment [3]. Therefore, it is the Guidelines for the Care and Use of Laboratory Ani- urgent to elucidate the mechanisms and causes of OCD. mals by the National Institutes of Health, and the animal OCD symptoms include obsessive (e.g., fear of con- experiment protocols have been approved by the Institu- tamination, the need to order things symmetrically, and tional Animal Care and Use Committee of Sun Yat-Sen aggressive, sexual, or religious thoughts) and compulsive University. To avoid the impact of estrogen changes in (e.g., excessive washing, checking, ordering, counting, the female mice on the behavior results, we only used and repeating) traits [4]. Besides, OCD is a highly het- male C57BJ6 mice (8–10 weeks old) in the study. 10–13 erogeneous disease, with which many patients experience mice were used in each group in the behavioral experi- anxiety and cognitive deficits additionally. The symptoms ments. Six mice in the 8-OH-DPAT group and MCPP vary widely among patients [5], and different dimensions group were used in the NOR test. In the cFos experiment, of OCD symptoms may be caused by distinct neurobio- three mice were used in each group. Three brain slices logical mechanisms [6]. Previous studies proposed that with different cross-sections were selected from the same the dysfunction of parallel, partly segregated cortico– brain area in each mouse. All experimental mice were striato-thalamo-cortical (CSTC) loops, including sero- reared under standard laboratory conditions (12-h light– toninergic, dopaminergic, and glutamatergic systems, dark cycle, lights on at 21:00, food and water freely avail- are related to different cognitive-affective processes in able, the temperature at 22 °C, humidity at 60%) in mouse OCD [4, 7–9]. For example, Mataix-Cols et al. used func- cages covered with corncob litter. Mice were reared for at tional magnetic resonance imaging (fMRI) to measure least 1 week to familiarize themselves with the environ- neurological activity in patients with different symptom ment before starting the formal experiments. Mice were dimensions of OCD. They found that the activities of the assigned randomly to experimental groups. bilateral ventromedial prefrontal regions and the right The environmental factors such as sound, light, and caudate nucleus were activated in patients with washing injection stimulation can profoundly impact behavior symptoms than in the control population. results and c-fos expression. To minimize these influ - Moreover, checking symptoms were accompanied by ences, we treated each group under the same conditions, increased activity of the putamen/globus pallidus, thala- including consistent light intensity, a quiet testing room, mus, and dorsal cortical areas, whereas the activities of and being handled in the same way for at least a week to the left precentral gyrus and right orbitofrontal cortex ensure the mouse familiar with the experimenter. in patients with hoarding symptoms were increased [6]. Therefore, each symptom may be mediated by relatively distinct brain regions or circuits. Targeted treatment on Chemicals relevant brain regions may enable us to develop precise RU24969 (HY-16688; MedChemExpress, Monmouth treatment, thereby improving treatment effectiveness in Junction, USA), 8-OH-DPAT (B6337, Houston, USA), OCD. and MCPP (125180; Sigma-Aldrich, Saint Louis, USA) Animal models are widely used to explore the physi- were dissolved in 0.9% saline. RU24969 and MCPP were ological and pathological characteristics of OCD. How- injected intraperitoneally, whereas 8-OH-DPAT was ever, the behavioral phenotypes of the OCD animal subcutaneously administered. Drug doses were selected model were quite different among different models. based on previous dose–response studies [11–15]. Additionally, CSTC circuit anomalies were only detected Drugs were injected at a volume of 20 ml/kg and 5 ml/ in some models [10, 11]. The studies that systematically kg for intraperitoneal and subcutaneous administration, compare the behavioral traits and abnormal brain circuits respectively. between different animal models are entirely lacking. Chen  et al. Behav Brain Funct (2021) 17:4 Page 3 of 14 Experimental procedures Marble‑burying test The MBT was carried out in a Experiment 1: behavioral experiments square box (31 cm × 23 cm × 16 cm), and a layer of 5 cm After adapting to the experimental environment for 30 corncob litter was laid on the bottom of this box, flattened, min, different groups of mice received acute injections and slightly compacted. Afterward, 20 black marbles were of RU24969 (10 mg/kg), 8-OH-DPAT (3 mg/kg), and evenly distributed on the surface of the corncob litter. MCPP (0.1 mg/kg), and their respective control groups During the experiment, mice were gently placed into the were injected with the same volume of saline. Differ - box and were quickly removed after 30 min. The number ent drugs have very different onset of action [12– 14, of buried marbles (more than two-thirds of the volume 16, 17]. Based on these literatures, 5 min (for RU24969 was buried in the corncob) was calculated [21]. and 8-OH-DPAT) or 20 min (for MCPP) after injec- tion, mice were tested in the circling behavior test, self- Novel object recognition test The NOR experiment was grooming test, induced-grooming test, open-field test performed in the open-field device described above. The (OFT), marble-burying test (MBT), and novel object NOR experiment was carried out over 3 consecutive days recognition test (NOR) on separate days, different [22, 23]. For habituation on the first day, mice were placed behavioral tests were conducted at least 2 days apart. in the open field without any objects for 5 min. For the Food and water were not present, and the luminance familiarization session after 24 h, two objects with the intensity was maintained at 5w during all the behavio- same shape and color were placed into the box, both 5 cm ral procedures. away from the wall. The mouse was placed into the box and removed after becoming familiar with these objects for 10 Circling behavior test An open field was used to evaluate min. After an additional 24 h, two objects were placed in the circling behavior. The apparatus consisted of a non- the box for the test session. One was the old object used porous plastic box with side lengths of 35 cm and a height in the previous familiarization session, whereas the other of 25 cm. The circling behavior was assessed as described was a novel object with a different shape, color, and tex - in a previous study [14]. The animals were placed in the ture. The mouse behavior was recorded for 5 min. The center of the open field, the bouts and durations of cir - interaction between the animal and the objects was meas- cling were determined for 20 min using TopScan Ver- ured according to the time the mouse spent sniffing the sion 3.0 (Clever Sys, Inc., Reston, USA) and SuperMaze novel object within a range of 2 cm. The discrimination (XR-Xmaze, Softmaze, Shanghai, Chian). Circling calcu- index calculated as the interaction time with the new lation program in TopScan was used to quantify circling object divided by the sum of the interaction times for the behavior. The mouse rotation angle equal to 360° within two objects was used to measure the interaction between the setting range of movement speed was recorded as one the mouse and the novel object. circling. Self‑grooming test The protocol of the self-grooming test Experiment 2: immunofluorescence was adapted from [18]. The mice were placed in a square Immunofluorescence staining was performed on dif- transparent mouse cage without food, water, and litter. ferent days with behavior tests. The procedures were After acclimation for 10 min, SuperMaze was used for described in previous work [24]. Briefly, 2 h after the video recording and TopScan for analyzing their groom- acute drug injection, the mice were anesthetized with ing behavior within 20 min. tribromoethanol (20 mg/kg), perfused with phos- phate-buffered saline (PBS), and 4% paraformalde- Induced‑grooming test The mouse was placed in a cage hyde (AR1068; Boster Biological Technology, Wuhan, as described for the self-grooming test. After 10 min, the China) for pre-fixation. The brain tissue was removed, mouse was gently sprayed with a watering can, and the placed in 4% paraformaldehyde, and stored at 4 °C subsequent grooming behavior of the mouse was recorded for 24 h, then dehydrated with 20% and 30% sucrose within 20 min for further analyses [19]. The grooming of solutions for 2 days. The OTC-embedded tissue was any part of the body was constituted a grooming event in cut into 40-μm sections using a freezing microtome Self-grooming and Induced-grooming test. (CM1950; Leica, Wetzlar, Germany). After permeabili- zation and blocking, the sections were incubated with Open‑field test An open field was used to evaluate anxi - primary anti-c-fos antibody (1:500, rabbit, #2250; Cell ety behavior [20]. Animals were placed in the center of Signaling Technology, Danvers, USA) at 4 °C for 20 the open field as described for the circling behavior test, h and then washed three times with PBS. Afterward, and the total time in the inner zone and the total distance the sections were incubated with the secondary Alexa covered were determined over 10 min. Fluor488-conjugated donkey anti-rabbit antibody Chen et al. Behav Brain Funct (2021) 17:4 Page 4 of 14 (1:500, A21208; Invitrogen, Carlsbad, USA) at room Results temperature for about 2 h and washed with PBS. Ru24969‑treated mice showed repetitive circling, memory To stain the nuclei, 4′,6′-diamidino-2-phenylindole impairment, and anxiety (DAPI, 0.1 μl/ml) was used for about 5 min. Sections In previous studies, pharmacological OCD models were were washed with PBS and covered on a glass slide. mainly derived from rats [10, 11]. We established OCD Brain slices were imaged using a confocal microscope mouse models by administration of RU24969, 8-OH- (LSM 880 with Airyscan; Zeiss, Jena, Germany). DPAT, and MCPP to complement these rat models. The effects of RU24969 treatment on mouse behav - ior were summarized in Fig.  1. We examined circling, Statistical analysis self-grooming, spray-induced grooming, and MBT in Prism software (GraphPad 8.0) was used for the sta- RU24969-treated mice. Acute treatment with RU24969 tistical analysis. c-fos expression was analyzed using increased both bouts (t = 2.68, p < 0.05) and dura- (20) the ImageJ software (National Institutes of Health, tion (t = 2.69, p < 0.05) of circling, compared with (20) Bethesda, MD, USA). The student’s t-test was used to saline-treated mice (Fig.  1a). RU24969-treated mice compare the statistical difference between two groups showed repeated circling around the edges of the open in behavioral experiments and immunofluorescence. field, whereas saline-treated mice moved in random In the NOR, the exploration time of the novel and the directions. These results suggest that RU24969 mice old object in the same group was compared using the exhibited repetitive, stereotyped behavior. Probably paired t-test. Data are expressed as the mean ± SE M. due to the long-term repeated movement in circles in Significant differences were defined as p < 0.05. the cage, and thus ignoring the marbles and reducing Fig. 1 Ru24969-treated mice showed repetitive circling behavior, memory impairments, and anxiety. Comparison between RU24969- and saline-treated mice regarding bouts and duration in the circling behavior (a), self-grooming (b), and spray-induced grooming (c) tests; the number of buried marbles in the MBT (d); bouts and duration in the OFT (e); and exploration time and discrimination index in the NOR (f). Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen  et al. Behav Brain Funct (2021) 17:4 Page 5 of 14 the grooming, RU24969-treated mice did not show administration led to the impairment of recognition increased OCD-like behavior in MBT and groom- memory. ing tests. Instead, the numbers of buried marbles and grooming bouts were decreased (Fig.  1b–d) (self- MCPP induced repetitive self‑grooming in mice grooming, bouts: t = 4.08, p < 0.001; self-grooming , Previous studies reported that MCPP induced repetitive (22) duration: t = 5.44, p < 0.0001; induced-grooming , self-grooming in rats [16, 18]. Could the same or distinct (22) bouts: t = 3.84, p < 0.001; induced-grooming , dura- behavioral phenotypes be induced in mice? To answer (22) tion: t = 3.89, p < 0.001; MBT: t = 4.82, p < 0.0001; this question, we performed behavioral tests in MCPP- (22) (22) Fig. 1b–d). treated mice. Our results showed that, similar to the rats, The OFT is frequently used to measure anxiety and MCPP-treated mice exhibited increased self-grooming locomotion levels in rodents [20]. In the OFT, the total (Fig.  2a–f). Both grooming bouts (t = 3.33, p < 0.01) (22) time in the inner zoom was reduced in the RU24969- and duration (t = 3.31, p < 0.01; Fig. 2b) were increased, (22) treated group (t = 3.49, p < 0.01), and the total dis- suggesting that MCPP could induce over-grooming in (22) tance was increased (t = 5.69, p < 0.0001; Fig .  1e), mice. However, MCPP-treated mice did not show a dif- (22) indicating that RU24969-treated mice exhibited anxi- ference in other behavioral tests than saline-treated mice ety and hyperlocomotion. In the NOR test, the control (Fig. 2a, c–f ). group mice showed a preference for novel objects in the test session (t = 4.35, p < 0.01), whereas the RU24969- 8‑OH‑DPAT‑treated mice exhibited excessive grooming (9) treated mice did not show this preference (t = 0.99, and memory impairment but not anxiety (11) p = 0.34; Fig .  1f). The discrimination index was sig - 8-OH-DPAT is a commonly used reagent to induce nificantly lower in the RU24969 group (t = 4.01, OCD-like behavior in the rat [10]. When we applied (20) p < 0.001; Fig .  1f ). These results suggest that RU24969 it to mice (Fig.  3a–f), we found that the duration of spray-induced grooming was significantly increased Fig. 2 MCPP induced repetitive self-grooming behavior but no other behavioral abnormalities in mice. Bouts and duration in the circling behavior (a), self-grooming (b), and induced-grooming (c) tests; the number of buried marbles in the MBT (d), bouts and duration in the OFT (e), and exploration time and discrimination index in the NOR (f) comparing MCPP- with saline-treated control mice. Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen et al. Behav Brain Funct (2021) 17:4 Page 6 of 14 Fig. 3 Excessive grooming and memory impairment, but no anxiety, in 8-OH-DPAT-treated mice. Bouts and duration in the circling behavior (a), self-grooming (b), and induced-grooming (c) tests; the number of buried marbles in the MBT (d), bouts and duration in the OFT (e), and exploration time and discrimination index in the NOR (f) comparing 8-OH-DPAT mice with saline-treated mice. Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: *p < 0.05, **p < 0.01, ****p < 0.0001 NOR; the discrimination index of 8-OH-DPAT-treated (t = 2.22, p < 0.05), although the number of bouts (23) mice was significantly lower than that of the saline- was not different compared with the saline-treated treated group (t = 2.42, p < 0.05; Fig. 3f ). group (t = 1.19, p = 0.25; Fig.  3c). Surprisingly, in self- (10) (23) grooming test, 8-OH-DPAT-treated mice did not exhibit Increased c‑fos expression in the OFC, ACC, PrL, IL, CPu, over-grooming (bouts: t = 0.44, p = 0.67; duration: (22) NAc, hypothalamus, BSTLD, and IPACL in RU24969‑treated t = 0.36, p = 0.72; Fig.  3b). Indeed, self-grooming and (22) mice spray-induced grooming represent very different groom - The distinct behavioral traits observed above suggest ing forms; anomalies in one form were not necessarily that each model might involve the activation of differ - accompanied by deficits in the other [25]. We found no ent brain regions. To validate this hypothesis, we used significant alteration in circling, either (bouts: t = 1.04, (21) c-fos expression to indicate the neuronal activation in p = 0.31; duration: t = 1.85, p = 0.08; Fig. 3a). (21) different brain regions. We found that RU24969 induced Notably, the number of buried marbles was decreased higher c-fos expression in many brain areas, includ- in the 8-OH-DPAT-treated group (t = 8.86, p < 0.0001; (22) ing OFC, ACC, PrL, IL, CPu, nucleus NAc, hypothala- Fig. 3d). Besides, 8-OH-DPAT also caused reduced loco- mus, BSTLD, and IPACL (Fig.  4a, b, Additional file  1: motion as shown in the OFT; the total distance was lower Fig. S1). Noteworthy, dysfunctions of the OFC, includ- in the 8-OH-DPAT group than in the control group ing the lateral orbital cortex (LO), ventral orbital cortex (t = 6.67, p < 0.0001), whereas the total time in the (21) (VO), and medial orbital cortex (MO), and ACC, have inner zone was increased (t = 2.86, p < 0.01; Fig.  3e). (21) been implicated in the etiology of OCD in previous This suggests the motion inhibition and lack of anxiety studies [11]. Consistently, all of these regions showed in 8-OH-DPAT-treated mice. Noteworthy, 8-OH-DPAT- increased c-fos expression in our RU24969-treated mice treated mice also presented memory impairments in the Chen  et al. Behav Brain Funct (2021) 17:4 Page 7 of 14 Fig. 4 Mapping c-fos expression in RU24969- and MCPP-treated mice. Representative immunofluorescence micrographs depicting c-fos expression in the OFC, PrL, IL, ACC, CPu, NAc, hypothalamus, BSTLD, and IPACL in RU24969-, MCPP- and saline-treated mice (Scale bars: 100 μm) the dysfunction in recognition memory, attention, and (LO + VO: t = 4.30, p < 0.001; MO: t = 2.78, p < 0.05; (16) (16) decision making [26]. In the CPu, c-fos was also highly Cg1: t = 3.42, p < 0.01; Cg2: t = 3.11, p < 0.01). The (16) (16) expressed in RU24969-treated mice, whereas hardly OFC and ACC are involved in many important neu- detectable in saline-treated mice (t = 6.92, p < 0.0001). ral functions, such as decision making, planning, inhi- (16) This was consistent with the findings that CPu’s func - bition of responses, and error monitoring. Indeed, in tions in learning, memory, action selection, and goal- OCD patients, almost all of these functions are impaired directed actions were impaired in OCD patients [5]. [5, 11]. The PrL and IL, components of the medial pre - The accumbens nucleus, shell (AcbSh) and accumbens frontal cortex (mPFC), showed increased c-fos expres- nucleus, core (AcbC) are two regions of the NAc and sion in the RU24969-treated group (PrL: t = 4.15, (16) participate in the regulation of reinforcement learning p < 0.001; IL: t = 2.30, p < 0.05), which may suggest (16) Chen et al. Behav Brain Funct (2021) 17:4 Page 8 of 14 Fig. 5 Quantification of the c-fos expression in RU24969- and MCPP-treated mice. a In RU24969-treated mice, c-fos expression levels were increased in the OFC, PrL, IL, CPu, NAc, hypothalamus, BSTLD, and IPACL. b There were no significant differences between MCPP- and saline-treated mice regarding the c-fos expression in the OFC, PrL, IL, CPu, NAc, hypothalamus, BSTLD, and IPACL. Data are expressed as the mean ± SEM. Unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen  et al. Behav Brain Funct (2021) 17:4 Page 9 of 14 (See figure on next page.) Fig. 6 The c-fos expression in the brain of 8-OH-DPAT-treated mice. a Representative immunofluorescence micrographs depicting the c-fos expression levels in the ACC, PrL, IL, OFC, AcbSh, and hypothalamus. Expression levels were increased in 8-OH-DPAT-treated mice compared with saline-treated control mice, whereas c-fos expression levels in the Acbc and CPu were not significantly different (Scale bars: 100 μm). b Quantification of the c-fos expression in the OFC, PrL, IL, ACC, CPu, NAc, and hypothalamus in 8-OH-DPAT and saline-treated mice. Data are expressed as the mean ± SEM. Unpaired t-test: ***p < 0.001, ****p < 0.0001 [27]. They also exhibited higher c-fos expression in OCD mouse models exhibited a distinct pattern RU24969-treated mice (AcbSh: t = 5.16, p < 0.0001; of the activated brain regions (16) AcbC: t = 3.94, p < 0.01). The BSTLD, a brain region To more intuitively compare the changes in the activa- (16) related to the regulation of anxiety and acute stress [28], tion of brain regions in the three OCD mouse models, showed increased c-fos expression in RU24969-treated we constructed a heatmap of c-fos expression (Fig.  7). mice (t = 5.73, p < 0.0001). Furthermore, we found The heatmap clearly illustrated the distinct pattern of (16) that c-fos was elevated in the IPACL in RU24969-treated the activated brain regions, in which increased c-fos mice (IPACL: t = 10.63, p < 0.0001; IL: t = 3.94, were detected in the OFC, ACC, PrL, IL, CPu, NAc, and (16) (16) p < 0.01). The IPACL receives projections from the hypothalamus in RU24969-treated mice, and the OFC, amygdala [29], but its functions related to OCD have ACC, PrL, IL, AcbSh, and hypothalamus in 8-OH-DPAT- not been clarified. Both control and RU24969-treated treated mice. In contrast, no significant increase was mice showed robust c-fos expression in the hypothala- detected in our interested brain regions in MCPP-treated mus, a brain region that responds to multiple stimula- mice. tions including stress and fear [30, 31], but the c-fos level was significantly higher in the RU24969-treated group Discussion (t = 6.93, p < 0.0001). Pharmacological animal models of OCD have been (16) widely used in previous studies [10, 11]. However, most of the published studies used rats instead of mice. In this report, three mouse models of OCD were established to Neuronal activity is not significantly increased provide more options for drug-induced OCD models and in MCPP‑treated mice offered more possibilities for researchers to explore the Next, we investigated MCPP-induced c-foc expression mechanisms of OCD. in mice brains. Surprisingly, after MCPP injection, we RU24969, 8-OH-DPAT, and MCPP all belong to sero- found that limited brain areas expressed c-fos with tonin receptor agonists. The choice of these reagents was a weak signal. When we compared it with the saline made based on previous clinical experience with OCD group, there were no statistically significant differences medications and hypothesized involvement of the sero- (Fig. 5b). No detectable changes were seen in the corti- tonin system in OCD. They all augment the sensitivity of cal brain areas that we examined. 5-HT receptor subtypes, which caused OCD [13]. The dysfunction of 5-HT receptor 1B (5-HT1B) in OCD has been suggested in previous studies. For example, in Increased c‑fos expression in the ACC, PrL, IL, OFC, AcbSh, pharmacological studies, the use of 5-HT1B agonists and hypothalamus in 8‑OH‑DPAT‑treated mice aggravated symptoms in patients with OCD, and muta- The brain regions expressing c-fos after 8-OH-DPAT tions of the serotonin transporter SCL6A4 were associ- administration were similar but not identical to those ated with OCD [14]. As serotonin 5-HT1A/1B receptor after RU24969 administration. In 8-OH-DPAT-treated agonist, RU24969 treatment in rats induced locomotor mice, higher c-fos expression were detected in the stereotypy, prepulse inhibition (PPI) deficits, and impair - ACC, PrL, IL, OFC, AcbSh, and hypothalamus (Cg1: ments in delayed alternation, all related to common signs t = 5.99, p < 0.0001; Cg2: t = 6.50, p < 0.0001; (16) (16) and symptoms in OCD patients [12]. Consistently, in the PrL: t = 5.02, p < 0.0001; IL: t = 5.25, p < 0.0001; (16) (16) present study, RU24969 administration induced repetitive LO + VO: t = 5.13, p < 0.0001; MO: t = 4.50, (16) (16) circling behavior in mice (Additional file  2: Fig. S2). More- p < 0.001; AcbSh: t = 6.32, p < 0.0001; hypothalamus : (16) over, the mice exhibited anxiety in the OFT. Impaired t = 7.15, p < 0.0001; Fig . 6a, b). These results indicate (16) memory is one of the cognitive dysfunctions of OCD [32]. that the excessive spay-induced grooming observed in In the NOR, RU24969-treated mice had a decreased time 8-OH-DPAT-treated mice might be mediated by the to explore novel objects indicative of memory impair- activation of these brain areas. ment, further confirming the validity of this model. Chen et al. Behav Brain Funct (2021) 17:4 Page 10 of 14 Chen  et al. Behav Brain Funct (2021) 17:4 Page 11 of 14 Fig. 7 Heatmap of the c-fos expression in the OCD models. The color range represents a logarithm of 2 for the ratio of each mouse to the each mouse of OCD models corresponding control average log . Red indicates high expression control average 8-OH-DPAT, a 5-HT receptor 1A (5-HT1A) ago- induced spray-induced grooming in mice. Interestingly, nist, could bind to its receptor with high selectivity self-grooming behaviors were not increased in 8-OH- [33]. 8-OH-DPAT-treated animals were commonly DPAT mice compared to control. This may be due to used as an OCD model. In the spontaneous alternation the necessity of a trigger to induce OCD-like behavior behavior test, the animals repeatedly selected the same in this model, similar to patients with excessive washing arm, similar to the OCD perseveration symptoms [34]. or cleaning behavior whose symptoms were triggered Importantly, treatment with selective serotonin reup- by contamination stimuli in clinical [4]. Thus, the OCD- take inhibitors could eliminate this repetitive behavior in like behaviors in 8-OH-DPAT-treated mice differed from the 8-OH-DPAT rat model [35]. However, other OCD- those in RU24969-treated mice. On the other side, simi- like behavioral tests have been rarely tested in the rat lar to RU24969-treated mice, 8-OH-DPAT-treated mice model. In the present study, 8-OH-DPAT administration showed recognition memory impairments in the NOR, Chen et al. Behav Brain Funct (2021) 17:4 Page 12 of 14 whereas anxiety was not detected in the OFT, which regions chosen in this study belong to the CSTC loop might be related to a lack of related stimuli in these (except for BSTLD and IPACL). Here, neuronal activ- experiments. ity was increased in the OFC, PrL, IL, ACC, CPu, NAc, Administration of MCPP, which binds to 5-HT1A and BSTLD, IPACL, and hypothalamus of RU24969-treated 5-HT2C receptors, could aggravate symptoms in patients mice. In 8-OH-DPAT-treated mice, activated brain with OCD [13, 36]. In a preclinical study, the administra- regions included the ACC, PrL, IL, OFC, and AcbSh tion of MCPP blocked the beneficial effects of fluoxetine (Additional file  3: Table S1). This was similar to previous for OCD treatment. Moreover, MCPP could induce the studies in mouse and rats [33, 43–45] that 5-HT1B recep- occurrence of repetitive stereotypes such as increasing tors are distributed in the striatum, cerebellum, and basal the number of buried marbles in the MBT [13], promot- ganglia, 5-HT1A receptors were distributed in the neo- ing directional persistence in spontaneous alternation cortex, olfactory areas, hippocampal formation, cortical behavior [11], and reinforcing self-grooming [16]. In the subplate, pallidum, hypothalamus, and mesencephalic current study, MCPP-induced OCD-like behaviors com- raphe nuclei, although the brain regions with increased prised excessive self-grooming behavior, whereas the neuronal activity in these two model mice did not com- induced-grooming test showed no significant differences pletely coincide with those expressing 5-HT1A or to control mice. These results contrasted with those in 5-HT1B receptors. One possible reason is that the OCD- 8-OH-DPAT mice. Self-grooming and spray-induced like behaviors were not induced by directly activating all grooming are different grooming forms [25]. Self-groom - relevant brain regions containing 5-HT1A or 5-HT1B ing is spontaneous; thus, excessive self-grooming in receptors, but acting within the same receptor-associated MCPP mice resembles trichotillomania (hair-pulling dis- brain region instead, therefore indirectly increasing the order) appearing spontaneously or under high pressure. neuronal activity of downstream brain regions and caus- Besides, the MCPP model presented no anxiety or mem- ing the occurrence of OCD-like behaviors. Notably, most ory impairment according to the OFT and NOR. Most of the activated brain regions were related to the CSTC patients with OCD chose to obey obsessive–compulsive loop elements corresponding to the postulated CSTC thoughts and perform obsessive–compulsive behaviors loop dysfunction in OCD. to alleviate the anxiety caused by obsessive–compulsive In mice and rats, the 5-HT receptor 2C (5-HT2C) is thoughts and impulses. Thus, the reduction of anxiety in mainly distributed in the choroid plexus and other areas, MCPP-treated mice may be due to the performance of including the nucleus accumbens, patches of the caudate- obsessive–compulsive behavior. putamen, the olfactory tubercle, claustrum, septum, cin- Together, the three newly established OCD mouse gulate cortex, amygdala, dentate gyrus, periaqueductal models exhibited distinct OCD-like behavioral traits gray, and entorhinal cortex [37, 44, 46]. Interestingly, after and presented different levels of anxiety and memory injection with the 5-HT2C agonist MCPP, we observed a dysfunction. weak c-fos expression in the mouse brains, with no differ - The expression of c-fos is one of the commonly used ence to the control group. Even after increasing the dose, indicators to measure the activity of neurons because we obtained the same results (data not shown). In this repetitive action potentials were often accompanied MCPP model, mice only showed excessive self-grooming by neuronal c-fos expression [37]]. Based on the drugs’ behavior, whereas all other behaviors appeared normal. pharmacological properties and the expression of the To a certain extent, this was consistent between behavio- receptors, we speculate that the increase in c-fos expres- ral and c-fos expression. We speculate that MCPP injec- sion was majorly raised from neurons. All three drugs tion might cause inhibition in some brain regions, which are 5-HT receptor agonists and can act on almost all could not be reflected by c-fos elevation. Indeed, previous neuronal cells. The distribution of their corresponding studies found that OCD patients have decreased activity receptors is wide. 5-HT 1A receptors were expressed in in certain brain regions [47, 48]. the pyramidal neurons of the cortex, hippocampus, and raphe nuclei and the cholinergic neurons in the septum Conclusion [38–40]. 5-HT 1B receptors were also expressed in the Here, we established three different OCD mouse mod - medium spiny neurons in the caudate putamen, probably els with distinct OCD-like behaviors, accompanying GABAergic [41, 42]. 5-HT 2C receptors were expressed dysfunctions in different brain regions. Compared to in most GABAergic cells. Astrocyte was also reported to controls, RU24969- and 8-OH-DPAT-treated mice express 5-HT receptors; thus, the astrocyte could also be demonstrated enhanced activation in brain regions a source for the c-fos signal. Previous studies proposed mostly belonging to the CSTC circuit, although some that the dysfunction of CSTC loops is related to differ - of the activated brain regions differed between the ent cognitive-affective processes in OCD. The brain Chen  et al. Behav Brain Funct (2021) 17:4 Page 13 of 14 Declarations two models. Neuronal activity was not significantly increased in MCPP-treated mice. These results implied Ethics approval and consent to participate that targeted and individualized treatment should be All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health, and performed in OCD patients with distinct symptoms, the animal experiment protocols have been approved by the Institutional and interventional treatment targeting the correspond- Animal Care and Use Committee of Sun Yat-Sen University. ing specific brain regions may improve the therapeutic Consent for publication effects. The datasets generated during or after the current study are available from the corresponding author on reasonable request. Abbreviations Competing interests 5-HT: 5-Hydroxytryptamine; 8-OH-DPAT: 8-Hydroxy-DPAT hydrobromide; AcbC: The authors declare no conflicts of interest. Accumbens nucleus, core; AcbSh: Accumbens nucleus, shell; ACC: Ant erior cingulate cortex; BSTLD: Bed nucleus of the stria terminalis, lateral division, Received: 20 January 2021 Accepted: 8 May 2021 intermediate part; Cg1/2: Cingulate cortex, area 1/2; CPu: Caudate-putamen; CSTC: Cortico–striato-thalamo-cortical; IL: Infralimbic cortex; IPACL: Interstitial nucleus of the posterior limb of the anterior commissure, lateral part; LO: Lateral orbital cortex; MBT: Marble-burying test; MCPP: 1-(3-Chlorophenyl) pip- erazine hydrochloride-99%; MO: Medial orbital cortex; mPFC: Medial prefrontal References cortex; NAc: Nucleus accumbens; NOR: Novel object recognition test; OCD: 1. Goodman WK, Grice DE, Lapidus KA, Coffey BJ. Obsessive–compulsive Obsessive–compulsive disorder; OFC: Orbitofrontal cortex; OFT: Open-field disorder. Psychiatr Clin. 2014;37(3):257–67. test; PBS: Phosphate-buffered saline; PPI: Prepulse inhibition; PrL: Prelimbic 2. Association AP. 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Distinct behavioral traits and associated brain regions in mouse models for obsessive–compulsive disorder

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Abstract

Background: Obsessive–compulsive disorder (OCD) is a mental disease with heterogeneous behavioral pheno- types, including repetitive behaviors, anxiety, and impairments in cognitive functions. The brain regions related to the behavioral heterogeneity, however, are unknown. Methods: We systematically examined the behavioral phenotypes of three OCD mouse models induced by pharma- cological reagents [RU24969, 8-hydroxy-DPAT hydrobromide (8-OH-DPAT ), and 1-(3-chlorophenyl) piperazine hydro- chloride-99% (MCPP)], and compared the activated brain regions in each model, respectively. Results: We found that the mouse models presented distinct OCD-like behavioral traits. RU24969-treated mice exhibited repetitive circling, anxiety, and impairments in recognition memory. 8-OH-DPAT-treated mice exhibited excessive spray-induced grooming as well as impairments in recognition memory. MCPP-treated mice showed only excessive self-grooming. To determine the brain regions related to these distinct behavioral traits, we examined c-fos expression to indicate the neuronal activation in the brain. Our results showed that RU24969-treated mice exhibited increased c-fos expression in the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), prelimbic cortex (PrL), infralimbic cortex (IL), nucleus accumbens (NAc), hypothalamus, bed nucleus of the stria terminalis, lateral division, intermediate part (BSTLD), and interstitial nucleus of the posterior limb of the anterior commissure, lateral part (IPACL), whereas in 8-OH-DPAT-treated mice showed increased c-fos expression in the ACC, PrL, IL, OFC, NAc shell, and hypo- thalamus. By contrast, MCPP did not induce higher c-fos expression in the cortex than control groups. Conclusion: Our results indicate that different OCD mouse models exhibited distinct behavioral traits, which may be mediated by the activation of different brain regions. Keywords: Obsessive–compulsive disorder, Behavior, Brain area, Neuronal activity Highlights • Three mouse models of obsessive–compulsive disor - *Correspondence: huangly55@mail.sysu.edu.cn; liboxing@mail.sysu.edu.cn; wenshl@mail.sysu.edu.cn der (OCD) were established. Department of Psychology, The Fifth Affiliated Hospital, Sun Yat-Sen • RU24969, 8-OH-DPAT, or MCPP administration University, No.52 Meihua West Road, Zhuhai 519000, Guangdong caused specific OCD-like behaviors. Province, China Neuroscience Program, Department of Physiology, Guangdong • Regional neuronal activity was assessed using c-fos Provincial Key Laboratory of Brain Function and Disease, Zhongshan expression levels. School of Medicine, Sun Yat-Sen University, Guangzhou 510810, China 3 • Expression of c-fos differed among the three newly Neuroscience Program, Department of Pathophysiology, Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan established OCD mouse models. School of Medicine, Sun Yat-Sen University, Guangzhou 510810, China © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Behav Brain Funct (2021) 17:4 Page 2 of 14 • Distinct OCD symptoms may be treated by region- In this study, we systematically examined the behav- specific targeted therapy. ioral phenotypes of three OCD mouse models induced by pharmacological reagents (RU24969, 8-OH-DPAT, and MCPP), and compared the activated brain regions in each model, respectively. Our results showed that dif- Introduction ferent OCD mouse models exhibited distinct behavioral Obsessive–compulsive disorder (OCD) is a mental dis- traits, which may be mediated by the activation of differ - order mainly characterized by obsessive and compul- ent brain regions. sive behaviors. The prevalence of OCD is ~ 2.3% in the population [1], and most of the patients develop symp- toms before 35 years old [2]. Selective serotonin reup- Materials and methods take inhibitors comprise the first-line treatment of OCD Animals patients, but approximately half of the OCD patients All experiments were performed in accordance with fail to fully respond to this treatment [3]. Therefore, it is the Guidelines for the Care and Use of Laboratory Ani- urgent to elucidate the mechanisms and causes of OCD. mals by the National Institutes of Health, and the animal OCD symptoms include obsessive (e.g., fear of con- experiment protocols have been approved by the Institu- tamination, the need to order things symmetrically, and tional Animal Care and Use Committee of Sun Yat-Sen aggressive, sexual, or religious thoughts) and compulsive University. To avoid the impact of estrogen changes in (e.g., excessive washing, checking, ordering, counting, the female mice on the behavior results, we only used and repeating) traits [4]. Besides, OCD is a highly het- male C57BJ6 mice (8–10 weeks old) in the study. 10–13 erogeneous disease, with which many patients experience mice were used in each group in the behavioral experi- anxiety and cognitive deficits additionally. The symptoms ments. Six mice in the 8-OH-DPAT group and MCPP vary widely among patients [5], and different dimensions group were used in the NOR test. In the cFos experiment, of OCD symptoms may be caused by distinct neurobio- three mice were used in each group. Three brain slices logical mechanisms [6]. Previous studies proposed that with different cross-sections were selected from the same the dysfunction of parallel, partly segregated cortico– brain area in each mouse. All experimental mice were striato-thalamo-cortical (CSTC) loops, including sero- reared under standard laboratory conditions (12-h light– toninergic, dopaminergic, and glutamatergic systems, dark cycle, lights on at 21:00, food and water freely avail- are related to different cognitive-affective processes in able, the temperature at 22 °C, humidity at 60%) in mouse OCD [4, 7–9]. For example, Mataix-Cols et al. used func- cages covered with corncob litter. Mice were reared for at tional magnetic resonance imaging (fMRI) to measure least 1 week to familiarize themselves with the environ- neurological activity in patients with different symptom ment before starting the formal experiments. Mice were dimensions of OCD. They found that the activities of the assigned randomly to experimental groups. bilateral ventromedial prefrontal regions and the right The environmental factors such as sound, light, and caudate nucleus were activated in patients with washing injection stimulation can profoundly impact behavior symptoms than in the control population. results and c-fos expression. To minimize these influ - Moreover, checking symptoms were accompanied by ences, we treated each group under the same conditions, increased activity of the putamen/globus pallidus, thala- including consistent light intensity, a quiet testing room, mus, and dorsal cortical areas, whereas the activities of and being handled in the same way for at least a week to the left precentral gyrus and right orbitofrontal cortex ensure the mouse familiar with the experimenter. in patients with hoarding symptoms were increased [6]. Therefore, each symptom may be mediated by relatively distinct brain regions or circuits. Targeted treatment on Chemicals relevant brain regions may enable us to develop precise RU24969 (HY-16688; MedChemExpress, Monmouth treatment, thereby improving treatment effectiveness in Junction, USA), 8-OH-DPAT (B6337, Houston, USA), OCD. and MCPP (125180; Sigma-Aldrich, Saint Louis, USA) Animal models are widely used to explore the physi- were dissolved in 0.9% saline. RU24969 and MCPP were ological and pathological characteristics of OCD. How- injected intraperitoneally, whereas 8-OH-DPAT was ever, the behavioral phenotypes of the OCD animal subcutaneously administered. Drug doses were selected model were quite different among different models. based on previous dose–response studies [11–15]. Additionally, CSTC circuit anomalies were only detected Drugs were injected at a volume of 20 ml/kg and 5 ml/ in some models [10, 11]. The studies that systematically kg for intraperitoneal and subcutaneous administration, compare the behavioral traits and abnormal brain circuits respectively. between different animal models are entirely lacking. Chen  et al. Behav Brain Funct (2021) 17:4 Page 3 of 14 Experimental procedures Marble‑burying test The MBT was carried out in a Experiment 1: behavioral experiments square box (31 cm × 23 cm × 16 cm), and a layer of 5 cm After adapting to the experimental environment for 30 corncob litter was laid on the bottom of this box, flattened, min, different groups of mice received acute injections and slightly compacted. Afterward, 20 black marbles were of RU24969 (10 mg/kg), 8-OH-DPAT (3 mg/kg), and evenly distributed on the surface of the corncob litter. MCPP (0.1 mg/kg), and their respective control groups During the experiment, mice were gently placed into the were injected with the same volume of saline. Differ - box and were quickly removed after 30 min. The number ent drugs have very different onset of action [12– 14, of buried marbles (more than two-thirds of the volume 16, 17]. Based on these literatures, 5 min (for RU24969 was buried in the corncob) was calculated [21]. and 8-OH-DPAT) or 20 min (for MCPP) after injec- tion, mice were tested in the circling behavior test, self- Novel object recognition test The NOR experiment was grooming test, induced-grooming test, open-field test performed in the open-field device described above. The (OFT), marble-burying test (MBT), and novel object NOR experiment was carried out over 3 consecutive days recognition test (NOR) on separate days, different [22, 23]. For habituation on the first day, mice were placed behavioral tests were conducted at least 2 days apart. in the open field without any objects for 5 min. For the Food and water were not present, and the luminance familiarization session after 24 h, two objects with the intensity was maintained at 5w during all the behavio- same shape and color were placed into the box, both 5 cm ral procedures. away from the wall. The mouse was placed into the box and removed after becoming familiar with these objects for 10 Circling behavior test An open field was used to evaluate min. After an additional 24 h, two objects were placed in the circling behavior. The apparatus consisted of a non- the box for the test session. One was the old object used porous plastic box with side lengths of 35 cm and a height in the previous familiarization session, whereas the other of 25 cm. The circling behavior was assessed as described was a novel object with a different shape, color, and tex - in a previous study [14]. The animals were placed in the ture. The mouse behavior was recorded for 5 min. The center of the open field, the bouts and durations of cir - interaction between the animal and the objects was meas- cling were determined for 20 min using TopScan Ver- ured according to the time the mouse spent sniffing the sion 3.0 (Clever Sys, Inc., Reston, USA) and SuperMaze novel object within a range of 2 cm. The discrimination (XR-Xmaze, Softmaze, Shanghai, Chian). Circling calcu- index calculated as the interaction time with the new lation program in TopScan was used to quantify circling object divided by the sum of the interaction times for the behavior. The mouse rotation angle equal to 360° within two objects was used to measure the interaction between the setting range of movement speed was recorded as one the mouse and the novel object. circling. Self‑grooming test The protocol of the self-grooming test Experiment 2: immunofluorescence was adapted from [18]. The mice were placed in a square Immunofluorescence staining was performed on dif- transparent mouse cage without food, water, and litter. ferent days with behavior tests. The procedures were After acclimation for 10 min, SuperMaze was used for described in previous work [24]. Briefly, 2 h after the video recording and TopScan for analyzing their groom- acute drug injection, the mice were anesthetized with ing behavior within 20 min. tribromoethanol (20 mg/kg), perfused with phos- phate-buffered saline (PBS), and 4% paraformalde- Induced‑grooming test The mouse was placed in a cage hyde (AR1068; Boster Biological Technology, Wuhan, as described for the self-grooming test. After 10 min, the China) for pre-fixation. The brain tissue was removed, mouse was gently sprayed with a watering can, and the placed in 4% paraformaldehyde, and stored at 4 °C subsequent grooming behavior of the mouse was recorded for 24 h, then dehydrated with 20% and 30% sucrose within 20 min for further analyses [19]. The grooming of solutions for 2 days. The OTC-embedded tissue was any part of the body was constituted a grooming event in cut into 40-μm sections using a freezing microtome Self-grooming and Induced-grooming test. (CM1950; Leica, Wetzlar, Germany). After permeabili- zation and blocking, the sections were incubated with Open‑field test An open field was used to evaluate anxi - primary anti-c-fos antibody (1:500, rabbit, #2250; Cell ety behavior [20]. Animals were placed in the center of Signaling Technology, Danvers, USA) at 4 °C for 20 the open field as described for the circling behavior test, h and then washed three times with PBS. Afterward, and the total time in the inner zone and the total distance the sections were incubated with the secondary Alexa covered were determined over 10 min. Fluor488-conjugated donkey anti-rabbit antibody Chen et al. Behav Brain Funct (2021) 17:4 Page 4 of 14 (1:500, A21208; Invitrogen, Carlsbad, USA) at room Results temperature for about 2 h and washed with PBS. Ru24969‑treated mice showed repetitive circling, memory To stain the nuclei, 4′,6′-diamidino-2-phenylindole impairment, and anxiety (DAPI, 0.1 μl/ml) was used for about 5 min. Sections In previous studies, pharmacological OCD models were were washed with PBS and covered on a glass slide. mainly derived from rats [10, 11]. We established OCD Brain slices were imaged using a confocal microscope mouse models by administration of RU24969, 8-OH- (LSM 880 with Airyscan; Zeiss, Jena, Germany). DPAT, and MCPP to complement these rat models. The effects of RU24969 treatment on mouse behav - ior were summarized in Fig.  1. We examined circling, Statistical analysis self-grooming, spray-induced grooming, and MBT in Prism software (GraphPad 8.0) was used for the sta- RU24969-treated mice. Acute treatment with RU24969 tistical analysis. c-fos expression was analyzed using increased both bouts (t = 2.68, p < 0.05) and dura- (20) the ImageJ software (National Institutes of Health, tion (t = 2.69, p < 0.05) of circling, compared with (20) Bethesda, MD, USA). The student’s t-test was used to saline-treated mice (Fig.  1a). RU24969-treated mice compare the statistical difference between two groups showed repeated circling around the edges of the open in behavioral experiments and immunofluorescence. field, whereas saline-treated mice moved in random In the NOR, the exploration time of the novel and the directions. These results suggest that RU24969 mice old object in the same group was compared using the exhibited repetitive, stereotyped behavior. Probably paired t-test. Data are expressed as the mean ± SE M. due to the long-term repeated movement in circles in Significant differences were defined as p < 0.05. the cage, and thus ignoring the marbles and reducing Fig. 1 Ru24969-treated mice showed repetitive circling behavior, memory impairments, and anxiety. Comparison between RU24969- and saline-treated mice regarding bouts and duration in the circling behavior (a), self-grooming (b), and spray-induced grooming (c) tests; the number of buried marbles in the MBT (d); bouts and duration in the OFT (e); and exploration time and discrimination index in the NOR (f). Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen  et al. Behav Brain Funct (2021) 17:4 Page 5 of 14 the grooming, RU24969-treated mice did not show administration led to the impairment of recognition increased OCD-like behavior in MBT and groom- memory. ing tests. Instead, the numbers of buried marbles and grooming bouts were decreased (Fig.  1b–d) (self- MCPP induced repetitive self‑grooming in mice grooming, bouts: t = 4.08, p < 0.001; self-grooming , Previous studies reported that MCPP induced repetitive (22) duration: t = 5.44, p < 0.0001; induced-grooming , self-grooming in rats [16, 18]. Could the same or distinct (22) bouts: t = 3.84, p < 0.001; induced-grooming , dura- behavioral phenotypes be induced in mice? To answer (22) tion: t = 3.89, p < 0.001; MBT: t = 4.82, p < 0.0001; this question, we performed behavioral tests in MCPP- (22) (22) Fig. 1b–d). treated mice. Our results showed that, similar to the rats, The OFT is frequently used to measure anxiety and MCPP-treated mice exhibited increased self-grooming locomotion levels in rodents [20]. In the OFT, the total (Fig.  2a–f). Both grooming bouts (t = 3.33, p < 0.01) (22) time in the inner zoom was reduced in the RU24969- and duration (t = 3.31, p < 0.01; Fig. 2b) were increased, (22) treated group (t = 3.49, p < 0.01), and the total dis- suggesting that MCPP could induce over-grooming in (22) tance was increased (t = 5.69, p < 0.0001; Fig .  1e), mice. However, MCPP-treated mice did not show a dif- (22) indicating that RU24969-treated mice exhibited anxi- ference in other behavioral tests than saline-treated mice ety and hyperlocomotion. In the NOR test, the control (Fig. 2a, c–f ). group mice showed a preference for novel objects in the test session (t = 4.35, p < 0.01), whereas the RU24969- 8‑OH‑DPAT‑treated mice exhibited excessive grooming (9) treated mice did not show this preference (t = 0.99, and memory impairment but not anxiety (11) p = 0.34; Fig .  1f). The discrimination index was sig - 8-OH-DPAT is a commonly used reagent to induce nificantly lower in the RU24969 group (t = 4.01, OCD-like behavior in the rat [10]. When we applied (20) p < 0.001; Fig .  1f ). These results suggest that RU24969 it to mice (Fig.  3a–f), we found that the duration of spray-induced grooming was significantly increased Fig. 2 MCPP induced repetitive self-grooming behavior but no other behavioral abnormalities in mice. Bouts and duration in the circling behavior (a), self-grooming (b), and induced-grooming (c) tests; the number of buried marbles in the MBT (d), bouts and duration in the OFT (e), and exploration time and discrimination index in the NOR (f) comparing MCPP- with saline-treated control mice. Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen et al. Behav Brain Funct (2021) 17:4 Page 6 of 14 Fig. 3 Excessive grooming and memory impairment, but no anxiety, in 8-OH-DPAT-treated mice. Bouts and duration in the circling behavior (a), self-grooming (b), and induced-grooming (c) tests; the number of buried marbles in the MBT (d), bouts and duration in the OFT (e), and exploration time and discrimination index in the NOR (f) comparing 8-OH-DPAT mice with saline-treated mice. Data are expressed as the mean ± SEM. Unpaired t-test, except for the exploration time in the NOR using the paired t-test: *p < 0.05, **p < 0.01, ****p < 0.0001 NOR; the discrimination index of 8-OH-DPAT-treated (t = 2.22, p < 0.05), although the number of bouts (23) mice was significantly lower than that of the saline- was not different compared with the saline-treated treated group (t = 2.42, p < 0.05; Fig. 3f ). group (t = 1.19, p = 0.25; Fig.  3c). Surprisingly, in self- (10) (23) grooming test, 8-OH-DPAT-treated mice did not exhibit Increased c‑fos expression in the OFC, ACC, PrL, IL, CPu, over-grooming (bouts: t = 0.44, p = 0.67; duration: (22) NAc, hypothalamus, BSTLD, and IPACL in RU24969‑treated t = 0.36, p = 0.72; Fig.  3b). Indeed, self-grooming and (22) mice spray-induced grooming represent very different groom - The distinct behavioral traits observed above suggest ing forms; anomalies in one form were not necessarily that each model might involve the activation of differ - accompanied by deficits in the other [25]. We found no ent brain regions. To validate this hypothesis, we used significant alteration in circling, either (bouts: t = 1.04, (21) c-fos expression to indicate the neuronal activation in p = 0.31; duration: t = 1.85, p = 0.08; Fig. 3a). (21) different brain regions. We found that RU24969 induced Notably, the number of buried marbles was decreased higher c-fos expression in many brain areas, includ- in the 8-OH-DPAT-treated group (t = 8.86, p < 0.0001; (22) ing OFC, ACC, PrL, IL, CPu, nucleus NAc, hypothala- Fig. 3d). Besides, 8-OH-DPAT also caused reduced loco- mus, BSTLD, and IPACL (Fig.  4a, b, Additional file  1: motion as shown in the OFT; the total distance was lower Fig. S1). Noteworthy, dysfunctions of the OFC, includ- in the 8-OH-DPAT group than in the control group ing the lateral orbital cortex (LO), ventral orbital cortex (t = 6.67, p < 0.0001), whereas the total time in the (21) (VO), and medial orbital cortex (MO), and ACC, have inner zone was increased (t = 2.86, p < 0.01; Fig.  3e). (21) been implicated in the etiology of OCD in previous This suggests the motion inhibition and lack of anxiety studies [11]. Consistently, all of these regions showed in 8-OH-DPAT-treated mice. Noteworthy, 8-OH-DPAT- increased c-fos expression in our RU24969-treated mice treated mice also presented memory impairments in the Chen  et al. Behav Brain Funct (2021) 17:4 Page 7 of 14 Fig. 4 Mapping c-fos expression in RU24969- and MCPP-treated mice. Representative immunofluorescence micrographs depicting c-fos expression in the OFC, PrL, IL, ACC, CPu, NAc, hypothalamus, BSTLD, and IPACL in RU24969-, MCPP- and saline-treated mice (Scale bars: 100 μm) the dysfunction in recognition memory, attention, and (LO + VO: t = 4.30, p < 0.001; MO: t = 2.78, p < 0.05; (16) (16) decision making [26]. In the CPu, c-fos was also highly Cg1: t = 3.42, p < 0.01; Cg2: t = 3.11, p < 0.01). The (16) (16) expressed in RU24969-treated mice, whereas hardly OFC and ACC are involved in many important neu- detectable in saline-treated mice (t = 6.92, p < 0.0001). ral functions, such as decision making, planning, inhi- (16) This was consistent with the findings that CPu’s func - bition of responses, and error monitoring. Indeed, in tions in learning, memory, action selection, and goal- OCD patients, almost all of these functions are impaired directed actions were impaired in OCD patients [5]. [5, 11]. The PrL and IL, components of the medial pre - The accumbens nucleus, shell (AcbSh) and accumbens frontal cortex (mPFC), showed increased c-fos expres- nucleus, core (AcbC) are two regions of the NAc and sion in the RU24969-treated group (PrL: t = 4.15, (16) participate in the regulation of reinforcement learning p < 0.001; IL: t = 2.30, p < 0.05), which may suggest (16) Chen et al. Behav Brain Funct (2021) 17:4 Page 8 of 14 Fig. 5 Quantification of the c-fos expression in RU24969- and MCPP-treated mice. a In RU24969-treated mice, c-fos expression levels were increased in the OFC, PrL, IL, CPu, NAc, hypothalamus, BSTLD, and IPACL. b There were no significant differences between MCPP- and saline-treated mice regarding the c-fos expression in the OFC, PrL, IL, CPu, NAc, hypothalamus, BSTLD, and IPACL. Data are expressed as the mean ± SEM. Unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Chen  et al. Behav Brain Funct (2021) 17:4 Page 9 of 14 (See figure on next page.) Fig. 6 The c-fos expression in the brain of 8-OH-DPAT-treated mice. a Representative immunofluorescence micrographs depicting the c-fos expression levels in the ACC, PrL, IL, OFC, AcbSh, and hypothalamus. Expression levels were increased in 8-OH-DPAT-treated mice compared with saline-treated control mice, whereas c-fos expression levels in the Acbc and CPu were not significantly different (Scale bars: 100 μm). b Quantification of the c-fos expression in the OFC, PrL, IL, ACC, CPu, NAc, and hypothalamus in 8-OH-DPAT and saline-treated mice. Data are expressed as the mean ± SEM. Unpaired t-test: ***p < 0.001, ****p < 0.0001 [27]. They also exhibited higher c-fos expression in OCD mouse models exhibited a distinct pattern RU24969-treated mice (AcbSh: t = 5.16, p < 0.0001; of the activated brain regions (16) AcbC: t = 3.94, p < 0.01). The BSTLD, a brain region To more intuitively compare the changes in the activa- (16) related to the regulation of anxiety and acute stress [28], tion of brain regions in the three OCD mouse models, showed increased c-fos expression in RU24969-treated we constructed a heatmap of c-fos expression (Fig.  7). mice (t = 5.73, p < 0.0001). Furthermore, we found The heatmap clearly illustrated the distinct pattern of (16) that c-fos was elevated in the IPACL in RU24969-treated the activated brain regions, in which increased c-fos mice (IPACL: t = 10.63, p < 0.0001; IL: t = 3.94, were detected in the OFC, ACC, PrL, IL, CPu, NAc, and (16) (16) p < 0.01). The IPACL receives projections from the hypothalamus in RU24969-treated mice, and the OFC, amygdala [29], but its functions related to OCD have ACC, PrL, IL, AcbSh, and hypothalamus in 8-OH-DPAT- not been clarified. Both control and RU24969-treated treated mice. In contrast, no significant increase was mice showed robust c-fos expression in the hypothala- detected in our interested brain regions in MCPP-treated mus, a brain region that responds to multiple stimula- mice. tions including stress and fear [30, 31], but the c-fos level was significantly higher in the RU24969-treated group Discussion (t = 6.93, p < 0.0001). Pharmacological animal models of OCD have been (16) widely used in previous studies [10, 11]. However, most of the published studies used rats instead of mice. In this report, three mouse models of OCD were established to Neuronal activity is not significantly increased provide more options for drug-induced OCD models and in MCPP‑treated mice offered more possibilities for researchers to explore the Next, we investigated MCPP-induced c-foc expression mechanisms of OCD. in mice brains. Surprisingly, after MCPP injection, we RU24969, 8-OH-DPAT, and MCPP all belong to sero- found that limited brain areas expressed c-fos with tonin receptor agonists. The choice of these reagents was a weak signal. When we compared it with the saline made based on previous clinical experience with OCD group, there were no statistically significant differences medications and hypothesized involvement of the sero- (Fig. 5b). No detectable changes were seen in the corti- tonin system in OCD. They all augment the sensitivity of cal brain areas that we examined. 5-HT receptor subtypes, which caused OCD [13]. The dysfunction of 5-HT receptor 1B (5-HT1B) in OCD has been suggested in previous studies. For example, in Increased c‑fos expression in the ACC, PrL, IL, OFC, AcbSh, pharmacological studies, the use of 5-HT1B agonists and hypothalamus in 8‑OH‑DPAT‑treated mice aggravated symptoms in patients with OCD, and muta- The brain regions expressing c-fos after 8-OH-DPAT tions of the serotonin transporter SCL6A4 were associ- administration were similar but not identical to those ated with OCD [14]. As serotonin 5-HT1A/1B receptor after RU24969 administration. In 8-OH-DPAT-treated agonist, RU24969 treatment in rats induced locomotor mice, higher c-fos expression were detected in the stereotypy, prepulse inhibition (PPI) deficits, and impair - ACC, PrL, IL, OFC, AcbSh, and hypothalamus (Cg1: ments in delayed alternation, all related to common signs t = 5.99, p < 0.0001; Cg2: t = 6.50, p < 0.0001; (16) (16) and symptoms in OCD patients [12]. Consistently, in the PrL: t = 5.02, p < 0.0001; IL: t = 5.25, p < 0.0001; (16) (16) present study, RU24969 administration induced repetitive LO + VO: t = 5.13, p < 0.0001; MO: t = 4.50, (16) (16) circling behavior in mice (Additional file  2: Fig. S2). More- p < 0.001; AcbSh: t = 6.32, p < 0.0001; hypothalamus : (16) over, the mice exhibited anxiety in the OFT. Impaired t = 7.15, p < 0.0001; Fig . 6a, b). These results indicate (16) memory is one of the cognitive dysfunctions of OCD [32]. that the excessive spay-induced grooming observed in In the NOR, RU24969-treated mice had a decreased time 8-OH-DPAT-treated mice might be mediated by the to explore novel objects indicative of memory impair- activation of these brain areas. ment, further confirming the validity of this model. Chen et al. Behav Brain Funct (2021) 17:4 Page 10 of 14 Chen  et al. Behav Brain Funct (2021) 17:4 Page 11 of 14 Fig. 7 Heatmap of the c-fos expression in the OCD models. The color range represents a logarithm of 2 for the ratio of each mouse to the each mouse of OCD models corresponding control average log . Red indicates high expression control average 8-OH-DPAT, a 5-HT receptor 1A (5-HT1A) ago- induced spray-induced grooming in mice. Interestingly, nist, could bind to its receptor with high selectivity self-grooming behaviors were not increased in 8-OH- [33]. 8-OH-DPAT-treated animals were commonly DPAT mice compared to control. This may be due to used as an OCD model. In the spontaneous alternation the necessity of a trigger to induce OCD-like behavior behavior test, the animals repeatedly selected the same in this model, similar to patients with excessive washing arm, similar to the OCD perseveration symptoms [34]. or cleaning behavior whose symptoms were triggered Importantly, treatment with selective serotonin reup- by contamination stimuli in clinical [4]. Thus, the OCD- take inhibitors could eliminate this repetitive behavior in like behaviors in 8-OH-DPAT-treated mice differed from the 8-OH-DPAT rat model [35]. However, other OCD- those in RU24969-treated mice. On the other side, simi- like behavioral tests have been rarely tested in the rat lar to RU24969-treated mice, 8-OH-DPAT-treated mice model. In the present study, 8-OH-DPAT administration showed recognition memory impairments in the NOR, Chen et al. Behav Brain Funct (2021) 17:4 Page 12 of 14 whereas anxiety was not detected in the OFT, which regions chosen in this study belong to the CSTC loop might be related to a lack of related stimuli in these (except for BSTLD and IPACL). Here, neuronal activ- experiments. ity was increased in the OFC, PrL, IL, ACC, CPu, NAc, Administration of MCPP, which binds to 5-HT1A and BSTLD, IPACL, and hypothalamus of RU24969-treated 5-HT2C receptors, could aggravate symptoms in patients mice. In 8-OH-DPAT-treated mice, activated brain with OCD [13, 36]. In a preclinical study, the administra- regions included the ACC, PrL, IL, OFC, and AcbSh tion of MCPP blocked the beneficial effects of fluoxetine (Additional file  3: Table S1). This was similar to previous for OCD treatment. Moreover, MCPP could induce the studies in mouse and rats [33, 43–45] that 5-HT1B recep- occurrence of repetitive stereotypes such as increasing tors are distributed in the striatum, cerebellum, and basal the number of buried marbles in the MBT [13], promot- ganglia, 5-HT1A receptors were distributed in the neo- ing directional persistence in spontaneous alternation cortex, olfactory areas, hippocampal formation, cortical behavior [11], and reinforcing self-grooming [16]. In the subplate, pallidum, hypothalamus, and mesencephalic current study, MCPP-induced OCD-like behaviors com- raphe nuclei, although the brain regions with increased prised excessive self-grooming behavior, whereas the neuronal activity in these two model mice did not com- induced-grooming test showed no significant differences pletely coincide with those expressing 5-HT1A or to control mice. These results contrasted with those in 5-HT1B receptors. One possible reason is that the OCD- 8-OH-DPAT mice. Self-grooming and spray-induced like behaviors were not induced by directly activating all grooming are different grooming forms [25]. Self-groom - relevant brain regions containing 5-HT1A or 5-HT1B ing is spontaneous; thus, excessive self-grooming in receptors, but acting within the same receptor-associated MCPP mice resembles trichotillomania (hair-pulling dis- brain region instead, therefore indirectly increasing the order) appearing spontaneously or under high pressure. neuronal activity of downstream brain regions and caus- Besides, the MCPP model presented no anxiety or mem- ing the occurrence of OCD-like behaviors. Notably, most ory impairment according to the OFT and NOR. Most of the activated brain regions were related to the CSTC patients with OCD chose to obey obsessive–compulsive loop elements corresponding to the postulated CSTC thoughts and perform obsessive–compulsive behaviors loop dysfunction in OCD. to alleviate the anxiety caused by obsessive–compulsive In mice and rats, the 5-HT receptor 2C (5-HT2C) is thoughts and impulses. Thus, the reduction of anxiety in mainly distributed in the choroid plexus and other areas, MCPP-treated mice may be due to the performance of including the nucleus accumbens, patches of the caudate- obsessive–compulsive behavior. putamen, the olfactory tubercle, claustrum, septum, cin- Together, the three newly established OCD mouse gulate cortex, amygdala, dentate gyrus, periaqueductal models exhibited distinct OCD-like behavioral traits gray, and entorhinal cortex [37, 44, 46]. Interestingly, after and presented different levels of anxiety and memory injection with the 5-HT2C agonist MCPP, we observed a dysfunction. weak c-fos expression in the mouse brains, with no differ - The expression of c-fos is one of the commonly used ence to the control group. Even after increasing the dose, indicators to measure the activity of neurons because we obtained the same results (data not shown). In this repetitive action potentials were often accompanied MCPP model, mice only showed excessive self-grooming by neuronal c-fos expression [37]]. Based on the drugs’ behavior, whereas all other behaviors appeared normal. pharmacological properties and the expression of the To a certain extent, this was consistent between behavio- receptors, we speculate that the increase in c-fos expres- ral and c-fos expression. We speculate that MCPP injec- sion was majorly raised from neurons. All three drugs tion might cause inhibition in some brain regions, which are 5-HT receptor agonists and can act on almost all could not be reflected by c-fos elevation. Indeed, previous neuronal cells. The distribution of their corresponding studies found that OCD patients have decreased activity receptors is wide. 5-HT 1A receptors were expressed in in certain brain regions [47, 48]. the pyramidal neurons of the cortex, hippocampus, and raphe nuclei and the cholinergic neurons in the septum Conclusion [38–40]. 5-HT 1B receptors were also expressed in the Here, we established three different OCD mouse mod - medium spiny neurons in the caudate putamen, probably els with distinct OCD-like behaviors, accompanying GABAergic [41, 42]. 5-HT 2C receptors were expressed dysfunctions in different brain regions. Compared to in most GABAergic cells. Astrocyte was also reported to controls, RU24969- and 8-OH-DPAT-treated mice express 5-HT receptors; thus, the astrocyte could also be demonstrated enhanced activation in brain regions a source for the c-fos signal. Previous studies proposed mostly belonging to the CSTC circuit, although some that the dysfunction of CSTC loops is related to differ - of the activated brain regions differed between the ent cognitive-affective processes in OCD. The brain Chen  et al. Behav Brain Funct (2021) 17:4 Page 13 of 14 Declarations two models. Neuronal activity was not significantly increased in MCPP-treated mice. These results implied Ethics approval and consent to participate that targeted and individualized treatment should be All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health, and performed in OCD patients with distinct symptoms, the animal experiment protocols have been approved by the Institutional and interventional treatment targeting the correspond- Animal Care and Use Committee of Sun Yat-Sen University. ing specific brain regions may improve the therapeutic Consent for publication effects. The datasets generated during or after the current study are available from the corresponding author on reasonable request. Abbreviations Competing interests 5-HT: 5-Hydroxytryptamine; 8-OH-DPAT: 8-Hydroxy-DPAT hydrobromide; AcbC: The authors declare no conflicts of interest. Accumbens nucleus, core; AcbSh: Accumbens nucleus, shell; ACC: Ant erior cingulate cortex; BSTLD: Bed nucleus of the stria terminalis, lateral division, Received: 20 January 2021 Accepted: 8 May 2021 intermediate part; Cg1/2: Cingulate cortex, area 1/2; CPu: Caudate-putamen; CSTC: Cortico–striato-thalamo-cortical; IL: Infralimbic cortex; IPACL: Interstitial nucleus of the posterior limb of the anterior commissure, lateral part; LO: Lateral orbital cortex; MBT: Marble-burying test; MCPP: 1-(3-Chlorophenyl) pip- erazine hydrochloride-99%; MO: Medial orbital cortex; mPFC: Medial prefrontal References cortex; NAc: Nucleus accumbens; NOR: Novel object recognition test; OCD: 1. Goodman WK, Grice DE, Lapidus KA, Coffey BJ. Obsessive–compulsive Obsessive–compulsive disorder; OFC: Orbitofrontal cortex; OFT: Open-field disorder. Psychiatr Clin. 2014;37(3):257–67. test; PBS: Phosphate-buffered saline; PPI: Prepulse inhibition; PrL: Prelimbic 2. Association AP. 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