Mglur3 Knockout Mice Show a Working Memory Defect and an Enhanced Response to MK-801 in the T- and Y-Maze Cognitive Tests

Behavioural Brain Research 266 (2014) 94–103

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Research report mGluR3 knockout mice show a working memory defect and an enhanced response to MK-801 in the T- and Y-maze cognitive tests

Mira Lainiola, Chiara Procaccini, Anni-Maija Linden ∗

Institute of Biomedicine, Pharmacology, Biomedicum Helsinki, P.O.B. 63, FIN-00014 University of Helsinki, Finland highlights

• Cognition of mGluR3-KO mice was analyzed in T- and Y-maze tests after MK-801. • T-maze spontaneous alternation was signiﬁcantly reduced in mGluR3-KO mice. • MK-801 enhanced locomotion more in mGluR3-KO than in WT mice in the Y-maze. • Both of these genotype effects depended on the side of forced or habituation arm. • Findings suggest side bias or left-right asymmetry in responses of mGluR3-KO mice. article info abstract

Article history: Polymorphisms in the metabotropic glutamate receptor 3 (mGluR3) encoding gene GRM3 have been Received 7 December 2013 linked to schizophrenia and cognitive performance in humans. Our aim was to analyze the role of Received in revised form 28 February 2014 mGluR3 in basal working memory and attentional processes, and also when these functions were Accepted 4 March 2014 distracted by the psychotomimetic N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine (MK- Available online 11 March 2014 801). mGluR3 knockout (KO) mice were used. Spontaneous alternation in a T-maze test was significantly reduced in mGluR3-KO mice compared to wildtype (WT) mice, particularly after a low dose of MK-801 Keywords: (0.03 mg/kg, i.p., 30 min). In a Y-maze novelty discrimination test, the locomotor stimulatory effect of mGlu3 receptor Mouse MK-801 (0.1 mg/kg) was enhanced in mGluR3-KO mice. Interestingly, mGluR3-KO mice showed the sig- Working memory nificantly reduced alternation in the spontaneous alternation T-maze test and the significantly enhanced Side bias sensitivity to MK-801 in the Y-maze test only when forced to enter the right arm first, not when the Left-right asymmetry forced arm was on the left. A side-biased response was also found in a rewarded alternation T-maze Dizocilpine test, where mGluR3-KO mice made significantly more incorrect visits to the left arm than the right arm after a 25-s delay. No genotype difference was found in the novelty discrimination in the Y-maze test, rewarded alternation with a 5-s delay, preference for left or right when free to enter either arm or in MK-801-induced circling. Our findings indicate cognitive disturbance and left-right asymmetry in certain behavioral responses of mGluR3-KO mice. This novel observation warrants further elucidation, and should also be considered in other studies of mGluR3 in brain functions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction psychiatric disorders. Pharmacological activation of mGluR2/3s shows antipsychotic and anxiolytic properties in preclinical animal Group II metabotropic glutamate receptors 2 and 3 (mGluR2 models [1,2] and also in clinical settings [3,4]. However, the antipsy- and mGluR3) have been implicated in brain functions related to chotic efficacy of mGluR2/3 agonists has not yet been confirmed in other clinical studies [5]. Both mGluR2 and mGluR3 modulate neuronal excitability by regulating glutamate (and GABA) release from presynaptic terminals, and postsynaptically by regulating activ- Abbreviations: ANOVA, analyses of variance; GRM3, mGluR3 gene; KO, knockout; ity of adenylate cyclase and various ion channels [6,7]. Although mGluR, metabotropic glutamate receptor; MK-801, dizocilpine; NMDA, N-methyl- mGluR3 is widely expressed in neurons and glia with high expres- d-aspartate; WT, wildtype. ∗ sion in the cerebral cortex, hippocampus, thalamus, striatum and Corresponding author at: Institute of Biomedicine, Pharmacology, University of substantia nigra [8–10] its role in neuronal and glial communica- Helsinki, P.O.B. 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. Tel.: +358 9 19125335; fax: +358 9 19125364. tion is poorly understood [11]. mGluR3s located in the astrocytes E-mail address: anni-maija.linden@helsinki.fi (A.-M. Linden). appear to be important in neuroprotection [12]. http://dx.doi.org/10.1016/j.bbr.2014.03.008 0166-4328/© 2014 Elsevier B.V. All rights reserved. M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103 95

Glutamate N-methyl-d-aspartate (NMDA) receptor antagonists The mice were maintained in same-sex groups of 3–8 (cage, [phencyclidine, ketamine and dizocilpine (MK-801)] have been 40 cm × 30 cm × 15 cm; Tecniplast, Buguggiate, Italy) with food used to model symptoms of schizophrenia in rodents because pellets (Harlan BV, Horst, Netherlands) and tap water available of their psychotomimetic effects [13–15]. Typical behavioral ad libitum at standard housing conditions (12 h light–dark cycle, responses to MK-801 include disruption of working memory and lights on at 6:00 A.M.; temperature, 20–23 ◦C; relative humidity, attention, as well as induction of locomotor activity and stereotypic 50–60%; aspen chip beddings). Males were housed alone (cage, behaviors. Sensitivity to the effects of MK-801 or phencyclidine 20 cm × 27 cm × 13 cm) if overt aggressive behavior was observed, is often analyzed in transgenic mice to elucidate functional roles and when on food restriction (only in the rewarded T-maze alterna- of targeted genes in schizophrenia related behaviors [16,17].In tion test). A wooden toy and a shelter were kept in each cage during preclinical models, antipsychotic drugs inhibit psychotomimetic housing. All behavioral tests and drug injections were performed effects of NMDA receptor antagonists. Also, activation of mGluR2/3s between 8 A.M. and noon at the light intensity of approximately has been shown to reverse phencyclidine induced hyperloco- 175 lux, unless otherwise stated. The mice were let to adapt to test- motion indicating the role of mGluR2/3s in reversing neuronal ing laboratory for at least 1 h before all experiments. The mice were processes elicited by NMDA receptor blockade [2]. This effect of the tested blindly for genotype. All animal procedures were approved mGluR2/3 agonists is mediated through activation of the mGluR2 by the Southern Finland Provincial Government. All efforts were as it is abolished in mGluR2-KO (knockout), but not in mGluR3- made to minimize the number of animals used. KO mice [18,19]. Although, these preclinical findings indicate the importance of mGluR2 in antipsychotic-like actions, several human genetic studies suggest association of the mGluR3 gene 2.2. Drugs (GRM3) with schizophrenia [11]. Schizophrenia-associated polymorphisms of GRM3 have been linked to changes in markers of (+)-MK-801 maleate (Asc-027, Ascent Scientific) was dissolved glutamate transmission, to poorer performance in cognitive tests in saline and injected (i.p.) at the volume of 10 ml/kg. Mice were and changes in mismatch negativity [20–23]. In addition, involve- injected with MK-801 or saline, returned to their home cages for ment of mGluR3 in schizophrenia-related functions is suggested by 30 min until transferred to the testing room and placed in the maze. studies where increased brain levels of N-acetylaspartylglutamate, an endogenous agonist for mGluR3 [24–27], have prevented psychotomimetic effects of phencyclidine and cognitive deficits 2.3. Spontaneous alternation T-maze test induced by MK-801 [28–30]. It is also important to note that we use here the same mGluR3-KO mouse line with Hsd:ICR (CD-1®) A spontaneous alternation T-maze test for analyzing spatial genetic background as utilized in a study showing the abolished working memory was performed as previously described [36,37]. antipsychotic effect of the elevated N-acetylaspartylglutamate lev- A T-maze was made of gray plastic and consisted of three arms els due to genetic disruption of mGluR3 [29]. Clearly, the role of (50 cm × 10 cm, walls 15 cm height). A door separated a 12-cm mGluR3 in basal and disrupted cognitive performance requires fur- start compartment, and two other doors separated 24-cm compart- ther investigation.mGluR2-KO and mGluR3-KO mouse lines have ments of the other arms. Vehicle or MK-801 (0.03–0.1 mg/kg, i.p.) been utilized to dissect which receptor mediates various behavioral was injected 30 min before the test and the mouse returned to its effects of mGluR2/3 agonists [12,18,19,29,31–33] and antagonists home cage until transferred to the testing room and placed in the [34]. However, very few basal phenotypic features have been start compartment for 5 s. Thereafter, the door was opened and the reported for either knockout line. Recently, it has been shown that latency for the mouse to visit the forced arm (another arm was mGluR2/3-double KO mice display deficits in spatial working mem- closed) and to return to the start arm was recorded (forced trial). ory and reference memory tests when motivated by food reward, A side of the forced arm was balanced by the treatment and geno- but not when motivated by avoidance of aversive situation [35]. type. Immediately after the forced trial, the test trial started and Specific roles of mGluR2 or mGluR3 were not addressed in that the mouse was allowed to visit either arm. When it entered one study. Our aim here was to analyze the role of mGluR3 in spatial arm, the opposite arm was closed, and when the mouse returned working memory and attentional processes in T- and Y-maze tests, to the start arm, both doors were again opened for the next trial. respectively, in basal conditions and after cognitive disturbance This was continued for 15 min. A video camera was located above with the NMDA receptor antagonist MK-801. the T-maze and the experimenter observed the mouse through a video monitor and remotely operated the doors from an adjacent room. The maze was cleaned after each mouse with water-wet paper towel and dried (as all other mazes and arenas described 2. Materials and methods below). A percentage of spontaneous alternation was calculated by dividing the number of alternated arm visits (visits to the other arm 2.1. mGluR3-KO mouse line than the previously visited) by the total number of arm visits × 100. A percentage of forced arm preference was calculated by dividing mGluR3-KO mouse line was generated by homologous recom- the number of visits to the forced arm by the total number of arm bination as described earlier [33]. For this study heterozygous visits × 100 (Table 1). mGluR3-KO mice obtained from Taconic Farms Inc. (Germantown, Experimentally naïve, age-matched WT and mGluR3-KO males NY, USA; kindly provided by Lilly Neuroscience, Eli Lilly and Com- and females were obtained from heterozygous breeding (Fig. 1) pany, Indianapolis, IN, USA) were further backcrossed with Hsd:ICR and for subsequent experiments from homozygous breeding (CD-1®; Harlan) mice at Helsinki University. Heterozygous breed- (Figs. 2 and 3). First two T-maze experiments were performed all ing provided mGluR3-KO and littermate WT mice after at least five in the same room (Room 1, Figs. 1 and 2) so that the position of the backcrosses for the first behavioral test. Homozygous breeding was T-maze was to the right from the door. As we realized that the side used to produce a larger amount of age-matched mice for behav- of the forced arm may affect the results, the next T-maze experi- ioral tests. Homozygous WT and KO breeding pairs were littermates ment was performed in another room (Room 2, Fig. 3) so that the of heterozygous parents after at least eight backcrosses to Hsd:ICR position of the T-maze was left from the door mirroring the posi- (CD-1®) strain. Age-matched WT and mGluR3-KO mice were 10–14 tion of the T-maze in Room 1. The number of animals used in each weeks of age unless otherwise stated. test is indicated in Table 1. 96 M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103

Fig. 1. Effect of MK-801 (0.03 mg/kg) on spontaneous alternation of mGluR3-KO mice in the T-maze test. % Spontaneous alternation during a 15-min testing period. (A) Combined data regardless of the side of the forced arm. Pooled male and female data are shown as means ± SEM (n is indicated above bars). (B and C) Data have been divided based on the forced arm side because a signiﬁcant interaction between genotype and forced side. G, p < 0.05, genotype effect; GF, p < 0.05, genotype-forced side interaction (ANOVA); **p < 0.01, *p < 0.05 (Bonferroni post test).

Fig. 2. Effect of MK-801 (0.06–0.1 mg/kg) on spontaneous alternation of mGluR3-KO mice in the T-maze test. % Spontaneous alternation during a 15-min testing period. (A) Combined data regardless of the side of the forced arm. Pooled male and female data are shown as means ± SEM (n is indicated above bars). (B and C) Data have been divided based on the forced arm side because signiﬁcant interactions of forced arm side were found in ANOVA. G, p < 0.05, genotype effect; D, p < 0.05, drug effect; FS, p < 0.05, forced side-sex interaction; GFS, p < 0.05, genotype-forced side-sex interactions (ANOVA). Due to sex-related ﬁndings male and female data are presented separately in the supplemental Fig. S2.

2.4. Continuous Y-maze novelty discrimination test walls 25 cm height) connected to a triangular central platform. Each arm contained an opaque removable door 1 cm from the central Attention and novelty preference was analyzed in a continu- platform. The maze was surrounded by outer walls with distinct ous Y-maze test as previously described [38,39]. A Y-maze was visual cues for the test arms (white horizontal or vertical lines in made of clear plastic and consisted of three arms (50 cm × 9cm, black background) and a black wall for the start arm. The outer

Table 1 The number of mice, dose of MK-801 and history of mice used in different tests.

Number of males and females in saline and MK-801 treatment groups MK-801 (mg/kg) Conditions

Spontaneous alternation T-maze tests ♂ Sal: WT 6, KO 6; MK: WT 6, KO 7; ♀ Sal: WT 7, KO 6; MK: WT 8, KO 6 0.03 Naïve ♂ Sal: WT 8, KO 6; MK: WT 7–8, KO 5–7; ♀ Sal: WT 10, KO 6; MK: WT 8–10, KO 6 0.06–0.1 Naïve ♂ Sal: WT 10, KO 10; MK: WT 10, KO 11; ♀ Sal: WT 9, KO 8; MK: WT 9, KO 9 0.1 Naïve

Continuous Y-maze novelty discrimination test ♂ Sal: WT 19, KO 16; MK: WT 20, KO 15; ♀ Sal: WT 15, KO 13; MK: WT 16, KO 14 0.1 Naïve

Free choice arm entries in T- and Y-maze tests ♂ Sal: WT 11, KO 11; ♀ Sal: WT 9, KO 9 Naïve ♂ Sal: WT 6, KO 6; MK: WT 6, KO 7; ♀ Sal: WT 7, KO 6; MK: WT 7, KO 7 0.1 Naïve

Rewarded and delayed T-maze alternation test ♂ WT 14, KO 14 – Retested

Circling behavior tests ♂ Sal: WT 4, KO 3; MK: WT 4, KO 3; ♀ Sal: WT 6, KO 4; MK: WT 7, KO 6 0.15 Retested ♂ Sal: WT 4, KO 4; MK: WT 9, KO 11; ♀ Sal: WT 6, KO 6; MK: WT 14, KO 13 0.3 Retested M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103 97

(0.1 mg/kg, 30 min) injections of experimentally naïve WT and mGluR3-KO male and female mice.

2.6. Rewarded T-maze alternation training and 25-s delayed alternation test

Spatial working memory of mGluR3-KO mice were tested in a rewarded alternation training followed by a delayed alternation test using the same T-maze as described above. WT and mGluR3- KO male mice (16 ± 0.4 weeks old) used here were tested 4 weeks earlier in a T-maze test once. Female mice were not used in this test. The test was performed essentially as described earlier [35,40]. After training delay time in the start arm was increased from 5 to 25 s to set higher demands on working memory. The mice were food restricted aiming to 90% of their initial weight and single housed because in preliminary tests food restriction increased fighting. Food restriction and habituation (20 min/day/mouse) began 3 days before training and included handling, familiarization to the T- maze and finding and eating food rewards (small pieces of cereal; Honey Cheerios, Nestle) from small dishes located on both arm ends and visually hidden by a low wall. An additional 5-min habituation was done on the first training day. Training and testing were per- Fig. 3. Effect of MK-801 (0.1 mg/kg) on spontaneous alternation of mGluR3-KO mice formed between noon and 6 P.M., after which the mice received forced to enter right arm first on the T-maze. % Spontaneous alternation during a 15- their daily food pellets. A training phase contained four blocks of min testing period. The forced arm was to the right for all animals. The T-maze was 10 trials (five trials/day with a 10-min interval) and was followed by located in Room 2, which was different from Figs. 1 and 2. Pooled male and female data are shown as means ± SEM (n is indicated above bars). G, p < 0.05, genotype a test of delayed alternation containing 10 trials (five trials/day). In effect; D, p < 0.05, drug effect (ANOVA). each trial, in a sample run, the mouse was first forced to enter either right or left goal arm where it consumed a food reward in approximately 5 s. Then it was returned to the closed start arm, and the walls with visual cues were changeable between the arms and corridor between the goal arms was quickly wiped to spread odor were alternated in a balanced way between left and right arms trail. After a 5-s or 25-s delay, the start arm door was opened for a as well as between different treatment groups. The test consisted choice run, and the mouse was free to enter either goal arms, but of a 5-min habituation phase and 2-min test phase. A side of the only the arm not visited previously was baited. Both arms were habituation arm was alternated in a balanced way. The habituation scented with powdered cereals. In block 3, the cereal was dropped phase was started by placing the mouse in the distal end of the to the dish after the correct arm entry to control that the mice do not start arm and allowing it to enter the habituation arm after which smell the reward. A side of the sample arm was balanced, pseudo- the start arm was closed. After 5 min in the habituation arm, the randomized and included the same amount of left and right runs in mouse was allowed to explore both familiar and unfamiliar arms the same order for each mouse. A video camera was located above (and the center area) for 2 min. The maze was not cleaned between the T-maze to help the experimenter observe the behavior through habituation and test phases. A CCD video camera was located above a video monitor and remotely operate the doors. The T-maze was the Y-maze for automatic behavioral analysis utilizing Ethovision placed in Room 1 (mirroring the position in spontaneous alterna- Color-Pro 3.0 software (Noldus Information Technology, Wagenin- tion tests), its position was not changed between trials, and fixed gen, Netherlands). A discrimination index (DI) was calculated using extra-maze visual cues were placed on the walls. The mice were − kept in the testing room in their individual home cages during inter an equation: DI = (tu tf)/(tu + tf) where tu was time spent in the trial intervals. unfamiliar arm and tf was time spent in the familiar arm. The Y-maze experiment was performed with two different cohorts of experimentally naïve, age-matched mGluR3-KO and WT 2.7. Circling behavior after MK-801 administration male and female mice, and in two different rooms so that the position of the Y-maze was right from the door in Room 1 (Cohort 1) Mice were injected with saline or MK-801 and returned to their and left from the door in Room 2 (Cohort 2). home cages for 30 min after which they were placed in visually iso- lated cylindrical buckets (gray plastic, diameter 18 cm, walls 24 cm height) for 20 min. A video camera was located above the buckets 2.5. Analysis of free choice arm entries in the T- and Y-mazes to analyze their locomotor activity using Ethovision Color-Pro 3.0 software and to record behavior for later observational analysis. A We next analyzed which arm WT and mGluR3-KO mice would 5-min observational analysis from recorded videos was performed prefer if free to enter either one when exploring the maze for using data acquisition software (Ethograph software 2.06, RITEC, St. the first time. A position of the T-maze in Room 3 was alternated Petersburg, Russia) and started 2 min after the mouse was placed between two mirroring positions (180◦ rotation) in a balanced in the bucket. A lower dose of MK-801 (0.15 mg/kg) did not induce manner. Preference to enter either right or left arm was analyzed of clear circling, and therefore, we analyzed duration of locomotion experimentally naïve WT and mGluR3-KO male and female mice. which was possible to classify as clockwise (right) or counterclock- Saline was injected 30 min before placing the mouse in the start wise (left). A higher dose of MK-801 (0.3 mg/kg) induced clear arm. circling, and it was possible to analyze the number and direction of A position of the Y-maze in Room 2 was alternated between full circles. Two different cohorts of age-matched WT controls and four positions (90◦ rotations) in a balanced manner. The outer mGluR3-KO mice were used (17 ± 0.8 weeks old males and females walls of the Y-maze were in this test similar (black). Preference to for 0.15 mg/kg dose; and 17 ± 3.6 weeks old males and 13 ± 0.4 enter either right or left arm was analyzed after saline or MK-801 weeks old females for 0.3 mg/kg dose). The mice were previously 98 M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103

Table 2 Additional behavioral parameters obtained in the spontaneous alternation T-maze tests after MK-801 administration (0.03–0.1 mg/kg, i.p., 30 min).

WT KO WT KO WT KO Statistics

Spontaneous alternation T-maze test (Fig. 1) Sal Sal MK 0.03 MK 0.03 ANOVA Forced arm %, combined 47 ± 2.4 53 ± 1.7 50 ± 2.6 48 ± 3.6 / Trials, combined 17 ± 1.1 17 ± 1.3 21 ± 1.1 19 ± 1.5 D, F, DS Forced right 19 ± 1.4 17 ± 2.1 22 ± 1.7 18 ± 2.4 / Forced left 14 ± 0.5 17 ± 1.7 19 ± 1.1 19 ± 1.7 D Forced trial duration (s), combined 35 ± 2.8 38 ± 4.3 38 ± 2.4 34 ± 2.2 /

Spontaneous alternation T-maze test (Fig. 2) Sal Sal MK 0.06 MK 0.06 MK 0.1 MK 0.1 Forced arm %, combined 47 ± 2.1 53 ± 4.2 54 ± 2.8 47 ± 4.2 49 ± 3.0 61 ± 4.0* GD, GFS Forced right 49 ± 4.0 50 ± 6.0 51 ± 2.9 43 ± 4.4 49 ± 5.3 73 ± 2.6* D, GD Forced left 45 ± 2.2 58 ± 5.6 57 ± 4.7 54 ± 8.2 50 ± 3.6 57 ± 4.6 GS Trials, combined 16 ± 0.8 15 ± 1.2 22 ± 1.3 21 ± 2.0 25 ± 1.9 26 ± 2.4 D, DFS Forced right 18 ± 0.9 15 ± 2.0 20 ± 1.5 23 ± 2.7 25 ± 3.0 23 ± 6.4 D, DS Forced left 15 ± 1.2 14 ± 1.0 24 ± 2.2 19 ± 3.1 26 ± 2.5 27 ± 2.6 D

Spontaneous alternation T-maze test; all mice forced right (Fig. 3) Sal Sal MK 0.1 MK 0.1 Forced arm % 51 ± 1.5 51 ± 1.6 51 ± 2.4 56 ± 1.8 / Trials 19 ± 0.9 20 ± 1.1 34 ± 2.1 31 ± 2.0 D, S

First values represent means ± SEM of combined values including data of both forced sides. The number of animals in each treatment group is indicated in the corresponding figures. If four-way ANOVA indicated significant effect or interaction for forced side, separate data for both sides are shown. If sex effect or interaction emerged in ANOVA, separate values for males and females are shown in the supplemental figures (Fig. S1–S3). ANOVA results (p < 0.05) are indicated by G, genotype effect; D, drug effect; F, forced side effect; combinations of letters indicate interactions. Slash line (/) indicates that none of the factors were found significant. Significant effects in Bonferroni post hoc tests: *p < 0.05, for difference between genotypes after the same treatment.

tested in other behavioral tests with at least 1 week washout period arm (genotype-forced side interaction, F1,44 = 4.94, p < 0.05; no sex before analyzing circling behavior. effect or interaction, males and females pooled, Fig. 1A) post hoc testing showing that mGluR3-KO mice alternated the arms signif- 2.8. Statistical analyses icantly less than WT mice after MK-801 treatment. To analyze the data further, we divided the data based on the side of the forced arm, Spontaneous alternation T-maze and novelty discrimination Y- and found that spontaneous alternation was significantly reduced maze data were analyzed by analyses of variance (ANOVA) to test only in mGluR3-KO mice which visited the right arm in the forced effects of genotype, drug, sex and forced side (SPSS 15.0 for Win- trial (genotype effect, F1,23 = 7.35, p < 0.05, Fig. 1B), not in those mice dows, SPSS Inc., Chicago, IL, USA). If sex-related findings emerged, starting the test by turning first to the left arm in the forced trial four-way ANOVA findings were reported, if not, males and females (Fig. 1C). were pooled for three-way ANOVA. When side-related findings Forced arm preference was not altered in KO mice or affected by emerged, forced side specific treatment groups were separately MK-801 treatment, forced side or sex (Table 2). MK-801 showed a tested in three-way ANOVA (genotype, drug, sex), if sex-related modest stimulatory effect by increasing the total number of trials findings emerged three-way ANOVA findings were reported, if in both genotypes (drug effect, F1,36 = 6.45, p < 0.05; Table 2). Also not males and females were pooled and findings from two-way the side of the forced arm influenced the overall number of trials ANOVA reported. ANOVA was followed by Bonferroni’s post hoc performed during the test (side effect, F1,36 = 5.28, p < 0.05) and the test (GraphPad Prism 5.00, San Diego, CA, USA). Rewarded alterna- increase in trials after MK-801 was observed only in the mice which tion T-maze training (% correct in each block) and genotype effects started the test by visiting the left arm first (drug effect, F1,21 = 5.92, were analyzed using repeated measures one-way ANOVA followed p < 0.05). Moreover, sex influenced this effect of MK-801 (drug–sex by Dunnett’s post hoc test, when errors by side data were analyzed a interaction, F1,36 = 5.34, p < 0.05; Table 2; Supplemental Fig. S1 for side was added as a factor and repeated measures two-way ANOVA separate male and female data). was used. Delayed alternation T-maze data (% correct) was analyzed using Student’s t-test, and errors by side data using two-way 3.2. Effect of MK-801 (0.06–0.1 mg/kg) on mGluR3-KO behavior ANOVA (genotype, erroneous side). For analysis of free choice arm in the spontaneous alternation T-maze test entries in the T- and Y-mazes by comparing percentages Chi-Square cross tabulation was used (SPSS). Circling data were analyzed by Next we repeated the T-maze alternation test with higher doses three-way ANOVA (genotype, drug, sex), if no sex-related findings of MK-801 (0.06 and 0.1 mg/kg). Alternation percentage was again emerged males and females were pooled and two-way ANOVA reduced in mGluR3-KO mice (genotype effect, F1,63 = 4.76, p < 0.05) findings reported. The statistical significance was set at p < 0.05. and by MK-801 treatment (drug effect, F2,63 = 3.67, p < 0.05), and Data are expressed as means ± standard errors of the mean (SEM). also the side of the forced arm influenced the results (genotype- side-sex interaction, F1,63 = 5.24, p < 0.05; side-sex interaction, 3. Results F1,63 = 4.06, p < 0.05; Fig. 2A). Male and female data were pooled in Fig. 2, but shown separately in the supplemental Fig. S2. However, 3.1. Effect of MK-801 (0.03 mg/kg) on mGluR3-KO behavior in the we did not analyze further sex-related effects because the number spontaneous alternation T-maze test of animals was too low in treatment groups if data would have been divided also by sex in addition to the side of the forced arm. Because Spatial working memory of mGluR3-KO mice was analyzed the side of the forced arm affected the results, the forced arm side- in the spontaneous alternation T-maze test 30 min after saline specific data sets were analyzed separately (Fig. 2B and C). No sex and MK-801 (0.03 mg/kg) injections. Spontaneous alternation was effect or interaction was observed in the side-specific data sets. reduced in mGluR3-KO mice depending on the side of the forced Spontaneous alternation was significantly reduced in mGluR3-KO M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103 99 mice forced to enter the right arm first (genotype effect, F1,35 = 6.22, mice approximately 5 months apart, and in two different rooms. p < 0.05, Fig. 2B), but not when the left arm was the forced arm When analyzing the effect of the cohort/room, it affected signifi- (Fig. 2C). The main effect of MK-801 treatment on the spontaneous cantly total movements during the habituation phase (F1,105 = 6.50, alternation was not significant in the side-specific data sets (Fig. 2B p < 0.05), latency to enter the habituation arm (F1,105 = 19.91, and C). p < 0.001) and total movements in the test phase (F1,108 = 6.70, Forced arm preference was increased by the highest dose of p < 0.05), but did not interact with other factors. Therefore, to MK-801 (0.1 mg/kg) in mGluR3-KO mice but not in WT mice increase the number of animals in each treatment group, we com- when all data were analyzed together (genotype-drug interaction, bined these two cohorts in subsequent statistical tests and figures F2,63 = 4.08, p < 0.05; Table 2) with genotype-side-sex interac- (Fig. 4). The separate data of two cohorts are shown in the supple- tion (F1,63 = 5.09, p < 0.05). Within the mice which were forced mental Fig. S4. Main statistical findings described below for the to enter the right arm first, mGluR3-KO mice showed higher combined cohorts (Fig. 4) were observed in both cohorts when preference for the forced arm compared to WT mice after treat- tested separately (Fig. S4). ment with 0.1 mg/kg MK-801 (drug effect, F2,29 = 4.07, p < 0.05; MK-801 treatment significantly reduced discrimination genotype–drug interaction, F2,29 = 4.03, p < 0.05; no sex effect or between novel and familiar arms (drug effect, F1,112 = 13.01, interaction, Table 2). Within the mice forced to enter the left p < 0.001; Fig. 4A). Discrimination index was not altered in mGluR3- arm first, the preference to the forced arm was influenced by KO mice compared to WT mice (no significant genotype, sex or side genotype–sex interaction (F1,34 = 4.36, p < 0.05, Table 2 and Fig. S2) effects, Fig. 4B and C). However, interestingly, MK-801 treatment without any significant main effects of genotype, drug or sex. How- increased significantly more locomotor activity in mGluR3-KO ever, the low number of KO mice especially in the 0.1-mg/kg group mice compared to WT mice during both habituation and test forced to turn right, due to a mistake in balancing the groups, warr- phases (Fig. 4D and G). Statistical analysis indicated that the side of anted replication of these findings (see below). the habituation arm influenced locomotion during the habituation MK-801 treatment significantly increased the number of trials phase (drug effect, F1,109 = 138.12, p < 0.001; genotype effect, performed during a 15-min testing period (drug effect, F2,63 = 17.15, F1,109 = 10.99, p < 0.01; drug-side interaction, F1,109 = 5.27, p < 0.05; p < 0.001, Table 2) with drug-side–sex interaction (F2,63 = 6.71, genotype-side interaction, F1,109 = 7.54, p < 0.01; genotype–drug- p < 0.01) when all data were analyzed together. This drug effect was side–sex interaction, F1,109 = 4.02, p < 0.05; Fig. 4D). Therefore, observed also in the forced arm-specific data sets (right forced arm, data obtained from the mice habituated in the right or left arm F2,29 = 5.21, p < 0.05, Fig. 2B; left forced arm, F2,34 = 14.78, p < 0.001, were analyzed separately. This revealed that MK-801 increased Table 2). Drug–sex interaction (F2,29 = 3.85, p < 0.05) emerged in the locomotion more in mGluR3-KO mice only when the mice were right forced arm data set without any significant main effects of habituated in the right arm (drug effect, F1,54 = 94.23, p < 0.001; genotype or sex and could be due to females showing stronger genotype, F1,54 = 15.18, p < 0.001; no sex related findings; Fig. 4E). response to the highest MK-801 dose regardless of the genotype When habituated in the left arm, MK-801 increased locomotion (Fig. S2). Latency to enter the forced arm and return to the start in both genotypes in the same way (drug effect, F1,63 = 58.93, arm was not affected by genotype, MK-801 treatment, side of the p < 0.001; Fig. 4F). forced arm or sex (data not shown). The same effect of MK-801 was also clear during the testing As the T-maze tests described above in Figs. 1 and 2 were per- phase when the mice were allowed to explore both arms; enhanced formed in the same room (Room 1) before the influence of the side sensitivity of KO mice to MK-801 depended on the side of the of the forced arm was noticed, and because the number of animals habituation arm (Fig. 4G–I). Total locomotor activity during the test in the 0.1-mg/kg group of mGluR3-KO mice forced to turn right was phase was significantly affected by drug (F1, 112 = 94.24, p < 0.001), low, we next aimed to replicate the findings and tested whether the genotype (F1,112 = 6.16, p < 0.05), habituation side (F1,112 = 5.41, forced right arm turn would affect mGluR3-KO behavior more than p < 0.05), also drug–genotype interaction (F1,112 = 4.66, p < 0.05) and WT behavior after 0.1 mg/kg MK-801 also in another room (Room genotype-side interaction (F1,112 = 7.61, p < 0.01) emerged. Thus, 2). In this test utilizing test naïve mice, MK-801 reduced signif- MK-801 increased test phase locomotion more in mGluR3-KO icantly spontaneous alternation (drug effect, F1,68 = 5.17, p < 0.05, mice only when the mice were habituated in the right arm Fig. 3), and mGluR3-KO mice alternated overall less than WT mice (drug effect, F1,56 = 62.98, p < 0.001; genotype effect, F1,56 = 13.54, (genotype effect, F1,68 = 5.05, p < 0.05, no significant sex effect or p < 0.001; drug–genotype interaction, F1,56 = 5.58, p < 0.05; Fig. 4H). interactions). However, in this cohort of mice, MK-801 did not When habituated in the left arm, the effect of MK-801 did not differ significantly affect forced arm preference in mGluR3-KO mice between genotypes (drug effect, F1,64 = 37.62, p < 0.001; Fig. 4I). as observed earlier (Table 2). As before, MK-801 increased sig- To assess details of these arm side-specific findings, other nificantly the number of trials in both genotypes (drug effect, behavioral parameters obtained from the Y-maze study were also F1,68 = 60.67, p < 0.001, Table 2) without any difference between analyzed (Fig. S5). Shortly, latency to leave the start arm and enter genotypes, but with a significant sex effect (F1,68 = 5.41, p < 0.05) the habituation arm for the first time was shorter in mGluR3-KO probably due to males showing higher activity than females after mice irrespective of the drug treatment, and no side effect was MK-801 treatment (Fig. S3). observed (genotype effect, F1,117 = 4.43, p < 0.05; Fig. S5A–C). We also analyzed locomotion separately in the unfamiliar and famil- 3.3. Effect of MK-801 (0.1 mg/kg) on mGluR3-KO behavior in the iar arms during the test phase to find out in which part of the continuous novelty discrimination Y-maze test maze KO mice moved more than WT mice. The difference between MK-801-treated KO and WT mice habituated in the right arm Because mGluR3-KO mice showed an increased preference for was significant in the unfamiliar arm (genotype effect, F1,56 = 5.95, the forced arm (i.e. the arm visited first) in the T-maze test after p < 0.05; drug effect, F1,56 = 22.66, p < 0.001, genotype–drug interac- 0.1 mg/kg dose of MK-801 in one study but not another (Table 2), tion, F1,56 = 5.82, p < 0.05; Fig. S5D), but not in the familiar arm (drug we analyzed their preference for familiar versus novel arm in an effect, F1,56 = 32.64, p < 0.001). Y-maze novelty discrimination test after MK-801 administration (0.1 mg/kg, 30 min). Also, the effect of the side of the first arm 3.4. Side of the first entered arm in free choice T- and Y-maze tests (habituation arm) the mice were forced to enter, was assessed because of the side-specific results in the earlier T-maze tests. Because the side of the forced arm in the T-maze or habitua- The Y-maze experiment was performed using two cohorts of tion arm in the Y-maze affected mGluR3-KO responses, we next 100 M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103

Table 3 Analysis of preferred arm of mGluR3-KO mice in the T- and Y-mazes when free to enter either.

WT Saline WT MK-801 KO saline KO MK-801 Statistics

Percentage of mice entering the right arm ﬁrst, % (n) Chi-Square T-maze 60 (20) – 55 (20) – / Y-maze 50 (13) 62 (13) 67 (12) 50 (14) /

Injections (i.p.) were 30 min before analysis. All mice were naïve to the mazes. In the T-maze test, mice received only saline injections. In the Y-maze test, mice received MK-801 (0.1 mg/kg) or saline. The number in parenthesis indicates the number of animals (males and females pooled). To compare percentages Chi-Square cross tabulation was used, saline- and MK-801-treated mice were tested separately. Slash line indicates non-signiﬁcant results.

from that of WT mice after saline injections in the T-maze or after saline or MK-801 (0.1 mg/kg) injections in the Y-maze (Table 3).

3.5. Rewarded T-maze alternation and delayed alternation behavior in mGluR3-KO mice

In addition to the spontaneous alternation test, spatial working memory of mGluR3-KO male mice was analyzed in a rewarded alternation test. In the training phase, both WT and mGluR3-KO mice successfully alternated arm entries and slightly increased the percentages of correct responses on the 3rd block (block effect, F3,78 = 4.85, p < 0.01; no genotype effect; Fig. 5A). Pooled data by genotype indicated that the performance was significantly better on the 3rd block compared to the 1st block (p < 0.05, Dunnett’s post hoc test). Genotype did not affect the number of errors made to right or left during training (block effect, F3,150 = 4.23, p < 0.01; block-side interaction, F3,78 = 6.13, p < 0.01, repeated measures two- way ANOVA; Fig. 5B). Body weights at the beginning of the food restriction did not significantly differ between WT (37.9 ± 0.7 g) and KO mice (39.8 ± 0.7 g). Neither did weight loss differ between genotypes during training or delayed alternation test (repeated measures two-way ANOVA; % weight of original weight being for example after the training, WT, 87.1 ± 0.5%, KO, 86.0 ± 0.3%, mean ± SEM; and after delayed alternation test, WT, 94.7 ± 0.6%, KO, 93.9 ± 0.4%). More demanding working memory was assessed by prolonging the delay between sample and test runs to 25 s. In this test, mGluR3-KO mice tended to alternate less than WT mice although this difference did not reach statistical significance (p = 0.082, Stu- dent’s t-test, Fig. 5C). When the numbers of left and right errors were analyzed in two-way ANOVA, the effect of genotype did not reach significance (F1,52 = 3.68, p = 0.06), however, the effect of side Fig. 4. Effect of MK-801 (0.1 mg/kg) on behavior of mGluR3-KO mice in the Y-maze was significant (F1,52 = 5.75, p < 0.05) and post hoc test indicated discrimination test depends on the side of the habituation phase. (A) Discrimination that mGluR3-KO mice performed significantly more incorrect left index of all animals. (B and C) Discrimination index of the mice habituated either in arm visits than incorrect right arm visits (Fig. 5D). the right or left arm. (D) Total locomotor activity in the habituation arm and center area during the habituation phase of all animals. (E and F) Total locomotor activity during the habituation of the mice habituated either in the right or left arm. (G) 3.6. Circling behavior in a round arena after MK-801 Total locomotor activity during the test phase in the unfamiliar arm, familiar arm administration and center. (H) and (I) Total locomotor activity during the test phase of the mice habituated either in the right or left arm. Pooled male and female data are shown A lower dose of MK-801 (0.15 mg/kg) increased locomotion as means ± SEM (n is indicated above bars; numbers differ in the habituation phase due to detection problems leading to missing automatically detected total move- which was possible to classify as clockwise (right) or counter- ments data of three animals). G, p < 0.05, genotype effect; D, p < 0.05, drug effect; clockwise (left) directed in a similar way in both genotypes (drug F, p < 0.05, habituation (forced) side effect; S, sex effect; combinations of letters effect, F1,33 = 30.7, p < 0.001; male and female data pooled, the Sup- indicate interactions, p < 0.05 (ANOVA); ***p < 0.001; **p < 0.01; *p < 0.05 (Bonferroni plemental Fig. S6A). Time spent turning right or left wise was post test). not significantly different between treatment groups (Fig. S6B). Increase in total locomotor activity analyzed automatically by Ethovision was not different between genotypes after MK-801 analyzed which arm they would choose if free to enter either one. treatment (drug effect, F1,29 = 47.3, p < 0.001; drug–sex interaction, New cohorts of naïve mice not exposed beforehand to any maze F1,29 = 4.22, p < 0.05; data not shown). or treatment were used. No side preference was found in the T- A higher dose of MK-801 (0.3 mg/kg) increased clear circling or Y-maze free choice tests (Table 3). The percentage of mGluR3- behavior in both genotypes (drug effect, F1,63 = 65.3, p < 0.001; Fig. KO mice entering first the right arm was not significantly different S6C). The number of right or left wise circles was not significantly M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103 101

that KO mice alternated less regardless of treatment and that the enhanced sensitivity to MK-801 of KO mice was not observed when higher doses were used. When mGluR3-KO behavior was analyzed in the Y-maze spatial novelty discrimination test after saline and MK-801 (0.1 mg/kg), no genotype difference was observed in the novelty discrimination index suggesting that neuronal functions involved in novelty detection and preference are not affected by the deletion of mGluR3. However, MK-801 increased locomotion significantly more in mGluR3-KO mice compared to WT mice in the Y-maze test. It was intriguing to find out that both the reduced alternation of mGluR3-KO mice in the T-maze tests, as well as the enhanced sensitivity of mGluR3-KO mice to MK-801 in the Y-maze test were dependent on the side of the forced or habituation arm. When mGluR3-KO mice were forced to enter the right arm first, the effect of genotype and the enhanced sensitivity to MK-801 were more pronounced. In contrast, when the mice started the tests by entering the left arm first, the behaviors of mGluR3-KO mice did not differ from that of WT mice. The major problem in interpreting of our results was a low number of animals per treatment group when they were divided based on the forced/habituation arm. This decreased the power of the statistical analyses. Also, it was not possible to analyze further sex-related findings because the number of animals would have become too low in treatment groups if the data would have been divided also by sex. To address these problems, we aimed to replicate the findings shown in Fig. 2, utilizing a new cohort of animals, the highest dose of MK-801 and performing the experiment in a different room. In this experiment (Fig. 3) we could repeat the findings (Figs. 1 and 2) that mGluR3-KO mice show reduced alternation compared to WT mice when forced to turn to the right arm. How- ever, the increased forced arm preference of mGluR3-KO mice after MK-801 (0.1 mg/kg) treatment observed in one experiment was not replicated in another test (Table 2). Although, the side biased behaviors in mGluR3-KO mice were observed in several different tests, it is clear, that all findings warrant replication with more animals in future studies. To mimic cognitive disturbances in schizophrenia acute administration of phencyclidine or MK-801 has been utilized in various hippocampus and prefrontal cortex-dependent working memory tests [14,15]. In our spontaneous alternation T-maze experiments, MK-801 reduced working memory performance as expected from Fig. 5. T-maze rewarded alternation and delayed alternation behavior of mGluR3- previous studies [41–43]. In our Y-maze experiments, MK-801 KO mice. (A) % Correct choices and (B) the number of erroneous visits to a left or right reduced discrimination of novelty at a closely similar magnitude arm during the training phase (males: WT, n = 14, KO, n = 14). (C) % Correct choices what has earlier been reported after chronic phencyclidine treat- and (D) the number of errors by side in the delayed alternation test. B, p < 0.01, block ments [39]. These effects of MK-801 indicate that our testing effect; BE, p < 0.001, block-erroneous side interaction (repeated measures ANOVA); #p < 0.05, difference to the 1st block of pooled WT and KO data (Dunnett’s post hoc protocols are capable of detecting deficits in cognitive functions. test); E, p < 0.05, erroneous side effect (ANOVA); *p < 0.05 (Bonferroni post test). There is no published report on altered cognitive phenotype of mGluR3-KO mice to our knowledge. We found that mGluR3- KO mice alternated less than WT mice in the test which is based different between treatments groups (Fig. S6D). Increase in total on spontaneous alternation in a novel environment. However, the locomotor activity analyzed automatically by Ethovision was not reward-motivated alternation of mGluR3-KO mice in the familiar different between genotypes after MK-801 treatment (drug effect, T-maze was not different from WT mice indicating that also nov- F = 108.7, p < 0.001; data not shown). 1,63 elty may play a role in the reduced alternation of mGluR3-KO in the spontaneous T-maze alternation test. We observed here that 4. Discussion mGluR3-KO mice performed the rewarded alternation task as well as WT mice, although Lyon and others have shown that an essen- We report here alterations in behavioral responses of mGluR3- tially similar task was clearly disrupted in the mGluR2/3-double KO KO mice in the T-maze and Y-maze tests measuring spatial working mice [35]. This suggests that mGluR2 might be more important in memory and attentional processes. Importantly, these alterations working memory requiring only a short retention of information, were more pronounced when the mice were forced to enter first and can completely compensate the lack of mGluR3. Nevertheless, the right arm of the maze. In the T-maze tests, spontaneous alterna- we found that mGluR3-KO mice tended to perform more errors tion was significantly reduced in mGluR3-KO mice compared to WT than WT mice when the alternation task was changed to more mice. This reduction was significant in post hoc comparisons after a demanding by prolonging the delay to 25 s. As stated above, also low dose of MK-801 (0.03 mg/kg). However, only a main genotype these findings warrant replication with more animals. effect of reduced alternation in mGluR3-KO mice was observed in Supporting our observation of the mild cognitive deficit and the T-maze experiments with the higher MK-801 doses, suggesting enhanced sensitivity to MK-801 in mGluR3-KO mice, it has been 102 M. Lainiola et al. / Behavioural Brain Research 266 (2014) 94–103 shown that pharmacologically increased activity of mGluR3 pre- no defect in the rewarded-alternation behavior with a short 5-s vented MK-801-induced deficits in a novel object discrimination delay or in the novelty discrimination was observed. However, the (recognition) test [30]. Also other findings support the beneficial genetic deletion of mGluR3 caused significantly reduced sponta- effects of the activation of mGluR3 on cognitive processes [44,45], neous alternation which was especially clear after the lowest dose but see [46]. These studies utilizing pharmacological enzyme of MK-801. Our main finding was that both the reduced alterna- inhibitors to reduce inactivation of the endogenous mGluR3 agonist tion of mGluR3-KO mice in the spontaneous T-maze test and the N-acetylaspartylglutamate [24] and our findings utilizing mGluR3- enhanced sensitivity to the stimulatory effects of MK-801 in the Y- KO mice are in agreement with human genetic, imaging and maze test were significantly more pronounced when the mice were functional studies indicating the involvement of mGluR3 in cog- forced to turn to the right arm as their first arm entry. Because nitive functions [20–23]. mGluR3-KO mice were also more prone to make incorrect visits In search for an explanation for the observed effect of the side of to the left than to the right arm after the prolonged delay time in the forced/habituation arm on mGluR3-KO behavior, we also ana- the rewarded alternation test, we hypothesize that neural functions lyzed the preferred circling direction in a round arena after MK-801 involved in sensory or motor processing of turning behaviors might treatment, but no preference to either direction was observed. This be abnormal in mGluR3-KO mice. Further studies are required to is consistent with the previous report of a lack of any direction elucidate the significance of this unexpected finding and to under- preference after MK-801-induced circling in mice [47]. In addition, stand mechanisms leading to the observed asymmetric behavioral phencyclidine-induced circling behavior of mGluR3-KO mice has responses of mGluR3-KO mice. been tested without finding any genotype difference, however it was not reported whether circling direction was analyzed [18].On Acknowledgments contrary to these findings, earlier rat studies indicated that MK- 80-induced circling can be biased to left, as 72% of all complete The study was supported by the Academy of Finland (AML) rotations in MK-801-treated female rats were directed to left [48]. (#128378) and Orion-Farmos Research Foundation (AML). We Similarly to the lack of circling preference after MK-801 adminis- thank Lilly Neuroscience, Eli Lilly and Company for providing the tration, we did not find any side preference or genotype difference breeding pairs for mGluR3-KO mice. 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