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Evidence that electrolytic median raphe lesions increase locomotion but not exploration
Physiology & Behavior, Vol. 28, pp. 749--754. Pergamon Press and Brain Research Publ., 1982. Printed in the U.S.A.
Evidence That Electrolytic Median Raphe Lesions Increase Locomotion but Not Exploration D A V I D W l R T S H A F T E R A N D K A R E N E. A S I N I
Department of Psychology, University of Illinois at Chicago Circle, Box 4348, Chicago, IL 60680 Received 10 October 1981 WlRTSHAFTER, D. AND K. E. ASIN. Evidence that electrolytic median raphe lesions increase locomotion but not exploration. PHYSIOL. BEHAV. 28(5) 749-754, 1982.--Electrolytic lesions of the median raphe nucleus were found to increase locomotion but decrease rearing in the open field. Additionally, these lesions reduced the amount of time that rats spent sniffing at a novel object placed in the open field on their first encounter with it. In a test of exploratory behavior in a T-maze, median raphe lesions eliminated the preference for entering a novel arm displayed by sham operated animals. These results suggest that, although median raphe lesions increase locomotion, they may actually decrease exploration.
Median raphe nucleus
Limbic-midbraincircuit
Hyperactivity
A NUMBER of authors have demonstrated that damage to the midbrain raphe nuclei produces a dramatic increase in locomotor activity in rats [2, 15, 16, 17, 20, 21, 22, 30, 33] and involvement of the median raphe nucleus appears to be primarily responsible for this effect [2, 14, 17, 29]. In other studies [33], we have observed that this hyperactivity results primarily from lesioned animals spending a larger proportion of their time in motion rather than from their moving at a greater velocity. Observation of the animals suggests that whereas intact rats frequently stop to sniff at or investigate various stimuli, lesioned subjects rarely do so. These informal observations suggest that although rats with median raphe damage are hyperactive, they may actually be exploring less than controls. A similar suggestion has been offered with respect to animals with hippocampal damage [24]. In the current studies we attempted to more directly investigate the exploratory behavior of rats with electrolytic median raphe lesions by examining their responses to novel stimuli. The first experiment examined the ability of a novel stimulus, presented in an environment to which rats had been given five minutes to habituate, to elicit investigatory behavior. The second and third experiments investigated the extent to which lesioned and sham operated animals would direct their locomotor behavior selectively at a novel, as compared to a familiar, location. EXPERIMENT 1 METHOD
Subjects Animals were 15 adult male, Sprague-Dawley derived rats
Exploration
Rearing
Open field
obtained from a colony maintained by the University of Illinois. At the time of surgery rats weighed about 300 g. Animals were housed in individual wire mesh cages with food and water available ad lib. Testing was conducted during the light phase of a 12:12 hour light:dark cycle. Eight rats received median raphe lesions and the remaining seven served as sham operated controls.
Surgery Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg). A stainless-steel electrode (0.23 mm in diameter), insulated except for 0.5 mm at the tip, was stereotaxically placed at coordinates of AP: -0.2, Lat: 0.0, H: -4.3 [25]. Electrodes were lowered in the sagittal plane following retraction of the superior sagittal sinus using the technique we have described elsewhere [31]. A I mA current was then passed for 8 sec between the electrode and a rectal cathode. In sham-operated control animals, a burr hole was drilled and the dura opened, but the electrode was not lowered.
Apparatus Activity was measured in a 152.5x91.5 cm open field enclosed by 47 cm high plywood walls. The floor of the field was divided into fifteen 30.5x30.5 cm squares. Lighting was provided by overhead fluorescent fixtures.
Procedure Rats were weighed daily during the recovery period, but were not otherwise handled. Open field testing was con-
1Current address: Kinsmen Laboratory for Neurological Research, Faculty of Medicine, University of British Columbia, 2075 Westbrook Mall, Vancouver, British Columbia, V6T lW5. During the preparation of this paper K.E.A. was supported by N.I.N.C.D.S. Postdoctoral Fellowship grant No. IF32NS06399-01. The authors would like to thank William Montana for technical assistance.
Copyright © 1982 Brain Research Publications Inc.--0031-9384/82/080749-06503.00/0
750
WIRTSHAFTF~R ANI) ASIN
ducted on day 12 following surgery. Animals were removed from their home cages, gently placed in the center square of the open field and covered by a plastic bucket 38 cm in diameter which was removed fifteen seconds later. Over the next five minutes, the number of squares entered and the number of rears made were recorded. An animal was considered to have entered a square when all four paws were placed within it. A rear was counted when a rat removed both forepaws from the floor and stretched upwards (i.e., an animal was not considered to have reared if he raised his forepaws in order to groom). In addition, the number of fecal boli deposited in the field was recorded. At the end of this 5-minute period, a hexagonal piece of white plastic 2.5 cm high and 5 cm on a side was placed in the center of a square, adjacent to one of the long edges of the open field equidistant from the corners of that side. The plastic object was placed on the side of the field farthest from the animals' location at the time of placement and was situated 12 cm from the edge of the field. For the next two times that each rat entered the square containing the new object, the amount of time spent sniffing at or in contact with the object was recorded to the nearest 0.1 second using a Hunter timer. Following the completion of behavioral study, lesioned animals were perfused transcardially, under deep pentobarbital anesthesia, with normal saline followed by 10% Formalin. Brains were removed at once and stored in Formalin for at least two weeks. Sixty-four micron frozen sections were then taken through the extent of the lesions. Photographs were made of unstained sections. RESULTS The median raphe lesions produced here were very similar to those we have described previously [2, 3, 5] and an example of a typical lesion is shown in Fig. 1. All lesions severely damaged the median raphe nucleus and in some cases extended out of its bounds to produce, usually minor, damage to the ventral tegmental nuclei of Gudden or the reticular tegmental nucleus of the pons. No obvious relations could be determined between lesion placements and behavioral results. Results of the open field test are shown in Table 1. It can be seen that, during the first five minutes of testing, animals with median raphe lesions entered significantly more squares than did controls (t(13)=5.613; p<0.01), but made significantly fewer rears (t(13)=3.580; p<0.01). Numbers of fecal boll did not differ between groups. Table 1 also shows the mean amounts of time spent sniffing at the novel object for lesioned and control animals during their first two entries to the square containing it. It can be seen that on their first opportunity to investigate the object,
FIG. 1. Photograph of an unstained section through the maximal extent of a typical median raphe lesion.
sham operated rats spent significantly more time sniffing at it than did lesioned animals (t(13)=3.467; p<0.01). Control subjects spent significantly less time sniffing at the object on their second than on their first encounter with it (t(6)=2.41; p <0.05). A similar trend towards habituation was not seen in lesioned animals, but this may have been due to a floor effect. DISCUSSION In accordance with many other reports, the above study demonstrates that electrolytic median raphe lesions produce a dramatic increase in open field activity [2, 14, 16, 33]. Additionally, in this experiment, rats with median raphe lesions displayed significantly less rearing than did controls. This result is not in agreement with those of other investigators who have reported no effect of median raphe lesions on rearing [20,30]. We have investigated rearing following median raphe damage on four other occasions in different groups of rats ([33] and unpublished observations). In two of these studies rearing was found to be reduced whereas in the other two no effect was found. We are as of yet uncertain as to the reasons for this variability. It is interesting to note that
TABLE 1 EFFECTS OF MEDIANRAPHE LESIONS ON OPEN FIELD ACTIVITYAND INVESTIGATIONOF A NOVEL OBJECT Sniffing time (sec)
Lesioned Control
Squares entered
Rears
Boli
1st encounter
2nd encounter
132.2 + 20.5 42.8 ± 6.4
5.5 _+ 1.2 14.7 ± 2.4
1.0 _+ 0.7 0.6 _+ 0.6
1.0 ± 0.7 5.7 -+ 1.2
1.3 ± 1.3 1.7 ± 1.1
RAPHE LESIONS INCREASE LOCOMOTION hippocampal and septal lesions have also been occasionally reported to reduce rearing [8, 11, 17, 18, 19]. In contrast, several other treatments which increase locomotion, such as amphetamine administration [26,28] or food deprivation [26], have been reported to increase rearing. The finding that median raphe lesions increase locomotion but not rearing suggests that raphe damage does not simply result in a nonspecific increase in motor behavior. The most important result of this study is that lesioned subjects spend less time than controls sniffing at a novel object placed in the open field on their first encounter with it. This finding may be related to our previous observation that lesioned subjects are less distracted than controls by novel stimuli presented during the execution of a food reinforced runaway response [1]. At least two explanations of the reduced investigation shown by animals with median raphe lesions are possible. Raphe damage might directly reduce exploratory tendencies or perhaps reduce attention to external stimuli. It is conceivable that the reduced rearing seen in lesioned animals in the current study might also be a reflection of reduced exploratory tendencies. Alternatively, it is possible that the reduced investigation might result from an inability of lesioned animals to suppress locomotion in order to sniff at the novel stimulus. It is difficult to dissociate these two possiblities experimentally, but at" any account, the current results suggests that raphe damage may reduce the investigation of novel sitmuli whether or not this effect is secondary to hyperactivity.
751
Apparatus Testing was conducted in a wooden T-maze with 10 cm high walls constructed so that the start arm could be removed from the goal arms. Two start arms, 5 9 c m long and 10.2 cm wide, were employed at different times. One of them was painted fiat black and the other flat white. The two goal arms of the maze were 51.2 cm long and 10.2 cm wide and were painted flat black. The entire maze was covered by clear Plexiglas. No doors were present.
Procedure Rats were removed from their home cages and gently placed in the left goal arm of the maze which, at this time, had the white start arm in place. Rats were allowed to freely move about the maze for a ten minute period during which time the number and sequence of arm entries were recorded. An animal was considered to have entered an arm when all four paws were placed within it. (The 10.2× 10.2 cm region formed by the junction of the start and goal arms was not considered to be a part of any arm). Rats were then removed to a holding cage for one minute during which time the white start arm was removed and replaced by a black one. Animals were then replaced in the left goal arm and allowed to move freely about the maze for five minutes. Arm entries were recorded as above. Following the completion of behavioral testing, histological verification of lesion placements was conducted as described in Experiment 1.
EXPERIMENT 2
RESULTS
In this study we attempted to examine the effects of median raphe lesions on exploration using a somewhat different methodology. Several workers (reviewed in [6]) have demonstrated that if normal rats are preexposed to an environment, removed from it, and then replaced with some part of the environment changed, they will tend to direct their locomotor behavior towards the altered, i.e., novel, aspect of the environment. If median raphe lesions produce a reduction in exploratory tendencies, one might expect that they would not show as large of a preference for entering novel aspects of the environment as do controls. To examine this possiblity, we preexposed lesioned and control animals for ten minutes to a T-maze in which the goal arms were painted black and the start arm white. Subjects were then briefly removed from the maze and returned to it after the white start arm had been removed and replaced by a black one. Thus, the maze on this second, test, trial was identical to that on the preexposure trial except for the change in the start arm. The black start arm, on the test trial, would therefore constitute a novel stimulus and one would expect that it would elicit approach behavior in normal rats. If subjects with median raphe lesions display reduced exploratory tendencies, one would expect that they would be less likely to enter the novel start arm on the test trial than would controls.
Results of this experiment are shown in Table 2. The left hand columns indicate the mean number of arms entered per minute for lesioned and sham operated animals during the two testing periods. It can be seen that median raphe lesioned animals were considerably more active than controls during both periods and that animals in both groups showed lower activity levels on their second than their first exposure to the T-maze. These conclusions are supported by the results of a 2 x 2 repeated measure analysis of variance (ANOVA) which indicated a significant effect of both lesion (F(1,14)=15.47, p<0.01) and of trials (F(1,14)=16.1; p<0.01). The lesion x trials interaction failed to approach significance ( F < l ) . The decline in motility in the second period probably reflects habituation and, therefore, the failure of the interaction to reach significance indicates that, although lesioned animals were more active than controls, both groups habituated at the same rate. In order to examine the sequence of arm entries, data were expressed as start arm entries per opportunity, i.e., the number of entries to the start arm divided by the number of entries to the other two arms. As can be seen from Table 2, the performance of lesioned and control rats did not differ during the preexposure period, subjects in both groups entering the start arm on about one-third of their opportunities. During the test trial, however, sham operated subjects showed a significantly greater tendency to enter the novel black start arm than did lesioned animals (t(14)=3.661; p<0.01). It is possible, since lesioned animals entered more arms during the second period than did controls, that they initially showed a strong preference for the novel arm, but that this preference disappeared with repeated exposures. To examine this possiblity, an analysis was conducted on start arm e~tries per opportunity considering only the first
METHOD
Subjects Subjects were 16 rats similar to those described in Experiment 1. Eight randomly selected animals received median raphe lesions and eight were sham operated. Testing was conducted on day 30 following surgery.
752
WIRTSHAFTER AND ASIN TABLE 2 EFFECTS OF MEDIAN RAPHE LESIONSON T-MAZEEXPLORATIONIN EXPERIMENT 2 WITHTHE STARTARM CHANGEDBETWEENTRIALS Arms entered per min
Start arm entries per opportunity
Preexposure
Test trial
Preexposure
Test trial
4.3 ± 0.4 2.4 ± 0.2
3.8 ± 0.4 1.9 ± 0.3
0.320 _+ 0.046 0.342 _+ 0.050
0.363 ± 0.042 0.648 + 0.075
Lesioned Control
TABLE 3 EFFECTS OF MEDIANRAPHE LESIONS ON T-MAZEEXPLORATIONIN EXPERIMENT 3 WITHTHE STARTARM UNCHANGEDBETWEENTRIALS Arms entered per min
Start arm entries per opportunity
Preexposure
Test trial
Preexposure
Test trial
4.8 ± 0.7 2.0 ± 0.2
3.1 ± 0.6 1.2 ± 0.5
0.298 ± 0.030 0.348 ± 0.015
0.265 -+ 0.082 0.292 ± 0.104
Lesioned Control
five arms entered. Under these conditions, with number of arm entries equated, sham operated animals still showed a significantly greater number of start arm entries per opportunity than did lesioned rats. (0.575+-0.045 vs 0.45+-0.033; t(14)=2.24, p<0.05). Histological examination revealed that the lesions produced here were very similar to those seen in the first experiment. EXPERIMENT 3 The results of Experiment 2 suggest that animals with electrolytic median raphe lesions show a reduced tendency to explore novel aspects of the environment. An alternative explanation of the observed results is possible, however. Sham operated rats may simply be more likely than lesioned animals to enter the start arm of an all black T-maze, regardless of any prior exposure to a maze with a white stem. Experiment 3 attempted to examine this possiblity by replicating the previous study with the exception that a black start arm was employed in both the preexposure and test periods. Under these conditions the black start arm would not be more novel than the goal arms during the test trial.
rate of movement declined significantly between trials F(1,8)=13.84, p<0.01) but the lesion by trial interaction again failed to reach significance (p<0.1). Examination of the right hand side of Table 3 shows that number of start arm entries made per opportunity did not differ between lesioned and sham operated subjects during either the preexposure or the test periods. Subjects in both groups entered the start arm on about one-third of their opportunities. This result demonstrates that the difference in start arm entries observed between lesioned and sham operated subjects on the test trial of Experiment 2 was indeed a result of the novelty of the start arm in that study. Furthermore, control animals during the test period of Experiment 3 were significantly less likely to enter the start arm than were the control subjects of Experiment 2 who had been preexposed to a maze with a white stem (t (11) = 3.086; p < 0.0 l). A similar difference was not present in lesioned rats. Histological examiniation showed that the lesions produced here were very similar to those described above. and no consistent differences could be observed between the size or placement of lesions in any of the three studies that have been reported. DISCUSSION
METHOD Subjects were ten rats similar to those described above. Five had received median raphe lesions and five were sham operated. As in the previous experiment, testing was conducted on day 30 following surgery. The procedure employed was identical to that of Experiment 2 except that the maze was equipped with a black start arm on both trials. RESULTS Results of this study are shown in Table 3. As was found in Experiment 2, lesioned animals were significantly more active than controls (F(1,8)=12.52, p<0.02). Again, the
Experiments 2 and 3 demonstrate that electrolytic median raphe lesions produce significant hypermotility in a T-maze. Median raphe lesion-induced hyperactivity is a very general phenomenon and has been observed in a wide variety of situations including open fields, stabilimeter and tilt cages, running wheels, hole boards, straight alleys ([2, 14, 16, 17, 20, 21, 22, 30, 33] and unpublished observations) and now T-mazes. These experiments also demonstrate that median raphe lesions reduce the tendency, displayed by intact rats, to selectively enter novel, as compared to familiar, parts of the environment. Thus, control subjects were more likely to
RAPHE LESIONS INCREASE LOCOMOTION
753
enter the stem of an all black T-maze when they had been preexposed to a maze with a white stem than when they had been preexposed to a maze with a black stem. A similar effect of preexposure was absent in lesioned subjects. Under the conditions of Experiment 2, in which animals had been preexposed to a maze with a white stem, lesioned subjects were significantly less likely than controls to enter the novel black start arm on the first trial. It is likely that this effect is related to the failure of animals with median raphe lesions to display spontaneous alternation [1,14], since the alternation of nonreinforced arm choices by normal rats probably results from their preference for entering the relatively more novel arm. It is interesting to note that septal and hippocampal lesions also disrupt spontaneous alternation [ 10], and Graffan [12] has found, in a task basically similar to that employed here, that hippocampal lesions disrupt the preference of rats for the arm of a T-maze which had been altered following an adaptation period. These similarities between the effects of hippocampal and median raphe lesions lend credence to the notion that the paramedian tegmentum may play an important role in the functioning of the limbic system. GENERAL DISCUSSION The current experiments demonstrate that although electrolytic lesions of the median raphe nucleus produce hyperactivity in a variety of situations, they appear, if anything, to reduce exploratory behavior. Thus, lesioned animals displayed a reduced tendency to enter a novel arm in a T-maze and initially spent less time sniffing at a novel object placed in the open field than did controls. The suggestion that median raphe lesions may reduce exploration is also supported by our finding that such lesions reduce the amount of time spent in a complex environment when animals are
offered a choice between simple and complex surroundings [33]. The reduced rearing observed in Experiment 1 might conceivably also be a reflection o f reduced exploratory tendencies. The possible relations between the hyperactivity and the apparent reduction in exploration shown by animals with median raphe lesions deserve comment. Three possibilities can be mentioned. (1) Median raphe lesions might produce an independent increase in activity and a decrease in exploration. (2) The reduced exploration of lesioned subjects might result from their hyperactivity, since an animal in continuous rapid motion might have less chance to notice, or respond to, salient aspects of the stimulus environment. (3) It is possible that the reduced tendency of lesioned animals to explore their surrounds might contribute to their hyperactivity since they might not be " d i s t r a c t e d " from their locomotion by "interesting" stimuli which normally elicit non-locomotor investigatory behavior. Regardless of whether reduced exploration is a primary effect of median raphe lesions or is secondary to the hyperactivity, the current results are in agreement with the suggestion, which we have offered elsewhere [5,33], that lesioned animals may attend less to environmental stimuli, unless they are highly salient, than do control. Finally, it should be noted that altough electrolytic median raphe lesions deplete certain forebraln regions of serotonin [14, 17, 29, 30], they also damage non-serotonergic cell bodies and fibers of passage. Thus, there is no reason to assume that serotonin depletion underlies the effects reported here, particularly since specific serotonin depletions following intraraphe injections of the neurotoxin 5,7dihydroxytryptamine, fail to produce open field hyperactivity [21 ]. The anatomical substrate of the dramatic behavioral syndrome produced by electrolytic median raphe lesions remains to be identified.
REFERENCES 1. Asin, K. E., D. Wirtshafter and E. W. Kent. An investigation of the role of the medial nucleus of the raphe in behavior. Soc. Neurosci. Abstr. 4: 267, 1978. 2. Asin, K. E., D. Wirtshafter and E. W. Kent. Straight alley acquisition and extinction and open field activity following discrete electrolytic lesions of the mesencephalic raphe nuclei. Behay. Neural Biol. 25: 242-256, 1979. 3. Asin, K. E., D. Wirtshafter and E. W. Kent. Impaired patterned responding in rats with electrolytic median raphe lesions. Physiol. Behav. 23: 803-806, 1979. 4. Asin, K. E., D. Wirtshafter and E. W. Kent. Discrimination learning and reversal following electrolytic median raphe lesions. Soc. Neurosci. Abstr. 5: 269, 1979. 5. Asin, K. E., D. Wirtshafter and E. W. Kent. The effects of electrolytic median raphe lesions on two measures of latent inhibition. Behav. Neural Biol. 2,8: 408-417, 1980. 6. Berlyne, D. E. Conflict, Arousal and Curiosity. New York: McGraw-Hill, 1960. 7. Bobillier, P., S. Seguin, F. Petijean, D. Salvert, M. Touret and M. Jouvet. The raphe nuclei of the cat bralnstem: A topographical atlas of their efferent projections as revealed by autoradiography. Brain Res. 113: 449--486, 1976. 8. Clark, C. V. H. Effect of hippocampal and neocortical ablation on scopolamine-induced activity in the rat. Psychopharmacology 17: 289-301, 1970. 9. Conrad, L. C. and D. W. Pfaff. Connections of the median and dorsal raphe nuclei in the rat: An autoradiographic and degeneration study. J. comp. Neurol. 156: 179-206, 1974.
10. Dalland, T. Response and stimulus perserveration in rats with septal and dorsal hippocampal lesions. J. comp. physiol. Psychol. 71: 114-118, 1970. 11. Donovick, P. J., R. G. Burright and M. A. Swidler. Presurgical rearing environment alters exploration, fluid consumption, and learning of septal lesioned and control rats. Physiol. Behav. 11: 543-553, 1973. 12. Graffan, D. Loss of recognition memory in rats with lesions of the fornix. Neuropsychology 10: 327-371, 1972. 13. Greyer, M. A., M. Puerto, D. B. Menkers, D. S. Segal and A. J. Mandell. Histologic and enzymatic studies of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 242256, 1976. 14. Geyer, M. A., A. Puerto, D. B. Menkes, D. S. Segal and A. J. Mandell. Behavioral studies following lesions of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 241256, 1976. 15. Jackson, W. J. The effect of hippocampal lesions upon activity and learning. Ph.D. thesis, Texas Technological College, 1967. Cited in: The Hippocampus as a Cognitive Map, J. O'Keefe and L. Nadel. Oxford: Clarendon Press, 1978, p. 257. 16. Jacobs, B. L. and A. Cohen. Differential behavioral effects of lesions of the median or dorsal raphe nuclei in rats: Open field and pain-elicited agression. J. comp. physiol. Psychol. 90: 102108, 1976. 17. Jacobs, B. L., W. D. Wise and K. M. Taylor. Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats. Brain Res. 79:353-361, 1974.
754 18. Kemble, E. D. and J. A. Nagel. Persistent depression of rearing behavior in rats after extensive septal lesions. ,I. comp. phy.~iol. Psychol. 83: 747-758, 1975. 19. Kemble, E. D., D. R. Studelska and J. A. Nagel. Rearing behavior of rats after amygdaloid, hippocampal, olfactory bulb, cortical or straital lesions. Bull. Psychon. Soc. 8: 163-166, 1976. 20. Kohler, C. and S. A. Lorens. Open field activity and avoidance behavior following serotonin depletion: A comparison of the effects of parachlorophenylalanine and electrolytic midbrain raphe lesions. Pharmac. Biochem. Behav. 8: 223-233, 1978. 21. Lorens, S. A., J. C. Guildberg, K. Hole, C. Kohler and B. Srebro. Activity, avoidance learning and regional 5-hydroxytryptamine following intra-brain stem 5,7-hydroxytryptamine and electrolytic midbrain raphe lesions in the rat. Brain Res. 108: 97-113, 1976. 22. Lorens, S. A., J. P. Sorenson and L. M. Yunger. Behavioral and neurochemical effects of lesions in the raphe system of the rat. ,I. comp. physiol. Psychol. 77: 48-52, 1971. 23. Nauta, W. J. H. Hippocampal projections and related neural pathways to the midbrain in the rat. Brain 81: 31%340, 1958. 24. O'Keefe, J. and L. Nadel. The Hippocampus as a Cognitive Map. Oxford: Clarendon Press, 1978. 25. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century-Crofts, 1967.
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26. Prescolt, R. G. W. Some behavioral effects of variables which influence the "'general level of~activity" of rat~. Anita. Bcha!' 18: 791-796, 1970. 27. Ray, O. and S. Hockhauser. Growth hormone and environ~ mental complexity effects on behavior in the ral. I)(,vl Psv~hol. I: 311-317, 1969. 28. Schioring, E. Amphetamine induced selective stimulation of certain behavior items with concurrent inhibition of others in an open-field test with rats. Behaviour 39" 1-17. 1971. 29. Solomon, P. R., G. L. Nichols, R. S. Kamer and L. J. Kaplan. Differential effects of lesions in medial and dorsal raphe of the rat: Latent inhibition and septohippocampal serotonin levels. J. comp. physiol. Psychol. 94: 145-154, 1980. 30. Srebro, B. and S. A. Lorens. Behavioral effects of selective midbrain raphe lesions in the rat. Brain Res. 89: 303-325, 1975. 31. Wirtshafter, D., K. E. Asin and E. W. Kent. A simple technique for midline stereotaxic surgery in the rat. t'hysi~l. Behav. 23: 40%410, 1979. 32. Wirtshafter, D., K. E. Asin and E. W. Kent. Median raphe lesions impair the acquisition and performance of an 8-arm maze task. Soc. Neurosci. Abstr. 5: 285, 1979. 33. Wirtshafter, D., K. E. Asin and E. W. Kent. An analysis of open field hyperactivity following electrolytic median raphe lesion in the rat. Soc. Neurosci. Abstr. 6: 422, 1980.
Evidence That Electrolytic Median Raphe Lesions Increase Locomotion but Not Exploration D A V I D W l R T S H A F T E R A N D K A R E N E. A S I N I
Department of Psychology, University of Illinois at Chicago Circle, Box 4348, Chicago, IL 60680 Received 10 October 1981 WlRTSHAFTER, D. AND K. E. ASIN. Evidence that electrolytic median raphe lesions increase locomotion but not exploration. PHYSIOL. BEHAV. 28(5) 749-754, 1982.--Electrolytic lesions of the median raphe nucleus were found to increase locomotion but decrease rearing in the open field. Additionally, these lesions reduced the amount of time that rats spent sniffing at a novel object placed in the open field on their first encounter with it. In a test of exploratory behavior in a T-maze, median raphe lesions eliminated the preference for entering a novel arm displayed by sham operated animals. These results suggest that, although median raphe lesions increase locomotion, they may actually decrease exploration.
Median raphe nucleus
Limbic-midbraincircuit
Hyperactivity
A NUMBER of authors have demonstrated that damage to the midbrain raphe nuclei produces a dramatic increase in locomotor activity in rats [2, 15, 16, 17, 20, 21, 22, 30, 33] and involvement of the median raphe nucleus appears to be primarily responsible for this effect [2, 14, 17, 29]. In other studies [33], we have observed that this hyperactivity results primarily from lesioned animals spending a larger proportion of their time in motion rather than from their moving at a greater velocity. Observation of the animals suggests that whereas intact rats frequently stop to sniff at or investigate various stimuli, lesioned subjects rarely do so. These informal observations suggest that although rats with median raphe damage are hyperactive, they may actually be exploring less than controls. A similar suggestion has been offered with respect to animals with hippocampal damage [24]. In the current studies we attempted to more directly investigate the exploratory behavior of rats with electrolytic median raphe lesions by examining their responses to novel stimuli. The first experiment examined the ability of a novel stimulus, presented in an environment to which rats had been given five minutes to habituate, to elicit investigatory behavior. The second and third experiments investigated the extent to which lesioned and sham operated animals would direct their locomotor behavior selectively at a novel, as compared to a familiar, location. EXPERIMENT 1 METHOD
Subjects Animals were 15 adult male, Sprague-Dawley derived rats
Exploration
Rearing
Open field
obtained from a colony maintained by the University of Illinois. At the time of surgery rats weighed about 300 g. Animals were housed in individual wire mesh cages with food and water available ad lib. Testing was conducted during the light phase of a 12:12 hour light:dark cycle. Eight rats received median raphe lesions and the remaining seven served as sham operated controls.
Surgery Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg). A stainless-steel electrode (0.23 mm in diameter), insulated except for 0.5 mm at the tip, was stereotaxically placed at coordinates of AP: -0.2, Lat: 0.0, H: -4.3 [25]. Electrodes were lowered in the sagittal plane following retraction of the superior sagittal sinus using the technique we have described elsewhere [31]. A I mA current was then passed for 8 sec between the electrode and a rectal cathode. In sham-operated control animals, a burr hole was drilled and the dura opened, but the electrode was not lowered.
Apparatus Activity was measured in a 152.5x91.5 cm open field enclosed by 47 cm high plywood walls. The floor of the field was divided into fifteen 30.5x30.5 cm squares. Lighting was provided by overhead fluorescent fixtures.
Procedure Rats were weighed daily during the recovery period, but were not otherwise handled. Open field testing was con-
1Current address: Kinsmen Laboratory for Neurological Research, Faculty of Medicine, University of British Columbia, 2075 Westbrook Mall, Vancouver, British Columbia, V6T lW5. During the preparation of this paper K.E.A. was supported by N.I.N.C.D.S. Postdoctoral Fellowship grant No. IF32NS06399-01. The authors would like to thank William Montana for technical assistance.
Copyright © 1982 Brain Research Publications Inc.--0031-9384/82/080749-06503.00/0
750
WIRTSHAFTF~R ANI) ASIN
ducted on day 12 following surgery. Animals were removed from their home cages, gently placed in the center square of the open field and covered by a plastic bucket 38 cm in diameter which was removed fifteen seconds later. Over the next five minutes, the number of squares entered and the number of rears made were recorded. An animal was considered to have entered a square when all four paws were placed within it. A rear was counted when a rat removed both forepaws from the floor and stretched upwards (i.e., an animal was not considered to have reared if he raised his forepaws in order to groom). In addition, the number of fecal boli deposited in the field was recorded. At the end of this 5-minute period, a hexagonal piece of white plastic 2.5 cm high and 5 cm on a side was placed in the center of a square, adjacent to one of the long edges of the open field equidistant from the corners of that side. The plastic object was placed on the side of the field farthest from the animals' location at the time of placement and was situated 12 cm from the edge of the field. For the next two times that each rat entered the square containing the new object, the amount of time spent sniffing at or in contact with the object was recorded to the nearest 0.1 second using a Hunter timer. Following the completion of behavioral study, lesioned animals were perfused transcardially, under deep pentobarbital anesthesia, with normal saline followed by 10% Formalin. Brains were removed at once and stored in Formalin for at least two weeks. Sixty-four micron frozen sections were then taken through the extent of the lesions. Photographs were made of unstained sections. RESULTS The median raphe lesions produced here were very similar to those we have described previously [2, 3, 5] and an example of a typical lesion is shown in Fig. 1. All lesions severely damaged the median raphe nucleus and in some cases extended out of its bounds to produce, usually minor, damage to the ventral tegmental nuclei of Gudden or the reticular tegmental nucleus of the pons. No obvious relations could be determined between lesion placements and behavioral results. Results of the open field test are shown in Table 1. It can be seen that, during the first five minutes of testing, animals with median raphe lesions entered significantly more squares than did controls (t(13)=5.613; p<0.01), but made significantly fewer rears (t(13)=3.580; p<0.01). Numbers of fecal boll did not differ between groups. Table 1 also shows the mean amounts of time spent sniffing at the novel object for lesioned and control animals during their first two entries to the square containing it. It can be seen that on their first opportunity to investigate the object,
FIG. 1. Photograph of an unstained section through the maximal extent of a typical median raphe lesion.
sham operated rats spent significantly more time sniffing at it than did lesioned animals (t(13)=3.467; p<0.01). Control subjects spent significantly less time sniffing at the object on their second than on their first encounter with it (t(6)=2.41; p <0.05). A similar trend towards habituation was not seen in lesioned animals, but this may have been due to a floor effect. DISCUSSION In accordance with many other reports, the above study demonstrates that electrolytic median raphe lesions produce a dramatic increase in open field activity [2, 14, 16, 33]. Additionally, in this experiment, rats with median raphe lesions displayed significantly less rearing than did controls. This result is not in agreement with those of other investigators who have reported no effect of median raphe lesions on rearing [20,30]. We have investigated rearing following median raphe damage on four other occasions in different groups of rats ([33] and unpublished observations). In two of these studies rearing was found to be reduced whereas in the other two no effect was found. We are as of yet uncertain as to the reasons for this variability. It is interesting to note that
TABLE 1 EFFECTS OF MEDIANRAPHE LESIONS ON OPEN FIELD ACTIVITYAND INVESTIGATIONOF A NOVEL OBJECT Sniffing time (sec)
Lesioned Control
Squares entered
Rears
Boli
1st encounter
2nd encounter
132.2 + 20.5 42.8 ± 6.4
5.5 _+ 1.2 14.7 ± 2.4
1.0 _+ 0.7 0.6 _+ 0.6
1.0 ± 0.7 5.7 -+ 1.2
1.3 ± 1.3 1.7 ± 1.1
RAPHE LESIONS INCREASE LOCOMOTION hippocampal and septal lesions have also been occasionally reported to reduce rearing [8, 11, 17, 18, 19]. In contrast, several other treatments which increase locomotion, such as amphetamine administration [26,28] or food deprivation [26], have been reported to increase rearing. The finding that median raphe lesions increase locomotion but not rearing suggests that raphe damage does not simply result in a nonspecific increase in motor behavior. The most important result of this study is that lesioned subjects spend less time than controls sniffing at a novel object placed in the open field on their first encounter with it. This finding may be related to our previous observation that lesioned subjects are less distracted than controls by novel stimuli presented during the execution of a food reinforced runaway response [1]. At least two explanations of the reduced investigation shown by animals with median raphe lesions are possible. Raphe damage might directly reduce exploratory tendencies or perhaps reduce attention to external stimuli. It is conceivable that the reduced rearing seen in lesioned animals in the current study might also be a reflection of reduced exploratory tendencies. Alternatively, it is possible that the reduced investigation might result from an inability of lesioned animals to suppress locomotion in order to sniff at the novel stimulus. It is difficult to dissociate these two possiblities experimentally, but at" any account, the current results suggests that raphe damage may reduce the investigation of novel sitmuli whether or not this effect is secondary to hyperactivity.
751
Apparatus Testing was conducted in a wooden T-maze with 10 cm high walls constructed so that the start arm could be removed from the goal arms. Two start arms, 5 9 c m long and 10.2 cm wide, were employed at different times. One of them was painted fiat black and the other flat white. The two goal arms of the maze were 51.2 cm long and 10.2 cm wide and were painted flat black. The entire maze was covered by clear Plexiglas. No doors were present.
Procedure Rats were removed from their home cages and gently placed in the left goal arm of the maze which, at this time, had the white start arm in place. Rats were allowed to freely move about the maze for a ten minute period during which time the number and sequence of arm entries were recorded. An animal was considered to have entered an arm when all four paws were placed within it. (The 10.2× 10.2 cm region formed by the junction of the start and goal arms was not considered to be a part of any arm). Rats were then removed to a holding cage for one minute during which time the white start arm was removed and replaced by a black one. Animals were then replaced in the left goal arm and allowed to move freely about the maze for five minutes. Arm entries were recorded as above. Following the completion of behavioral testing, histological verification of lesion placements was conducted as described in Experiment 1.
EXPERIMENT 2
RESULTS
In this study we attempted to examine the effects of median raphe lesions on exploration using a somewhat different methodology. Several workers (reviewed in [6]) have demonstrated that if normal rats are preexposed to an environment, removed from it, and then replaced with some part of the environment changed, they will tend to direct their locomotor behavior towards the altered, i.e., novel, aspect of the environment. If median raphe lesions produce a reduction in exploratory tendencies, one might expect that they would not show as large of a preference for entering novel aspects of the environment as do controls. To examine this possiblity, we preexposed lesioned and control animals for ten minutes to a T-maze in which the goal arms were painted black and the start arm white. Subjects were then briefly removed from the maze and returned to it after the white start arm had been removed and replaced by a black one. Thus, the maze on this second, test, trial was identical to that on the preexposure trial except for the change in the start arm. The black start arm, on the test trial, would therefore constitute a novel stimulus and one would expect that it would elicit approach behavior in normal rats. If subjects with median raphe lesions display reduced exploratory tendencies, one would expect that they would be less likely to enter the novel start arm on the test trial than would controls.
Results of this experiment are shown in Table 2. The left hand columns indicate the mean number of arms entered per minute for lesioned and sham operated animals during the two testing periods. It can be seen that median raphe lesioned animals were considerably more active than controls during both periods and that animals in both groups showed lower activity levels on their second than their first exposure to the T-maze. These conclusions are supported by the results of a 2 x 2 repeated measure analysis of variance (ANOVA) which indicated a significant effect of both lesion (F(1,14)=15.47, p<0.01) and of trials (F(1,14)=16.1; p<0.01). The lesion x trials interaction failed to approach significance ( F < l ) . The decline in motility in the second period probably reflects habituation and, therefore, the failure of the interaction to reach significance indicates that, although lesioned animals were more active than controls, both groups habituated at the same rate. In order to examine the sequence of arm entries, data were expressed as start arm entries per opportunity, i.e., the number of entries to the start arm divided by the number of entries to the other two arms. As can be seen from Table 2, the performance of lesioned and control rats did not differ during the preexposure period, subjects in both groups entering the start arm on about one-third of their opportunities. During the test trial, however, sham operated subjects showed a significantly greater tendency to enter the novel black start arm than did lesioned animals (t(14)=3.661; p<0.01). It is possible, since lesioned animals entered more arms during the second period than did controls, that they initially showed a strong preference for the novel arm, but that this preference disappeared with repeated exposures. To examine this possiblity, an analysis was conducted on start arm e~tries per opportunity considering only the first
METHOD
Subjects Subjects were 16 rats similar to those described in Experiment 1. Eight randomly selected animals received median raphe lesions and eight were sham operated. Testing was conducted on day 30 following surgery.
752
WIRTSHAFTER AND ASIN TABLE 2 EFFECTS OF MEDIAN RAPHE LESIONSON T-MAZEEXPLORATIONIN EXPERIMENT 2 WITHTHE STARTARM CHANGEDBETWEENTRIALS Arms entered per min
Start arm entries per opportunity
Preexposure
Test trial
Preexposure
Test trial
4.3 ± 0.4 2.4 ± 0.2
3.8 ± 0.4 1.9 ± 0.3
0.320 _+ 0.046 0.342 _+ 0.050
0.363 ± 0.042 0.648 + 0.075
Lesioned Control
TABLE 3 EFFECTS OF MEDIANRAPHE LESIONS ON T-MAZEEXPLORATIONIN EXPERIMENT 3 WITHTHE STARTARM UNCHANGEDBETWEENTRIALS Arms entered per min
Start arm entries per opportunity
Preexposure
Test trial
Preexposure
Test trial
4.8 ± 0.7 2.0 ± 0.2
3.1 ± 0.6 1.2 ± 0.5
0.298 ± 0.030 0.348 ± 0.015
0.265 -+ 0.082 0.292 ± 0.104
Lesioned Control
five arms entered. Under these conditions, with number of arm entries equated, sham operated animals still showed a significantly greater number of start arm entries per opportunity than did lesioned rats. (0.575+-0.045 vs 0.45+-0.033; t(14)=2.24, p<0.05). Histological examination revealed that the lesions produced here were very similar to those seen in the first experiment. EXPERIMENT 3 The results of Experiment 2 suggest that animals with electrolytic median raphe lesions show a reduced tendency to explore novel aspects of the environment. An alternative explanation of the observed results is possible, however. Sham operated rats may simply be more likely than lesioned animals to enter the start arm of an all black T-maze, regardless of any prior exposure to a maze with a white stem. Experiment 3 attempted to examine this possiblity by replicating the previous study with the exception that a black start arm was employed in both the preexposure and test periods. Under these conditions the black start arm would not be more novel than the goal arms during the test trial.
rate of movement declined significantly between trials F(1,8)=13.84, p<0.01) but the lesion by trial interaction again failed to reach significance (p<0.1). Examination of the right hand side of Table 3 shows that number of start arm entries made per opportunity did not differ between lesioned and sham operated subjects during either the preexposure or the test periods. Subjects in both groups entered the start arm on about one-third of their opportunities. This result demonstrates that the difference in start arm entries observed between lesioned and sham operated subjects on the test trial of Experiment 2 was indeed a result of the novelty of the start arm in that study. Furthermore, control animals during the test period of Experiment 3 were significantly less likely to enter the start arm than were the control subjects of Experiment 2 who had been preexposed to a maze with a white stem (t (11) = 3.086; p < 0.0 l). A similar difference was not present in lesioned rats. Histological examiniation showed that the lesions produced here were very similar to those described above. and no consistent differences could be observed between the size or placement of lesions in any of the three studies that have been reported. DISCUSSION
METHOD Subjects were ten rats similar to those described above. Five had received median raphe lesions and five were sham operated. As in the previous experiment, testing was conducted on day 30 following surgery. The procedure employed was identical to that of Experiment 2 except that the maze was equipped with a black start arm on both trials. RESULTS Results of this study are shown in Table 3. As was found in Experiment 2, lesioned animals were significantly more active than controls (F(1,8)=12.52, p<0.02). Again, the
Experiments 2 and 3 demonstrate that electrolytic median raphe lesions produce significant hypermotility in a T-maze. Median raphe lesion-induced hyperactivity is a very general phenomenon and has been observed in a wide variety of situations including open fields, stabilimeter and tilt cages, running wheels, hole boards, straight alleys ([2, 14, 16, 17, 20, 21, 22, 30, 33] and unpublished observations) and now T-mazes. These experiments also demonstrate that median raphe lesions reduce the tendency, displayed by intact rats, to selectively enter novel, as compared to familiar, parts of the environment. Thus, control subjects were more likely to
RAPHE LESIONS INCREASE LOCOMOTION
753
enter the stem of an all black T-maze when they had been preexposed to a maze with a white stem than when they had been preexposed to a maze with a black stem. A similar effect of preexposure was absent in lesioned subjects. Under the conditions of Experiment 2, in which animals had been preexposed to a maze with a white stem, lesioned subjects were significantly less likely than controls to enter the novel black start arm on the first trial. It is likely that this effect is related to the failure of animals with median raphe lesions to display spontaneous alternation [1,14], since the alternation of nonreinforced arm choices by normal rats probably results from their preference for entering the relatively more novel arm. It is interesting to note that septal and hippocampal lesions also disrupt spontaneous alternation [ 10], and Graffan [12] has found, in a task basically similar to that employed here, that hippocampal lesions disrupt the preference of rats for the arm of a T-maze which had been altered following an adaptation period. These similarities between the effects of hippocampal and median raphe lesions lend credence to the notion that the paramedian tegmentum may play an important role in the functioning of the limbic system. GENERAL DISCUSSION The current experiments demonstrate that although electrolytic lesions of the median raphe nucleus produce hyperactivity in a variety of situations, they appear, if anything, to reduce exploratory behavior. Thus, lesioned animals displayed a reduced tendency to enter a novel arm in a T-maze and initially spent less time sniffing at a novel object placed in the open field than did controls. The suggestion that median raphe lesions may reduce exploration is also supported by our finding that such lesions reduce the amount of time spent in a complex environment when animals are
offered a choice between simple and complex surroundings [33]. The reduced rearing observed in Experiment 1 might conceivably also be a reflection o f reduced exploratory tendencies. The possible relations between the hyperactivity and the apparent reduction in exploration shown by animals with median raphe lesions deserve comment. Three possibilities can be mentioned. (1) Median raphe lesions might produce an independent increase in activity and a decrease in exploration. (2) The reduced exploration of lesioned subjects might result from their hyperactivity, since an animal in continuous rapid motion might have less chance to notice, or respond to, salient aspects of the stimulus environment. (3) It is possible that the reduced tendency of lesioned animals to explore their surrounds might contribute to their hyperactivity since they might not be " d i s t r a c t e d " from their locomotion by "interesting" stimuli which normally elicit non-locomotor investigatory behavior. Regardless of whether reduced exploration is a primary effect of median raphe lesions or is secondary to the hyperactivity, the current results are in agreement with the suggestion, which we have offered elsewhere [5,33], that lesioned animals may attend less to environmental stimuli, unless they are highly salient, than do control. Finally, it should be noted that altough electrolytic median raphe lesions deplete certain forebraln regions of serotonin [14, 17, 29, 30], they also damage non-serotonergic cell bodies and fibers of passage. Thus, there is no reason to assume that serotonin depletion underlies the effects reported here, particularly since specific serotonin depletions following intraraphe injections of the neurotoxin 5,7dihydroxytryptamine, fail to produce open field hyperactivity [21 ]. The anatomical substrate of the dramatic behavioral syndrome produced by electrolytic median raphe lesions remains to be identified.
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