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Behavioral characterization of a mutant mouse strain lacking d -amino acid oxidase activity
Behavioural Brain Research 217 (2011) 81–87
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Behavioral characterization of a mutant mouse strain lacking d-amino acid oxidase activity Min Zhang a,∗ , Michael E. Ballard a,1 , Ana M. Basso a , Natalie Bratcher a , Kaitlin E. Browman a , Pete Curzon a , Ryuichi Konno c , Axel H. Meyer b , Lynne E. Rueter a a
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park IL 60064, USA Abbott GmbH & CoKG, Knollstr.50, 67061 Ludwigshafen, Germany c Center for Medical Science, International University of Health and Welfare, Ohtawara, Tochigi 324-8501, Japan b
a r t i c l e
i n f o
Article history: Received 8 October 2009 Received in revised form 14 September 2010 Accepted 24 September 2010 Available online 7 October 2010 Keywords: d-serine d-amino acid oxidase NMDA Prepulse inhibition Cognition Anxiety DAO mutants
a b s t r a c t d-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO− ) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO−/− mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO−/− mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO−/− mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO−/− mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia. © 2010 Elsevier B.V. All rights reserved.
1. Introduction A unique feature of N-methyl-d-aspartate (NMDA) receptors (NMDAR) is that the channel only operates when both the glutamate- and glycine-site are occupied [5]. d-serine has been recognized as a major endogenous ligand acting as a co-agonist at the glycine site of NMDAR [10,19]. d-serine is mainly synthesized in glial cells [29,30]. The observations that d-serine-containing astrocytes ensheathe NMDA-bearing neurons and that levels of d-serine parallel the distribution of NMDAR suggest that d-serine plays an important role in modulating NMDA-mediated signaling via a glialneuronal interaction [25]. D-serine has recently drawn significant attention in the field of schizophrenia. Based on the NMDAR hypofunction theory of schizophrenia [14,22], it has been proposed that enhancing NMDAR function would have therapeutic potential. This has been tested
∗ Corresponding author. Tel.: +1 847 9381016, fax: +1 847 9380072. E-mail address: [email protected] (M. Zhang). 1 Note: Michael E. Ballard is now at Department of Psychiatry, University of Chicago, Chicago, IL 60637, USA. 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.09.030
in the clinic with d-serine, glycine, d-cycloserine and sarcosine [3,8,12,13,27]. Although results are not consistent, the therapeutic benefits on schizophrenia have been reported when these agents were used as add-on therapy to antipsychotics. These clinical proof-of-concept studies have provoked drug discovery efforts for novel antipsychotics with mechanisms to enhance glycine and dserine activity in the brain. One approach to increase d-serine is to interfere with the metabolism or re-uptake of these amino acids. Inhibiting d-amino acid oxidase (DAO), an enzyme responsible for degrading d-serine, has been proposed as one of the approaches to enhance d-serine in the brain. Several genetic linkage studies have shown an association of schizophrenia with single nucleotide polymorphisms in DAO and its activator (DAOA) [4,6,26], although contradictory results have also been reported [26]. A mouse strain (ddY/DAO− ) [15] has been identified lacking the activity of DAO due to a natural single point mutation (G181R) in DAO [24]. The mutant mice have been shown to have elevated levels of d-serine in the brain, serum, spinal cord and some peripheral organs [9,20,28]. As expected, NMDAR-mediated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons were significantly enhanced in DAO mutants [28]. The ability of d-serine to modulate NMDAR function is further supported by
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the findings that DAO mutants had enhanced LTP in hippocampus and improved performance in Morris water maze, an assay associated with spatial learning [18]. Two studies have shown that DAO mutants responded less to NMDA antagonist-induced effects on certain aspects of motoric behaviors [2,11]. Here we sought to further characterize DAO mutants in order to understand more about the behavioral consequences of lacking DAO and the therapeutic implications of manipulating DAO activity. In the current study, the mice were initially characterized in the paradigm of prepulse inhibition (PPI), an assay to assess sensory motor gating function. Baseline PPI and PPI responses to non-competitive and competitive NMDA antagonists, MK-801 and SDZ 220-581, were assessed. For the cognitive aspects, the mice were tested in the Barnes Maze paradigm for spatial learning and memory and in 24-h inhibitory avoidance (IA) for memory consolidation. In order to understand the activity pattern observed in Barnes Maze, the mice were then tested for locomotor activity in a setting identical to Barnes Maze but with the tunnel removed as well as in a protected open field. In order to furthermore understand the data, the mice were further investigated in two anxiety assays, elevated plus maze (EPM) and novelty suppressed feeding (NSF). The behavioral tests mentioned above were chosen based upon the relevance to the gating deficits and cognitive dysfunctions observed in schizophrenia as well as the translation of previously reported effects in LTP and Morris water maze into two further cognitive assays with a demonstrated hippocampal component, i.e. IA and Barnes Maze. Some of the assays were added, e.g. activity test in Barnes Maze (without the escape tunnel) and anxiety tests, in the course of the studies in order to better interpret the collected data. 2. Materials and methods 2.1. Animals ddY/DAO− mutants and the wild-type ddY/DAO+ mice were obtained from the laboratory of Dr. R. Konno in Japan. Male mice were used in the behavioral tests. All animals were group housed in climate-controlled animal facilities under 12:12 h light–dark cycle (lights on at 6:00 am) with free access to food and water in home cages except those used in the study of novelty suppressed feeding. All experiments were conducted in accordance with Abbott Animal Care and Use Committee and National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. 2.2. Behavioral testing, experimental design and data analysis 2.2.1. Prepulse inhibition (PPI) tests The mice were tested in their light phase in Hamilton Kinder PPI equipment (SM 100 version 4.1, Poway, CA, USA). The animals were given a 5 min acclimation period in the startle chambers during which a 65-decibel (dB) background noise was presented. This background noise remained throughout the entire test. Following the 5 min acclimation period, four successive trials of 40-millisecond (ms) noise bursts at 120 dB were presented. These trials were not included in data analysis. The mice were then exposed to five different types of acoustic stimuli: Pulse Alone (120 dB noise for 40 ms), No Stimulus (no stimulus was presented), and three Prepulse + Pulse with prepulse set at three sound levels of 70, 75 and 80 dB for 20 ms followed by 40 ms pulse at 120 dB. There was a 100 ms interval between the prepulse and the pulse. A total of 12 trials under each acoustic stimulus condition were presented with 20 s variable intervals. Finally, the test ended with four trials of 40 ms 120 dB pulse which were excluded from data analysis. The inclusion of four Pulse Alone trials in the beginning of the experiment were presented to help normalize the responses of the mice as there is rapid habituation to the startle response seen within the first few trials [7]. The four Pulse Alone trials at the end of the test session are part of the standard lab PPI program and were included to provide a means to assess startle reflex habituation to acoustic stimuli when it is of interest in a study. As we have not seen a differential effect of antipsychotics on habituation within these models [23], those eight trials were excluded from data analysis in the current experiments. Percent prepulse inhibition (%PPI) at each prepulse level was calculated as following: [1 − (startle response to Prepulse + Pulse)/(startle response to Pulse Alone)] × 100. Collapsed PPI data across prepulses were also calculated for data analysis. In the PPI studies, %PPI data were analyzed with a three-way ANOVA with genotype and drug treatment as between-subjects independent variables and prepulses as within-subjects variable followed by separate two-way ANOVA and posthoc
comparisons (Bonferroni test) when appropriate. In order to better compare the responses to NMDA antagonists, the collapsed %PPI data was transformed into %vehicle control calculated as following: individual %PPI value in the drug-treated group/average %PPI value of the vehicle-treated group in each genotype × 100%. The %vehicle control values in the water- and drug-treated groups were compared by using two-tailed unpaired t-test. The data for startle responses to s120 were analyzed with two-way ANOVA with treatment and genotype as between-subjects variables followed by Bonferrroni test when appropriate. 2.2.2. Barnes Maze test Our version of the Barnes Maze comprised a circular disk (122 cm in diameter × 2.54 cm thick) made of black acrylic with 40 holes (5 cm in diameter), located 5 cm from the perimeter (Piper Plastics, Inc. Libertyville, IL, USA). Underneath one of the holes there was a stainless steel escape tunnel (6.35 × 20.32 × 3.81 cm), the location of which was fixed. A video camera located above the maze was connected to a viewing monitor via which the experimenter observed the mice. There were cues on the walls of testing room for spatial orientation. Three rows of 16 halogen lamps (75 W each) were located approximately 160 cm above the maze. Mice were tested on the maze for one trial per day. On the first day of the experiment, mice were brought into the testing room, weighed, and left undisturbed for 60 min prior to testing. Following this habituation period, mice were removed from their home cages and were placed into the escape tunnel for 1 min. During this 1 min adaptation period, an opaque cylinder was placed around the hole leading to the tunnel to prevent the mouse from observing the visual cues through the open hole. Following this adaptation period the mouse was removed from the tunnel and placed in the center of the maze beneath a Styrofoam cover attached to a pulley system. After 10 s, the investigator remotely raised the cover using the pulley system, and the mouse was allowed to explore the maze for 5 min. If the mouse entered the tunnel, the session was stopped. If the mouse did not enter the tunnel within 5 min, the investigator gently guided the mouse to the escape tunnel. Once the mouse completely entered the tunnel, the mouse was placed back in their home cage. On days 2–4 the procedures were repeated as described for day 1, minus the tunnel adaptation period. Mice were tested for one trial per day. Latency to enter the tunnel and velocity of the movement were measured through EthoVision imaging system. The data were analyzed using two-way ANOVA with days as repeated measures and genotype as between-subjects factor followed by Bonferroni test if significant day × genotype interaction was identified. 2.2.3. Activity test on the Barnes Maze with the tunnel removed and activity test in enclosed boxes In order to better understand the data collected from the Barnes Maze study, two groups of mice were tested on the maze with the tunnel removed and in enclosed boxes with dimension of 60 × 60 × 35 cm (Piper Plastics, Inc. Libertyville, IL, USA), respectively, for the spontaneous activity measured by EthoVision for 10 min. In addition to total distance traveled measured in both arenas, time spent in the peripheral zone on the Barnes Maze without the tunnel was also measured. The peripheral zone was defined as a circular area with the edge of the maze as the outer circle and with an inner circle 16.5 cm from the edge of the maze. The data was analyzed by two-tailed unpaired t-test. 2.2.4. 24-h inhibitory avoidance test (IA) The mice were brought to the testing room and allowed to habituate to the room for about 2 h. Mice were then injected with sterile water intraperitoneally in a volume of 10 ml/kg, 30 min prior to training. Upon training initiation, mice were placed into the light side of a two-compartment chamber (San Diego Instruments, San Diego, CA, USA). The latency to enter the adjoining dark chamber was recorded, and an inescapable footshock (0.12–0.14 mA, 1 s duration) was presented to the mouse. 24 h later the mouse was tested using methods identical to those on the training day without being dosed or shocked. The data was analyzed by two-tailed unpaired t-test. 2.2.5. Elevated plus maze (EPM) and novel suppressed feeding (NSF) The EPM apparatus (Piper Plastics, Inc. Libertyville, IL, USA) was a plus (+) shaped structure elevated 50 cm above the ground, with two open arms (30 × 5 × 0.25 cm) and two enclosed arms (30 × 5 × 15 cm) separated by a central platform (5 × 5 cm). For testing, the mouse was placed into the center of the maze for a 5 min testing period, and %time and entries to open and closed arms were measured. The data was analyzed by two-tailed unpaired t-test. NSF was carried out in black plastic boxes with the dimension of 42 × 42 × 31 cm (Piper Plastics, Inc. Libertyville, IL, USA) covered with approximately 1.5 cm of bedding. Mice were food deprived for 24 h before the test with unlimited water access. During the test, 2 small pellets (2–3 g each) were placed in the center of the box over a small piece of paper. The mouse was placed in the corner of the cage and behavior was observed for 5 min. The parameters recorded were the latency to start eating the food pellets and the time spent interacting/eating the food. 2.3. Compounds and doses MK-801 (Sigma Chemical Co., St. Louis, MO, USA) and SDZ 220-581 (Tocris Bioscience, Ellisville, MS, USA) were purchased from commercial suppliers. In the PPI
M. Zhang et al. / Behavioural Brain Research 217 (2011) 81–87 study, both of the compounds were given intraperitoneally (ip) in a volume of 10 ml/kg, 15 min prior to the test. MK-801 was dissolved in water and SDZ 220-581 was dissolved in 10 N NaOH and was titrated to pH around 6.5 with HCL. MK-801 and SDZ 220-581 were given at 0.3 and 5 mg/kg, respectively. These doses could induce PPI deficit in our preliminary studies.
Table 1 Startle responses in PPI studies. Groups
3. Results 3.1. Prepulse inhibition studies In the study investigating MK-801-induced effects on PPI in both DAO+/+ and DAO−/− mice, three-way ANOVA revealed a significant main effect of treatment (F(1, 26) = 79.77, p < 0.01) and prepulse (F(2, 52) = 62.449, p < 0.01). No significant interaction of treatment, prepulse and genotype was detected. Follow-up twoway ANOVA revealed a nearly significant treatment and genotype interaction (F(1, 26) = 4.054, p = 0.0545). As shown in Fig. 1A, Bonferroni posthoc comparison showed vehicle-treated DAO−/− mice had significantly higher %PPI compared to vehicle-treated DAO+/+ mice (p < 0.01). MK-801 significantly disrupted PPI response in both genotypes (p < 0.05). The MK-801-induced PPI-disruptive effect in DAO−/− mice was not significantly different from that in DAO+/+ mice, when the data was expressed in %control (Fig. 1B). For startle responses, two-way ANOVA revealed a significant effect of treatment (F(1, 26) = 5.10, p < 0.01) and nearly significant treatment and genotype interaction (F(1, 26) = 4.073, p = 0.0540). As shown in Table 1, MK-801 only increased startle significantly in mutants (p < 0.01), revealed by Bonferroni posthoc test. In the study investigating the effect of SDZ 220-581 on PPI, three-way ANOVA revealed a significant main effect of treatment (F(1, 25) = 44.272, p < 0.01) and prepulse (F(2, 50) = 84.111, p < 0.01). No significant interaction of treatment, prepulse and genotype was detected. Follow-up two-way ANOVA revealed a significant treatment and phenotype interaction (F(1, 25) = 11.213, p < 0.01). 0
A
MK-801/PPI study Water-treated DAO+/+ MK-801-treated DAO+/+ Water-treated DAO−/− MK-801-treated DAO−/−
1.92 ± 0.17 2.0 ± 0.2 1.45 ± 0.17 2.4 ± 0.29**
SDZ/PPI study* Water-treated DAO+/+ SDZ-treated DAO+/+ Water-treated DAO−/− SDZ-treated DAO−/−
1.21 ± 0.14 1.59 ± 0.15 1.33 ± 0.15 1.89 ± 0.23
* **
3.2. Barnes Maze test Two-way ANOVA revealed a significant main effect of genotype (F(1, 16) = 25.92, p < 0.01) on latency to enter the tunnel and significant main effect of day (F(3, 48) = 4.83, p < 0.01). No significant interaction of treatment and day was detected. As shown in Fig. 2A, DAO−/− mice showed shortened latency to enter the tunnel. Two-
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p < 0.05, significant main effect of treatment. p < 0.01 compared to water-treated DAO+/+ mice.
As shown in Fig. 1C, Bonferroni posthoc comparison showed vehicle-treated DAO−/− mice had significantly higher %PPI compared to vehicle-treated DAO−/− mice (p < 0.05), and SDZ 220-581 significantly disrupted PPI response in both genotypes (p < 0.05 and p < 0.01 in DAO+/+ and DAO−/−, respectively). SDZ 220-581induced disruptive effect was stronger in DAO−/− when the data was expressed in %control (Fig. 1D; p < 0.01, unpaired t-test). For startle responses, two-way ANOVA only revealed a significant treatment effect (F(1, 25) = 7.615, p < 0.01) without significant treatment and genotype difference.
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Fig. 1. Effects of MK-801 (n = 7–8/group) and SDZ (n = 7–8/group) on PPI in DAO+/+ and DAO−/− mice. DAO−/− mice showed enhanced PPI compared to DAO+/+ following the treatment with water (* p < 0.05; A and C). MK-801 and SDZ significantly disrupted PPI in both DAO+/+ and DAO−/− (## p < 0.01, # p < 0.05, compared to water-treated group in each genotype; A and C). The effects seem to be more potent in DAO−/− mice (&& p < 0.01, compared to SDZ/DAO+/+ group) with SDZ reaching statistical significance (B and D).
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Fig. 2. In Barnes Maze test (n = 9/group), an assay involving spatial learning and memory, DAO−/− mice entered the tunnel faster than DAO+/+ mice (** p < 0.01, significant main effect of genotype on latency to enter the tunnel; A). The DAO−/− mice also had higher velocity (## p < 0.01, compared to DAO+/+ mice at day 3 and day 4; B).
way ANOVA showed a significant main effect of genotype (F(1, 16) = 11.6, p < 0.01) on velocity and significant main effect of day (F(3, 48) = 22.81, p < 0.01), as well as a significant genotype and day interaction (F(3, 48) = 7.02, p < 0.01). Follow-up Bonferroni posthoc comparison showed a significant increase of velocity at D3 and D4 (p < 0.01) in DAO−/− mice compared to DAO+/+ mice, as shown in Fig. 2B. 3.3. Activity test on the Barnes Maze with the tunnel removed and activity test in enclosed boxes As shown in Fig. 3B, DAO+/+ and DAO−/− mice were not significantly different from each other when total distance traveled was measured on the Barnes Maze arena (with tunnel removed). Interestingly, DAO−/− mice spent much more time on the periphery than DAO+/+ mice (p < 0.01, unpaired t-test; Fig. 3C). When the activity was measured in a protected open field (Fig. 3A), DAO−/− mice were much less active than DAO+/+ mice, expressed as a significant reduction of total distance traveled (p < 0.01, unpaired t-test).
3.4. Elevated plus maze (EPM) and novel suppressed feeding (NSF) As shown in Fig. 4, DAO−/− mice were not significantly different from DAO+/+ mice in the measure of %open time in EPM (Fig. 4A) as well as in the measure of latency to start eating or the time spent interacting with the food in NSF (Fig. 4C). Significant reduction of closed arm entries and total arm entries were observed in DAO−/− mice (Fig. 4B), a reflection of hypoactivity. 3.5. 24-h inhibitory avoidance test (IA) As shown in Fig. 5, there was a significant increase of transfer latency in DAO−/− mice during training (p < 0.05, unpaired t-test), but no difference was observed between DAO−/− and DAO+/+ mice 24 h later when the mice were tested for recall. 4. Discussion Our studies showed that DAO−/− mice showed enhanced PPI responses and increased sensitivity to the PPI-disruptive effect
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Fig. 3. DAO−/− mice were hypoactive when tested in enclosed boxes (** p < 0.01, compared to DAO+/+; n = 9/group; A). However, when they were tested on Barnes Maze with tunnel removed (n = 8/group), DAO−/− mice were as active as DAO+/+ (B) and spent more time in the peripheral area (** p < 0.01; C).
M. Zhang et al. / Behavioural Brain Research 217 (2011) 81–87
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Fig. 4. In anxiety tests, elevated plus maze (EPM; n = 22 or 25/group) and novelty suppressed feeding (NSF; n = 16/group), there were no genotype effects on anxiety-like measures (A and C), although DAO−/− mice made significantly less closed arm entries and total arm entries (B), a reflection of hypoactivity in EPM (** p < 0.01, compared to DAO+/+ mice).
of SDZ 220-581. MK-801-induced PPI deficit in DAO−/− mutant mice was not significantly different from that observed in DAO+/+ mice. It is notable in DAO+/+ mice, MK-801 treatment at 0.3 mg/kg reduced %PPI to 30% of the vehicle-treated group, while the effect of SDZ 220-581 on PPI in DAO+/+ was more moderate, reaching 60% of the corresponding vehicle-treated group. It would be interesting to test a lower dose of MK-801 which can produce an effect on PPI comparable to SDZ in DAO+/+ mice and then to see if a similar enhanced response to MK-801 treatment can be observed in DAO−/− mice. In the spatial learning and memory test, DAO−/− showed shortened latency to enter the escape tunnel of Barnes Maze, however the procognitive interpretation of the shortened latency could be confounded by the fact that DAO−/− spent more time in the periphery area of the maze. No genotype effect was observed in 24-h inhibitory avoidance test. DAO−/− mice were not different from DAO+/+ mice in the two models, EPM and NSF, for anxiety assessment. In contrast to a published study [2], we showed an increase of PPI in DAO−/− mice. As discussed by the authors, the protocol
Inhibitory Avoidance Transfer Latency (sec)
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Fig. 5. In 24-h inhibitory avoidance test (IA; n = 9 or 10/group), there was no genotype effect on recall, although DAO−/−mice showed significantly prolonged cross-over latency on training day due to hypoactivity.
used in the study by Almond et al. may not be sensitive enough to detect a genotype effect on PPI. It has been well documented that NMDA receptors are involved in sensory motor gating. The increase of PPI in DAO−/− mice and enhanced response to a NMDA antagonist in DAO−/− mice might suggest an altered NMDAR function in these mice. Enhanced NMDAR function in DAO mutants was observed in several studies. For instance, DAO−/− mice showed exaggerated responses to chronic treatment of nociceptive stimuli accompanied by enhanced NMDA receptor-mediated excitatory postsynaptic currents in dorsal horn neurons in spinal cord [28]. Increased LTP in hippocampal slices [18] was also reported. Almond et al. [2] demonstrated an enhanced NMDAR function in vivo by showing that harmaline-stimulated cerebellar cGMP increase was larger in DAO−/− [2]. It was interesting to notice that SDZ 220-581, a competitive NMDA antagonist binding to the glutamate-recognition site outside the channel, also induced a stronger effect on PPI in DAO−/− mice. This may indicate that changes of NR2 subunits might occur in DAO−/− mice in addition to an enhanced occupancy of the glycine-binding site located on the NR1 subunit due to elevated extracellular d-serine level [2]. Although we observed that DAO−/− mice tended to show enhanced response to NMDA antagonists in PPI, it is worthwhile to note that other studies have reported attenuated response to MK-801- or L-701324- (a glycine site antagonist) induced effects on motor activity especially on motor coordination [2,11]. Given that the increase of d-serine in the brain of DAO−/− mice is predominantly in cerebellum [20], it is reasonable to expect a protective role of d-serine against cerebellar ataxia induced by NMDAR disruption. As a matter of fact, DAO−/− mice showed better motor coordination in a beam-walking test [2] and a recent clinical study found dcycloserine, a partial agonist at glycine site, may relieve cerebellar ataxia [21]. However, it remains unknown why both augmentation and attenuation of NMDA antagonist-induced behavioral effects have been observed in DAO−/− mice, although it can be speculated that neural circuits underlying different types of behaviors can be fundamentally different, thus leading to behavior-dependent phenotype differences. Nevertheless changes of NMDA functions in
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DAO−/− mice seem to be suggested by several lines of studies as discussed above. The Barnes Maze study showed a shortened latency to enter the escape tunnel. Although DAO−/− mice were less active than DAO+/+ when tested in activity boxes, they showed faster velocity on the Barnes Maze. Taken together, the data seem to suggest DAO−/− mice may have a better spatial navigation capacity which enabled them to find the location of the escape tunnel faster than DAO+/+. This speculation was in line with a previous study reporting DAO−/− mice performed better than DAO+/+ in water maze [18]. However the control study with the escape tunnel removed showed that DAO−/− mice spent substantially more time in the peripheral area of the maze, suggesting, in the previous Barnes Maze test, the DAO−/− mice may be faster to find the escape tunnel underneath the edge because of the increased edge activity rather than better spatial learning and memory performance. In order to tease apart the increased edge activity and enhanced spatial navigation, it would be necessary to redesign the maze to make the location of escape tunnel far away from the edge. We did attempt to test the mice in water maze for spatial learning and memory, however, the data were confounded by the fact that the mice did not readily swim, but instead showed a hypoactive response and just floated in the water. It was interesting to note that, although the mutant mice were hypoactive when tested in activity boxes, they were equally active compared to the wild-type mice when tested on an unprotected and elevated open field such as on the Barnes Maze. The increased edge activity corresponded well with predominant increase of d-serine in cerebellum and enhanced motor coordination observed in DAO−/− mice [2,20]. An alternative interpretation of increased edge activity could be DAO−/− mice were less anxious on the edge so that they spent more time over there. However, we did not observe anxiolytic-like behaviors in two anxiety models. As a matter of fact, Labrie et al. has reported an increase of anxiety-like behaviors in mice with deficient DAO activity [16]. We further profiled the mice in another cognitive assay, 24-h IA, an assay assessing memory consolidation. No genotype effect on recall was observed, although DAO−/− mice showed a significant increase of latency during the training, probably due to hypoactivity. It appears that our data did not provide incontrovertible evidence suggesting DAO−/− mice had better cognitive performance in learning and memory. However, it is possible that DAO−/− mutants may have alterations in other cognitive functions. Labrie et al. has reported that mice lacking DAO activity showed enhanced cognitive flexibility in response to changing environmental contingency, although these mice were not different from the wild-types in the initial associative learning [17]. In summary, DAO−/− mice seem to have changes in NMDA function. In DAO−/− mice, we observed enhanced PPI responses and enhanced PPI-disruptive effects of NMDA antagonists. However, we didn’t see clear genotype effects on cognition and anxiety measures. The therapeutic utility of DAO inhibition can be better characterized by using pharmacological agents in animal models. So far only limited data suggested antipsychotic-like profile following treatment with DAO inhibitors [1]. Acknowledgements We thank Dr. Michael W. Decker who made valuable suggestions to improve the manuscript. The work was supported by Abbott Laboratories. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbr.2010.09.030.
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Genetic and physiological data implicating the new human gene G72 and the gene for d-amino acid oxidase in schizophrenia. Proc Natl Acad Sci USA 2002;99:13675–80. [5] Danysz W, Parsons AC. Glycine and N-methyl-d-aspartate receptors: physiological significance and possible therapeutic applications. Pharmacol Rev 1998;50:597–664. [6] Detera-Wadleigh SD, McMahon FJ. G72/G30 in schizophrenia and bipolar disorder: review and meta-analysis. Biol Psychiatry 2006;60:106–14. [7] Dulawa SC, Geyer MA. Effects of strain and serotonergic agents on prepulse inhibition and habituation in mice. Neuropharmacology 2000;39: 2170–9. [8] Goff DC, Herz L, Posever T, Shih V, Tsai G, Henderson DC, et al. A six-month, placebo-controlled trial of d-cycloserine co-administered with conventional antipsychotics in schizophrenia patients. Psychopharmacology (Berl) 2005;179:144–50. [9] Hashimoto A, Nishikawa T, Konno R, Niwa A, Yasumura Y, Oka T, et al. Free d-serine, d-aspartate and d-alanine in central nervous system and serum in mutant mice lacking d-amino acid oxidase. Neurosci Lett 1993;152:33–6. [10] Hashimoto A, Nishikawa T, Oka T, Takahashi K. Endogenous d-serine in rat brain: N-methyl-d-aspartate receptor-related distribution and aging. J Neurochem 1993;60:783–6. [11] Hashimoto A, Yoshikawa M, Niwa A, Konno R. Mice lacking d-amino acid oxidase activity display marked attenuation of stereotypy and ataxia induced by MK-801. Brain Res 2005;1033:210–5. [12] Heresco-Levy U, Ermilov M, Lichtenberg P, Bar G, Javitt DC. High-dose glycine added to olanzapine and risperidone for the treatment of schizophrenia. Biol Psychiatry 2004;55:165–71. [13] Heresco-Levy U, Javitt DC, Ebstein R, Vass A, Lichtenberg P, Bar G, et al. dserine efficacy as add-on pharmacotherapy to risperidone and olanzapine for treatment-refractory schizophrenia. Biol Psychiatry 2005;57:577–85. [14] Javitt DC, Zukin SR. 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[25] Schell MJ, Brady Jr RO, Molliver ME, Snyder SH. d-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors. J Neurosci 1997;17:1604–15. [26] Schumacher J, Jamra RA, Freudenberg J, Becker T, Ohlraun S, Otte AC, et al. Examination of G72 and d-amino-acid oxidase as genetic risk factors for schizophrenia and bipolar affective disorder. Mol Psychiatry 2004;9: 203–7. [27] Tsai G, Lane HY, Yang P, Chong MY, Lange N. Glycine transporter I inhibitor, N-methylglycine (sarcosine), added to antipsychotics for the treatment of schizophrenia. Biol Psychiatry 2004;55:452–6.
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Research report
Behavioral characterization of a mutant mouse strain lacking d-amino acid oxidase activity Min Zhang a,∗ , Michael E. Ballard a,1 , Ana M. Basso a , Natalie Bratcher a , Kaitlin E. Browman a , Pete Curzon a , Ryuichi Konno c , Axel H. Meyer b , Lynne E. Rueter a a
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park IL 60064, USA Abbott GmbH & CoKG, Knollstr.50, 67061 Ludwigshafen, Germany c Center for Medical Science, International University of Health and Welfare, Ohtawara, Tochigi 324-8501, Japan b
a r t i c l e
i n f o
Article history: Received 8 October 2009 Received in revised form 14 September 2010 Accepted 24 September 2010 Available online 7 October 2010 Keywords: d-serine d-amino acid oxidase NMDA Prepulse inhibition Cognition Anxiety DAO mutants
a b s t r a c t d-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO− ) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO−/− mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO−/− mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO−/− mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO−/− mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia. © 2010 Elsevier B.V. All rights reserved.
1. Introduction A unique feature of N-methyl-d-aspartate (NMDA) receptors (NMDAR) is that the channel only operates when both the glutamate- and glycine-site are occupied [5]. d-serine has been recognized as a major endogenous ligand acting as a co-agonist at the glycine site of NMDAR [10,19]. d-serine is mainly synthesized in glial cells [29,30]. The observations that d-serine-containing astrocytes ensheathe NMDA-bearing neurons and that levels of d-serine parallel the distribution of NMDAR suggest that d-serine plays an important role in modulating NMDA-mediated signaling via a glialneuronal interaction [25]. D-serine has recently drawn significant attention in the field of schizophrenia. Based on the NMDAR hypofunction theory of schizophrenia [14,22], it has been proposed that enhancing NMDAR function would have therapeutic potential. This has been tested
∗ Corresponding author. Tel.: +1 847 9381016, fax: +1 847 9380072. E-mail address: [email protected] (M. Zhang). 1 Note: Michael E. Ballard is now at Department of Psychiatry, University of Chicago, Chicago, IL 60637, USA. 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.09.030
in the clinic with d-serine, glycine, d-cycloserine and sarcosine [3,8,12,13,27]. Although results are not consistent, the therapeutic benefits on schizophrenia have been reported when these agents were used as add-on therapy to antipsychotics. These clinical proof-of-concept studies have provoked drug discovery efforts for novel antipsychotics with mechanisms to enhance glycine and dserine activity in the brain. One approach to increase d-serine is to interfere with the metabolism or re-uptake of these amino acids. Inhibiting d-amino acid oxidase (DAO), an enzyme responsible for degrading d-serine, has been proposed as one of the approaches to enhance d-serine in the brain. Several genetic linkage studies have shown an association of schizophrenia with single nucleotide polymorphisms in DAO and its activator (DAOA) [4,6,26], although contradictory results have also been reported [26]. A mouse strain (ddY/DAO− ) [15] has been identified lacking the activity of DAO due to a natural single point mutation (G181R) in DAO [24]. The mutant mice have been shown to have elevated levels of d-serine in the brain, serum, spinal cord and some peripheral organs [9,20,28]. As expected, NMDAR-mediated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons were significantly enhanced in DAO mutants [28]. The ability of d-serine to modulate NMDAR function is further supported by
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the findings that DAO mutants had enhanced LTP in hippocampus and improved performance in Morris water maze, an assay associated with spatial learning [18]. Two studies have shown that DAO mutants responded less to NMDA antagonist-induced effects on certain aspects of motoric behaviors [2,11]. Here we sought to further characterize DAO mutants in order to understand more about the behavioral consequences of lacking DAO and the therapeutic implications of manipulating DAO activity. In the current study, the mice were initially characterized in the paradigm of prepulse inhibition (PPI), an assay to assess sensory motor gating function. Baseline PPI and PPI responses to non-competitive and competitive NMDA antagonists, MK-801 and SDZ 220-581, were assessed. For the cognitive aspects, the mice were tested in the Barnes Maze paradigm for spatial learning and memory and in 24-h inhibitory avoidance (IA) for memory consolidation. In order to understand the activity pattern observed in Barnes Maze, the mice were then tested for locomotor activity in a setting identical to Barnes Maze but with the tunnel removed as well as in a protected open field. In order to furthermore understand the data, the mice were further investigated in two anxiety assays, elevated plus maze (EPM) and novelty suppressed feeding (NSF). The behavioral tests mentioned above were chosen based upon the relevance to the gating deficits and cognitive dysfunctions observed in schizophrenia as well as the translation of previously reported effects in LTP and Morris water maze into two further cognitive assays with a demonstrated hippocampal component, i.e. IA and Barnes Maze. Some of the assays were added, e.g. activity test in Barnes Maze (without the escape tunnel) and anxiety tests, in the course of the studies in order to better interpret the collected data. 2. Materials and methods 2.1. Animals ddY/DAO− mutants and the wild-type ddY/DAO+ mice were obtained from the laboratory of Dr. R. Konno in Japan. Male mice were used in the behavioral tests. All animals were group housed in climate-controlled animal facilities under 12:12 h light–dark cycle (lights on at 6:00 am) with free access to food and water in home cages except those used in the study of novelty suppressed feeding. All experiments were conducted in accordance with Abbott Animal Care and Use Committee and National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. 2.2. Behavioral testing, experimental design and data analysis 2.2.1. Prepulse inhibition (PPI) tests The mice were tested in their light phase in Hamilton Kinder PPI equipment (SM 100 version 4.1, Poway, CA, USA). The animals were given a 5 min acclimation period in the startle chambers during which a 65-decibel (dB) background noise was presented. This background noise remained throughout the entire test. Following the 5 min acclimation period, four successive trials of 40-millisecond (ms) noise bursts at 120 dB were presented. These trials were not included in data analysis. The mice were then exposed to five different types of acoustic stimuli: Pulse Alone (120 dB noise for 40 ms), No Stimulus (no stimulus was presented), and three Prepulse + Pulse with prepulse set at three sound levels of 70, 75 and 80 dB for 20 ms followed by 40 ms pulse at 120 dB. There was a 100 ms interval between the prepulse and the pulse. A total of 12 trials under each acoustic stimulus condition were presented with 20 s variable intervals. Finally, the test ended with four trials of 40 ms 120 dB pulse which were excluded from data analysis. The inclusion of four Pulse Alone trials in the beginning of the experiment were presented to help normalize the responses of the mice as there is rapid habituation to the startle response seen within the first few trials [7]. The four Pulse Alone trials at the end of the test session are part of the standard lab PPI program and were included to provide a means to assess startle reflex habituation to acoustic stimuli when it is of interest in a study. As we have not seen a differential effect of antipsychotics on habituation within these models [23], those eight trials were excluded from data analysis in the current experiments. Percent prepulse inhibition (%PPI) at each prepulse level was calculated as following: [1 − (startle response to Prepulse + Pulse)/(startle response to Pulse Alone)] × 100. Collapsed PPI data across prepulses were also calculated for data analysis. In the PPI studies, %PPI data were analyzed with a three-way ANOVA with genotype and drug treatment as between-subjects independent variables and prepulses as within-subjects variable followed by separate two-way ANOVA and posthoc
comparisons (Bonferroni test) when appropriate. In order to better compare the responses to NMDA antagonists, the collapsed %PPI data was transformed into %vehicle control calculated as following: individual %PPI value in the drug-treated group/average %PPI value of the vehicle-treated group in each genotype × 100%. The %vehicle control values in the water- and drug-treated groups were compared by using two-tailed unpaired t-test. The data for startle responses to s120 were analyzed with two-way ANOVA with treatment and genotype as between-subjects variables followed by Bonferrroni test when appropriate. 2.2.2. Barnes Maze test Our version of the Barnes Maze comprised a circular disk (122 cm in diameter × 2.54 cm thick) made of black acrylic with 40 holes (5 cm in diameter), located 5 cm from the perimeter (Piper Plastics, Inc. Libertyville, IL, USA). Underneath one of the holes there was a stainless steel escape tunnel (6.35 × 20.32 × 3.81 cm), the location of which was fixed. A video camera located above the maze was connected to a viewing monitor via which the experimenter observed the mice. There were cues on the walls of testing room for spatial orientation. Three rows of 16 halogen lamps (75 W each) were located approximately 160 cm above the maze. Mice were tested on the maze for one trial per day. On the first day of the experiment, mice were brought into the testing room, weighed, and left undisturbed for 60 min prior to testing. Following this habituation period, mice were removed from their home cages and were placed into the escape tunnel for 1 min. During this 1 min adaptation period, an opaque cylinder was placed around the hole leading to the tunnel to prevent the mouse from observing the visual cues through the open hole. Following this adaptation period the mouse was removed from the tunnel and placed in the center of the maze beneath a Styrofoam cover attached to a pulley system. After 10 s, the investigator remotely raised the cover using the pulley system, and the mouse was allowed to explore the maze for 5 min. If the mouse entered the tunnel, the session was stopped. If the mouse did not enter the tunnel within 5 min, the investigator gently guided the mouse to the escape tunnel. Once the mouse completely entered the tunnel, the mouse was placed back in their home cage. On days 2–4 the procedures were repeated as described for day 1, minus the tunnel adaptation period. Mice were tested for one trial per day. Latency to enter the tunnel and velocity of the movement were measured through EthoVision imaging system. The data were analyzed using two-way ANOVA with days as repeated measures and genotype as between-subjects factor followed by Bonferroni test if significant day × genotype interaction was identified. 2.2.3. Activity test on the Barnes Maze with the tunnel removed and activity test in enclosed boxes In order to better understand the data collected from the Barnes Maze study, two groups of mice were tested on the maze with the tunnel removed and in enclosed boxes with dimension of 60 × 60 × 35 cm (Piper Plastics, Inc. Libertyville, IL, USA), respectively, for the spontaneous activity measured by EthoVision for 10 min. In addition to total distance traveled measured in both arenas, time spent in the peripheral zone on the Barnes Maze without the tunnel was also measured. The peripheral zone was defined as a circular area with the edge of the maze as the outer circle and with an inner circle 16.5 cm from the edge of the maze. The data was analyzed by two-tailed unpaired t-test. 2.2.4. 24-h inhibitory avoidance test (IA) The mice were brought to the testing room and allowed to habituate to the room for about 2 h. Mice were then injected with sterile water intraperitoneally in a volume of 10 ml/kg, 30 min prior to training. Upon training initiation, mice were placed into the light side of a two-compartment chamber (San Diego Instruments, San Diego, CA, USA). The latency to enter the adjoining dark chamber was recorded, and an inescapable footshock (0.12–0.14 mA, 1 s duration) was presented to the mouse. 24 h later the mouse was tested using methods identical to those on the training day without being dosed or shocked. The data was analyzed by two-tailed unpaired t-test. 2.2.5. Elevated plus maze (EPM) and novel suppressed feeding (NSF) The EPM apparatus (Piper Plastics, Inc. Libertyville, IL, USA) was a plus (+) shaped structure elevated 50 cm above the ground, with two open arms (30 × 5 × 0.25 cm) and two enclosed arms (30 × 5 × 15 cm) separated by a central platform (5 × 5 cm). For testing, the mouse was placed into the center of the maze for a 5 min testing period, and %time and entries to open and closed arms were measured. The data was analyzed by two-tailed unpaired t-test. NSF was carried out in black plastic boxes with the dimension of 42 × 42 × 31 cm (Piper Plastics, Inc. Libertyville, IL, USA) covered with approximately 1.5 cm of bedding. Mice were food deprived for 24 h before the test with unlimited water access. During the test, 2 small pellets (2–3 g each) were placed in the center of the box over a small piece of paper. The mouse was placed in the corner of the cage and behavior was observed for 5 min. The parameters recorded were the latency to start eating the food pellets and the time spent interacting/eating the food. 2.3. Compounds and doses MK-801 (Sigma Chemical Co., St. Louis, MO, USA) and SDZ 220-581 (Tocris Bioscience, Ellisville, MS, USA) were purchased from commercial suppliers. In the PPI
M. Zhang et al. / Behavioural Brain Research 217 (2011) 81–87 study, both of the compounds were given intraperitoneally (ip) in a volume of 10 ml/kg, 15 min prior to the test. MK-801 was dissolved in water and SDZ 220-581 was dissolved in 10 N NaOH and was titrated to pH around 6.5 with HCL. MK-801 and SDZ 220-581 were given at 0.3 and 5 mg/kg, respectively. These doses could induce PPI deficit in our preliminary studies.
Table 1 Startle responses in PPI studies. Groups
3. Results 3.1. Prepulse inhibition studies In the study investigating MK-801-induced effects on PPI in both DAO+/+ and DAO−/− mice, three-way ANOVA revealed a significant main effect of treatment (F(1, 26) = 79.77, p < 0.01) and prepulse (F(2, 52) = 62.449, p < 0.01). No significant interaction of treatment, prepulse and genotype was detected. Follow-up twoway ANOVA revealed a nearly significant treatment and genotype interaction (F(1, 26) = 4.054, p = 0.0545). As shown in Fig. 1A, Bonferroni posthoc comparison showed vehicle-treated DAO−/− mice had significantly higher %PPI compared to vehicle-treated DAO+/+ mice (p < 0.01). MK-801 significantly disrupted PPI response in both genotypes (p < 0.05). The MK-801-induced PPI-disruptive effect in DAO−/− mice was not significantly different from that in DAO+/+ mice, when the data was expressed in %control (Fig. 1B). For startle responses, two-way ANOVA revealed a significant effect of treatment (F(1, 26) = 5.10, p < 0.01) and nearly significant treatment and genotype interaction (F(1, 26) = 4.073, p = 0.0540). As shown in Table 1, MK-801 only increased startle significantly in mutants (p < 0.01), revealed by Bonferroni posthoc test. In the study investigating the effect of SDZ 220-581 on PPI, three-way ANOVA revealed a significant main effect of treatment (F(1, 25) = 44.272, p < 0.01) and prepulse (F(2, 50) = 84.111, p < 0.01). No significant interaction of treatment, prepulse and genotype was detected. Follow-up two-way ANOVA revealed a significant treatment and phenotype interaction (F(1, 25) = 11.213, p < 0.01). 0
A
MK-801/PPI study Water-treated DAO+/+ MK-801-treated DAO+/+ Water-treated DAO−/− MK-801-treated DAO−/−
1.92 ± 0.17 2.0 ± 0.2 1.45 ± 0.17 2.4 ± 0.29**
SDZ/PPI study* Water-treated DAO+/+ SDZ-treated DAO+/+ Water-treated DAO−/− SDZ-treated DAO−/−
1.21 ± 0.14 1.59 ± 0.15 1.33 ± 0.15 1.89 ± 0.23
* **
3.2. Barnes Maze test Two-way ANOVA revealed a significant main effect of genotype (F(1, 16) = 25.92, p < 0.01) on latency to enter the tunnel and significant main effect of day (F(3, 48) = 4.83, p < 0.01). No significant interaction of treatment and day was detected. As shown in Fig. 2A, DAO−/− mice showed shortened latency to enter the tunnel. Two-
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As shown in Fig. 1C, Bonferroni posthoc comparison showed vehicle-treated DAO−/− mice had significantly higher %PPI compared to vehicle-treated DAO−/− mice (p < 0.05), and SDZ 220-581 significantly disrupted PPI response in both genotypes (p < 0.05 and p < 0.01 in DAO+/+ and DAO−/−, respectively). SDZ 220-581induced disruptive effect was stronger in DAO−/− when the data was expressed in %control (Fig. 1D; p < 0.01, unpaired t-test). For startle responses, two-way ANOVA only revealed a significant treatment effect (F(1, 25) = 7.615, p < 0.01) without significant treatment and genotype difference.
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Fig. 1. Effects of MK-801 (n = 7–8/group) and SDZ (n = 7–8/group) on PPI in DAO+/+ and DAO−/− mice. DAO−/− mice showed enhanced PPI compared to DAO+/+ following the treatment with water (* p < 0.05; A and C). MK-801 and SDZ significantly disrupted PPI in both DAO+/+ and DAO−/− (## p < 0.01, # p < 0.05, compared to water-treated group in each genotype; A and C). The effects seem to be more potent in DAO−/− mice (&& p < 0.01, compared to SDZ/DAO+/+ group) with SDZ reaching statistical significance (B and D).
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Fig. 2. In Barnes Maze test (n = 9/group), an assay involving spatial learning and memory, DAO−/− mice entered the tunnel faster than DAO+/+ mice (** p < 0.01, significant main effect of genotype on latency to enter the tunnel; A). The DAO−/− mice also had higher velocity (## p < 0.01, compared to DAO+/+ mice at day 3 and day 4; B).
way ANOVA showed a significant main effect of genotype (F(1, 16) = 11.6, p < 0.01) on velocity and significant main effect of day (F(3, 48) = 22.81, p < 0.01), as well as a significant genotype and day interaction (F(3, 48) = 7.02, p < 0.01). Follow-up Bonferroni posthoc comparison showed a significant increase of velocity at D3 and D4 (p < 0.01) in DAO−/− mice compared to DAO+/+ mice, as shown in Fig. 2B. 3.3. Activity test on the Barnes Maze with the tunnel removed and activity test in enclosed boxes As shown in Fig. 3B, DAO+/+ and DAO−/− mice were not significantly different from each other when total distance traveled was measured on the Barnes Maze arena (with tunnel removed). Interestingly, DAO−/− mice spent much more time on the periphery than DAO+/+ mice (p < 0.01, unpaired t-test; Fig. 3C). When the activity was measured in a protected open field (Fig. 3A), DAO−/− mice were much less active than DAO+/+ mice, expressed as a significant reduction of total distance traveled (p < 0.01, unpaired t-test).
3.4. Elevated plus maze (EPM) and novel suppressed feeding (NSF) As shown in Fig. 4, DAO−/− mice were not significantly different from DAO+/+ mice in the measure of %open time in EPM (Fig. 4A) as well as in the measure of latency to start eating or the time spent interacting with the food in NSF (Fig. 4C). Significant reduction of closed arm entries and total arm entries were observed in DAO−/− mice (Fig. 4B), a reflection of hypoactivity. 3.5. 24-h inhibitory avoidance test (IA) As shown in Fig. 5, there was a significant increase of transfer latency in DAO−/− mice during training (p < 0.05, unpaired t-test), but no difference was observed between DAO−/− and DAO+/+ mice 24 h later when the mice were tested for recall. 4. Discussion Our studies showed that DAO−/− mice showed enhanced PPI responses and increased sensitivity to the PPI-disruptive effect
A Total Distance (cm)
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Fig. 3. DAO−/− mice were hypoactive when tested in enclosed boxes (** p < 0.01, compared to DAO+/+; n = 9/group; A). However, when they were tested on Barnes Maze with tunnel removed (n = 8/group), DAO−/− mice were as active as DAO+/+ (B) and spent more time in the peripheral area (** p < 0.01; C).
M. Zhang et al. / Behavioural Brain Research 217 (2011) 81–87
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Fig. 4. In anxiety tests, elevated plus maze (EPM; n = 22 or 25/group) and novelty suppressed feeding (NSF; n = 16/group), there were no genotype effects on anxiety-like measures (A and C), although DAO−/− mice made significantly less closed arm entries and total arm entries (B), a reflection of hypoactivity in EPM (** p < 0.01, compared to DAO+/+ mice).
of SDZ 220-581. MK-801-induced PPI deficit in DAO−/− mutant mice was not significantly different from that observed in DAO+/+ mice. It is notable in DAO+/+ mice, MK-801 treatment at 0.3 mg/kg reduced %PPI to 30% of the vehicle-treated group, while the effect of SDZ 220-581 on PPI in DAO+/+ was more moderate, reaching 60% of the corresponding vehicle-treated group. It would be interesting to test a lower dose of MK-801 which can produce an effect on PPI comparable to SDZ in DAO+/+ mice and then to see if a similar enhanced response to MK-801 treatment can be observed in DAO−/− mice. In the spatial learning and memory test, DAO−/− showed shortened latency to enter the escape tunnel of Barnes Maze, however the procognitive interpretation of the shortened latency could be confounded by the fact that DAO−/− spent more time in the periphery area of the maze. No genotype effect was observed in 24-h inhibitory avoidance test. DAO−/− mice were not different from DAO+/+ mice in the two models, EPM and NSF, for anxiety assessment. In contrast to a published study [2], we showed an increase of PPI in DAO−/− mice. As discussed by the authors, the protocol
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Fig. 5. In 24-h inhibitory avoidance test (IA; n = 9 or 10/group), there was no genotype effect on recall, although DAO−/−mice showed significantly prolonged cross-over latency on training day due to hypoactivity.
used in the study by Almond et al. may not be sensitive enough to detect a genotype effect on PPI. It has been well documented that NMDA receptors are involved in sensory motor gating. The increase of PPI in DAO−/− mice and enhanced response to a NMDA antagonist in DAO−/− mice might suggest an altered NMDAR function in these mice. Enhanced NMDAR function in DAO mutants was observed in several studies. For instance, DAO−/− mice showed exaggerated responses to chronic treatment of nociceptive stimuli accompanied by enhanced NMDA receptor-mediated excitatory postsynaptic currents in dorsal horn neurons in spinal cord [28]. Increased LTP in hippocampal slices [18] was also reported. Almond et al. [2] demonstrated an enhanced NMDAR function in vivo by showing that harmaline-stimulated cerebellar cGMP increase was larger in DAO−/− [2]. It was interesting to notice that SDZ 220-581, a competitive NMDA antagonist binding to the glutamate-recognition site outside the channel, also induced a stronger effect on PPI in DAO−/− mice. This may indicate that changes of NR2 subunits might occur in DAO−/− mice in addition to an enhanced occupancy of the glycine-binding site located on the NR1 subunit due to elevated extracellular d-serine level [2]. Although we observed that DAO−/− mice tended to show enhanced response to NMDA antagonists in PPI, it is worthwhile to note that other studies have reported attenuated response to MK-801- or L-701324- (a glycine site antagonist) induced effects on motor activity especially on motor coordination [2,11]. Given that the increase of d-serine in the brain of DAO−/− mice is predominantly in cerebellum [20], it is reasonable to expect a protective role of d-serine against cerebellar ataxia induced by NMDAR disruption. As a matter of fact, DAO−/− mice showed better motor coordination in a beam-walking test [2] and a recent clinical study found dcycloserine, a partial agonist at glycine site, may relieve cerebellar ataxia [21]. However, it remains unknown why both augmentation and attenuation of NMDA antagonist-induced behavioral effects have been observed in DAO−/− mice, although it can be speculated that neural circuits underlying different types of behaviors can be fundamentally different, thus leading to behavior-dependent phenotype differences. Nevertheless changes of NMDA functions in
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DAO−/− mice seem to be suggested by several lines of studies as discussed above. The Barnes Maze study showed a shortened latency to enter the escape tunnel. Although DAO−/− mice were less active than DAO+/+ when tested in activity boxes, they showed faster velocity on the Barnes Maze. Taken together, the data seem to suggest DAO−/− mice may have a better spatial navigation capacity which enabled them to find the location of the escape tunnel faster than DAO+/+. This speculation was in line with a previous study reporting DAO−/− mice performed better than DAO+/+ in water maze [18]. However the control study with the escape tunnel removed showed that DAO−/− mice spent substantially more time in the peripheral area of the maze, suggesting, in the previous Barnes Maze test, the DAO−/− mice may be faster to find the escape tunnel underneath the edge because of the increased edge activity rather than better spatial learning and memory performance. In order to tease apart the increased edge activity and enhanced spatial navigation, it would be necessary to redesign the maze to make the location of escape tunnel far away from the edge. We did attempt to test the mice in water maze for spatial learning and memory, however, the data were confounded by the fact that the mice did not readily swim, but instead showed a hypoactive response and just floated in the water. It was interesting to note that, although the mutant mice were hypoactive when tested in activity boxes, they were equally active compared to the wild-type mice when tested on an unprotected and elevated open field such as on the Barnes Maze. The increased edge activity corresponded well with predominant increase of d-serine in cerebellum and enhanced motor coordination observed in DAO−/− mice [2,20]. An alternative interpretation of increased edge activity could be DAO−/− mice were less anxious on the edge so that they spent more time over there. However, we did not observe anxiolytic-like behaviors in two anxiety models. As a matter of fact, Labrie et al. has reported an increase of anxiety-like behaviors in mice with deficient DAO activity [16]. We further profiled the mice in another cognitive assay, 24-h IA, an assay assessing memory consolidation. No genotype effect on recall was observed, although DAO−/− mice showed a significant increase of latency during the training, probably due to hypoactivity. It appears that our data did not provide incontrovertible evidence suggesting DAO−/− mice had better cognitive performance in learning and memory. However, it is possible that DAO−/− mutants may have alterations in other cognitive functions. Labrie et al. has reported that mice lacking DAO activity showed enhanced cognitive flexibility in response to changing environmental contingency, although these mice were not different from the wild-types in the initial associative learning [17]. In summary, DAO−/− mice seem to have changes in NMDA function. In DAO−/− mice, we observed enhanced PPI responses and enhanced PPI-disruptive effects of NMDA antagonists. However, we didn’t see clear genotype effects on cognition and anxiety measures. The therapeutic utility of DAO inhibition can be better characterized by using pharmacological agents in animal models. So far only limited data suggested antipsychotic-like profile following treatment with DAO inhibitors [1]. Acknowledgements We thank Dr. Michael W. Decker who made valuable suggestions to improve the manuscript. The work was supported by Abbott Laboratories. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbr.2010.09.030.
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