- Email: [email protected]
Restoration of serotonergic innervation underlies the behavioral effects of raphe grafts
Brain Research, 566 (1991) 21-25 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50
21
BRES 17198
Restoration of serotonergic innervation underlies the behavioral effects of raphe grafts Gal Richter-Levin
and Menahem
Segal
Center for Neuroscience, The Weizmann Institute, Rehovot (Israel)
(Accepted 16 July 1991) Key words: Raphe; Graft; Serotonin; Hippocampus; Spatial memory
It has been previously demonstrated that an embryonic raphe grafted into a serotonin-depleted hippocampus restores normal scrotonin innervation of the hippocampus 1°'23'24 and behaviors associated with serotonin 14'Is'2°. To test the possibility that the behavioral effects of these grafts result from non-specific actions of the grafted tissue or the grafting procedure itselfs'l', we compared raphe grafts with septal grafts, in serotonin-depleted rats. We also compared the effects of a serotonin synthesis inhibitor, p-ch!orophenylalanine, on the behavior of normal, serotonin-depleted and raphe-grafted rats. The results indicate that the bulk of behavioral effects of raphe grafts are due to the serotonergic nature of the graft.
INTRODUCTION The possibility of restoring impaired brair~ functions by transplantation of neural tissue has attract!ed a great deal of attention over the past decade s'6't2. Tlms, it has been demonstrated that grafted neurons can ameliorate cognitive and motor deficits resulting from selective lesions in host mammalian brains s'6.t2. Embryonic raphe cells grafted into the hippocampus of serotonin-depleted, septal-lesioned rats hell:~ compensate for spatial memory deficits observed in these rats Is' 2o. While we would like to believe that a raphe graft is instrumental in restoring impaired functions because it contains serotonergic neurons, it is possible that the grafted tissue may affect the host brain in several nonspecific ways (e,g, as a source of trophic factors or as a 'bridge' to facilitate the growth of axons of intrinsic nerve cells) s'tt. These grafts restore the serotonergic innervation of the hippocampus and very often result in a hyper-serotonergic innervation at the areas surrounding the graft 10'20'23'24. Nevertheless, when staining for serotoner~ic immunoreactivity, it is obvious that only a small fraction of the grafted cells are serotonergic tg. Furthermore, the grafting procedure causes slight but noticeable damage to the host brain (e.g. Fig. 1). This may trigger some compensatory processes that are not dependent on the serotonergic nature of the graft. Thus, when evaluating the behavioral effects of the grafts, it is imperative to test the degree of specificity of the grafts. In the present study, we examined the involvement of
serotonin in the behavioral effects of raphe grafts. The effects of embryonic raphe tissue, rich with serotonincontaining cells, were compared with those of embryonic septal tissue. In another study, the ability of the serotonin synthesis blocker, p-chlorophenylalanine (PCPA) 13 to affect behavior of raphe-grafted rats was compared with that of control rats. The results suggest that, although grafted embryonic tissue of both septal and raphe origin may exert some non-specific effects, the improvement in the ability to perform the watermaze, observed in the raphe-grafted rats, depends largely on the presence of sufficient levels of serotonin in the hippocampus, EXPERIMENT 1: COMPARISON BETWEEN THE EFFECTS
OF RAPHE AND SEPTAL GRAFTS Materials and Methods
Adult (200-250 g) male Wistar rats from a local breeding colony were injected with 5,7.dihydroxytryptamine (5,7-DHT) (intraventricularly, bilaterally, 100/~gfree base in 10/~1of saline containing 0.2% ascorblc acid on each side) under chloral hydrate anesthesia (3.5% solution, 10 mg/kg, i.p.). Rats were pretreated with 30 mg/kg of desipramine (i.p., in 1 ml saline) 20 min prior to anesthesia. One week later 5,7.DHT-treated rats were grafted with either raphe (RG) or septal (SG) embryonic tissue (day 14 and day 17, respectively, bilaterally, into the hippocampus) as described19 (coordinates for transplantation: 4.5 mm posterior from bregma; 5.5 mm lateral; depth from 6 to 4.5 mm below the skull). Controls were naive male rats. Experiments were conducted 5-6 months after transplantation. The watermaze consisted of a round pool of water (130 cm di-
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute, Rehovot 76100, Israel.
22 ameter, 50 cm high rim and water level of 18 cm. A glass platform, l0 cm in diameter, was submerged in the water, 3 cm below the surface). Milk powder was added to the pool to achieve opaque water. Rats were placed in the pool and could escape it by climbing the platform, using extra-maze cues. Intertrial intervals were 15-20 rain. The escape latency was used as the measure of level of performance t~. Control (n = 5), 5.7-DHT injected (DHT, n = 5), DHT rats having a raphe graft in the hippocampus (RG, n = 9), and DHT rats having a septal graft in the hippocampus (SG, n = 5) were tested in the water maze as follows. Rats were placed in the watermaze facing the wall, and left there for 60 s or until they reached the platform. Rats were led to or left on the platform for 15 s before being taken hack to their home cages. Rats were given 8 trials a day. During the last two trials of the second day the room was darkened to reduce extra-maze cues and verify that rats do indeed use these cues to locate the platform. On the third day, all rats were treated with atropine (25 mg/kg, i.p.) 20 rain before the third trial. This low dose of atropine was administered in an attempt to reproduce the conditions under which serotonin-depleted rats exhibit poor performance in the water-maze task 16'zT'2s. At the end of behavioral experiments, rats were decapitated, their brains quickly removed and kept at -70 °C. The brains were sliced into 30-pro sections for histological studies. The presence of grafts was detected by Cresyl violet staining2°. In all rats examined (n = 14), except for one (which was excluded), the grafted tissue, of both rdphe and septai origin, could be easily detected by Cresyl violet staining (Fig. 1). in most cases, the graft was located within the medial-ventral hippocampus, most often in or near the dentate gyrus. In two cases, the grafts were located on the medial wall of the dentate gyrus. The size of the
grafts was 0,5-1 mm 2. Grafted tissue caused a slight distortion of the host hippocampal tissue only at the site of transplantation (Fig.
I). Statistical analysesutilized ANOVAfor repeated measurements. Results
There were no significant differences in performance between the groups during the first day, except for a tendency toward longer escape latencies of the SO rats during trials 2-3 (F -- 4.58, P < 0.04) (Fig. 2A). Likewise, there were no significant differences among the groups during the second day. To test for the possibility that under extreme conditions, of minimizing extra-maze cues, an adverse effect of serotonin depletion will become evident, the lights were dintmed during the last two trials of the second day. Reducing the illumination in the room affected all rats but there was still no significant difference between groups, due to an increase in variability in the behavior of the rats (Fig. 2B). On the third day o~ training, control and RG rats returned to their previous level of performance, whereas, in the first trial, the DHT and SG rats were still significantly worse (control vs DHT; F = 7.07, P < 0,01: control vs SG; F = 7.4, P < 0.01) (Fig. 2C). On the second retention trial (prior to administr~:tion of atropine) all the groups performed as before. Following atropine (Fig. 2C), DHT rats had significantly longer escape latencies compared to control (F = 12.4, P < 0,002) and to RG (F = 7.1, P < 0.01). The
Fig. 1. Cresyl violet sections of the hippocampus of a raphe-grafted rat. Arrows indicate the graft. DG, dentate gyms; BS, brainstem; S, subiculum. Scale bar = 0.5 ram.
performance of the SG group lay in between these groups. There was no significant difference between the SG rats and the DHT group, nor between them and the RG rats, but there was a significant difference between them and control rats (F = 5.46, P < 0.02) (Fig. 2C). In contrast, RG and DHT rats were significantly different (F - 6.01, P < 0.02) and there was no significant difference between them and control rats. The RG rats, however, exhibited a non-significant tendency to be slower thaa controls. This was particularly evident in the first trial after atropine injection.
Discussion The results of this experiment confirm earlier observations in suggesting that blockade of both serotonergic (by 5,7-DHT) and cholinergic (by atropine) neurotransmission but not each of them by itself, causes a severe retention deficit tf-ts'2s, Both DHT and SG rats were less affected than controis or RG rats by dimming the lights (Fig. 2B, trial 7). It is possible that these rats were depending less on extra-maze cues and that the improvement in their performance up to that stage was in part due to the development of a different searching strategy. Further studies, howev.~.r, are needed to clarify this possibility. The inability of septal grafts to mimic the effects of raphe i,rafts supports the assumption that the behavioral effects associated with the graft are unique to the raphe. These results are congruent with the previous observation that raphe but not septal grafts could restore the responses of the hippocampus to application of serotonin-releasing drugs, in serotonin-depleted rats tg.
23 A
m~.50
EXPERIMENT 2: THE EFFECTS OF RAPHE GRAFTS IN THE PRESENCE OF PCPA
Day 1
60
o---- control,n=5 ; DHT, n=5 = RG, n=9
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Materials and Methods
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Day 2 o---- control, n=5 : DHT, n=5 = RG, n=9
Three groups were used. Control rats (n = 7), DHT rats (n = 4) and RG rats (n = 12). Lesioning and grafting procedures were the same as in Expt. 1. In order to test the rats before and after administration of PCPA, at the same stage of retention, rats were first trained in the water-maze (6 days, 4 trials a day). At this stage, application of either atropine or scopolamine, at relatively high doses (40 mg/kg and 4 mg/kg, respectively), had no effect on the performance of any of the groups in the water maze (data not shown). They were then left in their home cages for 3 weeks after which they were tested in the following procedure: rats were given two trials in which the platform was placed in the same location as during training. They were then injected with scopolamine (2 mg/ kg, i.p.) 20 min before a third trial. The platform was then shifted to a new location and the rats were given 3 additional trials.
A
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control,n,.7 5,7-DHT,n,.4 RG, n-12
C
platform shift
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Trial No. Fig. 2. A comparison between the effects of raphe and septal grafts in the hippocampus of serotonin depleted rats. A: day 1, acquisition day. No significant differences were found between groups except for a tendency for a slower learning rate, in the SG group (trials 2-3, F = 4.58, P < 0.04). B: day 2, retention day and the effects of darkening the room. No significant differences were found during retention trials or after darkening the room. C: day 3, retention day and the effects of atropine (25 mg/kg, i.p.). In the first trial the DHT and SG rats exhibited a significantly poorer performance compared to control rats (control vs DHT, F = 7.07, P < 0.01; control vs SG, F = 7.4, P < 0.01). During the second trial, however, there were no significant differenccs between the groups. Following the administration of atropine (25 mg/kg i.p.) control rats performed significantly better than DHT rats (F = 12.4, P < 0.002) and SG rats (F = 6.1, P < 0.02). RG rats were not different from control. (Note differences in scale between A, B and C.)
Fig. 3. A compa~,'ison between the effects of raphe grafts before and after administration of PCPA. A: before PCPA. There were no significant differences between groups during the retention trials nor following the administration of scopolamine. Shifting the platform to a new location significantly disturbed DHT rats compared to control (F = 8.38, P < 0.008) and to RG (F = 5.61, P < 0.02). B: in the presence of PCPA. PCPA did not affect the performance of any of the groups when the platform was placed in the same location as during training. Scopolamine had no effect at this stage. After shifting the platform, control rats performed similar to their performance before administration of PCPA. DHT rats were affected by the shift of the platform as before. Contrary to their pre-PCPA performance RG rats were now significantly affected compared to control (F = 5.54, P < 0.02). There was still a significant difference between the RG and DHT rats (F = 4.15, P < 0.05).
24 The rats were then injected with PCPA (200 mg/kg, i.p.) and were tested again 3 days later, using the same procedure. Statistical analyses utilized ANOVA for repeated measurements. Results
There were no significant differences among any of the groups during the first 3 trials on the control day, even after the injection of scopolamine (Fig. 3A). However, when the platform was shifted to a new location, DHT rats had significantly longer escape latencies during the last two trials compared to the control (F = 8.38, e < 0.008) and the RG groups (F = 5.61, e < 0.02) (Fig. 3A). There were no significant differences among the 3 groups in the first 3 trials of the second testing day. Scopolamine did not have an effect on the performance of any of the groups. However, when the platform was shifted, control rats were only affected during the following trial but returned to previous levels of performance immediately thereafter, as they did on the first testing day. In contrast, both the DHT and the RG groups did not return to previous levels of performance until the end of the test (DHT vs control, F = 13.14, P < 0.001; RG vs control, F = 5.54, P < 0.02) (Fig. 3B), There was a significant difference between RG rats and the DHT group (F = 4.15, P < 0.05) as well. Discussion
The results indicate that the relatively better watermaze performance of raphe-grafted rats depends largely on the serotonergic component of these grafts. When serotonin synthesis is reduced, the ability of these rats to perform in the maze is impaired and becomes similar to that of the DHT rats. A relatively low dose of PCPA ~'tT'2s was used here in order to reduce possible significant effects of the combined treatment on control rats. Administration of PCPA did not affect significantly the performance of DHT rats either, thus enabling the evaluation of the contribution of the grafts,
REFERENCES 1 Airman, N.J. and Hormile, H.J., What is the nature of the role of the serotonergic nervous system in learning and memory: prospects for development of an effective treatment strategy for senile dementia, Neurobiol, Aging, 9 (1988) 627-638. 2 Altman, H.J., Normile, HJ., Galloway, M.P., Ram;,rez, A. and Azmitia, E.C., Enhanced spatial discrimination learning in rats following 5,7-DHT-induced serotonergic deafferent~tion of the hippocampus, Brain Research, 518 (1990) 61-66. 3 Airman, H.J., Ogren, S.O., Berman, R.F. and Normile, H.J., The effects of p-chloroamphetamine, a depletor of brain serotonin, on the performance of rats in two types of positively reinforced complex spatial discrimination tasks, Behav. Neural.
GENERAL DISCUSSION The present results indicate that raphe grafts have a unique effect on the ability of serotonin-depleted rats to perform well in the spatial memory water-maze task. These effects could not be mimicked by septal grafts and were absent in the raphe-grafted rats in which serotonin synthesis was reduced. The slower rate of acquisition observed in the SG group (Expt. 1, day 1) may indicate some adverse effects of the grafting procedure or the grafted tissue on the host brain, as was suggested before 7. Septal grafts, however, improved the performance of septal-lesioned rats in the water-maze t5'22, suggesting specificity of the effects of grafts. While the present results illustrate the importance of the serotonergic innervation of the hippocampus in spatial memory, the nature of its involvement remains unclear. Depletion of forebrain serotonin adversely affected the ability of septal-lesioned rats to perform the watermaze t6'2°, and yet, it increased the ability of rats to perform other learning tasks t. Furthermore, selective depletion of the serotonergic fibers innervating the hippocampus increased the ability of rats to perform some spatial memory tasks 2'3'2t. These conflicting results, while indicating an important influence of the serotonergic system on these behaviors, suggest that these are secondary effects of the serotonergic system on other neurotransmitter systems, The lack of effect of serotonin depletion alone on the performance of rats in the watermaze 4't6.t7 (Expt. 1, days 1-2) supports this view. The present results help in establishing the use of raphe grafts as a tool for further studies related to the behavioral roles of the serotonergic system.
Acknowledgements, We thank E, Pearl for her technical assistance, This research was supported by a grant from MINERVA Foundation, Munich, F,R,G., and by a research grant from MINERVA from the Mario-Negri lnstitute-Weizman Collaboration fund,
Biol., 52 (1989) 131-144. 4 Asin, K,E,, Wimhafter, D, and Fibiger, H.C., Electrolytic, but not $,7.dihydroxytryptamine, lesions of the nucleus raphe impair acquisition of a radial maze task, Behav. Neural. Biol., 44
(1985) 415-424, 5 Azmitia, E,C, and Bj6rklund, A,, Cell and Tissue Transplantation into the Adult Brain, Ann, N,Y. Acad. Sci., 495, (1987). 6 Bj0rklund, A, and Stenevi, U,, Neural Grafting in the Mammalian CNS, Fernstrom Foundation Series, Vol. 5, Elsevier, Amsterdam, 1985. 7 Buzs6ki, G., Freund, T., Bjgrklund, A. and Gage, F.H., Restoration and detrioration of function by brain grafts in the septohippocampal system. In D.M. Gash and J.R. Sladek, (Eds.), Progre~ in Brain Research Vol. 78, Elsevier, Amsterdam, 1988,
25 pp. 69-77. 8 Buzsfiki, G. and Gage, EH., Neural grafts: possible mechanisms of action. In T.L. Petit (Ed.), Neuronal Plasticity: a Lifespan Approach, Liss, New York, 1987. 9 Chaput, Y., Lesieur, P. and De Montigny, C., Effects of shortterm serotonin depletion on the efficacy of serotonin neurotransmission: Electrophysiological studies in the rat central nervous system, Synapse, 6 (1990) 328-337. 10 Daszuta, A., Strecker, R.E., Brundin, P. and Bj6rklund, A., Serotonin neurons grafted to the adult rat hippocampus. I. Time course of growth as studied by immunohistochemistry and biochemistry, Brain Research, 48 (1988) 1-19. 11 Freed, W.J., De Medinaceli, L., Wyatt, R.J., Promoting functional plasticity in the damaged nervous system, Science, 227 (1985) 1544-1552. 12 Gash, D.M. and Sladek, J.R., Progress in Brain Research, VoL 78, Elsevier, Amsterdam, 1988. 13 Koe, B.L. and Weissman, A., P-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. Exp. Ther., 154 (1966) 499-510. 14 Luine, V., Renner, K., Frankfurt, M. and Azmitia, E.C., Raphe transplants into hypothalamus reverse facilitation of sexual behavior in 5,7-dihydroxytryptamine-treated female rats: immunocytochemical, neurochemical and behavioral studies. In A. Bj6rklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Fernstrom Foundation Series, Vol. 5, Elsevier, Amsterdam, 1985, pp. 655-662. 15 Nilsson, O.G,, Shapiro, M.L., Gage, F.H., Oiton, D.S. and Bj6rklund, A,, Spatial learning and memory following fimbriafornix transection and grafting of fetal septal neurons to the hippocampus, Exp. Brain Res., 67 (1987) 195-215. 16 Nilssen, O.G., Strecker, R.E., Daszuta, A. and Bj0rklund, A., Combined cholinergic and serotonergic denervation of the forebrain produces severe deficits in a spatial learning task in the rat, Brain Research, 453 (1988) 235-246.
17 Richter-Levin, G. and Segal, M., Spatial performance is severely impaired in rats with combined reduction of serotonergic and cholinergic transmission, Brain Research, 477 (1989) 404407. 18 Richter-Levin, G. and Segal, M., Raphe cells grafted into the hippocampus can ameliorate spatial memory deficits in rats with combined serotonergic/cholinergic deficiencies, Brain Research, 478 (1989) 184-186. 19 Richter-Levin, G. and Segal, M., Grafting of midbrain neurons into the hippoc~mpus restores serotonergic modulation of .~ippocampal activity in the rat, Brain Research, 52i (1990) 1-6. 20 Richter-Levin, G. and Sega!, M., The effects of serotonin depletion and raphe grafts on hippoc~mpal electrophysiology and behavior, $. Neurosci., 11 (1991) 1585-1596. 21 3akurai, Y. and Wenk, G.L., The interaction of acetylcholinergic and serotonergic neural systems on performance in a continuous non-matching to sample task, Brain Research, 519 (1990) 118-121. 22 Segal, M., Greenberger, V. and Pearl, E., Septal transplants ameliorate spatial deficits and restore cholinergic functions in rats with a damaged septo-hippocampal connection, Brain Research, 500 (1989) 139-148. 23 Sharp, T. and Foster, G.A., In vivo measurement using microdialysis of the release and metabolism of 5-hydroxytryptamine in raphe neurons grafted to the rat hippocampus, J. Neu. rochem., 53 (1989) 303-306. 24 Steinbusch, H.W.M., Beek, A., Frankhuzen, A.L., Tonnaer, J.A.D.M., Gage, EH. and Bj6rklund A., Functional activity of raphe neurons transplanted to the hippocampus and caudateputamen. In E.C. Azmitia and A. BjOrklund, (Eds.), Cell and Tissue Transplantation into the Adult Brain, Ann. N. E Acad. Sci., Vol. 495, 1987, pp. 169-184. 25 Vanderwolf, C.H., Near-total loss of learning and memory as a result of combined cholinergic and serotonergic blockade in the rat, Behav. Brain Res., 23 (1987) 43-57.
21
BRES 17198
Restoration of serotonergic innervation underlies the behavioral effects of raphe grafts Gal Richter-Levin
and Menahem
Segal
Center for Neuroscience, The Weizmann Institute, Rehovot (Israel)
(Accepted 16 July 1991) Key words: Raphe; Graft; Serotonin; Hippocampus; Spatial memory
It has been previously demonstrated that an embryonic raphe grafted into a serotonin-depleted hippocampus restores normal scrotonin innervation of the hippocampus 1°'23'24 and behaviors associated with serotonin 14'Is'2°. To test the possibility that the behavioral effects of these grafts result from non-specific actions of the grafted tissue or the grafting procedure itselfs'l', we compared raphe grafts with septal grafts, in serotonin-depleted rats. We also compared the effects of a serotonin synthesis inhibitor, p-ch!orophenylalanine, on the behavior of normal, serotonin-depleted and raphe-grafted rats. The results indicate that the bulk of behavioral effects of raphe grafts are due to the serotonergic nature of the graft.
INTRODUCTION The possibility of restoring impaired brair~ functions by transplantation of neural tissue has attract!ed a great deal of attention over the past decade s'6't2. Tlms, it has been demonstrated that grafted neurons can ameliorate cognitive and motor deficits resulting from selective lesions in host mammalian brains s'6.t2. Embryonic raphe cells grafted into the hippocampus of serotonin-depleted, septal-lesioned rats hell:~ compensate for spatial memory deficits observed in these rats Is' 2o. While we would like to believe that a raphe graft is instrumental in restoring impaired functions because it contains serotonergic neurons, it is possible that the grafted tissue may affect the host brain in several nonspecific ways (e,g, as a source of trophic factors or as a 'bridge' to facilitate the growth of axons of intrinsic nerve cells) s'tt. These grafts restore the serotonergic innervation of the hippocampus and very often result in a hyper-serotonergic innervation at the areas surrounding the graft 10'20'23'24. Nevertheless, when staining for serotoner~ic immunoreactivity, it is obvious that only a small fraction of the grafted cells are serotonergic tg. Furthermore, the grafting procedure causes slight but noticeable damage to the host brain (e.g. Fig. 1). This may trigger some compensatory processes that are not dependent on the serotonergic nature of the graft. Thus, when evaluating the behavioral effects of the grafts, it is imperative to test the degree of specificity of the grafts. In the present study, we examined the involvement of
serotonin in the behavioral effects of raphe grafts. The effects of embryonic raphe tissue, rich with serotonincontaining cells, were compared with those of embryonic septal tissue. In another study, the ability of the serotonin synthesis blocker, p-chlorophenylalanine (PCPA) 13 to affect behavior of raphe-grafted rats was compared with that of control rats. The results suggest that, although grafted embryonic tissue of both septal and raphe origin may exert some non-specific effects, the improvement in the ability to perform the watermaze, observed in the raphe-grafted rats, depends largely on the presence of sufficient levels of serotonin in the hippocampus, EXPERIMENT 1: COMPARISON BETWEEN THE EFFECTS
OF RAPHE AND SEPTAL GRAFTS Materials and Methods
Adult (200-250 g) male Wistar rats from a local breeding colony were injected with 5,7.dihydroxytryptamine (5,7-DHT) (intraventricularly, bilaterally, 100/~gfree base in 10/~1of saline containing 0.2% ascorblc acid on each side) under chloral hydrate anesthesia (3.5% solution, 10 mg/kg, i.p.). Rats were pretreated with 30 mg/kg of desipramine (i.p., in 1 ml saline) 20 min prior to anesthesia. One week later 5,7.DHT-treated rats were grafted with either raphe (RG) or septal (SG) embryonic tissue (day 14 and day 17, respectively, bilaterally, into the hippocampus) as described19 (coordinates for transplantation: 4.5 mm posterior from bregma; 5.5 mm lateral; depth from 6 to 4.5 mm below the skull). Controls were naive male rats. Experiments were conducted 5-6 months after transplantation. The watermaze consisted of a round pool of water (130 cm di-
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute, Rehovot 76100, Israel.
22 ameter, 50 cm high rim and water level of 18 cm. A glass platform, l0 cm in diameter, was submerged in the water, 3 cm below the surface). Milk powder was added to the pool to achieve opaque water. Rats were placed in the pool and could escape it by climbing the platform, using extra-maze cues. Intertrial intervals were 15-20 rain. The escape latency was used as the measure of level of performance t~. Control (n = 5), 5.7-DHT injected (DHT, n = 5), DHT rats having a raphe graft in the hippocampus (RG, n = 9), and DHT rats having a septal graft in the hippocampus (SG, n = 5) were tested in the water maze as follows. Rats were placed in the watermaze facing the wall, and left there for 60 s or until they reached the platform. Rats were led to or left on the platform for 15 s before being taken hack to their home cages. Rats were given 8 trials a day. During the last two trials of the second day the room was darkened to reduce extra-maze cues and verify that rats do indeed use these cues to locate the platform. On the third day, all rats were treated with atropine (25 mg/kg, i.p.) 20 rain before the third trial. This low dose of atropine was administered in an attempt to reproduce the conditions under which serotonin-depleted rats exhibit poor performance in the water-maze task 16'zT'2s. At the end of behavioral experiments, rats were decapitated, their brains quickly removed and kept at -70 °C. The brains were sliced into 30-pro sections for histological studies. The presence of grafts was detected by Cresyl violet staining2°. In all rats examined (n = 14), except for one (which was excluded), the grafted tissue, of both rdphe and septai origin, could be easily detected by Cresyl violet staining (Fig. 1). in most cases, the graft was located within the medial-ventral hippocampus, most often in or near the dentate gyrus. In two cases, the grafts were located on the medial wall of the dentate gyrus. The size of the
grafts was 0,5-1 mm 2. Grafted tissue caused a slight distortion of the host hippocampal tissue only at the site of transplantation (Fig.
I). Statistical analysesutilized ANOVAfor repeated measurements. Results
There were no significant differences in performance between the groups during the first day, except for a tendency toward longer escape latencies of the SO rats during trials 2-3 (F -- 4.58, P < 0.04) (Fig. 2A). Likewise, there were no significant differences among the groups during the second day. To test for the possibility that under extreme conditions, of minimizing extra-maze cues, an adverse effect of serotonin depletion will become evident, the lights were dintmed during the last two trials of the second day. Reducing the illumination in the room affected all rats but there was still no significant difference between groups, due to an increase in variability in the behavior of the rats (Fig. 2B). On the third day o~ training, control and RG rats returned to their previous level of performance, whereas, in the first trial, the DHT and SG rats were still significantly worse (control vs DHT; F = 7.07, P < 0,01: control vs SG; F = 7.4, P < 0.01) (Fig. 2C). On the second retention trial (prior to administr~:tion of atropine) all the groups performed as before. Following atropine (Fig. 2C), DHT rats had significantly longer escape latencies compared to control (F = 12.4, P < 0,002) and to RG (F = 7.1, P < 0.01). The
Fig. 1. Cresyl violet sections of the hippocampus of a raphe-grafted rat. Arrows indicate the graft. DG, dentate gyms; BS, brainstem; S, subiculum. Scale bar = 0.5 ram.
performance of the SG group lay in between these groups. There was no significant difference between the SG rats and the DHT group, nor between them and the RG rats, but there was a significant difference between them and control rats (F = 5.46, P < 0.02) (Fig. 2C). In contrast, RG and DHT rats were significantly different (F - 6.01, P < 0.02) and there was no significant difference between them and control rats. The RG rats, however, exhibited a non-significant tendency to be slower thaa controls. This was particularly evident in the first trial after atropine injection.
Discussion The results of this experiment confirm earlier observations in suggesting that blockade of both serotonergic (by 5,7-DHT) and cholinergic (by atropine) neurotransmission but not each of them by itself, causes a severe retention deficit tf-ts'2s, Both DHT and SG rats were less affected than controis or RG rats by dimming the lights (Fig. 2B, trial 7). It is possible that these rats were depending less on extra-maze cues and that the improvement in their performance up to that stage was in part due to the development of a different searching strategy. Further studies, howev.~.r, are needed to clarify this possibility. The inability of septal grafts to mimic the effects of raphe i,rafts supports the assumption that the behavioral effects associated with the graft are unique to the raphe. These results are congruent with the previous observation that raphe but not septal grafts could restore the responses of the hippocampus to application of serotonin-releasing drugs, in serotonin-depleted rats tg.
23 A
m~.50
EXPERIMENT 2: THE EFFECTS OF RAPHE GRAFTS IN THE PRESENCE OF PCPA
Day 1
60
o---- control,n=5 ; DHT, n=5 = RG, n=9
"t ~
v
=
Materials and Methods
=
o>, 40 ie al, ao 8. 2o
o"
"=' lO 0
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o
Trial No.
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A
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v
Day 2 o---- control, n=5 : DHT, n=5 = RG, n=9
Three groups were used. Control rats (n = 7), DHT rats (n = 4) and RG rats (n = 12). Lesioning and grafting procedures were the same as in Expt. 1. In order to test the rats before and after administration of PCPA, at the same stage of retention, rats were first trained in the water-maze (6 days, 4 trials a day). At this stage, application of either atropine or scopolamine, at relatively high doses (40 mg/kg and 4 mg/kg, respectively), had no effect on the performance of any of the groups in the water maze (data not shown). They were then left in their home cages for 3 weeks after which they were tested in the following procedure: rats were given two trials in which the platform was placed in the same location as during training. They were then injected with scopolamine (2 mg/ kg, i.p.) 20 min before a third trial. The platform was then shifted to a new location and the rats were given 3 additional trials.
A
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30
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control,n,.7 5,7-DHT,n,.4 RG, n-12
C
platform shift
,, 20'
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t~10' 0
~darkness
0
0
Trial No.
0
Trial No.
C
Day 3
20"
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B
---.-o--- control,n-5 •.---e.--- DHT,n..5
j,
control,n,,7
platform e h i l t ~
80"
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Trial No. '~
6
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Trial No. Fig. 2. A comparison between the effects of raphe and septal grafts in the hippocampus of serotonin depleted rats. A: day 1, acquisition day. No significant differences were found between groups except for a tendency for a slower learning rate, in the SG group (trials 2-3, F = 4.58, P < 0.04). B: day 2, retention day and the effects of darkening the room. No significant differences were found during retention trials or after darkening the room. C: day 3, retention day and the effects of atropine (25 mg/kg, i.p.). In the first trial the DHT and SG rats exhibited a significantly poorer performance compared to control rats (control vs DHT, F = 7.07, P < 0.01; control vs SG, F = 7.4, P < 0.01). During the second trial, however, there were no significant differenccs between the groups. Following the administration of atropine (25 mg/kg i.p.) control rats performed significantly better than DHT rats (F = 12.4, P < 0.002) and SG rats (F = 6.1, P < 0.02). RG rats were not different from control. (Note differences in scale between A, B and C.)
Fig. 3. A compa~,'ison between the effects of raphe grafts before and after administration of PCPA. A: before PCPA. There were no significant differences between groups during the retention trials nor following the administration of scopolamine. Shifting the platform to a new location significantly disturbed DHT rats compared to control (F = 8.38, P < 0.008) and to RG (F = 5.61, P < 0.02). B: in the presence of PCPA. PCPA did not affect the performance of any of the groups when the platform was placed in the same location as during training. Scopolamine had no effect at this stage. After shifting the platform, control rats performed similar to their performance before administration of PCPA. DHT rats were affected by the shift of the platform as before. Contrary to their pre-PCPA performance RG rats were now significantly affected compared to control (F = 5.54, P < 0.02). There was still a significant difference between the RG and DHT rats (F = 4.15, P < 0.05).
24 The rats were then injected with PCPA (200 mg/kg, i.p.) and were tested again 3 days later, using the same procedure. Statistical analyses utilized ANOVA for repeated measurements. Results
There were no significant differences among any of the groups during the first 3 trials on the control day, even after the injection of scopolamine (Fig. 3A). However, when the platform was shifted to a new location, DHT rats had significantly longer escape latencies during the last two trials compared to the control (F = 8.38, e < 0.008) and the RG groups (F = 5.61, e < 0.02) (Fig. 3A). There were no significant differences among the 3 groups in the first 3 trials of the second testing day. Scopolamine did not have an effect on the performance of any of the groups. However, when the platform was shifted, control rats were only affected during the following trial but returned to previous levels of performance immediately thereafter, as they did on the first testing day. In contrast, both the DHT and the RG groups did not return to previous levels of performance until the end of the test (DHT vs control, F = 13.14, P < 0.001; RG vs control, F = 5.54, P < 0.02) (Fig. 3B), There was a significant difference between RG rats and the DHT group (F = 4.15, P < 0.05) as well. Discussion
The results indicate that the relatively better watermaze performance of raphe-grafted rats depends largely on the serotonergic component of these grafts. When serotonin synthesis is reduced, the ability of these rats to perform in the maze is impaired and becomes similar to that of the DHT rats. A relatively low dose of PCPA ~'tT'2s was used here in order to reduce possible significant effects of the combined treatment on control rats. Administration of PCPA did not affect significantly the performance of DHT rats either, thus enabling the evaluation of the contribution of the grafts,
REFERENCES 1 Airman, N.J. and Hormile, H.J., What is the nature of the role of the serotonergic nervous system in learning and memory: prospects for development of an effective treatment strategy for senile dementia, Neurobiol, Aging, 9 (1988) 627-638. 2 Altman, H.J., Normile, HJ., Galloway, M.P., Ram;,rez, A. and Azmitia, E.C., Enhanced spatial discrimination learning in rats following 5,7-DHT-induced serotonergic deafferent~tion of the hippocampus, Brain Research, 518 (1990) 61-66. 3 Airman, H.J., Ogren, S.O., Berman, R.F. and Normile, H.J., The effects of p-chloroamphetamine, a depletor of brain serotonin, on the performance of rats in two types of positively reinforced complex spatial discrimination tasks, Behav. Neural.
GENERAL DISCUSSION The present results indicate that raphe grafts have a unique effect on the ability of serotonin-depleted rats to perform well in the spatial memory water-maze task. These effects could not be mimicked by septal grafts and were absent in the raphe-grafted rats in which serotonin synthesis was reduced. The slower rate of acquisition observed in the SG group (Expt. 1, day 1) may indicate some adverse effects of the grafting procedure or the grafted tissue on the host brain, as was suggested before 7. Septal grafts, however, improved the performance of septal-lesioned rats in the water-maze t5'22, suggesting specificity of the effects of grafts. While the present results illustrate the importance of the serotonergic innervation of the hippocampus in spatial memory, the nature of its involvement remains unclear. Depletion of forebrain serotonin adversely affected the ability of septal-lesioned rats to perform the watermaze t6'2°, and yet, it increased the ability of rats to perform other learning tasks t. Furthermore, selective depletion of the serotonergic fibers innervating the hippocampus increased the ability of rats to perform some spatial memory tasks 2'3'2t. These conflicting results, while indicating an important influence of the serotonergic system on these behaviors, suggest that these are secondary effects of the serotonergic system on other neurotransmitter systems, The lack of effect of serotonin depletion alone on the performance of rats in the watermaze 4't6.t7 (Expt. 1, days 1-2) supports this view. The present results help in establishing the use of raphe grafts as a tool for further studies related to the behavioral roles of the serotonergic system.
Acknowledgements, We thank E, Pearl for her technical assistance, This research was supported by a grant from MINERVA Foundation, Munich, F,R,G., and by a research grant from MINERVA from the Mario-Negri lnstitute-Weizman Collaboration fund,
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