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Intrahippocampal administration of a glycine site antagonist impairs working memory performance of rats
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European Journal of Pharmacology 253 (1994) 183-187
Short communication
Intrahippocampal administration of a glycine site antagonist impairs working memory performance of rats Masuo Ohno *, Tsuneyuki Yamamoto, Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812, Japan (Received 13 December 1993; accepted 28 December 1993)
Abstract
The effects of 7-chlorokynurenic acid, an antagonist at the glycine site associated with the NMDA receptor/channel complex, on memory functions were investigated using a three-panel runway task. In a working memory task, 7-chlorokynurenic acid at a dose of 3.2/zg/side, injected bilaterally into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). The working memory deficit induced by intrahippocampal 7-chlorokynurenic acid (3.2 /~g/side) was reversed by concurrent injection (32 /zg/side) of D-serine, the glycine site agonist. In a reference memory task, 7-chlorokynurenic acid had no effect on the number of errors when injected into the hippocampus at doses up to 3.2/~g/side. These results suggest that activation of the hippocampal glycine site coupled to the NMDA receptor is required for working memory function in rats. Key words: Glycine site; 7-Chlorokynurenic acid; Hippocampus; Working memory; Reference memory
1. Introduction
It is well documented that N M D A receptors play a crucial role in the induction of long-term potentiation in the hippocampus, which is hypothesized to be a neural basis of memory formation (Collingridge and Bliss, 1987). The N M D A r e c e p t o r / c h a n n e l complex contains a strychnine-insensitive glycine modulatory site; glycine is essential for activation of N M D A receptors (Johnson and Ascher, 1987; Thomson, 1990). Some investigators recently reported that the glycine site selective antagonist 7-chlorokynurenic acid blocked the induction of hippocampal long-term potentiation, and that this effect of 7-chlorokynurenic acid was attenuated by co-application of glycine or the glycine site agonist D-serine, suggesting the requirement of coactivation of the glycine site on the N M D A receptor complex in this phenomenon (Bashir et al., 1990; Izumi et al., 1990; Oliver et al., 1990; Watanabe et al., 1992).
* Corresponding author. Fax (81) (92) 632-2752. Elsevier Science B.V.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 0 0 9 - V
In contrast to considerable evidence that processes mediated by hippocampal N M D A receptors are involved in learning and memory (Morris et al., 1989; Davis et al., 1992; Ohno et al., 1992, 1993), there are few behavioral studies concerning the role of the glycine site in memory functions (Watanabe et al., 1992). It has been proposed that there are two different aspects of memory functions in experimental animals: working memory and reference memory (Olton et al., 1979). Working memory allows animals to remember information that is useful for a single session of an experiment but not for subsequent sessions, whereas reference memory is defined as the holding of information that is of continued value across all sessions. We previously reported that a three-panel runway task allows us to distinguish between working and reference memory, and thus provides a means to determine the mnemonic specificity of any performance change observed (Ohno et al., 1992, 1993). In this study, we examined the effects of intrahippocampal injection of 7-chlorokynurenic acid on working and reference memory in the three-panel runway task, to clarify the role of hippocampal glycine sites in these behaviors.
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M. Ohno et al. / European Journal of Pharmacology 253 (1994) 183-187
2. Materials and methods
2.3. Surgery and experimental procedures
2.1. Animals
The rats that achieved the learning criterion were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and were implanted bilaterally with guide cannulae for microinjection of drugs into the hippocampus, as described previously (Ohno et al., 1992). The position of the injection cannula tip, which protruded 1.0 mm below the tip of the guide cannula, was aimed at the dorsal hippocampus (3.8 mm posterior to the bregrna, 2.2 mm lateral to the midline, 3.2 mm ventral to the surface of the skull measured at the bregma) according to the brain atlas of Paxinos and Watson (1982). The rats were allowed at least 5 days of postoperative recovery before runway sessions were resumed. The rats were used after it was confirmed that they met the learning criterion after the surgical manipulations. 7-Chlorokynurenic acid (Sigma Chemical Co.) was dissolved in 0.1 N NaOH solution, after which the pH was adjusted to approximately 7.4 with an appropriate amount of HCI. D-Serine (Sigma Chemical Co.) was dissolved in saline. Two microliters of drug solution or saline was injected into the dorsal hippocampus through the injection cannula, which was connected to a 5-/zl Hamilton syringe via a polyethylene tube. The rate of injection was 0.5 /xl/min. The injection cannula was left in place for 1 min after completion of the injection to facilitate diffusion of the drug. The runway test was given 10 min after the drug injection. When microinjections were made repeatedly into a single rat, a minimum of 3 days was allowed between microinjections. Performance in the three-panel runway task during non-injected sessions was not affected by repeated intrahippocampal injections and the rats met the learning criterion.
Eight- to ten-week-old male rats of the Wistar strain were obtained from Japan SLC. The initial free-feeding weights were 230-250 g, but the rats were placed on a deprivation schedule to maintain their weights at approximately 80% of their weights before the experiment. The rats were housed in groups of four per cage at a constant temperature (23 + 2°C), with a 12-h light-dark cycle (light period: 07:00-19:00 h), and with water freely available. 2.2. Three-panel runway task Working memory and reference memory were assessed with a three-panel runway set-up, as described in our previous reports (Ohno et al., 1992, 1993). In brief, this apparatus (175 x 36 x 25 cm) was composed of a start box, a goal box and four consecutive choice points between them. Each choice point consisted of a gate with three panels (12 × 25 cm). The rats Were prevented from passing through two of the three panels in the gate by front stoppers; they were prevented from returning to the start box or to a previous choice point by rear stoppers fixed to each of the panels in all the gates. When the rats reached the goal box, they received two food pellets (about 50 mg each; Muromachi Kikai) as positive reinforcement. The rats were made to run the task in six consecutive trials (defined as one session) per day with removal of the front stopper of only one of the three panel-gates (the correct panel-gate) at each choice point. Trials were run at 2-min intervals, and water was freely available between trials in the home cage. Separate groups of animals were used for the working and reference memory experiments. In the working memory procedure, the locations of the correct panel-gates were held constant within a session, but were changed from one session to the next. Twelve different patterns of correct panel-gate locations were used, as described previously (Ohno et al., 1993). In the reference memory procedure, the correct panel-gate locations were kept constant, both within a session and in succeeding sessions. The number of times an animal attempted to pass through an incorrect panel-gate (defined as errors) and the time required for the animal to obtain food pellets (defined as latency) were recorded for each rat during each trial of a session. The learning criterion was fewer than 12 and fewer than 6 errors summed across the six trials of a session in the working and reference memory tasks, respectively. A rat was used in the experiment if it achieved this criterion throughout three consecutive sessions.
2. 4. Histology
After completion of the experiment, each rat was deeply anesthetized with ether and then perfused with saline followed by 10% formalin through the left cardiac ventricle. After the brain was removed, 50-/zm thick sections were stained with Cresyl violet to verify the injection site histologically, as described previously (Ohno et al., 1992). The stained sections showed that the tips of the injection cannulae were located in the dorsal hippocampus in all cases. Several animals were injected with 2 /~l of Cresyl violet dye in order to estimate the extent of dye diffusion. Ten minutes after this microinjection, the rats were anesthetized and prepared for cardiac perfusion. After the brain was removed rapidly and sliced, the diffusion of dye was confirmed by visual inspection. The dye was confined to the dorsal hippocampus, ranging from 2.8 to 4.8 mm posterior to the bregma.
M. Ohno et aL / European Journal of Pharmacology 253 (1994) 183-187
185
30"
2.5. Data analysis
25-
In the working memory task, the number of errors and the latency summed from the second to the sixth trial of a session were important for evaluating the ability of rats to remember new correct panel-gate locations. Thus, these parameters are presented separately from those recorded in the first trial. In the reference memory task, both these parameters were summed across all six trials of a session since this task was given in order to evaluate the ability of rats to retain the constant location of correct panel-gates. The significance of the differences between the groups was determined by a one-way analysis of variance (ANOVA) followed by Dunnett's test when F ratios reached significance ( P < 0.05).
.o 20"6
15" 10"
Z
50.32
1.0
Saline
32
3.2
7-Chlorokynurenic acid D-Serine 32 pgl,~de
100-
~6 o - ~
*
80-
8 40-
3. Results
In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. In the working memory task, the number of errors made from the second to the sixth trial (working memory errors) markedly decreased with repeated training, whereas the errors in the first trial remained constant at approximately four. Approximately 20-30 training sessions were required for the rats to reach the criterion of fewer than 12 errors summed across the six trials of a session. Latency was also reduced during repeated sessions and was stable from the 10th session on. In the reference memory task, the number of errors and latency in all six trials of a session decreased with repeated training. The rats performed the task within the six-error criterion summed across six trials after they had about 10 training sessions. 7-Chlorokynurenic acid at doses of 0.32-3.2 /zg/side, administered bilaterally into the dorsal hippocampus, increased the number of errors in the working memory task ( F ( 3 , 1 8 ) = 9.12, P < 0.01), an effect that reached significance for the 3.2 /zg/side dose, while it had no effect on the number of errors made in the first trial (Fig. 1). The latency recorded in the first trial (F(3,18) = 4.91, P < 0.05) and that recorded from the second to the sixth trial of a session (F(3,18) = 3.74, P < 0.05) were significantly prolonged when rats were given intrahippocampal 7-chlorokynurenic acid at 3.2 /zg/side. Intrahippocampal injection of 32 /~g/side D-serine by itself did not affect the number of working memory errors (7.0 + 1.7, n = 4) or the latencies recorded in the first trial and from the second to sixth trial (9.8 + 1.0 s and 28.8 + 3.5 s, respectively). The increase in working memory errors induced by intrahippocampal 7-chlorokynurenic acid at 3 . 2 / z g / s i d e was significantly attenuated by concurrent injection of D-serine at 32 tzg/side (F(1,8) = 6.89, P < 0.05).
200 0.32
1.0
Saline
3.2
3.2
/~g/side
7-Chlorokynurenic acid D-Serine 32/~J/Side
Fig. 1. Effects of intrahippocampal 7-chlorokynurenic acid injection on the number of errors and latency in a test of working memory. T~e runway test was given 10 rain after drugs were administered. o-Scrine was co-injected intrahippocampally with 7-chlorokynurenic acid. Each column represents the mean + S.E. of errors and latcncies for 5 - 6 animals recorded in the first trial (open columns) and those summed from the second to the sixth trial of a session (hatched columns). The significance of the differences from the saline-injected group (* P < 0.05, ** P < 0.01) and from the 3.2 ~ g / s i d e 7-chlorokynurenic acid-injected group (# P < 0.05) was determined by means of a one-way A N O V A followed by Dunnett's test.
In the reference memory task, intrahippocampal injection of 7-chlorokynurenic acid at doses up to 3.2 /zg/side had no effect on the number of errors or on the latency across all six trials of a session (Table 1).
Table 1 Effects of intrahippocampal 7-chlorokynurenic acid injection on the number of errors and latency in a test of reference memory Drug
/zg/ side
n
Number of errors Trial 1-6
Latency (s)
Saline 7-Chlorokynurenic acid
1.0 3.2
5 5 5
2.2 + 1.0 4.2 + 1.5 3.4 + 1.2
37.8 + 5.6 36.2 + 3.0 38.4 + 2.5
Trial 1-6
The runway test was given 10 min after 7-chlorokynurenic acid was administered. Values are m e a n s + S . E , of errors and latencies summed across all six trials of a session.
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M. Ohno et al. / European Journal of Pharmacology 253 (1994) 183-187
4. D i s c u s s i o n
Recently, Watanabe et al. (1992) reported that intracerebroventricular injection of 7-chlorokynurenic acid, an antagonist at the glycine site associated with the N M D A receptor complex, before daily training impaired the acquisition of spatial memory in the Morris water maze, which is highly sensitive to hippocampal damage (Morris et al., 1982). These findings suggest that the glycine site in the hippocampus may be involved in spatial learning. Working memory performance in the three-panel runway task is also sensitive to disruption by hippocampal lesions (Kitajima et al., 1992) and is thus suitable for the investigation of memory processes mediated by this structure. The present study clearly showed that microinjection of 7-chlorokynurenic acid into the hippocampus was sufficient to impair working memory in this task. That blockade of hippocampal glycine sites was responsible for the effect of 7-chlorokynurenic acid was confirmed by the result that the working memory deficit following intrahippocampal 7-chlorokynurenic acid was reversed by concurrent injection of D-serine, a glycine site agohist. Therefore, it is conceivable that the hippocampal glycine site is involved in working memory performance, i.e., acquisition of new and variable information that is useful for only one session. In contrast, intrahippocampal injection of 7-chlorokynurenic acid did not affect reference memory, which is resistant to hippocampal lesions (Kitajima et al., 1992). Thus, it is suggested that the hippocampal glycine site does not contribute to reference memory performance, i.e., retention and retrieval of previously acquired constant information. On the other hand, both working and reference memory tasks require equal motor performance and motivational and perceptual abilities. Since no effects on reference memory performance were found, we can exclude the possibility that the deficit in working memory performance following intrahippocampal 7-chlorokynurenic acid injection is related to an alteration of non-mnemonic functions. The distribution of glycine binding sites is similar to that of N M D A receptors in the brain, and the hippocampus is one of the structures that contain the greatest density of both binding sites (Bristow et al., 1986; Thomson, 1990). Activation of N M D A receptors is an absolute requirement for the induction of hippocampal long-term potentiation, a cellular model of learning and memory in the mammalian nervous system (Collingridge and Bliss, 1987). Morris et al. (1989) and Davis et al. (1992) have reported that intracerebroventricular and intrahippocampal infusion of the N M D A receptor antagonist 2-amino-5-phosphonovaleric acid (AP5) impairs spatial learning in the water maze across a dose range comparable to that required to block induction of hippocampal long-term potentia-
tion, suggesting a close link between long-term potentiation and memory processing in the brain. We also reported that intrahippocampal injections of N M D A receptor antagonists, such as AP5, CGS 19755 (c/s-4phosphonomethyl-2-piperidine carboxylic acid) and 3[( ± )-2-carboxypiperazin-4yl]propyl-1-phosphonic acid (CPP), impaired working memory without affecting reference memory in the three-panel runway task, suggesting an important role of hippocampal N M D A receptor-dependent mechanisms in working memory (Ohno et al., 1992). Agonist action at the glycine regulatory site is required for opening of the N M D A receptor channel (Johnson and Ascher, 1987; Thomson, 1990) and is also essential for induction of hippocampal long-term potentiation since 7-chlorokynurenic acid attenuates long-term potentiation in rat hippocampal slices (Bashir et al., 1990; Izumi et al., 1990; Oliver et al., 1990; Watanabe et al., 1992). Thus, interruption of hippocampal N M D A receptor-mediated processes accounts for the working memory deficit induced by blockade of hippocampal glycine sites. The normal concentration of brain glycine is generally thought to exceed the concentration required to saturate the glycine site on the N M D A receptor complex (Thomson, 1990). Therefore, it is not certain that hippocampal glycine by itself plays a modulatory role in memory processes, although support by endogeneous glycine of hippocampal N M D A receptor activation is necessary for working memory performance.
5. A c k n o w l e d g e m e n t
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
6. References
Bashir, Z.I., B. Tam and G.L. Collingridge, 1990, Activation of the glycine site in the NMDA receptor is necessaryfor the induction of LTP, Neurosci. Lett. 108, 261. Bristow, D.R., N.G. Boweryand G.N. Woodruff, 1986, Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain, Eur. J. Pharmacol. 126, 303. Collingridge, G.L. and T.V.P. Bliss, 1987, NMDA receptors - their role in long-term potentiation, Trends Neurosci. 10, 288. Davis, S., S.P. Butcher and R.G.M. Morris, 1992, The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate(D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro, J. Neurosci. 12, 21. Izumi, Y., D.B. Clifford and C.F. Zorumski, 1990, Glycine antagonists block the induction of long-term potentiation in CA1 of rat hippocampal slices, Neurosci. Lett. 112, 251. Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature 325, 529.
M. Ohno et al. / European Journal of Pharmacology 253 (1994) 183-187 Kitajima, I., T. Yamamoto, M. Ohno and S. Ueki, 1992, Working and reference memory in rats in the three-panel runway task following dorsal hippocampal lesions, Jpn. J. Pharmacol. 58, 175. Morris, R.G.M., P. Garrud, J.N.P. Rawlins and J. O'Keefe, 1982, Place navigation impaired in rats with hippocampal lesions, Nature 297, 681. Morris, R.G.M., R.F. Halliwell and N. Bowery, 1989, Synaptic plasticity and leaning. II: Do different kinds of plasticity underlie different kinds of learning?, Neuropsychologia 27, 41. Ohno, M., T. Yamamoto and S. Watanabe, 1992, Effects of intrahippocampal injections of N-methyl-D-aspartate receptor antagonists and scopolamine on working and reference memory assessed in rats by a three-panel runway task, J. Pharmacol. Exp. Ther. 263, 943. Ohno, M., T. Yamamoto and S. Watanabe, 1993, Amygdaloid NMDA
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and muscarinic receptors involved in working memory performance of rats, Physiol. Behav. 54, 993. Oliver, M.W., M. Kessler, J. Larson, F. Schottler and G. Lynch, 1990, Glycine site associated with the NMDA receptor modulates long-term potentiation, Synapse 5, 265. Olton, D.S., J.T. Becker and G.E. Handelmann, 1979, Hippocampus, space, and memory, Behav. Brain Sci. 2, 313. Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotaxic Coordinates (Academic Press, New York). Thomson, A.M., 1990, Glycine is a coagonist at the NMDA receptor/channel complex, Prog. Neurobiol. 35, 53. Watanabe, Y., T. Himi, H. Saito and K. Abe, 1992, Involvement of glycine site associated with the NMDA receptor in hippocampal long-term potentiation and acquisition of spatial memory in rats, Brain Res. 582, 58.
European Journal of Pharmacology 253 (1994) 183-187
Short communication
Intrahippocampal administration of a glycine site antagonist impairs working memory performance of rats Masuo Ohno *, Tsuneyuki Yamamoto, Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812, Japan (Received 13 December 1993; accepted 28 December 1993)
Abstract
The effects of 7-chlorokynurenic acid, an antagonist at the glycine site associated with the NMDA receptor/channel complex, on memory functions were investigated using a three-panel runway task. In a working memory task, 7-chlorokynurenic acid at a dose of 3.2/zg/side, injected bilaterally into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). The working memory deficit induced by intrahippocampal 7-chlorokynurenic acid (3.2 /~g/side) was reversed by concurrent injection (32 /zg/side) of D-serine, the glycine site agonist. In a reference memory task, 7-chlorokynurenic acid had no effect on the number of errors when injected into the hippocampus at doses up to 3.2/~g/side. These results suggest that activation of the hippocampal glycine site coupled to the NMDA receptor is required for working memory function in rats. Key words: Glycine site; 7-Chlorokynurenic acid; Hippocampus; Working memory; Reference memory
1. Introduction
It is well documented that N M D A receptors play a crucial role in the induction of long-term potentiation in the hippocampus, which is hypothesized to be a neural basis of memory formation (Collingridge and Bliss, 1987). The N M D A r e c e p t o r / c h a n n e l complex contains a strychnine-insensitive glycine modulatory site; glycine is essential for activation of N M D A receptors (Johnson and Ascher, 1987; Thomson, 1990). Some investigators recently reported that the glycine site selective antagonist 7-chlorokynurenic acid blocked the induction of hippocampal long-term potentiation, and that this effect of 7-chlorokynurenic acid was attenuated by co-application of glycine or the glycine site agonist D-serine, suggesting the requirement of coactivation of the glycine site on the N M D A receptor complex in this phenomenon (Bashir et al., 1990; Izumi et al., 1990; Oliver et al., 1990; Watanabe et al., 1992).
* Corresponding author. Fax (81) (92) 632-2752. Elsevier Science B.V.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 0 0 9 - V
In contrast to considerable evidence that processes mediated by hippocampal N M D A receptors are involved in learning and memory (Morris et al., 1989; Davis et al., 1992; Ohno et al., 1992, 1993), there are few behavioral studies concerning the role of the glycine site in memory functions (Watanabe et al., 1992). It has been proposed that there are two different aspects of memory functions in experimental animals: working memory and reference memory (Olton et al., 1979). Working memory allows animals to remember information that is useful for a single session of an experiment but not for subsequent sessions, whereas reference memory is defined as the holding of information that is of continued value across all sessions. We previously reported that a three-panel runway task allows us to distinguish between working and reference memory, and thus provides a means to determine the mnemonic specificity of any performance change observed (Ohno et al., 1992, 1993). In this study, we examined the effects of intrahippocampal injection of 7-chlorokynurenic acid on working and reference memory in the three-panel runway task, to clarify the role of hippocampal glycine sites in these behaviors.
184
M. Ohno et al. / European Journal of Pharmacology 253 (1994) 183-187
2. Materials and methods
2.3. Surgery and experimental procedures
2.1. Animals
The rats that achieved the learning criterion were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and were implanted bilaterally with guide cannulae for microinjection of drugs into the hippocampus, as described previously (Ohno et al., 1992). The position of the injection cannula tip, which protruded 1.0 mm below the tip of the guide cannula, was aimed at the dorsal hippocampus (3.8 mm posterior to the bregrna, 2.2 mm lateral to the midline, 3.2 mm ventral to the surface of the skull measured at the bregma) according to the brain atlas of Paxinos and Watson (1982). The rats were allowed at least 5 days of postoperative recovery before runway sessions were resumed. The rats were used after it was confirmed that they met the learning criterion after the surgical manipulations. 7-Chlorokynurenic acid (Sigma Chemical Co.) was dissolved in 0.1 N NaOH solution, after which the pH was adjusted to approximately 7.4 with an appropriate amount of HCI. D-Serine (Sigma Chemical Co.) was dissolved in saline. Two microliters of drug solution or saline was injected into the dorsal hippocampus through the injection cannula, which was connected to a 5-/zl Hamilton syringe via a polyethylene tube. The rate of injection was 0.5 /xl/min. The injection cannula was left in place for 1 min after completion of the injection to facilitate diffusion of the drug. The runway test was given 10 min after the drug injection. When microinjections were made repeatedly into a single rat, a minimum of 3 days was allowed between microinjections. Performance in the three-panel runway task during non-injected sessions was not affected by repeated intrahippocampal injections and the rats met the learning criterion.
Eight- to ten-week-old male rats of the Wistar strain were obtained from Japan SLC. The initial free-feeding weights were 230-250 g, but the rats were placed on a deprivation schedule to maintain their weights at approximately 80% of their weights before the experiment. The rats were housed in groups of four per cage at a constant temperature (23 + 2°C), with a 12-h light-dark cycle (light period: 07:00-19:00 h), and with water freely available. 2.2. Three-panel runway task Working memory and reference memory were assessed with a three-panel runway set-up, as described in our previous reports (Ohno et al., 1992, 1993). In brief, this apparatus (175 x 36 x 25 cm) was composed of a start box, a goal box and four consecutive choice points between them. Each choice point consisted of a gate with three panels (12 × 25 cm). The rats Were prevented from passing through two of the three panels in the gate by front stoppers; they were prevented from returning to the start box or to a previous choice point by rear stoppers fixed to each of the panels in all the gates. When the rats reached the goal box, they received two food pellets (about 50 mg each; Muromachi Kikai) as positive reinforcement. The rats were made to run the task in six consecutive trials (defined as one session) per day with removal of the front stopper of only one of the three panel-gates (the correct panel-gate) at each choice point. Trials were run at 2-min intervals, and water was freely available between trials in the home cage. Separate groups of animals were used for the working and reference memory experiments. In the working memory procedure, the locations of the correct panel-gates were held constant within a session, but were changed from one session to the next. Twelve different patterns of correct panel-gate locations were used, as described previously (Ohno et al., 1993). In the reference memory procedure, the correct panel-gate locations were kept constant, both within a session and in succeeding sessions. The number of times an animal attempted to pass through an incorrect panel-gate (defined as errors) and the time required for the animal to obtain food pellets (defined as latency) were recorded for each rat during each trial of a session. The learning criterion was fewer than 12 and fewer than 6 errors summed across the six trials of a session in the working and reference memory tasks, respectively. A rat was used in the experiment if it achieved this criterion throughout three consecutive sessions.
2. 4. Histology
After completion of the experiment, each rat was deeply anesthetized with ether and then perfused with saline followed by 10% formalin through the left cardiac ventricle. After the brain was removed, 50-/zm thick sections were stained with Cresyl violet to verify the injection site histologically, as described previously (Ohno et al., 1992). The stained sections showed that the tips of the injection cannulae were located in the dorsal hippocampus in all cases. Several animals were injected with 2 /~l of Cresyl violet dye in order to estimate the extent of dye diffusion. Ten minutes after this microinjection, the rats were anesthetized and prepared for cardiac perfusion. After the brain was removed rapidly and sliced, the diffusion of dye was confirmed by visual inspection. The dye was confined to the dorsal hippocampus, ranging from 2.8 to 4.8 mm posterior to the bregma.
M. Ohno et aL / European Journal of Pharmacology 253 (1994) 183-187
185
30"
2.5. Data analysis
25-
In the working memory task, the number of errors and the latency summed from the second to the sixth trial of a session were important for evaluating the ability of rats to remember new correct panel-gate locations. Thus, these parameters are presented separately from those recorded in the first trial. In the reference memory task, both these parameters were summed across all six trials of a session since this task was given in order to evaluate the ability of rats to retain the constant location of correct panel-gates. The significance of the differences between the groups was determined by a one-way analysis of variance (ANOVA) followed by Dunnett's test when F ratios reached significance ( P < 0.05).
.o 20"6
15" 10"
Z
50.32
1.0
Saline
32
3.2
7-Chlorokynurenic acid D-Serine 32 pgl,~de
100-
~6 o - ~
*
80-
8 40-
3. Results
In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. In the working memory task, the number of errors made from the second to the sixth trial (working memory errors) markedly decreased with repeated training, whereas the errors in the first trial remained constant at approximately four. Approximately 20-30 training sessions were required for the rats to reach the criterion of fewer than 12 errors summed across the six trials of a session. Latency was also reduced during repeated sessions and was stable from the 10th session on. In the reference memory task, the number of errors and latency in all six trials of a session decreased with repeated training. The rats performed the task within the six-error criterion summed across six trials after they had about 10 training sessions. 7-Chlorokynurenic acid at doses of 0.32-3.2 /zg/side, administered bilaterally into the dorsal hippocampus, increased the number of errors in the working memory task ( F ( 3 , 1 8 ) = 9.12, P < 0.01), an effect that reached significance for the 3.2 /zg/side dose, while it had no effect on the number of errors made in the first trial (Fig. 1). The latency recorded in the first trial (F(3,18) = 4.91, P < 0.05) and that recorded from the second to the sixth trial of a session (F(3,18) = 3.74, P < 0.05) were significantly prolonged when rats were given intrahippocampal 7-chlorokynurenic acid at 3.2 /zg/side. Intrahippocampal injection of 32 /~g/side D-serine by itself did not affect the number of working memory errors (7.0 + 1.7, n = 4) or the latencies recorded in the first trial and from the second to sixth trial (9.8 + 1.0 s and 28.8 + 3.5 s, respectively). The increase in working memory errors induced by intrahippocampal 7-chlorokynurenic acid at 3 . 2 / z g / s i d e was significantly attenuated by concurrent injection of D-serine at 32 tzg/side (F(1,8) = 6.89, P < 0.05).
200 0.32
1.0
Saline
3.2
3.2
/~g/side
7-Chlorokynurenic acid D-Serine 32/~J/Side
Fig. 1. Effects of intrahippocampal 7-chlorokynurenic acid injection on the number of errors and latency in a test of working memory. T~e runway test was given 10 rain after drugs were administered. o-Scrine was co-injected intrahippocampally with 7-chlorokynurenic acid. Each column represents the mean + S.E. of errors and latcncies for 5 - 6 animals recorded in the first trial (open columns) and those summed from the second to the sixth trial of a session (hatched columns). The significance of the differences from the saline-injected group (* P < 0.05, ** P < 0.01) and from the 3.2 ~ g / s i d e 7-chlorokynurenic acid-injected group (# P < 0.05) was determined by means of a one-way A N O V A followed by Dunnett's test.
In the reference memory task, intrahippocampal injection of 7-chlorokynurenic acid at doses up to 3.2 /zg/side had no effect on the number of errors or on the latency across all six trials of a session (Table 1).
Table 1 Effects of intrahippocampal 7-chlorokynurenic acid injection on the number of errors and latency in a test of reference memory Drug
/zg/ side
n
Number of errors Trial 1-6
Latency (s)
Saline 7-Chlorokynurenic acid
1.0 3.2
5 5 5
2.2 + 1.0 4.2 + 1.5 3.4 + 1.2
37.8 + 5.6 36.2 + 3.0 38.4 + 2.5
Trial 1-6
The runway test was given 10 min after 7-chlorokynurenic acid was administered. Values are m e a n s + S . E , of errors and latencies summed across all six trials of a session.
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4. D i s c u s s i o n
Recently, Watanabe et al. (1992) reported that intracerebroventricular injection of 7-chlorokynurenic acid, an antagonist at the glycine site associated with the N M D A receptor complex, before daily training impaired the acquisition of spatial memory in the Morris water maze, which is highly sensitive to hippocampal damage (Morris et al., 1982). These findings suggest that the glycine site in the hippocampus may be involved in spatial learning. Working memory performance in the three-panel runway task is also sensitive to disruption by hippocampal lesions (Kitajima et al., 1992) and is thus suitable for the investigation of memory processes mediated by this structure. The present study clearly showed that microinjection of 7-chlorokynurenic acid into the hippocampus was sufficient to impair working memory in this task. That blockade of hippocampal glycine sites was responsible for the effect of 7-chlorokynurenic acid was confirmed by the result that the working memory deficit following intrahippocampal 7-chlorokynurenic acid was reversed by concurrent injection of D-serine, a glycine site agohist. Therefore, it is conceivable that the hippocampal glycine site is involved in working memory performance, i.e., acquisition of new and variable information that is useful for only one session. In contrast, intrahippocampal injection of 7-chlorokynurenic acid did not affect reference memory, which is resistant to hippocampal lesions (Kitajima et al., 1992). Thus, it is suggested that the hippocampal glycine site does not contribute to reference memory performance, i.e., retention and retrieval of previously acquired constant information. On the other hand, both working and reference memory tasks require equal motor performance and motivational and perceptual abilities. Since no effects on reference memory performance were found, we can exclude the possibility that the deficit in working memory performance following intrahippocampal 7-chlorokynurenic acid injection is related to an alteration of non-mnemonic functions. The distribution of glycine binding sites is similar to that of N M D A receptors in the brain, and the hippocampus is one of the structures that contain the greatest density of both binding sites (Bristow et al., 1986; Thomson, 1990). Activation of N M D A receptors is an absolute requirement for the induction of hippocampal long-term potentiation, a cellular model of learning and memory in the mammalian nervous system (Collingridge and Bliss, 1987). Morris et al. (1989) and Davis et al. (1992) have reported that intracerebroventricular and intrahippocampal infusion of the N M D A receptor antagonist 2-amino-5-phosphonovaleric acid (AP5) impairs spatial learning in the water maze across a dose range comparable to that required to block induction of hippocampal long-term potentia-
tion, suggesting a close link between long-term potentiation and memory processing in the brain. We also reported that intrahippocampal injections of N M D A receptor antagonists, such as AP5, CGS 19755 (c/s-4phosphonomethyl-2-piperidine carboxylic acid) and 3[( ± )-2-carboxypiperazin-4yl]propyl-1-phosphonic acid (CPP), impaired working memory without affecting reference memory in the three-panel runway task, suggesting an important role of hippocampal N M D A receptor-dependent mechanisms in working memory (Ohno et al., 1992). Agonist action at the glycine regulatory site is required for opening of the N M D A receptor channel (Johnson and Ascher, 1987; Thomson, 1990) and is also essential for induction of hippocampal long-term potentiation since 7-chlorokynurenic acid attenuates long-term potentiation in rat hippocampal slices (Bashir et al., 1990; Izumi et al., 1990; Oliver et al., 1990; Watanabe et al., 1992). Thus, interruption of hippocampal N M D A receptor-mediated processes accounts for the working memory deficit induced by blockade of hippocampal glycine sites. The normal concentration of brain glycine is generally thought to exceed the concentration required to saturate the glycine site on the N M D A receptor complex (Thomson, 1990). Therefore, it is not certain that hippocampal glycine by itself plays a modulatory role in memory processes, although support by endogeneous glycine of hippocampal N M D A receptor activation is necessary for working memory performance.
5. A c k n o w l e d g e m e n t
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
6. References
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