Series of Selected Papers from Chun-Tsung Scholars,Peking University (2002).

Roles of oxytocin in spatial learning in the nucleus basalis of Meynert in rats

Wei Wu , Long-Chuan Yu
College of Life Sciences, Peking University, Beijing 100871, China

Abstract

Oxytocin (OT) has been implicated to play an important role in learning and memory in the central nervous system. The present study was performed to explore the effect of OT administered to the nucleus basalis magnocellularis (NBM) on the spatial learning and memory in rats. The rat was trained in the Morris water maze after intra-NBM administration of different doses of OT. The latency of spatial learning was impaired after intra-NBM injection of 2 or 10 nmol of OT, but not 0.2 nmol of OT, compared with the control group tested by Morris water maze. Furthermore, the impaired effect of OT on the latency of spatial learning was inhibited after intra-NBM administration of the OT receptor antagonist CAP 518. There were no significant changes in the swimming speed of rats during 5 days after OT injected into NBM of rats.

These results suggest that OT could play a modulatory role in the NBM by acting on the NBM Oxytocin receptor and thereby impair spatial learning.

Key words: OT, CAP 518, NBM, spatial learning, Oxytocin receptor, Morris water maze.

1.Introduction

Since the pioneering work of de Wied and his colleagues, the neuropeptide oxytocin (OT) has been thought to play a pivotal role in behavioral regulation in general, and in learning and memory in particular [10]. OT was considered to be an amnestic peptide as claimed by Kovacs and Telegdy [22], however, the exact role of it remains to be answered [14]. OT was shown to facilitate the extinction of avoidance reaction [5,11, 21] and to attenuate the storage of verbal memory [7]. The effect of OT on the social memory is widely reported. OT had an impairing effect on the social memory in male rats[9]. However, Arletti et al found that intraperitoneally injected OT to male rats significantly improved social memory [2]. Depending on the dose, social memory was shown to be attenuated or facilitated by OT and derivatives as reported by Popik et al [31].

The important role of the cholinergic system in learning and memory has been suggested by many findings [12, 15,16,23, 26, 27, 35]. The nucleus basalis magnocellularis (NBM) provides the primary cholinergic projection to the cerebral cortex [4, 33, 34, 37]. Lesions to the NBM impair memory formation in various learning tasks [1, 3, 6, 8, 12, 13,15, 17,20,25, 37]. Recent evidence has shown that NBM lesions in rats can induce permanent chlinergic hypofunction, as well as severe cognitive dysfunction [28, 33]. The present study was performed to investigate the role of OT in the modulation of spatial learning and memory in the NBM of rats. Furthermore, the involvement of oxytocin receptors in OT-induced impairment of spatial learning was also studied.

2.Experimental procedures

2.1Materials

OT (oxytocin acetate,PolyPeptide Laboratories, A/S, Herredsvejen 2-3400, Hillerod, Denmark) and CAP 518 were prepared with sterilized saline (0.9%).

2.2 Animals

All experiments were performed on freely moving male Wistar rats (200-300 g; Experiment Animal Center of Peking University, Beijing, China). The rats were housed in cages with free access to food and water, and maintained in a room temperature of 24±2℃with a 12 h light-dark cycle. Every effort was made to minimize animal suffering and the animal number.

2.3 Surgical procedure

Rats were anaesthetized by intraperitoneal pentobarbital (50 mg/kg) and were mounted on a stereotaxic instrument. A stainless steel guide cannula of 0.8 mm outside diameter was directed to the NBM (AP 1.4, L 3.0, H 7.0 mm from the surface of the skull) according to Paxinos and Watson [18] and was fixed to the skull by dental acrylic. After surgery a dummy cannula was put into the guide cammula to reduce the risk of infection, and the animals were allowed two days of recovery.

2.4 Apparatus

A circular water tank, 120 cm in diameter and 60 cm in depth was used in the Morris Water Task procedure. The water tank was filled with powdered milk, kept at 20±1℃, and the escape platform was submerged 1 cm below the water surface. The water tank was located in a room, which contained several visual cues, such as a small round mirror and a poster on the wall and a daylight lamp on the ceiling. All cues were kept constant throughout the experiment. The test parameters were measured using a digital TV system connected to a computer.

2.5 The Morris water task

The rats were randomly allocated to 8 experimental groups included in two experiments.

Experiment 1. One microliter of 0.9% saline was given to the NBM as a control (n=10); 0.2 nmol (n=8), 2 nmol (n=7) or 10 nmol (n=7) of OT was administered to the NBM.

Experiment 2. One microliter of 0.9% saline and 2 nmol of OT were administered to the NBM (n=10); 2 nmol of CAP 518 and 1 ul of 0.9% saline were given to the NBM (n=10); 2 nmol of CAP 518 and 2 nmol of OT were given to the NBM (n=6); 2 ul of 0.9% saline was given to the NBM (n=10).

All animals were allowed to habituate in the experimental room for 1 h prior to testing. Saline alone or with OT/CAP 518 was bilaterally infused in the NBM using a microinfusion pump. The injection needle with 0.4 mm diameter was inserted directly into the guide cannula, with 1 mm beyond the tip of the latter. One microliter of solution was thereafter infused into the NBM over 1min. Testing began at 15 min after the infusion of OT or saline in Experiment 1. However in Experiment 2, rats received the NBM injection of the first substance (CAP 518 or saline), followed 5 min later by the NBM injection of the second substance (OT or saline). Testing began 15 min after the infusion of the second substance.

During training the submerged escape platform was located in the center of the southeast quadrant. All rats of the 8 test groups were given four trials per day for five consecutive days. In each trial the rat was placed in the water facing the pool wall at one of four selected starting points (east, north, west and south pole). The animals always started in the first trial of each day at east pole, and the second trial at north pole, the third trail at west pole, the fourth at south pole. Once the rats located the platform, it was allowed to stay on it for 30 s. If the rat did not find the platform within 65 s, it was gently directed to the platform by hand and it was allowed to remain there for 30 s. During each trail, the rat’s escape latency, swim speed and swim length were measured using a computer connected to a video camera.

Retention test. One week after the last trial on day 5, a retention test was done. In this test, the platform was removed from the pool and the animals were allowed to swim freely for a 65 s period. The rat was released into the water tank from a position opposite to the quadrant where the platform had been during the training sessions.

2.6 Histology

Immediately after the retention test, all rats were killed by a high dose of pentobarbital (80 mg/kg) and the rat heads were fixed in 10% formalin for l week with the injecting rube in situ before section. The location of the tip of the injecting tube was verified and all the tips of the injecting tube were in the NBM area of rats in the present study.

2.7 Statistics

At the conclusion of the experiments, the location of the tip of the injection tube was verified. Only the results from the Morris Water Task where the tips of the injection tube were within the NBM were used for statistical analysis. The data were presented as the mean±S.E.M. The difference between groups was determined by two-way analysis of variance (ANOVA). The post hoc comparisons and Newman-Keuls test was performed for further analysis. *P<0.05, **P<0.01 and ***P<0.001 were considered as significant differences.

  1. Results
  1. Effects of intra-NBM injections of OT on spatial learning

Four groups of rats received intra-NBM injection of 0.2 (n=8), 2 (n=7), 10(n=7) nmol of OT or 1ul of 0.9% saline (n=10)as a control. The results were shown in Fig. 1A. For all the rats hadn’t been trained in the Morris water maze before the first experimental day, all the rats were found to search the platform merely by chance on the first day. So only the data (latencies, lengths and speeds included) on day 2-5 are considered in ANOVA analysis.

Rats received the NBM injection of 2 nmol OT showed a significantly (F=16.24, P<0.002) retarded learning compared to the control group. Also the 10 nmol dose impaired the acquisition of the spatial learning test and prolonged the latency significantly (F=7.48, P<0.05) compared to the control group. However when the lowest dose 0.2 nmol was examined, no significant differences in latency were found. (F=0.002, P>0.9)

There was no significant change in the mean swim speed between days during the study period, in any of the four groups (Fig. 1B). Neither was there any difference in swim speed between the four groups.

  1. Effects of intra-NBM injections of CAP 518 and OT on spatial learning

Two groups of rats were given 1 ul of 0.9% saline and 2 nmol of OT (n=10) or 2 ul of 0.9% saline (n=10). The results were showed in Fig.2A. 2 nmol OT caused a statistically significant (F=16.35, P<0.001) impairment of acquisition. This impairment was completely (F=6.73, P<0.05) blocked by CAP 518. (Fig. 2B). However, CAP 518 alone did not have any significant (F=2.91, P>0.1) effect on learning performance compared with the control group. (Fig. 2C)

There was no significant change in the mean swim speed between days during the study period, in any of the four groups (Fig. 2D). Neither was there any difference in swim speed between the four groups.

Fig.1 A: Effect of intra-NBM injection of OT on spatial navigation in the Morris Water Task. B: Effect of intra-NBM injection of OT on swim speed. The data were expressed as mean±S.E.M.(averaged over four trials per session). A significant change of latency compared to control was marked (*P<0.05, **P<0.01).

Fig 2. A BC: Effects of intra-NBM injections of OT or CAP 518 on spatial navigation in the Morris Water Task D: Effect of intra-NBM OT and CAP 518 injections on swim speed The data were expressed as mean±S.E.M.(averaged over four trials per session). A significant change of latency compared to control was marked (*P<0.05, **P<0.01, ***P<0.001).

  1. Effects of intra-NBM injections of CAP 518 and OT on memory retention

Retention performance was examined in all rats seven days after the last trail, using a probe trial procedure. No significant difference between the groups with regard to time spent in target quadrant or swim length in target quadrant was found. An analysis of various retention measures in shown in Table 1.

Table 1. Effects of OT and CAP 518 on memory retention in the spatial learning task

Experiment / Prior treatment / Dose(nmol) / Time spent in target quadrant(%)±S.E.M. / Swim-length in target quadrant (%)±S.E.M.
Saline / 32.4±3.2 / 31.8±3.2
1 / OT / 0.2 / 32.1±3.9 / 30.6±3.4
2 / 29.0±4.7 / 28.3±4.1
10 / 32.4±5.7 / 31.1±5.2
2 / Saline+saline / 32.4±3.2 / 31.8±3.2
Saline+OT / 2 / 28.2±2.7 / 28.0±1.9
CAP 518+OT / 2+2 / 28.3±2.6 / 26.9±2.8
CAP 518+saline / 2 / 38.1±2.7 / 35.6±2.9

In the retention procedure, the animals received the last training trial seven days prior to the probe trial and they did not receive any further treatment before the retention test. One trial was run in the probe test. The results are expressed as of time (%)±S.E.M. of the total time (65s) and swim length (%)±S.E.M of the total length, performed in the target quadrant. No significant in retention performance between the groups were observed.

  1. Effects of intra-NBM injections of CAP 518 and OT on swim paths

Swim paths recorded by the computer during trail 3 on day3 of acquisition by representative animals in the different treated groups, are shown in Fig.3. The results show that animals treated with 2 nmol and 10 nmol of OT swam a significantly longer distance in search of the platform than those in the control group, which indicates a more random search pattern compared to control.

A B C

D E F

Fig 3. Swim paths taken during trial 3 on day 3 of training by representative animails in the different groups: (A) Control (saline); (B) 0.2 nmol of OT; (C) 2 nmol of OT; (D) 10 nmol of OT; (E) 2 nmol of CAP 518; (F) 2 nmol of CAP 518 + 2 nmol of OT.

  1. Discussion

The present study found that intra-NBM administration of OT induced increases in latencys of the Morris Water Task, indicating that OT produced an impairment effect of spatial learning in the NBM of rats. The OT-induced impairment in spatial learning was blocked by intra-NBM injection of the oxytocin receptor antagonist CAP 518, indicating an involvement of oxytocin receptor in the OT-induced impairment in spatial learning.

A significant impairment of acquisition was observed at the 2 nmol of OT and this impairment was not increased when the dose of OT was increased five times. This finding may suggest that there is a threshold level of OT activity beyond which no further impairment is seen. It is also notable that the spatial learning ability was not completely blocked, since the latency in the treated group decreased over the five consecutive days. An interpretation of the above finding is OT acts on a limited number of receptors and not all relevant receptors in the NBM are reached.

Uvnas-Moberg K et al. reported that in the rats treated with low doses of oxytocin (1-4 mg/kg), there was a decrease in peripheral locomotor activity. With increasing doses (250-1000 mg/kg), there were clear signs of sedative effects as indicated by a suppression of locomotor activity and rearing [36]. Therefore, an evaluation of motor activity of the different groups was performed. The swim speed of the treated groups compared to control is shown in Fig.1B and Fig.2D. The results show that there is no significant effect in swim speed (motor activity) of intra-NBM injections of OT.

The swim paths for individual animals representative for each group are shown in Fig.3. The results indicate different search patterns for the groups. The control animals found the platform in a distinctive way without extensive searching. However, the animals treated with OT had to search the platform for a significantly longer time period than the control animals, which indicates a more random search pattern compared to control. The animals treated with CAP 518 found the platform quite fast, which indicates that this group had a good recollection of the task. Rats treated with CAP 518 and OT found the platform faster than the OT treated animals without any increase in swim speed.

The retention performance was examined in a probe trial seven days after the last trail in the acquisition experiment. The data in the retention test show that the rats remember the location of the platform at the time of retention testing. The time and swim length in target quadrant was above the 25% expected by chance. It may seem paradoxical that the retention performance was seemingly not affected by OT treatment (Table 1). The interpretation of this discrepancy given by J.Sandin et al., when they studied the role of hippocampal dynorphin B in spatial learning, is the rats of the control group actually had a very good memory of the platform-location, but after a short search they left the target quadrant probably due to lack of motivation [32].

Recent studies have found that the effect of OT in learning and memory is related with central cholinergicsystem. It’s suggested that oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response [5].The situation in NBM may be similar. Autoradiographic study has found high density of OT binding sites in this area [19,24]. It’s possible that OT impairs spatial learning through the interaction with cholinergic system, since this system of NBM has been demonstrated to play a very important role in learning and memory [28,29,30]. However this interaction need to be proved with further study.

In conclusion, our results show that intra-NBM injections of OT impair spatial learning in rats. These findings suggest that the NBM OT system plays a significant role in learning processes and that OT has a regulatory function in memory acquisition and the early stages of memory formation.

Acknowledgements

The study was supported by funds from the National Natural Science Foundation of China (NSFC) and Hui-Chun Chin and Tsung-Dao Lee Chinese Undergraduate Research Endowment.

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