- Email: [email protected]
d -Cycloserine enhances spatial memory in spontaneous place recognition in rats
Neuroscience Letters 509 (2012) 13–16
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
d-Cycloserine enhances spatial memory in spontaneous place recognition in rats Takaaki Ozawa a,b , Minae Kumeji a , Kazuo Yamada a , Yukio Ichitani a,∗ a b
Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Laboratory for Neural Circuitry of Memory, RIKEN Brain Science Institute, Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 19 November 2011 Accepted 18 December 2011 Key words: NMDA receptor d-Cycloserine Spatial memory Spontaneous place recognition Rat
a b s t r a c t The effects of d-cycloserine (DCS), an exogenous partial agonist of N-methyl-d-aspartate (NMDA) receptor-associated glycine site, on spatial memory were investigated using spontaneous place recognition (SPR) test. In SPR test, rats were allowed to explore an arena where two identical objects were presented for 5 min (sample phase), and after a delay of 24 h they were placed again in the same arena but one of the two objects was moved to a novel place (test phase, 4 min). Systemic administration of DCS before the sample phase, immediately after the sample phase, and before the test phase caused rats’ significant preference for the object in a novel place in the test phase, although in this condition rats without DCS treatment did not show any preference. DCS affected neither total object exploration time nor locomotor activity in the arena during testing. Results suggest the possibility that DCS can facilitate various processes of spatial memory including encoding, consolidation and retrieval, and that NMDA receptors play an important role in these memory processes. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction N-methyl-d-aspartate (NMDA) receptors, a subclass of glutamate receptors, are one of the key neurotransmitter receptors that are involved in learning and memory in the central nervous system. It has been repeatedly confirmed that NMDA receptors are required for the induction of long-term potentiation (LTP) in the brain [7,12], which is considered as a candidate of physiological basis of learning and memory. Actually, it has been demonstrated that antagonism of NMDA receptors disrupts memory task performance in rats [9,12]. Furthermore, several studies have suggested that NMDA receptors selectively play an important role in some specific memory processes including encoding, consolidation and retrieval [19,22]. d-Cycloserine (DCS) is an exogenous partial agonist of NMDA receptor-associated glycine site which modulates the activation of NMDA receptors [8]. Previous studies have suggested that activation of the glycine site is required for the induction of hippocampal LTP in rats [20], and that the administration of DCS facilitates the hippocampal LTP in adult and aged rats [3,16]. In behavioral studies, it has been reported that administration of DCS attenuates memory deficits induced by brain lesions [17], drug treatment [6,10] and aging [2]. DCS improves learning and/or memory even in
∗ Corresponding author at: Institute of Psychology, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. Tel.: +81 29 853 6094; fax: +81 29 853 6094. E-mail address: [email protected] (Y. Ichitani). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.031
normal animals [11,15]. Depending on these cumulative evidence, DCS is expected to serve as a therapeutic agent for mental disorders accompanied by memory deficits [4,18]. A few studies have previously investigated which memory processes DCS can affect using inhibitory avoidance [11] and two-unit linear maze tasks [15]. However, in these studies, the possibility still remains that DCS influenced general anxiety, responsiveness to aversive stimuli, or rule learning ability rather than memory itself. Spontaneous place recognition (SPR) test has been developed for the assessment of spatial memory. This test takes advantage of the rats’ innate tendency to spontaneously explore objects in a novel place [5]. In the standard procedure of this task, a rat is allowed to freely explore an open-field arena with two identical objects for several minutes (sample phase). After a delay interval, the rat is returned to the arena with the same two objects, but one object is placed in a novel location while the other is in its original location (test phase). During this phase, if the rat spends more time exploring the object in a novel location, then this rat is considered to have spatial memory of the original positions of the two objects during the sample phase. This test has several advantages: (1) animals are not required to learn task rules, thus animals’ ability of spatial memory rather than the ability to acquire the rules of the task can be directly assessed. (2) Aversive stimuli are not used, thus the effects of experimental treatment on cognitive function rather than emotional and motivational aspects can be determined. (3) The mechanism of each memory process can be examined separately by the drug injection in various timings within a trial. In the present study, we investigated the effects of DCS on various processes of spatial memory in SPR test. For this purpose, we
14
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
systemically administered DCS before the sample phase, immediately after the sample phase, or before the test phase, and examined the effects on discriminative performance between two objects in the test phase.
2. Materials and methods Thirty-two male Wistar-Imamichi rats (8 weeks old) were used as subjects. Mean body weight at the start of experiment was 286 g. Rats were housed in individual cages on a 12:12 h light–dark cycle (light on: 0800–2000) with free access to food and water throughout the experiment. Animal experiments were approved by the University of Tsukuba Committee on Animal Research. All efforts were made to minimize the number of animals used and their suffering. d-Cycloserine (Sigma, MO) was freshly dissolved in saline (SAL) in the concentration of 7.5, 15 or 30 mg/ml on the day of dug test. An open-field arena (90 cm × 90 cm × 45 cm) made of polyvinyl chloride was used, and its walls were colored in black, while the floor was gray. On one of the walls, a white–black vertically striped pattern was placed as an absolute spatial cue. The objects employed were black and white triangular cast metals, black and white cylinders of cast metal, cans of juice, pink plastic cups and brown china bowls. All objects were heavy enough or fixed to a heavy metal plate so that the rat could not move them. A video camera was suspended above the arena, and the image was projected to a monitor to allow the experimenter to observe the animal’s behavior. Handling and habituation to the apparatus preceded SPR test. Rats received 5-min handling and 10-min habituation to the apparatus for 5 days. One trial of SPR test consisted of a sample phase (5 min) and a test phase (4 min), and these phases were separated by a delay interval (24 h) (Fig. 1A). In the sample phase, two identical objects were placed diagonally in the arena (the center of each object was 22.5 cm from adjacent two walls), and a rat was released in the center of the arena and left to freely explore the arena. The rat was then removed from the arena and returned to its home cage (delay interval). After the delay interval, the rat was returned to the arena (test phase). In this phase, one object was placed in the same original position as in the sample phase (object F), while the other was moved to a different position (object N), which was 30 cm away from object F and 22.5 cm from a sidewall (two locations were possible). After each phase, the floor of the arena and the objects were cleaned with 70% ethanol. Different pairs of objects and different locations in the arena were randomly used in each trial. In each phase, we measured the time rats spent exploring the objects. Exploration was defined as the rat directing its nose toward each object within a distance of 2 cm. We previously showed that sample phase and delay length affect the discrimination performance in the test phase of SPR test, and confirmed that rats did not show any preference in the test phase under the sample phase and delay length conditions we used here [14]. In addition, locomotor activity was analyzed by the program of Image OF for open field test (O’Hara & Co. Ltd., Tokyo) based on the public domain NIH Image program (developed at the U.S. National Institutes of Health). In the drug test, the effect of intraperitoneal administration of DCS (7.5, 15 or 30 mg/kg) on the performance in SPR test was examined. DCS or SAL was infused at one of three timings within a trial, 30 min before the sample phase (timing I), immediately after the sample phase (timing II), and 30 min before the test phase (timing III) (Fig. 1A). Rats were assigned to three groups based on the timing of drug infusion (timing I, n = 11; timing II, n = 10; timing III, n = 11). Each drug treatment was tested in a random order using a withinsubject design. There was a 72 h minimum interval between drug tests.
Fig. 1. Schematic drawing of the spontaneous place recognition (SPR) test (A), and effects of systemic administration of DCS on the sample phase performance of SPR test (B and C). (A) In SPR test, a white–black striped pattern was put on a sidewall as an absolute spatial cue. The sample phase (5 min) and the test phase (4 min) were separated by a delay interval of 24 h. Two identical objects were used through a trial. In the test phase, one of the objects was moved to a novel position in the arena from the sample phase. Roman numerals above each arrow show drug injection timings. I: 30 min before the sample phase; II: immediately after the sample phase; and III: 30 min before the test phase. (B) Time spent in the exploration of each object in the sample phase. “Familiar” and “Novel” mean the object that will be in the same place and the object that will be in a novel place in the test phase, respectively. (C) Total moving distance in the arena in 5 min of the sample phase. Data are presented as mean ± SEM.
In the sample and test phases, the exploration time to each object was analyzed by a three-way ANOVA for repeated measures (Object × Dose × Timing) followed by post hoc comparisons using a Bonferroni test. In the test phase, for the assessment of discrimination between two objects, the discrimination ratio (DR) was additionally calculated by dividing the amount of exploration for the object N by the total amount of exploration for the two objects (object F + N). DRs were analyzed by a two-way ANOVA for repeated measure (Dose × Timing). Further, each DR was compared to the theoretical chance level (50%) using a one-sample t-test. Total moving distance in the sample and test phases was also analyzed by a two-way ANOVA for repeated measure (Dose × Timing).
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
3. Results Exploration time of each object in the sample phase is shown in Fig. 1B. A three-way ANOVA revealed that there were no significant main effects of three factors suggesting that differences of DR under each drug condition in the test phase were not results from differences of exploration time in the sample phase. Total moving distance in the sample phase is shown in Fig. 1C. According to a two-way ANOVA, there was a significant main effect of Dose (F(3,87) = 4.94, p < 0.01), and subsequent post hoc test revealed that the moving distance under DCS 30 mg/kg condition was higher than that under the 15 mg/kg condition (p < 0.05). However, the difference was not found when SAL and other conditions were compared. Exploration time of each object in the test phase is shown in Fig. 2A. Treatment of DCS provided the expression of preference to the object in a novel place. A three-way ANOVA revealed a
15
significant main effect of Object (F(1,29) = 9.52, p < 0.01) and a significant interaction between Object and Dose conditions (F(3,87) = 2.85, p < 0.05). Subsequent post hoc test revealed that under DCS 15 mg/kg condition, exploration for the object in a novel place was higher than that for the object in a familiar place (p < 0.05). DRs in the test phase are shown in Fig. 2B. Treatment of DCS increased DRs, and led to the expression of preference. According to a two-way ANOVA, there was a significant main effect of Dose condition (F(3,87) = 6.30, p < 0.01), and a post hoc test revealed that treatment of DCS 15, 30 mg/kg increased DRs (p < 0.01 and p < 0.05, respectively). Moreover, it was found that DRs under DCS 7.5, 15 mg/kg conditions were higher than chance level in timing I (p < 0.05), and DR under DCS 15 mg/kg condition was also higher than chance level in timing II and III (p < 0.05). Total moving distance in the test phase is shown in Fig. 2C. A two-way ANOVA showed that there were no significant effects suggesting that DCS did not affect the locomotor activity in the test phase.
4. Discussion
Fig. 2. Effects of DCS on the test phase performance of SPR test. (A) Time spent exploration for each object. (B) Discrimination ratios, which were calculated by dividing the amount of exploration for the object in a novel place by the total amount of object exploration. (C) Total moving distance in the arena in 4 min of the test phase. Data are presented as mean ± SEM. *p < 0.05 vs. chance (50%).
In the present study, systemic administration of DCS before the sample phase, after the sample phase, and before the test phase all yielded significant preference to the object in a novel place in the test phase of SPR test, in which a short-sample phase and a longdelay condition was employed and thus control rats could not show any evidence for discrimination between two objects. On the other hand, DCS treatment changed neither exploration time in the sample phase nor total moving distance in the sample and test phases. These results strongly suggest that DCS improves rats’ spatial memory in SPR test without affecting exploratory behavior for objects itself and locomotor activity. In a previous study using a SPR test in mice [1], systemic treatment of DCS before the sample phase facilitated the discrimination performance in the test phase suggesting that DCS enhanced the acquisition or early stage of consolidation. Our present study, on the other hand, showed all of three timings of treatment produced better performance in the test phase. Thus, it was suggested that DCS has potential to improve multiple processes in SPR probably including encoding, consolidation, and retrieval of place information of objects in the arena. Since it has been suggested that DCS is a short acting drug with half-lives no more than 180 min [21], the effect of DCS treated before/after the sample phase (timing I and II) and before the test phase (timing III) could be assessed separately at least in this study using 24 h delay. The pre-sample phase treatment is considered to have effect on encoding and/or consolidation process. The post-sample phase treatment is assumed to have effect on consolidation or retention process. Furthermore, pre-test phase treatment reflects the effect of DCS specifically on retrieval process. Thus, the present study suggests the possibility of enhancement of all these processes by DCS. This kind of facilitative effect of DCS on broad memory processes has been reported in several studies using other memory tasks, such as passive avoidance response [11] and two-unit linear maze tasks [15]. We demonstrated that DCS could enhance various spatial memory processes in the task that does not include conditioning procedure or rule learning. The roles of NMDA receptors in each process of spatial memory have been investigated by assessing the effects of a receptor antagonist (AP5) in various spatial memory tasks including water maze and radial maze tasks [19,22]. However, conclusions on this matter have been inconsistent. In SPR test, on the other hand, the role of NMDA receptors in spatial memory processes has not been examined. In this view, the present results that post-sample phase and
16
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
pre-test phase DCS treatment resulted in appearance of preferred exploration to the novel place object suggest that NMDA receptors play important roles in various processes after consolidation of spatial memory in SPR test. Additionally, memory-enhancing effect of pre-sample phase injection of DCS indicates possibility that NMDA receptors play a role in encoding process of spatial memory as well, although it is still difficult to decide this effect was not on consolidation but on encoding process. We examined the effect of DCS using three doses, 7.5, 15, 30 mg/kg, and found that DCS improved performance in SPR test according to a reversed U-shaped dose–response curve with maximum effect of 15 mg/kg. A similar tendency of effect of DCS has been reported in previous studies [11,15]. There are some possible mechanisms that can explain this dose–response relationship of effect of DCS on memory test performance. It has been reported in vitro that although low concentration (5 and 10 M, respectively) of glycine and d-serine, endogenous co-agonists of glycine binding site of NMDA receptors, caused persistent increase of NMDA receptor-mediated field excitatory postsynaptic potential (fEPSP) amplitude in the hippocampus, these substances at high concentration (100 M) induced only a transient increase of fEPSP amplitude [23]. Furthermore, Nong et al. [13] have suggested that high concentration of glycine and d-serine (100 and 30 M, respectively) primes the internalization of hippocampal NMDA receptors. In the present study, we found that the reversed U-shaped dose–response curve could be observed in all memory processes tested, suggesting the importance of dose determination of DCS in a clinical usage. Further studies are needed to reveal its underlying mechanism. 5. Conclusions The present study suggests that DCS can facilitate various processes of spatial memory including encoding, consolidation and retrieval, and that NMDA receptors play an important role in these memory processes. Our results support utilization of DCS as a cognitive enhancer. Acknowledgment This study was supported in part by grants from Japan Society for the Promotion of Science (21530759, 21530758). References [1] F.L. Assini, M. Duzzioni, R.N. Takahashi, Object location memory in mice: pharmacological validation and further evidence of hippocampal CA1 participation, Behav. Brain Res. 204 (2009) 206–211. [2] M.G. Baxter, T.H. Lanthorn, K.M. Frick, S. Golski, R.Q. Wan, D.S. Olton, dCycloserine, a novel cognitive enhancer, improves spatial memory in aged rats, Neurobiol. Aging 15 (1994) 207–213.
[3] J.M. Billard, E. Rouaud, Deficit of NMDA receptor activation in CA1 hippocampal area of aged rats is rescued by d-cycloserine, Eur. J. Neurosci. 25 (2007) 2260–2268. [4] D.A. Butterfield, C.B. Pocernich, The glutamatergic system and Alzheimer’s disease: therapeutic implications, CNS Drugs 17 (2003) 641–652. [5] A. Ennaceur, N. Neave, J.P. Aggleton, Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulate cortices, the medial prefrontal cortex, the cingulum bundle and the fornix, Exp. Brain Res. 113 (1997) 509–519. [6] R.J. Fishkin, E.S. Ince, W.A. Carlezon Jr., R.W. Dunn, d-Cycloserine attenuates scopolamine-induced learning and memory deficits in rats, Behav. Neural Biol. 59 (1993) 150–157. [7] E.W. Harris, A.H. Ganong, C.W. Cotman, Long-term potentiation in the hippocampus involves activation of N-methyl-d-aspartate receptors, Brain Res. 323 (1984) 132–137. [8] W.F. Hood, R.P. Compton, J.B. Monahan, d-Cycloserine: a ligand for the N-methyl-d-aspartate coupled glycine receptor has partial agonist characteristics, Neurosci. Lett. 98 (1989) 91–95. [9] K. Kawabe, Y. Ichitani, T. Iwasaki, Effects of intrahippocampal AP5 treatment on radial-arm maze performance in rats, Brain Res. 781 (1998) 300–306. [10] K. Kawabe, T. Yoshihara, Y. Ichitani, T. Iwasaki, Intrahippocampal d-cycloserine improves MK-801-induced memory deficits: radial-arm maze performance in rats, Brain Res. 814 (1998) 226–230. [11] J.B. Monahan, G.E. Handelmann, W.F. Hood, A.A. Cordi, d-Cycloserine, a positive modulator of the N-methyl-d-aspartate receptor, enhances performance of learning tasks in rats, Pharmacol. Biochem. Behav. 34 (1989) 649–653. [12] R.G. Morris, Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N-methyl-daspartate receptor antagonist AP5, J. Neurosci. 9 (1989) 3040–3057. [13] Y. Nong, Y.Q. Huang, W. Ju, L.V. Kalia, G. Ahmadian, Y.T. Wang, M.W. Salter, Glycine binding primes NMDA receptor internalization, Nature 422 (2003) 302–307. [14] T. Ozawa, K. Yamada, Y. Ichitani, Long-term object location memory in rats: effects of sample phase and delay length in spontaneous place recognition test, Neurosci. Lett. 497 (2011) 37–41. [15] D. Quartermain, J. Mower, M.F. Rafferty, R.L. Herting, T.H. Lanthorn, Acute but not chronic activation of the NMDA-coupled glycine receptor with dcycloserine facilitates learning and retention, Eur. J. Pharmacol. 257 (1994) 7–12. [16] E. Rouaud, J.M. Billard, d-Cycloserine facilitates synaptic plasticity but impairs glutamatergic neurotransmission in rat hippocampal slices, Br. J. Pharmacol. 140 (2003) 1051–1056. [17] G.M. Schuster, W.J. Schmidt, d-Cycloserine reverses the working memory impairment of hippocampal-lesioned rats in a spatial learning task, Eur. J. Pharmacol. 224 (1992) 97–98. [18] S.S. Shim, M.D. Hammonds, B.S. Kee, Potentiation of the NMDA receptor in the treatment of schizophrenia: focused on the glycine site, Eur. Arch. Psychiatry Clin. Neurosci. 258 (2008) 16–27. [19] R.J. Steele, R.G. Morris, Delay-dependent impairment of a matching-to-place task with chronic and intrahippocampal infusion of the NMDA-antagonist DAP5, Hippocampus 9 (1999) 118–136. [20] E. Thiels, D.J. Weisz, T.W. Berger, In vivo modulation of N-methyl-d-aspartate receptor-dependent long-term potentiation by the glycine modulatory site, Neuroscience 46 (1992) 501–509. [21] P. Wlaz, H. Baran, W. Loscher, Effect of the glycine/NMDA receptor partial agonist, d-cycloserine, on seizure threshold and some pharmacodynamic effects of MK-801 in mice, Eur. J. Pharmacol. 257 (1994) 217–225. [22] T. Yoshihara, Y. Ichitani, Hippocampal N-methyl-d-aspartate receptormediated encoding and retrieval processes in spatial working memory: delay-interposed radial maze performance in rats, Neuroscience 129 (2004) 1–10. [23] Z. Zhang, N. Gong, W. Wang, L. Xu, T.L. Xu, Bell-shaped d-serine actions on hippocampal long-term depression and spatial memory retrieval, Cereb. Cortex 18 (2008) 2391–2401.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
d-Cycloserine enhances spatial memory in spontaneous place recognition in rats Takaaki Ozawa a,b , Minae Kumeji a , Kazuo Yamada a , Yukio Ichitani a,∗ a b
Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan Laboratory for Neural Circuitry of Memory, RIKEN Brain Science Institute, Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 19 November 2011 Accepted 18 December 2011 Key words: NMDA receptor d-Cycloserine Spatial memory Spontaneous place recognition Rat
a b s t r a c t The effects of d-cycloserine (DCS), an exogenous partial agonist of N-methyl-d-aspartate (NMDA) receptor-associated glycine site, on spatial memory were investigated using spontaneous place recognition (SPR) test. In SPR test, rats were allowed to explore an arena where two identical objects were presented for 5 min (sample phase), and after a delay of 24 h they were placed again in the same arena but one of the two objects was moved to a novel place (test phase, 4 min). Systemic administration of DCS before the sample phase, immediately after the sample phase, and before the test phase caused rats’ significant preference for the object in a novel place in the test phase, although in this condition rats without DCS treatment did not show any preference. DCS affected neither total object exploration time nor locomotor activity in the arena during testing. Results suggest the possibility that DCS can facilitate various processes of spatial memory including encoding, consolidation and retrieval, and that NMDA receptors play an important role in these memory processes. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction N-methyl-d-aspartate (NMDA) receptors, a subclass of glutamate receptors, are one of the key neurotransmitter receptors that are involved in learning and memory in the central nervous system. It has been repeatedly confirmed that NMDA receptors are required for the induction of long-term potentiation (LTP) in the brain [7,12], which is considered as a candidate of physiological basis of learning and memory. Actually, it has been demonstrated that antagonism of NMDA receptors disrupts memory task performance in rats [9,12]. Furthermore, several studies have suggested that NMDA receptors selectively play an important role in some specific memory processes including encoding, consolidation and retrieval [19,22]. d-Cycloserine (DCS) is an exogenous partial agonist of NMDA receptor-associated glycine site which modulates the activation of NMDA receptors [8]. Previous studies have suggested that activation of the glycine site is required for the induction of hippocampal LTP in rats [20], and that the administration of DCS facilitates the hippocampal LTP in adult and aged rats [3,16]. In behavioral studies, it has been reported that administration of DCS attenuates memory deficits induced by brain lesions [17], drug treatment [6,10] and aging [2]. DCS improves learning and/or memory even in
∗ Corresponding author at: Institute of Psychology, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. Tel.: +81 29 853 6094; fax: +81 29 853 6094. E-mail address: [email protected] (Y. Ichitani). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.031
normal animals [11,15]. Depending on these cumulative evidence, DCS is expected to serve as a therapeutic agent for mental disorders accompanied by memory deficits [4,18]. A few studies have previously investigated which memory processes DCS can affect using inhibitory avoidance [11] and two-unit linear maze tasks [15]. However, in these studies, the possibility still remains that DCS influenced general anxiety, responsiveness to aversive stimuli, or rule learning ability rather than memory itself. Spontaneous place recognition (SPR) test has been developed for the assessment of spatial memory. This test takes advantage of the rats’ innate tendency to spontaneously explore objects in a novel place [5]. In the standard procedure of this task, a rat is allowed to freely explore an open-field arena with two identical objects for several minutes (sample phase). After a delay interval, the rat is returned to the arena with the same two objects, but one object is placed in a novel location while the other is in its original location (test phase). During this phase, if the rat spends more time exploring the object in a novel location, then this rat is considered to have spatial memory of the original positions of the two objects during the sample phase. This test has several advantages: (1) animals are not required to learn task rules, thus animals’ ability of spatial memory rather than the ability to acquire the rules of the task can be directly assessed. (2) Aversive stimuli are not used, thus the effects of experimental treatment on cognitive function rather than emotional and motivational aspects can be determined. (3) The mechanism of each memory process can be examined separately by the drug injection in various timings within a trial. In the present study, we investigated the effects of DCS on various processes of spatial memory in SPR test. For this purpose, we
14
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
systemically administered DCS before the sample phase, immediately after the sample phase, or before the test phase, and examined the effects on discriminative performance between two objects in the test phase.
2. Materials and methods Thirty-two male Wistar-Imamichi rats (8 weeks old) were used as subjects. Mean body weight at the start of experiment was 286 g. Rats were housed in individual cages on a 12:12 h light–dark cycle (light on: 0800–2000) with free access to food and water throughout the experiment. Animal experiments were approved by the University of Tsukuba Committee on Animal Research. All efforts were made to minimize the number of animals used and their suffering. d-Cycloserine (Sigma, MO) was freshly dissolved in saline (SAL) in the concentration of 7.5, 15 or 30 mg/ml on the day of dug test. An open-field arena (90 cm × 90 cm × 45 cm) made of polyvinyl chloride was used, and its walls were colored in black, while the floor was gray. On one of the walls, a white–black vertically striped pattern was placed as an absolute spatial cue. The objects employed were black and white triangular cast metals, black and white cylinders of cast metal, cans of juice, pink plastic cups and brown china bowls. All objects were heavy enough or fixed to a heavy metal plate so that the rat could not move them. A video camera was suspended above the arena, and the image was projected to a monitor to allow the experimenter to observe the animal’s behavior. Handling and habituation to the apparatus preceded SPR test. Rats received 5-min handling and 10-min habituation to the apparatus for 5 days. One trial of SPR test consisted of a sample phase (5 min) and a test phase (4 min), and these phases were separated by a delay interval (24 h) (Fig. 1A). In the sample phase, two identical objects were placed diagonally in the arena (the center of each object was 22.5 cm from adjacent two walls), and a rat was released in the center of the arena and left to freely explore the arena. The rat was then removed from the arena and returned to its home cage (delay interval). After the delay interval, the rat was returned to the arena (test phase). In this phase, one object was placed in the same original position as in the sample phase (object F), while the other was moved to a different position (object N), which was 30 cm away from object F and 22.5 cm from a sidewall (two locations were possible). After each phase, the floor of the arena and the objects were cleaned with 70% ethanol. Different pairs of objects and different locations in the arena were randomly used in each trial. In each phase, we measured the time rats spent exploring the objects. Exploration was defined as the rat directing its nose toward each object within a distance of 2 cm. We previously showed that sample phase and delay length affect the discrimination performance in the test phase of SPR test, and confirmed that rats did not show any preference in the test phase under the sample phase and delay length conditions we used here [14]. In addition, locomotor activity was analyzed by the program of Image OF for open field test (O’Hara & Co. Ltd., Tokyo) based on the public domain NIH Image program (developed at the U.S. National Institutes of Health). In the drug test, the effect of intraperitoneal administration of DCS (7.5, 15 or 30 mg/kg) on the performance in SPR test was examined. DCS or SAL was infused at one of three timings within a trial, 30 min before the sample phase (timing I), immediately after the sample phase (timing II), and 30 min before the test phase (timing III) (Fig. 1A). Rats were assigned to three groups based on the timing of drug infusion (timing I, n = 11; timing II, n = 10; timing III, n = 11). Each drug treatment was tested in a random order using a withinsubject design. There was a 72 h minimum interval between drug tests.
Fig. 1. Schematic drawing of the spontaneous place recognition (SPR) test (A), and effects of systemic administration of DCS on the sample phase performance of SPR test (B and C). (A) In SPR test, a white–black striped pattern was put on a sidewall as an absolute spatial cue. The sample phase (5 min) and the test phase (4 min) were separated by a delay interval of 24 h. Two identical objects were used through a trial. In the test phase, one of the objects was moved to a novel position in the arena from the sample phase. Roman numerals above each arrow show drug injection timings. I: 30 min before the sample phase; II: immediately after the sample phase; and III: 30 min before the test phase. (B) Time spent in the exploration of each object in the sample phase. “Familiar” and “Novel” mean the object that will be in the same place and the object that will be in a novel place in the test phase, respectively. (C) Total moving distance in the arena in 5 min of the sample phase. Data are presented as mean ± SEM.
In the sample and test phases, the exploration time to each object was analyzed by a three-way ANOVA for repeated measures (Object × Dose × Timing) followed by post hoc comparisons using a Bonferroni test. In the test phase, for the assessment of discrimination between two objects, the discrimination ratio (DR) was additionally calculated by dividing the amount of exploration for the object N by the total amount of exploration for the two objects (object F + N). DRs were analyzed by a two-way ANOVA for repeated measure (Dose × Timing). Further, each DR was compared to the theoretical chance level (50%) using a one-sample t-test. Total moving distance in the sample and test phases was also analyzed by a two-way ANOVA for repeated measure (Dose × Timing).
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
3. Results Exploration time of each object in the sample phase is shown in Fig. 1B. A three-way ANOVA revealed that there were no significant main effects of three factors suggesting that differences of DR under each drug condition in the test phase were not results from differences of exploration time in the sample phase. Total moving distance in the sample phase is shown in Fig. 1C. According to a two-way ANOVA, there was a significant main effect of Dose (F(3,87) = 4.94, p < 0.01), and subsequent post hoc test revealed that the moving distance under DCS 30 mg/kg condition was higher than that under the 15 mg/kg condition (p < 0.05). However, the difference was not found when SAL and other conditions were compared. Exploration time of each object in the test phase is shown in Fig. 2A. Treatment of DCS provided the expression of preference to the object in a novel place. A three-way ANOVA revealed a
15
significant main effect of Object (F(1,29) = 9.52, p < 0.01) and a significant interaction between Object and Dose conditions (F(3,87) = 2.85, p < 0.05). Subsequent post hoc test revealed that under DCS 15 mg/kg condition, exploration for the object in a novel place was higher than that for the object in a familiar place (p < 0.05). DRs in the test phase are shown in Fig. 2B. Treatment of DCS increased DRs, and led to the expression of preference. According to a two-way ANOVA, there was a significant main effect of Dose condition (F(3,87) = 6.30, p < 0.01), and a post hoc test revealed that treatment of DCS 15, 30 mg/kg increased DRs (p < 0.01 and p < 0.05, respectively). Moreover, it was found that DRs under DCS 7.5, 15 mg/kg conditions were higher than chance level in timing I (p < 0.05), and DR under DCS 15 mg/kg condition was also higher than chance level in timing II and III (p < 0.05). Total moving distance in the test phase is shown in Fig. 2C. A two-way ANOVA showed that there were no significant effects suggesting that DCS did not affect the locomotor activity in the test phase.
4. Discussion
Fig. 2. Effects of DCS on the test phase performance of SPR test. (A) Time spent exploration for each object. (B) Discrimination ratios, which were calculated by dividing the amount of exploration for the object in a novel place by the total amount of object exploration. (C) Total moving distance in the arena in 4 min of the test phase. Data are presented as mean ± SEM. *p < 0.05 vs. chance (50%).
In the present study, systemic administration of DCS before the sample phase, after the sample phase, and before the test phase all yielded significant preference to the object in a novel place in the test phase of SPR test, in which a short-sample phase and a longdelay condition was employed and thus control rats could not show any evidence for discrimination between two objects. On the other hand, DCS treatment changed neither exploration time in the sample phase nor total moving distance in the sample and test phases. These results strongly suggest that DCS improves rats’ spatial memory in SPR test without affecting exploratory behavior for objects itself and locomotor activity. In a previous study using a SPR test in mice [1], systemic treatment of DCS before the sample phase facilitated the discrimination performance in the test phase suggesting that DCS enhanced the acquisition or early stage of consolidation. Our present study, on the other hand, showed all of three timings of treatment produced better performance in the test phase. Thus, it was suggested that DCS has potential to improve multiple processes in SPR probably including encoding, consolidation, and retrieval of place information of objects in the arena. Since it has been suggested that DCS is a short acting drug with half-lives no more than 180 min [21], the effect of DCS treated before/after the sample phase (timing I and II) and before the test phase (timing III) could be assessed separately at least in this study using 24 h delay. The pre-sample phase treatment is considered to have effect on encoding and/or consolidation process. The post-sample phase treatment is assumed to have effect on consolidation or retention process. Furthermore, pre-test phase treatment reflects the effect of DCS specifically on retrieval process. Thus, the present study suggests the possibility of enhancement of all these processes by DCS. This kind of facilitative effect of DCS on broad memory processes has been reported in several studies using other memory tasks, such as passive avoidance response [11] and two-unit linear maze tasks [15]. We demonstrated that DCS could enhance various spatial memory processes in the task that does not include conditioning procedure or rule learning. The roles of NMDA receptors in each process of spatial memory have been investigated by assessing the effects of a receptor antagonist (AP5) in various spatial memory tasks including water maze and radial maze tasks [19,22]. However, conclusions on this matter have been inconsistent. In SPR test, on the other hand, the role of NMDA receptors in spatial memory processes has not been examined. In this view, the present results that post-sample phase and
16
T. Ozawa et al. / Neuroscience Letters 509 (2012) 13–16
pre-test phase DCS treatment resulted in appearance of preferred exploration to the novel place object suggest that NMDA receptors play important roles in various processes after consolidation of spatial memory in SPR test. Additionally, memory-enhancing effect of pre-sample phase injection of DCS indicates possibility that NMDA receptors play a role in encoding process of spatial memory as well, although it is still difficult to decide this effect was not on consolidation but on encoding process. We examined the effect of DCS using three doses, 7.5, 15, 30 mg/kg, and found that DCS improved performance in SPR test according to a reversed U-shaped dose–response curve with maximum effect of 15 mg/kg. A similar tendency of effect of DCS has been reported in previous studies [11,15]. There are some possible mechanisms that can explain this dose–response relationship of effect of DCS on memory test performance. It has been reported in vitro that although low concentration (5 and 10 M, respectively) of glycine and d-serine, endogenous co-agonists of glycine binding site of NMDA receptors, caused persistent increase of NMDA receptor-mediated field excitatory postsynaptic potential (fEPSP) amplitude in the hippocampus, these substances at high concentration (100 M) induced only a transient increase of fEPSP amplitude [23]. Furthermore, Nong et al. [13] have suggested that high concentration of glycine and d-serine (100 and 30 M, respectively) primes the internalization of hippocampal NMDA receptors. In the present study, we found that the reversed U-shaped dose–response curve could be observed in all memory processes tested, suggesting the importance of dose determination of DCS in a clinical usage. Further studies are needed to reveal its underlying mechanism. 5. Conclusions The present study suggests that DCS can facilitate various processes of spatial memory including encoding, consolidation and retrieval, and that NMDA receptors play an important role in these memory processes. Our results support utilization of DCS as a cognitive enhancer. Acknowledgment This study was supported in part by grants from Japan Society for the Promotion of Science (21530759, 21530758). References [1] F.L. Assini, M. Duzzioni, R.N. Takahashi, Object location memory in mice: pharmacological validation and further evidence of hippocampal CA1 participation, Behav. Brain Res. 204 (2009) 206–211. [2] M.G. Baxter, T.H. Lanthorn, K.M. Frick, S. Golski, R.Q. Wan, D.S. Olton, dCycloserine, a novel cognitive enhancer, improves spatial memory in aged rats, Neurobiol. Aging 15 (1994) 207–213.
[3] J.M. Billard, E. Rouaud, Deficit of NMDA receptor activation in CA1 hippocampal area of aged rats is rescued by d-cycloserine, Eur. J. Neurosci. 25 (2007) 2260–2268. [4] D.A. Butterfield, C.B. Pocernich, The glutamatergic system and Alzheimer’s disease: therapeutic implications, CNS Drugs 17 (2003) 641–652. [5] A. Ennaceur, N. Neave, J.P. Aggleton, Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulate cortices, the medial prefrontal cortex, the cingulum bundle and the fornix, Exp. Brain Res. 113 (1997) 509–519. [6] R.J. Fishkin, E.S. Ince, W.A. Carlezon Jr., R.W. Dunn, d-Cycloserine attenuates scopolamine-induced learning and memory deficits in rats, Behav. Neural Biol. 59 (1993) 150–157. [7] E.W. Harris, A.H. Ganong, C.W. Cotman, Long-term potentiation in the hippocampus involves activation of N-methyl-d-aspartate receptors, Brain Res. 323 (1984) 132–137. [8] W.F. Hood, R.P. Compton, J.B. Monahan, d-Cycloserine: a ligand for the N-methyl-d-aspartate coupled glycine receptor has partial agonist characteristics, Neurosci. Lett. 98 (1989) 91–95. [9] K. Kawabe, Y. Ichitani, T. Iwasaki, Effects of intrahippocampal AP5 treatment on radial-arm maze performance in rats, Brain Res. 781 (1998) 300–306. [10] K. Kawabe, T. Yoshihara, Y. Ichitani, T. Iwasaki, Intrahippocampal d-cycloserine improves MK-801-induced memory deficits: radial-arm maze performance in rats, Brain Res. 814 (1998) 226–230. [11] J.B. Monahan, G.E. Handelmann, W.F. Hood, A.A. Cordi, d-Cycloserine, a positive modulator of the N-methyl-d-aspartate receptor, enhances performance of learning tasks in rats, Pharmacol. Biochem. Behav. 34 (1989) 649–653. [12] R.G. Morris, Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N-methyl-daspartate receptor antagonist AP5, J. Neurosci. 9 (1989) 3040–3057. [13] Y. Nong, Y.Q. Huang, W. Ju, L.V. Kalia, G. Ahmadian, Y.T. Wang, M.W. Salter, Glycine binding primes NMDA receptor internalization, Nature 422 (2003) 302–307. [14] T. Ozawa, K. Yamada, Y. Ichitani, Long-term object location memory in rats: effects of sample phase and delay length in spontaneous place recognition test, Neurosci. Lett. 497 (2011) 37–41. [15] D. Quartermain, J. Mower, M.F. Rafferty, R.L. Herting, T.H. Lanthorn, Acute but not chronic activation of the NMDA-coupled glycine receptor with dcycloserine facilitates learning and retention, Eur. J. Pharmacol. 257 (1994) 7–12. [16] E. Rouaud, J.M. Billard, d-Cycloserine facilitates synaptic plasticity but impairs glutamatergic neurotransmission in rat hippocampal slices, Br. J. Pharmacol. 140 (2003) 1051–1056. [17] G.M. Schuster, W.J. Schmidt, d-Cycloserine reverses the working memory impairment of hippocampal-lesioned rats in a spatial learning task, Eur. J. Pharmacol. 224 (1992) 97–98. [18] S.S. Shim, M.D. Hammonds, B.S. Kee, Potentiation of the NMDA receptor in the treatment of schizophrenia: focused on the glycine site, Eur. Arch. Psychiatry Clin. Neurosci. 258 (2008) 16–27. [19] R.J. Steele, R.G. Morris, Delay-dependent impairment of a matching-to-place task with chronic and intrahippocampal infusion of the NMDA-antagonist DAP5, Hippocampus 9 (1999) 118–136. [20] E. Thiels, D.J. Weisz, T.W. Berger, In vivo modulation of N-methyl-d-aspartate receptor-dependent long-term potentiation by the glycine modulatory site, Neuroscience 46 (1992) 501–509. [21] P. Wlaz, H. Baran, W. Loscher, Effect of the glycine/NMDA receptor partial agonist, d-cycloserine, on seizure threshold and some pharmacodynamic effects of MK-801 in mice, Eur. J. Pharmacol. 257 (1994) 217–225. [22] T. Yoshihara, Y. Ichitani, Hippocampal N-methyl-d-aspartate receptormediated encoding and retrieval processes in spatial working memory: delay-interposed radial maze performance in rats, Neuroscience 129 (2004) 1–10. [23] Z. Zhang, N. Gong, W. Wang, L. Xu, T.L. Xu, Bell-shaped d-serine actions on hippocampal long-term depression and spatial memory retrieval, Cereb. Cortex 18 (2008) 2391–2401.