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Ameliorative effects of a cognitive enhancer, T-588, on place learning deficits induced by transient forebrain ischemia in rats
Physiology & Behavior 74 (2001) 227 – 235
Ameliorative effects of a cognitive enhancer, T-588, on place learning deficits induced by transient forebrain ischemia in rats Yasushi Nakada, Ryoi Tamura, Jun-Ichi Kuriwaki, Tatsuo Kimura, Teruko Uwano, Hisao Nishijo, Taketoshi Ono* Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan Received 2 January 2001; received in revised form 23 May 2001; accepted 24 May 2001
Abstract In the present study, we investigated the effect of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a newly synthesized cognitive enhancer, on place learning deficits in rats with damage selective to the hippocampal CA1 subfield induced by transient forebrain ischemia. Three weeks after the ischemic insult, T-588 was daily administered (0.3 or 3.0 mg/kg/day po). Place learning was tested in a task in which the rat was required to alternatively visit two places located diametrically opposite each other in an open field. The ischemic rats without the treatment of T-588 displayed severe learning impairment in this task; their performance level was significantly inferior to that of the sham-operated rats. The treatment of T-588 improved dose-dependently the task performance in ischemic rats, although no apparent protective effects on ischemic damage were found histologically. These results suggested that T-588 has ameliorative effects on learning deficits induced by brain ischemia, which could be produced through enhancement of residual cognitive functions. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Transient forebrain ischemia; Hippocampus; Spatial memory; Rats; Learning
1. Introduction With the increase in senescent population, senile dementia of the Alzheimer type (SDAT) and cerebrovascular dementia are among the most urgent public health problems, and effective therapeutics for these diseases are required. In the patients suffering from these diseases, cognitive dysfunction and memory impairment can range from more focal deficits to an overall intellectual decline [9,20]. The pathophysiological changes in senile dementia can occur in various parts of the brain including the frontal lobe, temporal lobe, and basal nucleus of Meynert to a variety of severities. Especially, the hippocampal formation (HF), a part of the medial temporal lobe, was indicated to be one of the areas, which are most affected at very early stage of SDAT [10,19]. Furthermore, the HF is known to be one of the most vulnerable brain areas to ischemic insult [16,17,36,37,40,42]. * Corresponding author. Tel.: +81-76-434-7220; fax: +81-76-4345013. E-mail address: [email protected] (T. Ono).
Dysfunction of various neurotransmitter systems reportedly occurs in the senile dementia [3,19]. Especially, the involvement of deficits in the central cholinergic systems in senile dementia has been extensively studied. For example, a massive loss of cholinergic neurons is frequently observed in the basal nucleus of Meynert in the SDAT patients [2]. Cholinergic deficits in the HF cause memory impairment [7,8,22] that is an early, prominent manifestation of SDAT. Development of drugs for treatment of both SDAT and cerebrovascular dementia has mainly aimed at activation of the cholinergic system in the brain. Acetylcholinesterase inhibitors and muscarinic receptor agonists have recently been investigated as agents of potential therapeutic value [27,38,39,41,46]. T-588, (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (Fig. 1), has recently been developed as a cognitive enhancer [29,33]. T-588 crosses the blood-brain barrier and it shows a good penetration into the brain. The level of T-588 in the brain was about 10 times higher than that in the serum [30]. The halflife of T-588 in the serum was 1.4 h (10 mg/kg po); T-588 concentration in the brain roughly paralleled that in the
0031-9384/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 1 ) 0 0 5 7 6 - 5
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Fig. 1. Chemical structure of T-588.
blood. A previous in vivo dialysis study demonstrated that treatment of T-588 increased acetylcholine (ACh) and noradrenaline release from the synaptic terminals in both the HF and frontal cortex [32]. T-588 has also been reported to have an inhibitory effect on potassium channels of CA1 pyramidal neurons in the rat HF [15]. Furthermore, T-588 was indicated to protect neuronal damage in excessive neuronal activation [34]. T-588 is known to have some side effects such as vomiting, sedation, and other inhibitory action on the central nervous system. However, these side effects are produced at doses higher than 100 mg/kg po. These suggest that T-588 can be used as the therapeutic drug for senile dementia. However, no studies have demonstrated the effect of T-588 on cognitive functions (e.g., spatial memory and working memory) in behaving animals. Transient forebrain ischemia produces a neuronal death selective to the CA1 subfield in gerbils, rats, monkeys, and humans [16,36,43,45,48,49]. The rat exposed to 15-min transient forebrain ischemia is used as an animal model for cerebrovascular dementia since it displays clear disorientation (impairment in spatial learning and memory) [11,13,23,25,28]. We have recently developed a behavioral protocol to evaluate spatial learning in rats [6,18]. Using this protocol, we could demonstrate that a calcium entry blocker, preventing neuronal death in the CA1 subfield, ameliorated spatial learning deficits in rats exposed to 15-min transient forebrain ischemia [44]. We have also demonstrated that red ginseng, a herb used in traditional Asian medicine, ameliorated spatial learning deficits in the rats exposed to 15-min transient forebrain ischemia and aged rats [47]. In the present study, we evaluated the effect of T-588 on place learning (PL) deficits in the rat exposed to transient forebrain ischemia using our behavioral protocol. The results have already been published elsewhere in an abstract form [24].
2. Methods 2.1. Subjects Thirty-four male albino Wistar rats, supplied by Japan SLC, Inc. (Hamamatsu, Japan), were used. The rats weighed 260 – 300 g at the time of ischemic surgery and were individually housed with standard rat pellets and water available ad lib. All rats were treated in strict compliance with the U.S. Public Health Service Policy on Human Care
and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with guiding principles for the care and use of animals in the field of physiological sciences recommended by the Physiological Society of Japan. Each rat was assigned to one of the following four groups: (1) Sham-operated (n = 12), (2) Ischemia control (n = 7), (3) Ischemia + low-dose T-588 (n = 7), and (4) Ischemia + high-dose T-588 (n = 8). 2.2. Surgery for transient forebrain ischemia Transient forebrain ischemia was induced by the fourvessel occlusion method reported previously by Pulsinelli and Brierley [36] with some modifications. In brief, a rat was anesthetized with pentobarbital sodium (40 mg/kg ip) and fixed in a stereotaxic apparatus. The bilateral vertebral arteries were permanently electrocauterized at the first vertebra. The common carotid arteries were exposed at both sides of the neck and isolated from the surrounding tissue. Atraumatic clasps (surgical silk strings) were placed loosely around each common carotid artery without interrupting the blood flow. On the following day, for the rats in the ischemic groups (Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588), the bilateral common carotid arteries were reexposed under slight anesthesia with halothane and the carotid arteries were simultaneously occluded with aneurysm clips. Fifteen minutes after the onset of ischemia, the clips were released. During ischemia, the temperature of the temporal muscle was maintained at 36.5 – 37.5 C by warming lamps. Any rats that failed to demonstrate pupillary dilatation or a loss of righting reflex during the 15-min ischemia were excluded from the experiment. For rats in the Sham-operated group, the same surgical procedure was performed except for the occlusion of the common carotid arteries. 2.3. Surgery for intracranial self-stimulation (ICSS) Two weeks after ischemia, each rat was anesthetized with pentobarbital sodium (40 mg/kg ip) and implanted bilaterally with monopolar stimulating electrodes (polyurethane insulated, 0.1-mm diameter, stainless steel) for ICSS reward. The electrodes were placed in the medial forebrain bundle at the level of the lateral hypothalamus (4.3 mm caudal from bregma, 1.6 mm lateral from the midline, and 8.7 – 8.9 mm ventral from the skull surface) according to the atlas of Paxinos and Watson [35]. The electrodes were attached to the skull with dental acrylic and stainless steel screws, one of which also served as the indifferent electrode for stimulation. 2.4. Self-stimulation screening After 1 week of recovery from the surgery for ICSS electrode implantation, the rat was screened for self-stimu-
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lation in an operant chamber (30 30 33 cm) equipped with a lever on the wall. Each lever press triggered the delivery of a 0.5-s train of 0.3-ms negative square wave pulses at 100 Hz. The current intensity for stimulation was set to produce 40– 70 lever presses per minute. Out of the two stimulating electrodes implanted, the electrode that produced a stronger reward effect for the same current intensity was selected for each rat (ICSS rewards were given to the rat through this electrode in subsequent behavioral tests). The rat was trained daily to self-stimulate in a 30 –60-min session for 3 –16 days (average: 7 days) until stable lever pressing was achieved. The current intensity, which was determined in this period for each rat, ranged from 0.1 to 0.2 mA and was used throughout the following behavioral tasks in the open field. The use of ICSS as a reward provided the following advantages over natural reinforcers such as food and water: (1) rapid learning of a task, (2) lack of satiation, and (3) absence of external sensory information (visual, auditory, olfactory, etc.). 2.5. Apparatus for spatial behavior Spatial behavior of the rat was investigated in a 1.5-m diameter cylindrical chamber (open field) with a 0.45-m wall, painted black on the inside (Fig. 2A). The experimenter could manually move or rotate the open field on casters attached to the bottom. The open field was enclosed by a black curtain (1.8-m diameter and 2.0-m high). The ceiling of the enclosure contained four small speakers mounted near the circumference, spaced 90 apart, and four incandescent lamps (40 W) individually mounted near the inner edge of each speaker. A charge coupled device (CCD) camera with a brightnesstracking interface (BTA-2A, Muromachi Kikai, Tokyo, Japan) was mounted on the center. The incandescent lamp at the 3 o’clock position was turned on, and the speaker at the 9 o’clock position continuously emitted white noise. A small light bulb was mounted on the head of the rat, and the horizontal motion of the light bulb was tracked by the CCD camera. A laboratory microcomputer (PC-9801, NEC, Tokyo, Japan) received the X and Y coordinates of the position of the head through an RS-232C serial port at 20 frames per second. A program delimited circular areas (reward places) in the open field. The program triggered the delivery of ICSS when the rat entered the reward place. The experimenter monitored the locations of the reward place and the rat on a display screen. No distinctive cues marked the reward location for the rat. In addition, special care was taken so that no salient local landmark cues (such as dust, smell, etc. on the floor of the open field) indicated the location of the reward places by frequently rotating the open field, wiping and cleaning up the floor of the open field, and so on. 2.6. Control for locomotion activity Since difference of spontaneous locomotion activity could affect the results of PL test, spontaneous locomotion
Fig. 2. Schema of the experimental setup (A) and behavioral paradigm (B). (A) An open field (1.5 m in diameter) containing a rat was viewed from top center by a CCD camera with brightness-tracking interface that signaled the rat’s position in Cartesian coordinates. The open field was enclosed by a black curtain. The incandescent lamp at the 3 o’clock position and the speaker at the 9 o’clock position on the ceiling of the enclosure were turned on. The video signal was sent to a conventional TV monitor directly and the tracking interface sent the X and Y coordinates of a miniature electric light bulb attached to the head of the rat every 50 ms through an RS-232C serial port to a microcomputer. The microcomputer plotted the trail of the rat, compared the rat’s behavior with present criteria, and gated rewarding ICSS delivery from a stimulator when the criteria were met. (B) Behavioral paradigm. In random reward place search task (a), a computer program delimited a circular reward place (small circle; 0.7-m diameter) at some randomly selected coordinate. The rat was rewarded with ICSS when it entered the reward place, which was then made inactive. After a 5-s interval, the reward place was moved to a different nonoverlapping location and reactivated. In place learning task (b), the rat received rewarding ICSS in two target areas (two small circles; 0.4-m diameter) when it returned to one reward place after a visit to the other reward place. Large circles, open field; START, location of the rat at start of a session; line, locomotion trail of the rat; each dot, the location of reward delivery.
activity was controlled as follows (see Ref. [6] for details of this procedure). (1) The rat was trained to perform a criterion distance task in which ICSS was delivered when the cumulative distance traveled by the rat reached a given distance. The purpose of this training was to get the rat in the habit of moving in the open field continuously during test sessions. The initial distance was 0.8 m, and this was increased progressively to 1.2 or 1.6 m. The rat was trained for 1 h/day until it learned to travel the open field continuously. (2) The rat was then trained in a random reward place search (RRPS) task (Fig. 2Ba). The purpose of this task was to encourage the rat to visit all parts of the open field. In this task, a reward place (0.7-m diameter) was
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delimited with its center chosen at random in the open field. The rat was rewarded with ICSS when it entered the reward place, which was then made inactive. After a 5.0-s interval, the reward place was reactivated in a different location that was set randomly in the open field. Each session was terminated after 50 rewards had been delivered or 10 min had elapsed, whichever occurred first. We continued this training for each rat (five sessions per day) until it achieved a criterion of more than 25 reward acquisition over three sessions in a day.
in distilled water and administered orally in a volume of 0.5 ml/100 g body weight 60 min before the daily training for the ICSS lever press, the RRPS task, and the PL task. The Ischemia + low-dose T-588 group received 0.3 mg/kg of T-588; the Ischemia + high-dose T-588 group received 3 mg/kg of T-588. We selected these doses because these were in the range of putative therapeutic dose without serious side effects (0.1 – 30 mg/kg po) [30 –33]. To control for the T-588 administration, the rats in the Sham-operated and Ischemia control groups received an equal amount of distilled water.
2.7. Evaluation of spatial learning ability 2.9. Histology The PL task (Fig. 2Bb) was used for the evaluation of the rat spatial learning ability. In the protocol of the PL task, two 0.4-m diameter reward places were set diametrically opposite one another in the open field. The locations of these reward places were fixed to the environment (experiment room) throughout the test period, one in the northern part of and the other in the southern part of the open field (Fig. 2Bb). The rat was required to alternatively visit the two reward places. When the rat entered the reward place and stayed there for more than 1.0 s (delay), the rat was rewarded. This delay was intended to ensure that the rat identified the reward place as such and was not rewarded for simply being brought into the reward place by automatic locomotion. In this task, the white noise from the speaker at the 9 o’clock position and the light from the lamp at the 3 o’clock position in the ceiling were thought to serve as salient landmark cues for the rat, since (1) the rat did not get ICSS rewards efficiently for, at least, several sessions in the PL task when these landmark cues were removed, and (2) hippocampal place cells changed the spatial correlates [6]. At the start of a session, an ICSS reward was delivered to the rat as a priming stimulation. Each session was terminated after 50 rewards had been delivered or 10 min had elapsed, whichever occurred first. The rat was trained by this task in five sessions per day. For the rats in the Shamoperated group, the training was continued until they achieved the criterion of 50 reward acquisition in three or more sessions a day for 3 successive days (in the present study, all the rats in the Sham-operated group fulfilled this criterion within 15 days). The rats in the Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588 groups were tested in this task for 30 days. In the present behavioral testing, the experimenters were not blind to the animal groups. However, we think there was little room for experimenter’s bias, since the data acquisition and analyses were all automatic. 2.8. Drug T-588 was provided by the Research Laboratory of Toyama Chemical Co., Ltd. This compound fulfilled quality control criteria for purity ( > 99%) as assayed by highperformance liquid chromatography. T-588 was dissolved
After completion of the PL task, the rat was deeply anesthetized with pentobarbital sodium (50 mg/kg ip). They were sacrificed by transcardial perfusion with 0.9% saline containing heparin followed by 10% buffered formalin. The whole brain was then removed and immersed in the same fixative for 2 – 3 days. After fixation, the brain was dehydrated with graded ethanol, passed through xylene, and embedded in paraffin. Serial 6-mm thick sections (coronal sections), which included the dorsal HF, were made. The sections were stained with cresyl violet and examined under a light microscope. Histological evaluation was performed by different experimenter in order to avoid the experimenter biases in selection of animals. 2.10. Data analysis The behavioral data of the PL task were analyzed on the following four parameters: (1) the locomotion pattern, (2) the mean number of rewards acquired in a session, (3) the mean period of time for rats to complete a session, and (4) the mean locomotion distance between the two reward places. A two-way analysis of variance (ANOVA) was performed to test main effect of group between the three (Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588) groups; Scheffe’s post hoc test was employed for pairwise comparisons. To evaluate the influence of transient forebrain ischemia and/or T-588 treatment on motivational level and spontaneous locomotion activity, a one-way ANOVA test was performed on the data of (1) ICSS current intensity, (2) the number of rewards acquired and the locomotion distance traveled in the last day of the RRPS task training, and (3) the 10-min locomotion activity (see above). The number of pyramidal cells in the CA1 subfield at two antero-posterior levels (3.5 and 4.5 mm posterior from the bregma) was counted in a 0.2-mm wide and 1-mm long strip of tissue under a light microscope. A two-way ANOVA test was performed on these data with three (Ischemia control, Ischemia + low-dose T-588, and Ischemia + highdose T-588) groups and antero-posterior levels as independent variables.
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The statistical tests were performed using Statview 5.0J (Abacus Concepts, Berkeley, CA). Significance level employed for all tests was P < .05.
3. Results 3.1. ICSS behavior and rats’ locomotion activity Since effectiveness of ICSS and spontaneous motor activity could strongly affect the performance of the PL task, these parameters were checked before the training of the PL task. The results are summarized in Table 1. There were no significant group differences of ICSS current intensities [one-way ANOVA, F(3,30) = 0.091, P >.1] or 10-min spontaneous motor activity [ F(3,30) = 2.21, P >.1]. Furthermore, in the RRPS task, the locomotion pattern of the rats was similar in all the four groups at the end of this task training: the rats visited all parts of the open field homogeneously (examples are shown in Fig. 3Aa, Ba, and Ca). The data on each behavioral parameter in the RRPS task are summarized in Table 2. There were no significant group differences in the mean number of rewards acquired in a session [one-way ANOVA, F(3,30) = 0.559, P >.1], the mean locomotion distance in a session [ F(3,30) = 1.30, P >.1], and the mean locomotion distance between one reward place and the next [ F(3,30) = 2.19, P >.1] in the last day of the RRPS task training. 3.2. Place learning 3.2.1. Typical examples of locomotion patterns in the PL task The typical locomotion patterns of rats in the Shamoperated, Ischemia control, and Ischemia + high-dose T-588 groups during the PL task are shown in Fig. 3Ab, Bb, and Cb. On the first day of the PL task training, the rats in the three groups randomly moved in the open field to the same extent and acquired only a small number of rewards. The rat in the Sham-operated group began to show shuttle behavior (direct back-and-forth movement between the two reward places) on the fourth day of the training (Fig. 3Ab); this Table 1 Effects of T-588 on ICSS current intensity and spontaneous motor activity Group Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
T-588 (mg/kg)
n
ICSS current intensity (mA)
Spontaneous motor activity (m)
0 0 0.3
12 7 7
0.177 ± 0.013 0.184 ± 0.014 0.174 ± 0.011
45.22 ± 5.51 46.14 ± 11.71 63.17 ± 7.39
8
0.182 ± 0.016
66.83 ± 6.93
3
Values, mean ± S.E.M. of ICSS current intensity at which the rats achieved a given criterion (more than 40 bar presses per minute). The rat’s spontaneous motor activity was evaluated as movement distance in the open field 10 min before the training of the RRPS task.
231
behavior dominated progressively in the course of the training. As mentioned in Methods, rats were required to stay in the reward place more than 1 s in order to get ICSS reward; otherwise, they could not get the reward even though they entered the reward place. We sometimes observed that the rat in the Sham-operated group immediately came back to the reward place when it passed the reward place without getting ICSS reward (we called this behavior ‘‘returning’’). In contrast to the Sham-operated group, the rat in the Ischemia control group did not show shuttle behavior during the 30-day test period (Fig. 3Bb). Rather, it developed a locomotion pattern of moving along the wall of the open field, although it showed weak tendency of shortcut later in the training period. We seldom observed the ‘‘returning’’ in this rat. The rat in the Ischemia + highdose T-588 group showed similar pattern to that of Shamoperated group (Fig. 3Cb). The rat began to show shuttle behavior around the fifth day and established it during the rest of the training period. 3.2.2. The mean number of rewards acquired in a session In the present study, the maximum time for a session was 10 min and the maximum number of rewards acquired in a session was 50 (see Methods). So, the more efficiently and quickly the rat moved between the two reward places in the PL task, the more rewards it acquired, approaching the maximum. We used this parameter as an index to evaluate learning. As shown in the Fig. 4A, the number of rewards acquired in a session increased rapidly for the rats in the Sham-operated group. The rats in this group reached the criterion (50 reward acquisition in three or more sessions) by the fifth day (5.3 ± 0.8), and this performance level was maintained throughout the rest of the test period. The increase in this parameter for the Ischemia control group was slow (16.0 ± 3.5), whereas for the Ischemia + high-dose T-588 group, it was analogous to that of the Sham-operated group. The rats in the Ischemia + low-dose T-588 group reached the criterion on the 16th day (15.7 ± 3.4). The rats in the Ischemia + high-dose T-588 group reached the criterion on the seventh day (7.3 ± 1.5). There were significant differences in this parameter between the three ischemic groups [two-way ANOVA, F(2,570) = 41.91, P < .01, Days 1– 30]. Post hoc comparisons revealed that there was a significant difference between the Ischemia + high-dose T-588 and Ischemia control groups and between the Ischemia + highdose T-588 and Ischemia + low-dose T-588 groups. There was no Group Day interaction [ F(58,570) = 0.59, P >.1]. 3.2.3. The mean period of time for rats to complete a session Since a session could finish before 10 min has passed if the rat acquired 50 rewards, the more efficiently and quickly the rat performed the PL task, the shorter the session time. As shown in Fig. 4B, the rats in the Sham-operated group learned the task rapidly so that this parameter decreased steeply in the early sessions (Days 1 –10). In contrast, the rats
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Fig. 3. Examples of change in locomotion pattern of rat in the Sham-operated group (A), the Ischemia control group (B), and the Ischemia + high-dose T-588 group (C) during test period of RRPS task (a) and PL task (b). Other descriptions as for Fig. 2B.
in the Ischemia control group displayed a slow decrease in the value of this parameter during the 30-day test period. The Ischemia + high-dose T-588 group reduced the value of this parameter more rapidly than the Ischemia control group. There were statistically significant differences in this parameter between the three ischemic groups [two-way ANOVA, F(2,570) = 113.2, P < .01, Days 1– 30]. Post hoc comparisons revealed that, to complete a session, the rats in the Ischemia + high-dose T-588 group took less time than the rats in the Ischemia + low-dose T-588 and Ischemia control groups, and the rats in the Ischemia + low-dose T-588 group took less time than the rats in the Ischemia control group. There was no Group Day interaction [ F(58,570) = 0.53, P >.1]. 3.2.4. The mean of the locomotion distance between the two reward places The most efficient strategy in the PL task was shuttling between the two reward places. So, the mean distance
traveled between the two reward places would be minimal if the rat developed this strategy. As shown in Fig. 4C, the value of this parameter decreased rapidly in the early sessions (Days 1 – 9) for the rats in the Sham-operated group. In contrast, the rats in the Ischemia control group displayed a slow decrease in this parameter. Furthermore, a clear fluctuation was observed in this group even in the later sessions of the training. There were statistically significant differences in this parameter between the three ischemic groups [twoway ANOVA, F(2,570) = 18.86, P < .01, Days 1– 30]. Post hoc comparisons revealed that the value of this parameter in the Ischemia + high-dose T-588 group was significantly shorter than those in the Ischemia control and Ischemia + low-dose T-588 groups. The data on the Ischemia + highdose T-588 and Ischemia + low-dose T-588 groups were less fluctuated compared with those on the Ischemia control group in the later sessions of the training. There was no Group Day interaction [ F(58,570) = 0.73, P >.1].
Table 2 Effects of T-588 on the behavioral parameters of the RRPS task
Group
T-588 (mg/kg)
Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
0 0 0.3 3
n
No. of rewards acquired in a session
Locomotion distance in a session (m)
Locomotion distance while entering the reward places (m)
12 7 7 8
31.5 ± 2.2 29.2 ± 2.9 29.6 ± 1.4 27.9 ± 1.6
116.36 ± 9.13 102.73 ± 9.59 97.23 ± 6.07 95.08 ± 9.55
3.86 ± 0.14 3.58 ± 0.11 3.35 ± 0.13 3.53 ± 0.19
Values, mean ± S.E.M. of each behavioral parameter at which the rats achieved a given criterion (more than 25 rewards acquisition over the three sessions per day) in the training of the RRPS task.
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Fig. 4. Effects of T-588 on each behavioral parameter of the place learning task. (A) The mean number of rewards acquired in a session. (B) The mean of the period of time for rats to complete a session. (C) The mean of the locomotion distance between the two reward places traveled by rats in each group. Open circles, Sham-operated group; closed circles, Ischemia control group; closed squares, Ischemia + low-dose T-588 group; closed triangles, Ischemia + high-dose T-588 group. Abscissa, day.
3.3. Histology Brain sections both from the anterior and posterior hippocampi showed selective CA1 neuronal loss in the Ischemia control group. The number of pyramidal cells in each CA1 subfield, which were counted in a strip 0.2-mm wide by 1-mm long, are shown in Table 3. The number of pyramidal neurons with normal appearance in the Ischemia control group was less than 3% of that in the Sham-operated group. Transient forebrain ischemia also produced CA1 neuronal loss to the same extent in the other two ischemic groups: there were no significant differences in the three ischemic groups [two-way ANOVA, F(2,38) = 0.013, P >.1].
Table 3 Number of pyramidal cells in the CA1 subfield of the rat hippocampus A – P level Group Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
T-588 (mg/kg)
n
3.5 mm from bregma
4.5 mm from bregma
0 0 0.3
9 7 7
87.6 ± 5.3** 2.2 ± 1.5 7.7 ± 4.0
89.6 ± 3.5** 0.3 ± 0.3 8.6 ± 5.3
3
8
9.6 ± 5.1
9.9 ± 3.8
Values, mean ± S.E.M. (neurons/1 mm strip). Pyramidal cells in the CA1 subfield were counted under a light microscope in a 1-mm strip of pyramidal cell layer in the CA1 subfield. ** P < .01 vs. Ischemia control group (Scheffe’s test).
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4. Discussion 4.1. Influence of transient forebrain ischemia on PL In the present study, 15-min transient forebrain ischemia produced selective neuronal loss in the CA1 subfield of the HF, which was accompanied with substantial learning deficits in the PL task. That is, while the Sham-operated rats altered the behavioral strategy from inefficient pattern (random movement) to the efficient (shuttle movement) in the early stage of the PL task, most Ischemia control rats did not display such a behavioral alteration. Rather, these rats tended to display different behavior, such as traveling along the wall of the open field. Furthermore, as described in Results, the rats in the Sham-operated group often showed ‘‘returning’’ when they passed the reward place, whereas the rats in the Ischemia control group did not. These findings suggest that the Ischemia control rats did not recognize the location of the reward places in the PL task. The three behavioral parameters in the PL task also indicated the impaired performance of the Ischemia control rats. These results were consistent with the previous studies in which the same behavioral paradigm was used for the rats exposed to transient forebrain ischemia [25,26], indicating the reliability of the present behavioral protocol. 4.2. Effects of T-588 on PL The principal finding of the present study was that the administration of high-dose T-588 significantly ameliorated the impaired task performance of the rats exposed to transient forebrain ischemia. This effect appeared at relatively early stage of the PL task training so that the task performance of the rats in the Ischemia + high-dose T-588 group was comparable to that of the rats in the Sham-operated group within a week. If a drug would have an influence on locomotion or rewarding efficacy, such an action could also influence on learning. However, in the present study, it is implausible that the ameliorative effect of T-588 occurred via changes in locomotor ability or rewarding efficacy since there were no significant effects of T-588 on spontaneous locomotion activity or ICSS current intensity. In the present study, there were no significant differences in degree of the neuronal loss observed in the CA1 subfield between the three groups exposed to transient forebrain ischemia. Although T-588 is known to have a neuroprotective effect [34], this is not surprising because T-588 administration began 3 weeks after the ischemic manipulation when the initial phase of neuronal death completed. This, however, does not rule out the possibilities that T-588 acts to prevent the chronic development of ischemia-induced neuronal damages and that T-588 acts to maintain residual cell bodies and neuropils in the damaged CA1 subfield. Indeed, administration of T-588 changed the activity in the HF of the ischemic rats in a previous study using functional magnetic resonance imaging [24]. Although these facts
indicate that T-588 activates the function in the residual hippocampal subfields, compensatory activation in other brain areas could also contribute to the ameliorative action of T-588. More studies are necessary to elucidate this point. The involvement of ACh in spatial learning and memory has been reported in several previous reports (e.g., Refs. [4,12,20,21]). The cholinergic dysfunction in the ischemic brain is remarkable [5,14,40]. Activation of cholinergic system with acetylcholinesterase inhibitors or muscarinic agonists improves spatial memory deficits following cerebral ischemia [1,4,45]. These findings indicate that reduced cholinergic action in the HF is involved in spatial memory deficits accompanied with ischemic damage. T-588 reportedly ameliorates memory impairment in rats produced by basal forebrain lesions, which result in a selective loss of cortical cholinergic markers [33]. A previous study showed that oral administration of T-588 at a dose of 3 mg/kg causes a significant increase in ACh release in the frontal cortex and HF [32]. These studies, together with other reports [29 – 31], suggest that T-588 has cholinergic stimulant actions, which is a possible transmitter mechanism underlying the therapeutic effects of T-588 observed in the present study.
Acknowledgments This work was supported partly by Grant-in-Aid for Scientific Research on Priority Areas (A)-Research for Comprehensive Promotion of Study of Brain (12050220), Priority Areas (C)-Advanced Brain Science Project (12210009), and Scientific Research (11308033 and 11680805) from Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Ameliorative effects of a cognitive enhancer, T-588, on place learning deficits induced by transient forebrain ischemia in rats Yasushi Nakada, Ryoi Tamura, Jun-Ichi Kuriwaki, Tatsuo Kimura, Teruko Uwano, Hisao Nishijo, Taketoshi Ono* Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan Received 2 January 2001; received in revised form 23 May 2001; accepted 24 May 2001
Abstract In the present study, we investigated the effect of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a newly synthesized cognitive enhancer, on place learning deficits in rats with damage selective to the hippocampal CA1 subfield induced by transient forebrain ischemia. Three weeks after the ischemic insult, T-588 was daily administered (0.3 or 3.0 mg/kg/day po). Place learning was tested in a task in which the rat was required to alternatively visit two places located diametrically opposite each other in an open field. The ischemic rats without the treatment of T-588 displayed severe learning impairment in this task; their performance level was significantly inferior to that of the sham-operated rats. The treatment of T-588 improved dose-dependently the task performance in ischemic rats, although no apparent protective effects on ischemic damage were found histologically. These results suggested that T-588 has ameliorative effects on learning deficits induced by brain ischemia, which could be produced through enhancement of residual cognitive functions. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Transient forebrain ischemia; Hippocampus; Spatial memory; Rats; Learning
1. Introduction With the increase in senescent population, senile dementia of the Alzheimer type (SDAT) and cerebrovascular dementia are among the most urgent public health problems, and effective therapeutics for these diseases are required. In the patients suffering from these diseases, cognitive dysfunction and memory impairment can range from more focal deficits to an overall intellectual decline [9,20]. The pathophysiological changes in senile dementia can occur in various parts of the brain including the frontal lobe, temporal lobe, and basal nucleus of Meynert to a variety of severities. Especially, the hippocampal formation (HF), a part of the medial temporal lobe, was indicated to be one of the areas, which are most affected at very early stage of SDAT [10,19]. Furthermore, the HF is known to be one of the most vulnerable brain areas to ischemic insult [16,17,36,37,40,42]. * Corresponding author. Tel.: +81-76-434-7220; fax: +81-76-4345013. E-mail address: [email protected] (T. Ono).
Dysfunction of various neurotransmitter systems reportedly occurs in the senile dementia [3,19]. Especially, the involvement of deficits in the central cholinergic systems in senile dementia has been extensively studied. For example, a massive loss of cholinergic neurons is frequently observed in the basal nucleus of Meynert in the SDAT patients [2]. Cholinergic deficits in the HF cause memory impairment [7,8,22] that is an early, prominent manifestation of SDAT. Development of drugs for treatment of both SDAT and cerebrovascular dementia has mainly aimed at activation of the cholinergic system in the brain. Acetylcholinesterase inhibitors and muscarinic receptor agonists have recently been investigated as agents of potential therapeutic value [27,38,39,41,46]. T-588, (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (Fig. 1), has recently been developed as a cognitive enhancer [29,33]. T-588 crosses the blood-brain barrier and it shows a good penetration into the brain. The level of T-588 in the brain was about 10 times higher than that in the serum [30]. The halflife of T-588 in the serum was 1.4 h (10 mg/kg po); T-588 concentration in the brain roughly paralleled that in the
0031-9384/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 1 ) 0 0 5 7 6 - 5
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Fig. 1. Chemical structure of T-588.
blood. A previous in vivo dialysis study demonstrated that treatment of T-588 increased acetylcholine (ACh) and noradrenaline release from the synaptic terminals in both the HF and frontal cortex [32]. T-588 has also been reported to have an inhibitory effect on potassium channels of CA1 pyramidal neurons in the rat HF [15]. Furthermore, T-588 was indicated to protect neuronal damage in excessive neuronal activation [34]. T-588 is known to have some side effects such as vomiting, sedation, and other inhibitory action on the central nervous system. However, these side effects are produced at doses higher than 100 mg/kg po. These suggest that T-588 can be used as the therapeutic drug for senile dementia. However, no studies have demonstrated the effect of T-588 on cognitive functions (e.g., spatial memory and working memory) in behaving animals. Transient forebrain ischemia produces a neuronal death selective to the CA1 subfield in gerbils, rats, monkeys, and humans [16,36,43,45,48,49]. The rat exposed to 15-min transient forebrain ischemia is used as an animal model for cerebrovascular dementia since it displays clear disorientation (impairment in spatial learning and memory) [11,13,23,25,28]. We have recently developed a behavioral protocol to evaluate spatial learning in rats [6,18]. Using this protocol, we could demonstrate that a calcium entry blocker, preventing neuronal death in the CA1 subfield, ameliorated spatial learning deficits in rats exposed to 15-min transient forebrain ischemia [44]. We have also demonstrated that red ginseng, a herb used in traditional Asian medicine, ameliorated spatial learning deficits in the rats exposed to 15-min transient forebrain ischemia and aged rats [47]. In the present study, we evaluated the effect of T-588 on place learning (PL) deficits in the rat exposed to transient forebrain ischemia using our behavioral protocol. The results have already been published elsewhere in an abstract form [24].
2. Methods 2.1. Subjects Thirty-four male albino Wistar rats, supplied by Japan SLC, Inc. (Hamamatsu, Japan), were used. The rats weighed 260 – 300 g at the time of ischemic surgery and were individually housed with standard rat pellets and water available ad lib. All rats were treated in strict compliance with the U.S. Public Health Service Policy on Human Care
and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with guiding principles for the care and use of animals in the field of physiological sciences recommended by the Physiological Society of Japan. Each rat was assigned to one of the following four groups: (1) Sham-operated (n = 12), (2) Ischemia control (n = 7), (3) Ischemia + low-dose T-588 (n = 7), and (4) Ischemia + high-dose T-588 (n = 8). 2.2. Surgery for transient forebrain ischemia Transient forebrain ischemia was induced by the fourvessel occlusion method reported previously by Pulsinelli and Brierley [36] with some modifications. In brief, a rat was anesthetized with pentobarbital sodium (40 mg/kg ip) and fixed in a stereotaxic apparatus. The bilateral vertebral arteries were permanently electrocauterized at the first vertebra. The common carotid arteries were exposed at both sides of the neck and isolated from the surrounding tissue. Atraumatic clasps (surgical silk strings) were placed loosely around each common carotid artery without interrupting the blood flow. On the following day, for the rats in the ischemic groups (Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588), the bilateral common carotid arteries were reexposed under slight anesthesia with halothane and the carotid arteries were simultaneously occluded with aneurysm clips. Fifteen minutes after the onset of ischemia, the clips were released. During ischemia, the temperature of the temporal muscle was maintained at 36.5 – 37.5 C by warming lamps. Any rats that failed to demonstrate pupillary dilatation or a loss of righting reflex during the 15-min ischemia were excluded from the experiment. For rats in the Sham-operated group, the same surgical procedure was performed except for the occlusion of the common carotid arteries. 2.3. Surgery for intracranial self-stimulation (ICSS) Two weeks after ischemia, each rat was anesthetized with pentobarbital sodium (40 mg/kg ip) and implanted bilaterally with monopolar stimulating electrodes (polyurethane insulated, 0.1-mm diameter, stainless steel) for ICSS reward. The electrodes were placed in the medial forebrain bundle at the level of the lateral hypothalamus (4.3 mm caudal from bregma, 1.6 mm lateral from the midline, and 8.7 – 8.9 mm ventral from the skull surface) according to the atlas of Paxinos and Watson [35]. The electrodes were attached to the skull with dental acrylic and stainless steel screws, one of which also served as the indifferent electrode for stimulation. 2.4. Self-stimulation screening After 1 week of recovery from the surgery for ICSS electrode implantation, the rat was screened for self-stimu-
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lation in an operant chamber (30 30 33 cm) equipped with a lever on the wall. Each lever press triggered the delivery of a 0.5-s train of 0.3-ms negative square wave pulses at 100 Hz. The current intensity for stimulation was set to produce 40– 70 lever presses per minute. Out of the two stimulating electrodes implanted, the electrode that produced a stronger reward effect for the same current intensity was selected for each rat (ICSS rewards were given to the rat through this electrode in subsequent behavioral tests). The rat was trained daily to self-stimulate in a 30 –60-min session for 3 –16 days (average: 7 days) until stable lever pressing was achieved. The current intensity, which was determined in this period for each rat, ranged from 0.1 to 0.2 mA and was used throughout the following behavioral tasks in the open field. The use of ICSS as a reward provided the following advantages over natural reinforcers such as food and water: (1) rapid learning of a task, (2) lack of satiation, and (3) absence of external sensory information (visual, auditory, olfactory, etc.). 2.5. Apparatus for spatial behavior Spatial behavior of the rat was investigated in a 1.5-m diameter cylindrical chamber (open field) with a 0.45-m wall, painted black on the inside (Fig. 2A). The experimenter could manually move or rotate the open field on casters attached to the bottom. The open field was enclosed by a black curtain (1.8-m diameter and 2.0-m high). The ceiling of the enclosure contained four small speakers mounted near the circumference, spaced 90 apart, and four incandescent lamps (40 W) individually mounted near the inner edge of each speaker. A charge coupled device (CCD) camera with a brightnesstracking interface (BTA-2A, Muromachi Kikai, Tokyo, Japan) was mounted on the center. The incandescent lamp at the 3 o’clock position was turned on, and the speaker at the 9 o’clock position continuously emitted white noise. A small light bulb was mounted on the head of the rat, and the horizontal motion of the light bulb was tracked by the CCD camera. A laboratory microcomputer (PC-9801, NEC, Tokyo, Japan) received the X and Y coordinates of the position of the head through an RS-232C serial port at 20 frames per second. A program delimited circular areas (reward places) in the open field. The program triggered the delivery of ICSS when the rat entered the reward place. The experimenter monitored the locations of the reward place and the rat on a display screen. No distinctive cues marked the reward location for the rat. In addition, special care was taken so that no salient local landmark cues (such as dust, smell, etc. on the floor of the open field) indicated the location of the reward places by frequently rotating the open field, wiping and cleaning up the floor of the open field, and so on. 2.6. Control for locomotion activity Since difference of spontaneous locomotion activity could affect the results of PL test, spontaneous locomotion
Fig. 2. Schema of the experimental setup (A) and behavioral paradigm (B). (A) An open field (1.5 m in diameter) containing a rat was viewed from top center by a CCD camera with brightness-tracking interface that signaled the rat’s position in Cartesian coordinates. The open field was enclosed by a black curtain. The incandescent lamp at the 3 o’clock position and the speaker at the 9 o’clock position on the ceiling of the enclosure were turned on. The video signal was sent to a conventional TV monitor directly and the tracking interface sent the X and Y coordinates of a miniature electric light bulb attached to the head of the rat every 50 ms through an RS-232C serial port to a microcomputer. The microcomputer plotted the trail of the rat, compared the rat’s behavior with present criteria, and gated rewarding ICSS delivery from a stimulator when the criteria were met. (B) Behavioral paradigm. In random reward place search task (a), a computer program delimited a circular reward place (small circle; 0.7-m diameter) at some randomly selected coordinate. The rat was rewarded with ICSS when it entered the reward place, which was then made inactive. After a 5-s interval, the reward place was moved to a different nonoverlapping location and reactivated. In place learning task (b), the rat received rewarding ICSS in two target areas (two small circles; 0.4-m diameter) when it returned to one reward place after a visit to the other reward place. Large circles, open field; START, location of the rat at start of a session; line, locomotion trail of the rat; each dot, the location of reward delivery.
activity was controlled as follows (see Ref. [6] for details of this procedure). (1) The rat was trained to perform a criterion distance task in which ICSS was delivered when the cumulative distance traveled by the rat reached a given distance. The purpose of this training was to get the rat in the habit of moving in the open field continuously during test sessions. The initial distance was 0.8 m, and this was increased progressively to 1.2 or 1.6 m. The rat was trained for 1 h/day until it learned to travel the open field continuously. (2) The rat was then trained in a random reward place search (RRPS) task (Fig. 2Ba). The purpose of this task was to encourage the rat to visit all parts of the open field. In this task, a reward place (0.7-m diameter) was
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delimited with its center chosen at random in the open field. The rat was rewarded with ICSS when it entered the reward place, which was then made inactive. After a 5.0-s interval, the reward place was reactivated in a different location that was set randomly in the open field. Each session was terminated after 50 rewards had been delivered or 10 min had elapsed, whichever occurred first. We continued this training for each rat (five sessions per day) until it achieved a criterion of more than 25 reward acquisition over three sessions in a day.
in distilled water and administered orally in a volume of 0.5 ml/100 g body weight 60 min before the daily training for the ICSS lever press, the RRPS task, and the PL task. The Ischemia + low-dose T-588 group received 0.3 mg/kg of T-588; the Ischemia + high-dose T-588 group received 3 mg/kg of T-588. We selected these doses because these were in the range of putative therapeutic dose without serious side effects (0.1 – 30 mg/kg po) [30 –33]. To control for the T-588 administration, the rats in the Sham-operated and Ischemia control groups received an equal amount of distilled water.
2.7. Evaluation of spatial learning ability 2.9. Histology The PL task (Fig. 2Bb) was used for the evaluation of the rat spatial learning ability. In the protocol of the PL task, two 0.4-m diameter reward places were set diametrically opposite one another in the open field. The locations of these reward places were fixed to the environment (experiment room) throughout the test period, one in the northern part of and the other in the southern part of the open field (Fig. 2Bb). The rat was required to alternatively visit the two reward places. When the rat entered the reward place and stayed there for more than 1.0 s (delay), the rat was rewarded. This delay was intended to ensure that the rat identified the reward place as such and was not rewarded for simply being brought into the reward place by automatic locomotion. In this task, the white noise from the speaker at the 9 o’clock position and the light from the lamp at the 3 o’clock position in the ceiling were thought to serve as salient landmark cues for the rat, since (1) the rat did not get ICSS rewards efficiently for, at least, several sessions in the PL task when these landmark cues were removed, and (2) hippocampal place cells changed the spatial correlates [6]. At the start of a session, an ICSS reward was delivered to the rat as a priming stimulation. Each session was terminated after 50 rewards had been delivered or 10 min had elapsed, whichever occurred first. The rat was trained by this task in five sessions per day. For the rats in the Shamoperated group, the training was continued until they achieved the criterion of 50 reward acquisition in three or more sessions a day for 3 successive days (in the present study, all the rats in the Sham-operated group fulfilled this criterion within 15 days). The rats in the Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588 groups were tested in this task for 30 days. In the present behavioral testing, the experimenters were not blind to the animal groups. However, we think there was little room for experimenter’s bias, since the data acquisition and analyses were all automatic. 2.8. Drug T-588 was provided by the Research Laboratory of Toyama Chemical Co., Ltd. This compound fulfilled quality control criteria for purity ( > 99%) as assayed by highperformance liquid chromatography. T-588 was dissolved
After completion of the PL task, the rat was deeply anesthetized with pentobarbital sodium (50 mg/kg ip). They were sacrificed by transcardial perfusion with 0.9% saline containing heparin followed by 10% buffered formalin. The whole brain was then removed and immersed in the same fixative for 2 – 3 days. After fixation, the brain was dehydrated with graded ethanol, passed through xylene, and embedded in paraffin. Serial 6-mm thick sections (coronal sections), which included the dorsal HF, were made. The sections were stained with cresyl violet and examined under a light microscope. Histological evaluation was performed by different experimenter in order to avoid the experimenter biases in selection of animals. 2.10. Data analysis The behavioral data of the PL task were analyzed on the following four parameters: (1) the locomotion pattern, (2) the mean number of rewards acquired in a session, (3) the mean period of time for rats to complete a session, and (4) the mean locomotion distance between the two reward places. A two-way analysis of variance (ANOVA) was performed to test main effect of group between the three (Ischemia control, Ischemia + low-dose T-588, and Ischemia + high-dose T-588) groups; Scheffe’s post hoc test was employed for pairwise comparisons. To evaluate the influence of transient forebrain ischemia and/or T-588 treatment on motivational level and spontaneous locomotion activity, a one-way ANOVA test was performed on the data of (1) ICSS current intensity, (2) the number of rewards acquired and the locomotion distance traveled in the last day of the RRPS task training, and (3) the 10-min locomotion activity (see above). The number of pyramidal cells in the CA1 subfield at two antero-posterior levels (3.5 and 4.5 mm posterior from the bregma) was counted in a 0.2-mm wide and 1-mm long strip of tissue under a light microscope. A two-way ANOVA test was performed on these data with three (Ischemia control, Ischemia + low-dose T-588, and Ischemia + highdose T-588) groups and antero-posterior levels as independent variables.
Y. Nakada et al. / Physiology & Behavior 74 (2001) 227–235
The statistical tests were performed using Statview 5.0J (Abacus Concepts, Berkeley, CA). Significance level employed for all tests was P < .05.
3. Results 3.1. ICSS behavior and rats’ locomotion activity Since effectiveness of ICSS and spontaneous motor activity could strongly affect the performance of the PL task, these parameters were checked before the training of the PL task. The results are summarized in Table 1. There were no significant group differences of ICSS current intensities [one-way ANOVA, F(3,30) = 0.091, P >.1] or 10-min spontaneous motor activity [ F(3,30) = 2.21, P >.1]. Furthermore, in the RRPS task, the locomotion pattern of the rats was similar in all the four groups at the end of this task training: the rats visited all parts of the open field homogeneously (examples are shown in Fig. 3Aa, Ba, and Ca). The data on each behavioral parameter in the RRPS task are summarized in Table 2. There were no significant group differences in the mean number of rewards acquired in a session [one-way ANOVA, F(3,30) = 0.559, P >.1], the mean locomotion distance in a session [ F(3,30) = 1.30, P >.1], and the mean locomotion distance between one reward place and the next [ F(3,30) = 2.19, P >.1] in the last day of the RRPS task training. 3.2. Place learning 3.2.1. Typical examples of locomotion patterns in the PL task The typical locomotion patterns of rats in the Shamoperated, Ischemia control, and Ischemia + high-dose T-588 groups during the PL task are shown in Fig. 3Ab, Bb, and Cb. On the first day of the PL task training, the rats in the three groups randomly moved in the open field to the same extent and acquired only a small number of rewards. The rat in the Sham-operated group began to show shuttle behavior (direct back-and-forth movement between the two reward places) on the fourth day of the training (Fig. 3Ab); this Table 1 Effects of T-588 on ICSS current intensity and spontaneous motor activity Group Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
T-588 (mg/kg)
n
ICSS current intensity (mA)
Spontaneous motor activity (m)
0 0 0.3
12 7 7
0.177 ± 0.013 0.184 ± 0.014 0.174 ± 0.011
45.22 ± 5.51 46.14 ± 11.71 63.17 ± 7.39
8
0.182 ± 0.016
66.83 ± 6.93
3
Values, mean ± S.E.M. of ICSS current intensity at which the rats achieved a given criterion (more than 40 bar presses per minute). The rat’s spontaneous motor activity was evaluated as movement distance in the open field 10 min before the training of the RRPS task.
231
behavior dominated progressively in the course of the training. As mentioned in Methods, rats were required to stay in the reward place more than 1 s in order to get ICSS reward; otherwise, they could not get the reward even though they entered the reward place. We sometimes observed that the rat in the Sham-operated group immediately came back to the reward place when it passed the reward place without getting ICSS reward (we called this behavior ‘‘returning’’). In contrast to the Sham-operated group, the rat in the Ischemia control group did not show shuttle behavior during the 30-day test period (Fig. 3Bb). Rather, it developed a locomotion pattern of moving along the wall of the open field, although it showed weak tendency of shortcut later in the training period. We seldom observed the ‘‘returning’’ in this rat. The rat in the Ischemia + highdose T-588 group showed similar pattern to that of Shamoperated group (Fig. 3Cb). The rat began to show shuttle behavior around the fifth day and established it during the rest of the training period. 3.2.2. The mean number of rewards acquired in a session In the present study, the maximum time for a session was 10 min and the maximum number of rewards acquired in a session was 50 (see Methods). So, the more efficiently and quickly the rat moved between the two reward places in the PL task, the more rewards it acquired, approaching the maximum. We used this parameter as an index to evaluate learning. As shown in the Fig. 4A, the number of rewards acquired in a session increased rapidly for the rats in the Sham-operated group. The rats in this group reached the criterion (50 reward acquisition in three or more sessions) by the fifth day (5.3 ± 0.8), and this performance level was maintained throughout the rest of the test period. The increase in this parameter for the Ischemia control group was slow (16.0 ± 3.5), whereas for the Ischemia + high-dose T-588 group, it was analogous to that of the Sham-operated group. The rats in the Ischemia + low-dose T-588 group reached the criterion on the 16th day (15.7 ± 3.4). The rats in the Ischemia + high-dose T-588 group reached the criterion on the seventh day (7.3 ± 1.5). There were significant differences in this parameter between the three ischemic groups [two-way ANOVA, F(2,570) = 41.91, P < .01, Days 1– 30]. Post hoc comparisons revealed that there was a significant difference between the Ischemia + high-dose T-588 and Ischemia control groups and between the Ischemia + highdose T-588 and Ischemia + low-dose T-588 groups. There was no Group Day interaction [ F(58,570) = 0.59, P >.1]. 3.2.3. The mean period of time for rats to complete a session Since a session could finish before 10 min has passed if the rat acquired 50 rewards, the more efficiently and quickly the rat performed the PL task, the shorter the session time. As shown in Fig. 4B, the rats in the Sham-operated group learned the task rapidly so that this parameter decreased steeply in the early sessions (Days 1 –10). In contrast, the rats
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Fig. 3. Examples of change in locomotion pattern of rat in the Sham-operated group (A), the Ischemia control group (B), and the Ischemia + high-dose T-588 group (C) during test period of RRPS task (a) and PL task (b). Other descriptions as for Fig. 2B.
in the Ischemia control group displayed a slow decrease in the value of this parameter during the 30-day test period. The Ischemia + high-dose T-588 group reduced the value of this parameter more rapidly than the Ischemia control group. There were statistically significant differences in this parameter between the three ischemic groups [two-way ANOVA, F(2,570) = 113.2, P < .01, Days 1– 30]. Post hoc comparisons revealed that, to complete a session, the rats in the Ischemia + high-dose T-588 group took less time than the rats in the Ischemia + low-dose T-588 and Ischemia control groups, and the rats in the Ischemia + low-dose T-588 group took less time than the rats in the Ischemia control group. There was no Group Day interaction [ F(58,570) = 0.53, P >.1]. 3.2.4. The mean of the locomotion distance between the two reward places The most efficient strategy in the PL task was shuttling between the two reward places. So, the mean distance
traveled between the two reward places would be minimal if the rat developed this strategy. As shown in Fig. 4C, the value of this parameter decreased rapidly in the early sessions (Days 1 – 9) for the rats in the Sham-operated group. In contrast, the rats in the Ischemia control group displayed a slow decrease in this parameter. Furthermore, a clear fluctuation was observed in this group even in the later sessions of the training. There were statistically significant differences in this parameter between the three ischemic groups [twoway ANOVA, F(2,570) = 18.86, P < .01, Days 1– 30]. Post hoc comparisons revealed that the value of this parameter in the Ischemia + high-dose T-588 group was significantly shorter than those in the Ischemia control and Ischemia + low-dose T-588 groups. The data on the Ischemia + highdose T-588 and Ischemia + low-dose T-588 groups were less fluctuated compared with those on the Ischemia control group in the later sessions of the training. There was no Group Day interaction [ F(58,570) = 0.73, P >.1].
Table 2 Effects of T-588 on the behavioral parameters of the RRPS task
Group
T-588 (mg/kg)
Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
0 0 0.3 3
n
No. of rewards acquired in a session
Locomotion distance in a session (m)
Locomotion distance while entering the reward places (m)
12 7 7 8
31.5 ± 2.2 29.2 ± 2.9 29.6 ± 1.4 27.9 ± 1.6
116.36 ± 9.13 102.73 ± 9.59 97.23 ± 6.07 95.08 ± 9.55
3.86 ± 0.14 3.58 ± 0.11 3.35 ± 0.13 3.53 ± 0.19
Values, mean ± S.E.M. of each behavioral parameter at which the rats achieved a given criterion (more than 25 rewards acquisition over the three sessions per day) in the training of the RRPS task.
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Fig. 4. Effects of T-588 on each behavioral parameter of the place learning task. (A) The mean number of rewards acquired in a session. (B) The mean of the period of time for rats to complete a session. (C) The mean of the locomotion distance between the two reward places traveled by rats in each group. Open circles, Sham-operated group; closed circles, Ischemia control group; closed squares, Ischemia + low-dose T-588 group; closed triangles, Ischemia + high-dose T-588 group. Abscissa, day.
3.3. Histology Brain sections both from the anterior and posterior hippocampi showed selective CA1 neuronal loss in the Ischemia control group. The number of pyramidal cells in each CA1 subfield, which were counted in a strip 0.2-mm wide by 1-mm long, are shown in Table 3. The number of pyramidal neurons with normal appearance in the Ischemia control group was less than 3% of that in the Sham-operated group. Transient forebrain ischemia also produced CA1 neuronal loss to the same extent in the other two ischemic groups: there were no significant differences in the three ischemic groups [two-way ANOVA, F(2,38) = 0.013, P >.1].
Table 3 Number of pyramidal cells in the CA1 subfield of the rat hippocampus A – P level Group Sham-operated Ischemia control Ischemia + low-dose T-588 Ischemia + high-dose T-588
T-588 (mg/kg)
n
3.5 mm from bregma
4.5 mm from bregma
0 0 0.3
9 7 7
87.6 ± 5.3** 2.2 ± 1.5 7.7 ± 4.0
89.6 ± 3.5** 0.3 ± 0.3 8.6 ± 5.3
3
8
9.6 ± 5.1
9.9 ± 3.8
Values, mean ± S.E.M. (neurons/1 mm strip). Pyramidal cells in the CA1 subfield were counted under a light microscope in a 1-mm strip of pyramidal cell layer in the CA1 subfield. ** P < .01 vs. Ischemia control group (Scheffe’s test).
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4. Discussion 4.1. Influence of transient forebrain ischemia on PL In the present study, 15-min transient forebrain ischemia produced selective neuronal loss in the CA1 subfield of the HF, which was accompanied with substantial learning deficits in the PL task. That is, while the Sham-operated rats altered the behavioral strategy from inefficient pattern (random movement) to the efficient (shuttle movement) in the early stage of the PL task, most Ischemia control rats did not display such a behavioral alteration. Rather, these rats tended to display different behavior, such as traveling along the wall of the open field. Furthermore, as described in Results, the rats in the Sham-operated group often showed ‘‘returning’’ when they passed the reward place, whereas the rats in the Ischemia control group did not. These findings suggest that the Ischemia control rats did not recognize the location of the reward places in the PL task. The three behavioral parameters in the PL task also indicated the impaired performance of the Ischemia control rats. These results were consistent with the previous studies in which the same behavioral paradigm was used for the rats exposed to transient forebrain ischemia [25,26], indicating the reliability of the present behavioral protocol. 4.2. Effects of T-588 on PL The principal finding of the present study was that the administration of high-dose T-588 significantly ameliorated the impaired task performance of the rats exposed to transient forebrain ischemia. This effect appeared at relatively early stage of the PL task training so that the task performance of the rats in the Ischemia + high-dose T-588 group was comparable to that of the rats in the Sham-operated group within a week. If a drug would have an influence on locomotion or rewarding efficacy, such an action could also influence on learning. However, in the present study, it is implausible that the ameliorative effect of T-588 occurred via changes in locomotor ability or rewarding efficacy since there were no significant effects of T-588 on spontaneous locomotion activity or ICSS current intensity. In the present study, there were no significant differences in degree of the neuronal loss observed in the CA1 subfield between the three groups exposed to transient forebrain ischemia. Although T-588 is known to have a neuroprotective effect [34], this is not surprising because T-588 administration began 3 weeks after the ischemic manipulation when the initial phase of neuronal death completed. This, however, does not rule out the possibilities that T-588 acts to prevent the chronic development of ischemia-induced neuronal damages and that T-588 acts to maintain residual cell bodies and neuropils in the damaged CA1 subfield. Indeed, administration of T-588 changed the activity in the HF of the ischemic rats in a previous study using functional magnetic resonance imaging [24]. Although these facts
indicate that T-588 activates the function in the residual hippocampal subfields, compensatory activation in other brain areas could also contribute to the ameliorative action of T-588. More studies are necessary to elucidate this point. The involvement of ACh in spatial learning and memory has been reported in several previous reports (e.g., Refs. [4,12,20,21]). The cholinergic dysfunction in the ischemic brain is remarkable [5,14,40]. Activation of cholinergic system with acetylcholinesterase inhibitors or muscarinic agonists improves spatial memory deficits following cerebral ischemia [1,4,45]. These findings indicate that reduced cholinergic action in the HF is involved in spatial memory deficits accompanied with ischemic damage. T-588 reportedly ameliorates memory impairment in rats produced by basal forebrain lesions, which result in a selective loss of cortical cholinergic markers [33]. A previous study showed that oral administration of T-588 at a dose of 3 mg/kg causes a significant increase in ACh release in the frontal cortex and HF [32]. These studies, together with other reports [29 – 31], suggest that T-588 has cholinergic stimulant actions, which is a possible transmitter mechanism underlying the therapeutic effects of T-588 observed in the present study.
Acknowledgments This work was supported partly by Grant-in-Aid for Scientific Research on Priority Areas (A)-Research for Comprehensive Promotion of Study of Brain (12050220), Priority Areas (C)-Advanced Brain Science Project (12210009), and Scientific Research (11308033 and 11680805) from Ministry of Education, Culture, Sports, Science and Technology, Japan.
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