BEHAVIORAL AND NEURAL BIOLOGY 25, 242--256 (1979)
Straight Alley Acquisition and Extinction and Open Field Activity following Discrete Electrolytic Lesions of the Mesencephalic Raphe Nuclei KAREN E. A S I N , DAVID W1RTSHAFTER, AND ERNEST W . K E N T 1
Department of Psychology, University of Illinois-Chicago Circle, Chicago, Illinois 60680 Open field behavior and the acquisition and extinction of a food-rewarded straight alley task were examined in rats with discrete electrolytic lesions of the midbrain raphe nuclei. Increased open field activity was seen following lesions of either the median or dorsal nucleus of the raphe, although the effect of median lesions was much more pronounced. Acquisition and extinction of a runway task were impaired following lesions of the median, but not dorsal, nucleus when trials were separated by 8 min. Animals with combined lesions of the dorsal and median raphe were behaviorally indistinguishable from those subjects with median raphe lesions alone. In a second experiment, it was determined that the acquisition deficit could be eliminated by a decrease in the intertrial interval, suggesting that the original deficit was not due to a motor impairment following median raphe lesions. Although other investigators have reported raphe involvement in aversively motivated behaviors the current study provides the first demonstration that the median raphe is also involved in the performance of tasks which are appetitively motivated. Additionally, we provide the first evidence that deficits in food-rewarded behaviors similar to those seen following damage to certain limbic structures can be produced by Lesions within the midbrain.
Of late, numerous studies have focused on the biochemical and behavioral effects of lesions of the nuclei of the raphe. The midbrain raphe nuclei, in particular, have generated a copious amount of literature. These nuclei are included in what Nauta has termed the "qimbic-midbrain circuit" and are profusely interconnected with limbic forebrain structures (Nauta, 1960). Additionally, the median nucleus of the raphe has been identified as the source of serotonergic efferents to the frontal cortex and most limbic structures (Bobillier, Seguin, Petijean, Salvert, Touret, & Jouvet, 1976; Conrad, Leonard, & Pfaff, 1974; Geyer, Puerto, Dawsey, 1 Portions of this study were presented at the Sixth and Seventh Annual Meetings of the Society for Neuroscience, 1976 and 1977. We thank R. Rimas for technical assistance. 242 0163-1047/79/020242-15502.00/0 Copyright© 1979by AcademicPress,Inc. All rightsof reproductionin any form reserved.
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Knapp, Bullard, & Mandell, 1976a; Lorens & Guldberg, 1974) while the dorsal nucleus of the raphe appears to send serotonergic projections to the striatal nuclei and portions of the substantia nigra (Bobillier et al., 1976; Conrad et al., 1974; Geyer et al., 1976a; Lorens & Guldberg, 1974; Miller, Richardson, Fibiger, & McLennan, 1975; Pierce, Foote, & Hobson, 1976; Roberge, Parent, & Boulay, 1976). Alterations in behavior have largely been attributed to the destruction of the median raphe nucleus (Geyer, Puerto, Menkers, Segall, & Mandell, 1976b; Jacobs, Wise, & Taylor, 1974; Srebro & Lorens, 1975) which may reflect the importance of the projections to limbic structures; in fact, many of the behavioral changes which have been reported in hippocampal-lesioned animals have also been reported for median raphelesioned animals. Although raphe lesions have been found to affect the performance of aversively motivated behaviors (Geyer et al., 1976b; Hole & Lorens, 1975; Lorens, 1973; Lorens, Guldberg, Hole, Kohler, & Srebro, 1976; Lorens, Sorenson, & Yunger, 1971; Srebro & Lorens 1975), little work has been done on their possible effects on tasks which are appetitively motivated. Therefore, Experiment I investigates the effects of selective damage to the mesencephalic raphe nuclei on the acquisition and extinction of a food-rewarded straight alley task and also reports their effects on open field activity. Experiment I1 further examines the alley performance of median raphe-lesioned rats by assessing the influence of the intertrial interval. EXPERIMENT
I
Methods Subjects. Twenty-four male, Sprague-Dawley-derived rats, obtained from a colony maintained by the University of Illinois, were used as subjects. The rats weighed approximately 300 g at the time of surgery. Six rats were randomly assigned to each of the following four treatment groups: median raphe (MR) lesions, dorsal raphe (DR) lesions, combined median and dorsal raphe (MDR) lesions, or sham-operated controls. Food and water were available ad lib. except during alley training, as noted below. Surgery. Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg) following treatment with atrophine sulfate (0.08 mg in 0.2 ml per rat). A stainless-steel electrode, 0.23 mm in diameter and insulated except for 0.5 mm at the tip, was stereotaxically placed (Pelligrino & Cushman, 1967) at coordinates AP: +0.2, Lat: 0, H: -4.3 for MR lesions, or AP: -0.3, Lat: 0, H: - 2 . 0 for DR lesions. A 1-mA current was then passed for 8 secs between the electrode and a rectal cathode. Lesions were placed in both of the above locations in MDR animals. In all cases the electrode was lowered through the center of the superior sagittal sinus. In sham-operated subjects a burr hole was drilled and the sinus was
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punctured but the electrode was not lowered. Animals were allowed 10 days to recover from surgery prior to testing. Open field behavior. Activity was measured in a 152.5 x 91.5-cm open field enclosed by 47-cm-high plywood walls. The floor of the field was demarcated into fifteen 30.5 x 30.5-cm squares. Lighting was provided by overhead fluorescent fixtures. Animals were tested in the open field 10 days following surgery. Subjects were gently placed in the center square of the field and the number of squares entered per minute was recorded over a 5-min period. A subject was considered to have entered a square when all four paws had been placed within it. As a measure of thigmotaxis, note was taken of the ratio of central squares entered (i.e., those which did not adjoin the plywood boundary of the field) to those entered in the periphery. Additionally, the total number of fecal boli deposited was recorded as a measure of emotionality. Straight alley acquisition and extinction.The alley was constructed of clear Plexiglas except for the floor, which was composed of metal rods 2 mm in diameter and spaced 10 mm apart. The start box (16 x 11.5 x 10 cm) and the goal box (30.3 x 15 x 10 cm) could be isolated from the remainder of the alley (110 x 15 x 10 cm) by metal guillotine doors. Latencies were evaluated by means of two photocells situated 14 and 103 cm from the start box which were, in turn, connected to a Hunter Klockounter. Interruption of the first beam started the clock which ran until the second photobeam was broken. Times were measured to the nearest 0.01 sec. Beginning the day after open field testing, animals were maintained on a restricted feeding schedule and were reduced to approximately 85% of their body weights. After 8 days on the diet, each animal was placed in the alley and was allowed to explore it for 5 rain. The following day, rats were placed in the goal box and were confined there until they had consumed one Froot Loop (Kellogg) which had been deposited in a glass coaster in the rear corner of the goal box. Acquisition training was begun 24 hr later. An animal was placed in the start box facing away from the lowered partition, which was raised when the animal oriented towards it. After entrance into the goal box, the door to that compartment was lowered to prevent retracing. Following Froot Loop ingestion, the animal was placed in a holding cage for 8-10 rain while five other animals were run. Each subject was run for six acquisition trials a day and was then allowed to feed. Training continued for 6 consecutive days. On the seventh day, animals were given one rewarded trial in the alley, followed by twelve nonreinforced extinction trials. During these trials, animals were retained in the goal box for 10 sec after entering it. Histological verification. Rats were given an overdose of sodium pen-
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tobarbital and were perfused intracardially with a 0.9% saline solution, followed by 10% formalin. Brains were removed from the skull and were stored in formalin for a minimum of 2 weeks. Sixty-four-micrometer sections were made on a freezing microtome and every third section through the extent of the lesion was kept. Samples were photomicrographed and then stained with cresyl violet.
Results Openfield. Results of the open field test are depicted in Fig. 1. It can be seen that while lesions of either the median or dorsal raphe nucleus increased activity, the median lesions were the more effective of the two. A 2 × 2 factorial analysis of variance (median raphe lesions x dorsal raphe lesions) conducted on the total number of squares entered indicated a significant median lesion effect IF(I, 20) = 10.2, p < .01] and a significant median lesion × dorsal lesion interaction [F(I, 20) = 4.63, p < .05J. A simple main effects analysis indicated that although dorsal raphe lesions increased activity relative to sham-operated controls, they did not further elevate the hyperactivity seen in animals with median raphe lesions. These results indicate that all operated subjects were more active than controls, with the activity of MR- and MDR-lesioned animals equivalent in magnitude and surpassing that of the other groups. It is possible that a ceiling effect may have prevented dorsal raphe lesions from further augmenting the increased locomotion of MR-lesioned rats. Neither lesion influenced the latency to leave the initial square, ratio of central to peripheral squares entered, or the number of fecal boli deposited in the field. ContrOl
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During open field testing it was noted that several MR- and MDRlesioned subjects displayed a Straub tail which was never seen in DRlesioned or control animals. This may reflect the release of pontine and medullary serotonin-containing neurons from inhibition following median raphe lesions (Jacobs & Klemfuss, 1975; Morgane & Stern 1974). Straight alley. Mean speeds (1/latencies) for the four groups in the alley during acquisition and extinction are shown in Fig. 2. As is apparent from the figure and confirmed by statistical analysis, MR- and MDR-lesioned animals ran more slowly during acquisition than did either control or DR-lesioned subjects. A median raphe lesion x dorsal raphe lesion x trials analysis of variance conducted on running speeds during acquisition revealed significant trial IF (5, 100) = 76,p < .01] and median raphe lesion effects IF(I, 20) = 9.01, < .01], and a significant median lesions x trials interaction IF(5, 100) = 11.81,p < .01]. The latter result indicates that the difference in running speeds between animals with and without median raphe lesions increased across trials. Neither the effect of dorsal lesions nor any of the remaining interactions approached statistical significance (p > 0.25). The failure of the median lesion x dorsal lesion interaction to attain significance suggests that the impaired acquisition seen in MDRlesioned subjects is primarily attributable to median raphe damage. Figure 2 also shows that MR- and MDR-lesioned subjects extinguished more slowly than control or DR-lesioned rats. This conclusion is confirmed by a median lesion x dorsal lesion x trials analysis of variance. The effect of trials was found to be significant [F(3,60) = 27.1, p < .01], as Control DR Lesion MR Lesion DR & MR Lesions
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was the median raphe lesion effect iF(l, 20) = 10.2, p < .01] and the median lesion x trials interaction IF(3, 60) = 4.3; p < .01]. As in acquisition, neither the dorsal lesion effect nor any of the remaining interactions a p p r o a c h e d significance (F < 1). Histology. The extent of the largest and smallest lesions seen in MR, DR, and M D R animals is shown in Fig. 3 and examples of typical lesions m a y be seen in Fig. 4. In all cases, median raphe lesions severely damaged the nucleus and invaded the immediately surrounding reticular formation. The ventral tegmental nuclei of Gudden were intact in all animals. Dorsal raphe lesions damaged the dorsal raphe and the adjacent central gray, the trochlear nuclei, and the medial longitudinal fasciculi. Although this lesion also encroached on the anterior pole of the dorsal tegmental nuclei of Gudden in two animals, these subjects did not differ behaviorally from other DR-lesioned animals. In MDR-lesioned subjects the two lesions fused through m o s t of their extent so as to damage structures between the MEDIAN
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two raphe nuclei. Thus, in addition to the damage mentioned above, these lesions infringed on the ventral tegmental nuclei, the nucleus linearis caudalis and, on occasion, the decussation of the brachia conjunctiva. E X P E R I M E N T II The results of the first experiment indicate that lesions of the MR can impair both the acquisition and extinction of a food-rewarded runway task. In the following experiment we attempted to specify more precisely the nature of these deficits by examining alley performance as a function of the intertrial interval. Additionally, we sought to determine whether any deficits in acquisition would persist through an extended number of training trials.
Methods Subjects. Subjects and their living conditions were similar to those reported in the first experiment. Surgery. Surgical procedures were virtually identical to those in Experiment I except there were only two groups, MR lesions (N = 16) or sham operated (N = 16). Alley acquisition and extinction. The alley apparatus was the same as that used in the first experiment. Following the 10-day surgical recovery period, rats were placed on the restricted feeding schedule and were reduced to approximately 85% of their body weights. Eight and nine days later animals were given the same preliminary introduction to the alley as those in Experiment I, again using Froot Loops as a reward. Acquisition training began the following day. Animals were assigned to one of four squads: MS or CS--median raphe-lesioned or shamoperated rats, respectively, trained under a 15-rain intertrial interval; or MM or CM--median raphe-lesioned or sham-operated animals whose training trials were immediately successive (virtually no intertrial interval). Within each treatment group, animals were randomly assigned to a squad. Each subject was given 6 trials a day for 12 days under a continuous reinforcement schedule. Training was continued for 12 consecutive days. The following day, animals received one rewarded trial followed by 12 extinction trials, still under their respective interval times. Histology. Techniques were the same as those in the first experiment.
Results Results of this experiment may be seen in Fig. 5. A 2 x 2 analysis of variance (lesion x intertrial interval) was conducted on mean running speeds across the last 5 days of acquisition. The analysis revealed a significant lesion x intertrial interval interaction iF(l, 28) = 5.7; p < .05]; a simple main effects analysis indicated that raphe-lesioned rats showed significantly slower running speeds under the spaced, but not massed, conditions [F(1, 28) -- 10.06, p < .01].
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F1G. 4. Unstained brain sections showing typical electrolytic lesions of the median raphe (a), dorsal raphe (b), or both raphe nuclei (c) in rats.
A lesions x intertrial interval x trials analysis of variance performed on mean running speeds during extinction indicated only a significant trials effect [F(3, 84) = 12.3, p < .01], although animals tended to extinguish faster under massed conditions. Although the analysis failed to indicate a significant lesion effect, examination of Fig. 5 suggests that lesioned rats run under spaced conditions tended to extinguish slower than the control group, as indicated by the rapid convergence of the two extinction curves. The failure of the analysis to indicate a significant lesion effect under spaced conditions may be due to the large difference in terminal acquisition running speeds between the control and lesioned groups. A comparison of the decline in running speeds during extinction (i.e., terminal acquisition speed minus terminal extinction speed) indicated that under spaced conditions control rats showed a significantly larger decline in
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FIG. 4-----Continued
running speeds than did MR-lesioned animals [t(14) = 2.3, p < .05]. A similar comparison under massed conditions was not significant. However, unless an actual crossover in running speeds occurs, as in the first experiment, it is difficult to interpret extinction rates when terminal acquisition speeds differ (Mackintosh, 1974). Histology. Median raphe lesions were found to be similar to those described in the first experiment.
GENERAL DISCUSSION Lesions of the dorsal raphe nucleus were found to produce a slight but significant increase in open field activity. Although significant open field hyperactivity following destruction of this nucleus has not previously
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F]o. 5. Mean running speeds during the acquisition and extinction of a runway task by control or median raphe-lesioned subjects under massed or spaced (15-rain intertrial interval) conditions. Abbreviations: CM, control subjects trained under massed conditions; CS, controls trained under spaced conditions; MM, median raphe-lesioned rats trained under massed conditions; MS, lesioned rats trained under spaced conditions. 251
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ASIN, WIRTSHAFTER, AND KENT
been reported, a trend in this direction has been observed (Jacobs & Cohen, 1976, Srebro & Lorens, 1975) and Grabowska (1974) has reported increased photocell cage activity following dorsal raphe lesions. Additionally, coronal knife cuts through the central gray substance immediately posterior to the dorsal raphe have been reported to increase open field activity (Wirtshafter, Pociask, & Kent, 1975). It is possible that differences in lesion size and placement account for the discrepant reports regarding dorsal raphe lesions and activity. In accordance with the reports of other investigators (Geyer et al., 1976b; Jacobs & Cohen, 1976; Lorens et al., 1971) lesions of the median raphe, either alone or in combination with dorsal lesions, were found to produce increased locomotion in the open field. This hyperactivity was of a much greater magnitude than that seen following dorsal raphe damage. We have also observed that similar median raphe lesions increase activity in tilt, photocell and stabilimeter cages (personal observations, also see Jacobs et al., 1974; Lorens et al., 1971). It is unlikely that the hyperactivity seen in the open field reflects serotonin depletion, since attempts to deplete the brain of serotonin with p-chlorophenylalanine (PCPA) or with intracerebral injections of 5,7dihydroxytryptamine have resulted in either unchanged or decreased levels of locomotion in the open field (Brody, 1970; Lorens et al., 1976; MacKenzie, Norelli, Trulson, & Hoebel, 1977; Marsden & Curzon, 1976; Tenen, 1967). Since the effects of median raphe lesions on open field activity are not in accordance with these findings, the hyperactivity produced by the lesions may reflect damage to nonserotonergic elements contained within the raphe (Aghajanian & Asher, 1971). Additionally, fiber systems of the tegmental nuclei of Gudden and of the interpeduncular nucleus course in close proximity to the median raphe and might inadvertently be damaged by lesions of this nucleus. We have observed that coronal knife cuts designed to interrupt projections of the dorsal tegmental nuclei (dorsal and caudal to the median raphe) result in hyperactivity which is at least as pronounced as that seen following median raphe lesions (in preparation). It is possible that damage to one of these systems might subserve hyperactivity in the open field after lesions of the raphe (Lorens, Kohler, & Guldberg, 1975). The first experiment also demonstrated that under certain conditions rats with lesions of the median, but not dorsal, raphe are impaired in both acquisition and extinction of a food-rewarded straight alley task. Although available evidence is limited, it should be noted that pharmacological antagonism of serotonin has been reported to have effects on alley acquisition and extinction similar to those observed here (Rosen & Buga, 1973; Rosen & Cohen, 1973), suggesting that serotonin depletion may underlie the effects of MR lesions on alley performance. Further work is needed, however, to substantiate this claim.
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In the second experiment, it was demonstrated that the conditions of training may effect the behavior of median raphe-lesioned rats. Thus, it was found that MR-lesioned animals are deficient in acquiring an alley task when the training trials are separated by 15 min, but not when they are immediately successive; furthermore, the deficits under spaced conditions persist through an extended number of training trials. These results suggest that the impaired acquisition of MR-lesioned rats trained under spaced conditions cannot be due to motor difficulties but may rather reflect alterations in attentional or motivational factors. The second experiment also obtained some evidence for impaired extinction under spaced but not massed conditions. The impairment was not as robust as that in the first experiment, however, where an actual crossover in running speeds in control and lesioned rats occurred. The absence of a dramatic extinction effect may have been due to the increased number of training trials in Experiment II. The number of acquisition trials has been reported to be an important determinant of extinction rate (Mackintosh, 1974) and has, on occasion, been found to interact with brain lesions (Henke, 1974). Based on available data, it is impossible to specify the precise nature of the deficits underlying the effects of median raphe lesions on alley performance. Anatomical considerations suggest, however, that a comparison of the effects of median raphe and limbic lesions may be profitable. Using fiber degeneration techniques, Nauta (1960) has described profuse interconnections between forebrain limbic structures and the paramedian tegmentum of the midbrain, including the median raphe, and has further suggested a functional relation between the two areas. More recent biochemical techniques have identified serotonergic projections from the MR to limbic structures, including the cingulum and hippocampus (Geyer et al., 1976a, Moore, Halaris, & Jones, 1978), and many of the behavioral effects of lesions of these structures resemble those following MR lesions. Hippocampal and raphe lesions have similar effects on avoidance behaviors (Douglas, 1967; Geyer et al., 1976b; Hole & Lorens, 1975; Isaacson 1974; Lorens, 1973; Lorens et al., 1975; Olton, 1973; Srebro & Lorens, 1975), latent inhibition (Ackil, Mellgren, Halgren, & Frommer, 1969; Asin, Wirtshafter, & Kent, 1978), and spontaneous alternation (Asin, Wirtshafter, & Kent, 1977; Dalland, 1970; Geyer et al., 1976b). X-ray induced agenesis of the dentate gyrus granule cell layer or electrolytic lesion of the cingulate gyrus impairs both the acquisition and extinction of a runway task (Bruner, Haggbloom, & Gazzara, 1974; Glass, Ison, & Thomas, 1969), a pattern of deficits similar to those reported here. Furthermore, like median raphe-lesioned animals, rats with lesions of the hippocampus display impaired acquisition and extinction when trained under long, but not short, intertrial intervals (Jarrard, Isaacson, & Wickelgren, 1964; Means, Woodruff, & Isaacson, 1972). Since the median
254 raphe appears tures, further destruction of median raphe parallels.
ASIN, WIRTSHAFTER, AND KENT to h a v e a b u n d a n t a n a t o m i c a l c o n n e c t i o n s w i t h l i m b i c s t r u c comparisons of the behavioral changes seen following the t h e s e a r e a s m a y p r o v e f r u i t f u l in e l u c i d a t i n g t h e r o l e o f t h e in b e h a v i o r . W e a r e c u r r e n t l y i n v e s t i g a t i n g s u c h p o s s i b l e
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Roberge, A. G., Parent, A., & Boulay, M. (1976). Demonstration d'une relation inversement proportionnelle entre la dopamine et la serotonine dans certaines structures cerebrales" Aspects neurochimique et morphologique. Journal of Neurochemistry, 26, 591-595. Rosen, A. J., & Buga, J. 0973). Effects of lysergic acid diethylamide on simple instrumental conditioning, extinction and discrimination learning in the rat. Pharmacology, Biochemistry, and Behavior, 1, 619-627. Rosen, A. J., & Cohen, M. E. (1973). The effects of cinanserin, a potent serotonin antagonist on the acquisition of a runway response in the rat. Neuropharmacology, 12, 501-508. Srebro, B., & Lorens, S. A. (1975). Behavioral effects of selective midbrain lesions in the rat. Brain Research, 89, 303-325. Tenen, S. S. (1967). The effects of p-chlorophenylalanine, a serotonin depletor, on avoidance acquisition, pain sensitivity and related behavior in the rat. Psychopharmacologia (Berlin), 10, 204-219. Wirtshafter, D., Pociask, R., & Kent, E. W. (1975). Effects of central grey transections at various rostro-caudal levels. Neuroscience Abstracts, 1, 471.
Straight Alley Acquisition and Extinction and Open Field Activity following Discrete Electrolytic Lesions of the Mesencephalic Raphe Nuclei KAREN E. A S I N , DAVID W1RTSHAFTER, AND ERNEST W . K E N T 1
Department of Psychology, University of Illinois-Chicago Circle, Chicago, Illinois 60680 Open field behavior and the acquisition and extinction of a food-rewarded straight alley task were examined in rats with discrete electrolytic lesions of the midbrain raphe nuclei. Increased open field activity was seen following lesions of either the median or dorsal nucleus of the raphe, although the effect of median lesions was much more pronounced. Acquisition and extinction of a runway task were impaired following lesions of the median, but not dorsal, nucleus when trials were separated by 8 min. Animals with combined lesions of the dorsal and median raphe were behaviorally indistinguishable from those subjects with median raphe lesions alone. In a second experiment, it was determined that the acquisition deficit could be eliminated by a decrease in the intertrial interval, suggesting that the original deficit was not due to a motor impairment following median raphe lesions. Although other investigators have reported raphe involvement in aversively motivated behaviors the current study provides the first demonstration that the median raphe is also involved in the performance of tasks which are appetitively motivated. Additionally, we provide the first evidence that deficits in food-rewarded behaviors similar to those seen following damage to certain limbic structures can be produced by Lesions within the midbrain.
Of late, numerous studies have focused on the biochemical and behavioral effects of lesions of the nuclei of the raphe. The midbrain raphe nuclei, in particular, have generated a copious amount of literature. These nuclei are included in what Nauta has termed the "qimbic-midbrain circuit" and are profusely interconnected with limbic forebrain structures (Nauta, 1960). Additionally, the median nucleus of the raphe has been identified as the source of serotonergic efferents to the frontal cortex and most limbic structures (Bobillier, Seguin, Petijean, Salvert, Touret, & Jouvet, 1976; Conrad, Leonard, & Pfaff, 1974; Geyer, Puerto, Dawsey, 1 Portions of this study were presented at the Sixth and Seventh Annual Meetings of the Society for Neuroscience, 1976 and 1977. We thank R. Rimas for technical assistance. 242 0163-1047/79/020242-15502.00/0 Copyright© 1979by AcademicPress,Inc. All rightsof reproductionin any form reserved.
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Knapp, Bullard, & Mandell, 1976a; Lorens & Guldberg, 1974) while the dorsal nucleus of the raphe appears to send serotonergic projections to the striatal nuclei and portions of the substantia nigra (Bobillier et al., 1976; Conrad et al., 1974; Geyer et al., 1976a; Lorens & Guldberg, 1974; Miller, Richardson, Fibiger, & McLennan, 1975; Pierce, Foote, & Hobson, 1976; Roberge, Parent, & Boulay, 1976). Alterations in behavior have largely been attributed to the destruction of the median raphe nucleus (Geyer, Puerto, Menkers, Segall, & Mandell, 1976b; Jacobs, Wise, & Taylor, 1974; Srebro & Lorens, 1975) which may reflect the importance of the projections to limbic structures; in fact, many of the behavioral changes which have been reported in hippocampal-lesioned animals have also been reported for median raphelesioned animals. Although raphe lesions have been found to affect the performance of aversively motivated behaviors (Geyer et al., 1976b; Hole & Lorens, 1975; Lorens, 1973; Lorens, Guldberg, Hole, Kohler, & Srebro, 1976; Lorens, Sorenson, & Yunger, 1971; Srebro & Lorens 1975), little work has been done on their possible effects on tasks which are appetitively motivated. Therefore, Experiment I investigates the effects of selective damage to the mesencephalic raphe nuclei on the acquisition and extinction of a food-rewarded straight alley task and also reports their effects on open field activity. Experiment I1 further examines the alley performance of median raphe-lesioned rats by assessing the influence of the intertrial interval. EXPERIMENT
I
Methods Subjects. Twenty-four male, Sprague-Dawley-derived rats, obtained from a colony maintained by the University of Illinois, were used as subjects. The rats weighed approximately 300 g at the time of surgery. Six rats were randomly assigned to each of the following four treatment groups: median raphe (MR) lesions, dorsal raphe (DR) lesions, combined median and dorsal raphe (MDR) lesions, or sham-operated controls. Food and water were available ad lib. except during alley training, as noted below. Surgery. Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg) following treatment with atrophine sulfate (0.08 mg in 0.2 ml per rat). A stainless-steel electrode, 0.23 mm in diameter and insulated except for 0.5 mm at the tip, was stereotaxically placed (Pelligrino & Cushman, 1967) at coordinates AP: +0.2, Lat: 0, H: -4.3 for MR lesions, or AP: -0.3, Lat: 0, H: - 2 . 0 for DR lesions. A 1-mA current was then passed for 8 secs between the electrode and a rectal cathode. Lesions were placed in both of the above locations in MDR animals. In all cases the electrode was lowered through the center of the superior sagittal sinus. In sham-operated subjects a burr hole was drilled and the sinus was
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punctured but the electrode was not lowered. Animals were allowed 10 days to recover from surgery prior to testing. Open field behavior. Activity was measured in a 152.5 x 91.5-cm open field enclosed by 47-cm-high plywood walls. The floor of the field was demarcated into fifteen 30.5 x 30.5-cm squares. Lighting was provided by overhead fluorescent fixtures. Animals were tested in the open field 10 days following surgery. Subjects were gently placed in the center square of the field and the number of squares entered per minute was recorded over a 5-min period. A subject was considered to have entered a square when all four paws had been placed within it. As a measure of thigmotaxis, note was taken of the ratio of central squares entered (i.e., those which did not adjoin the plywood boundary of the field) to those entered in the periphery. Additionally, the total number of fecal boli deposited was recorded as a measure of emotionality. Straight alley acquisition and extinction.The alley was constructed of clear Plexiglas except for the floor, which was composed of metal rods 2 mm in diameter and spaced 10 mm apart. The start box (16 x 11.5 x 10 cm) and the goal box (30.3 x 15 x 10 cm) could be isolated from the remainder of the alley (110 x 15 x 10 cm) by metal guillotine doors. Latencies were evaluated by means of two photocells situated 14 and 103 cm from the start box which were, in turn, connected to a Hunter Klockounter. Interruption of the first beam started the clock which ran until the second photobeam was broken. Times were measured to the nearest 0.01 sec. Beginning the day after open field testing, animals were maintained on a restricted feeding schedule and were reduced to approximately 85% of their body weights. After 8 days on the diet, each animal was placed in the alley and was allowed to explore it for 5 rain. The following day, rats were placed in the goal box and were confined there until they had consumed one Froot Loop (Kellogg) which had been deposited in a glass coaster in the rear corner of the goal box. Acquisition training was begun 24 hr later. An animal was placed in the start box facing away from the lowered partition, which was raised when the animal oriented towards it. After entrance into the goal box, the door to that compartment was lowered to prevent retracing. Following Froot Loop ingestion, the animal was placed in a holding cage for 8-10 rain while five other animals were run. Each subject was run for six acquisition trials a day and was then allowed to feed. Training continued for 6 consecutive days. On the seventh day, animals were given one rewarded trial in the alley, followed by twelve nonreinforced extinction trials. During these trials, animals were retained in the goal box for 10 sec after entering it. Histological verification. Rats were given an overdose of sodium pen-
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tobarbital and were perfused intracardially with a 0.9% saline solution, followed by 10% formalin. Brains were removed from the skull and were stored in formalin for a minimum of 2 weeks. Sixty-four-micrometer sections were made on a freezing microtome and every third section through the extent of the lesion was kept. Samples were photomicrographed and then stained with cresyl violet.
Results Openfield. Results of the open field test are depicted in Fig. 1. It can be seen that while lesions of either the median or dorsal raphe nucleus increased activity, the median lesions were the more effective of the two. A 2 × 2 factorial analysis of variance (median raphe lesions x dorsal raphe lesions) conducted on the total number of squares entered indicated a significant median lesion effect IF(I, 20) = 10.2, p < .01] and a significant median lesion × dorsal lesion interaction [F(I, 20) = 4.63, p < .05J. A simple main effects analysis indicated that although dorsal raphe lesions increased activity relative to sham-operated controls, they did not further elevate the hyperactivity seen in animals with median raphe lesions. These results indicate that all operated subjects were more active than controls, with the activity of MR- and MDR-lesioned animals equivalent in magnitude and surpassing that of the other groups. It is possible that a ceiling effect may have prevented dorsal raphe lesions from further augmenting the increased locomotion of MR-lesioned rats. Neither lesion influenced the latency to leave the initial square, ratio of central to peripheral squares entered, or the number of fecal boli deposited in the field. ContrOl
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During open field testing it was noted that several MR- and MDRlesioned subjects displayed a Straub tail which was never seen in DRlesioned or control animals. This may reflect the release of pontine and medullary serotonin-containing neurons from inhibition following median raphe lesions (Jacobs & Klemfuss, 1975; Morgane & Stern 1974). Straight alley. Mean speeds (1/latencies) for the four groups in the alley during acquisition and extinction are shown in Fig. 2. As is apparent from the figure and confirmed by statistical analysis, MR- and MDR-lesioned animals ran more slowly during acquisition than did either control or DR-lesioned subjects. A median raphe lesion x dorsal raphe lesion x trials analysis of variance conducted on running speeds during acquisition revealed significant trial IF (5, 100) = 76,p < .01] and median raphe lesion effects IF(I, 20) = 9.01, < .01], and a significant median lesions x trials interaction IF(5, 100) = 11.81,p < .01]. The latter result indicates that the difference in running speeds between animals with and without median raphe lesions increased across trials. Neither the effect of dorsal lesions nor any of the remaining interactions approached statistical significance (p > 0.25). The failure of the median lesion x dorsal lesion interaction to attain significance suggests that the impaired acquisition seen in MDRlesioned subjects is primarily attributable to median raphe damage. Figure 2 also shows that MR- and MDR-lesioned subjects extinguished more slowly than control or DR-lesioned rats. This conclusion is confirmed by a median lesion x dorsal lesion x trials analysis of variance. The effect of trials was found to be significant [F(3,60) = 27.1, p < .01], as Control DR Lesion MR Lesion DR & MR Lesions
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RAPHE LESIONS AND RUNWAY PERFORMANCE
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was the median raphe lesion effect iF(l, 20) = 10.2, p < .01] and the median lesion x trials interaction IF(3, 60) = 4.3; p < .01]. As in acquisition, neither the dorsal lesion effect nor any of the remaining interactions a p p r o a c h e d significance (F < 1). Histology. The extent of the largest and smallest lesions seen in MR, DR, and M D R animals is shown in Fig. 3 and examples of typical lesions m a y be seen in Fig. 4. In all cases, median raphe lesions severely damaged the nucleus and invaded the immediately surrounding reticular formation. The ventral tegmental nuclei of Gudden were intact in all animals. Dorsal raphe lesions damaged the dorsal raphe and the adjacent central gray, the trochlear nuclei, and the medial longitudinal fasciculi. Although this lesion also encroached on the anterior pole of the dorsal tegmental nuclei of Gudden in two animals, these subjects did not differ behaviorally from other DR-lesioned animals. In MDR-lesioned subjects the two lesions fused through m o s t of their extent so as to damage structures between the MEDIAN
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two raphe nuclei. Thus, in addition to the damage mentioned above, these lesions infringed on the ventral tegmental nuclei, the nucleus linearis caudalis and, on occasion, the decussation of the brachia conjunctiva. E X P E R I M E N T II The results of the first experiment indicate that lesions of the MR can impair both the acquisition and extinction of a food-rewarded runway task. In the following experiment we attempted to specify more precisely the nature of these deficits by examining alley performance as a function of the intertrial interval. Additionally, we sought to determine whether any deficits in acquisition would persist through an extended number of training trials.
Methods Subjects. Subjects and their living conditions were similar to those reported in the first experiment. Surgery. Surgical procedures were virtually identical to those in Experiment I except there were only two groups, MR lesions (N = 16) or sham operated (N = 16). Alley acquisition and extinction. The alley apparatus was the same as that used in the first experiment. Following the 10-day surgical recovery period, rats were placed on the restricted feeding schedule and were reduced to approximately 85% of their body weights. Eight and nine days later animals were given the same preliminary introduction to the alley as those in Experiment I, again using Froot Loops as a reward. Acquisition training began the following day. Animals were assigned to one of four squads: MS or CS--median raphe-lesioned or shamoperated rats, respectively, trained under a 15-rain intertrial interval; or MM or CM--median raphe-lesioned or sham-operated animals whose training trials were immediately successive (virtually no intertrial interval). Within each treatment group, animals were randomly assigned to a squad. Each subject was given 6 trials a day for 12 days under a continuous reinforcement schedule. Training was continued for 12 consecutive days. The following day, animals received one rewarded trial followed by 12 extinction trials, still under their respective interval times. Histology. Techniques were the same as those in the first experiment.
Results Results of this experiment may be seen in Fig. 5. A 2 x 2 analysis of variance (lesion x intertrial interval) was conducted on mean running speeds across the last 5 days of acquisition. The analysis revealed a significant lesion x intertrial interval interaction iF(l, 28) = 5.7; p < .05]; a simple main effects analysis indicated that raphe-lesioned rats showed significantly slower running speeds under the spaced, but not massed, conditions [F(1, 28) -- 10.06, p < .01].
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F1G. 4. Unstained brain sections showing typical electrolytic lesions of the median raphe (a), dorsal raphe (b), or both raphe nuclei (c) in rats.
A lesions x intertrial interval x trials analysis of variance performed on mean running speeds during extinction indicated only a significant trials effect [F(3, 84) = 12.3, p < .01], although animals tended to extinguish faster under massed conditions. Although the analysis failed to indicate a significant lesion effect, examination of Fig. 5 suggests that lesioned rats run under spaced conditions tended to extinguish slower than the control group, as indicated by the rapid convergence of the two extinction curves. The failure of the analysis to indicate a significant lesion effect under spaced conditions may be due to the large difference in terminal acquisition running speeds between the control and lesioned groups. A comparison of the decline in running speeds during extinction (i.e., terminal acquisition speed minus terminal extinction speed) indicated that under spaced conditions control rats showed a significantly larger decline in
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FIG. 4-----Continued
running speeds than did MR-lesioned animals [t(14) = 2.3, p < .05]. A similar comparison under massed conditions was not significant. However, unless an actual crossover in running speeds occurs, as in the first experiment, it is difficult to interpret extinction rates when terminal acquisition speeds differ (Mackintosh, 1974). Histology. Median raphe lesions were found to be similar to those described in the first experiment.
GENERAL DISCUSSION Lesions of the dorsal raphe nucleus were found to produce a slight but significant increase in open field activity. Although significant open field hyperactivity following destruction of this nucleus has not previously
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F]o. 5. Mean running speeds during the acquisition and extinction of a runway task by control or median raphe-lesioned subjects under massed or spaced (15-rain intertrial interval) conditions. Abbreviations: CM, control subjects trained under massed conditions; CS, controls trained under spaced conditions; MM, median raphe-lesioned rats trained under massed conditions; MS, lesioned rats trained under spaced conditions. 251
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ASIN, WIRTSHAFTER, AND KENT
been reported, a trend in this direction has been observed (Jacobs & Cohen, 1976, Srebro & Lorens, 1975) and Grabowska (1974) has reported increased photocell cage activity following dorsal raphe lesions. Additionally, coronal knife cuts through the central gray substance immediately posterior to the dorsal raphe have been reported to increase open field activity (Wirtshafter, Pociask, & Kent, 1975). It is possible that differences in lesion size and placement account for the discrepant reports regarding dorsal raphe lesions and activity. In accordance with the reports of other investigators (Geyer et al., 1976b; Jacobs & Cohen, 1976; Lorens et al., 1971) lesions of the median raphe, either alone or in combination with dorsal lesions, were found to produce increased locomotion in the open field. This hyperactivity was of a much greater magnitude than that seen following dorsal raphe damage. We have also observed that similar median raphe lesions increase activity in tilt, photocell and stabilimeter cages (personal observations, also see Jacobs et al., 1974; Lorens et al., 1971). It is unlikely that the hyperactivity seen in the open field reflects serotonin depletion, since attempts to deplete the brain of serotonin with p-chlorophenylalanine (PCPA) or with intracerebral injections of 5,7dihydroxytryptamine have resulted in either unchanged or decreased levels of locomotion in the open field (Brody, 1970; Lorens et al., 1976; MacKenzie, Norelli, Trulson, & Hoebel, 1977; Marsden & Curzon, 1976; Tenen, 1967). Since the effects of median raphe lesions on open field activity are not in accordance with these findings, the hyperactivity produced by the lesions may reflect damage to nonserotonergic elements contained within the raphe (Aghajanian & Asher, 1971). Additionally, fiber systems of the tegmental nuclei of Gudden and of the interpeduncular nucleus course in close proximity to the median raphe and might inadvertently be damaged by lesions of this nucleus. We have observed that coronal knife cuts designed to interrupt projections of the dorsal tegmental nuclei (dorsal and caudal to the median raphe) result in hyperactivity which is at least as pronounced as that seen following median raphe lesions (in preparation). It is possible that damage to one of these systems might subserve hyperactivity in the open field after lesions of the raphe (Lorens, Kohler, & Guldberg, 1975). The first experiment also demonstrated that under certain conditions rats with lesions of the median, but not dorsal, raphe are impaired in both acquisition and extinction of a food-rewarded straight alley task. Although available evidence is limited, it should be noted that pharmacological antagonism of serotonin has been reported to have effects on alley acquisition and extinction similar to those observed here (Rosen & Buga, 1973; Rosen & Cohen, 1973), suggesting that serotonin depletion may underlie the effects of MR lesions on alley performance. Further work is needed, however, to substantiate this claim.
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In the second experiment, it was demonstrated that the conditions of training may effect the behavior of median raphe-lesioned rats. Thus, it was found that MR-lesioned animals are deficient in acquiring an alley task when the training trials are separated by 15 min, but not when they are immediately successive; furthermore, the deficits under spaced conditions persist through an extended number of training trials. These results suggest that the impaired acquisition of MR-lesioned rats trained under spaced conditions cannot be due to motor difficulties but may rather reflect alterations in attentional or motivational factors. The second experiment also obtained some evidence for impaired extinction under spaced but not massed conditions. The impairment was not as robust as that in the first experiment, however, where an actual crossover in running speeds in control and lesioned rats occurred. The absence of a dramatic extinction effect may have been due to the increased number of training trials in Experiment II. The number of acquisition trials has been reported to be an important determinant of extinction rate (Mackintosh, 1974) and has, on occasion, been found to interact with brain lesions (Henke, 1974). Based on available data, it is impossible to specify the precise nature of the deficits underlying the effects of median raphe lesions on alley performance. Anatomical considerations suggest, however, that a comparison of the effects of median raphe and limbic lesions may be profitable. Using fiber degeneration techniques, Nauta (1960) has described profuse interconnections between forebrain limbic structures and the paramedian tegmentum of the midbrain, including the median raphe, and has further suggested a functional relation between the two areas. More recent biochemical techniques have identified serotonergic projections from the MR to limbic structures, including the cingulum and hippocampus (Geyer et al., 1976a, Moore, Halaris, & Jones, 1978), and many of the behavioral effects of lesions of these structures resemble those following MR lesions. Hippocampal and raphe lesions have similar effects on avoidance behaviors (Douglas, 1967; Geyer et al., 1976b; Hole & Lorens, 1975; Isaacson 1974; Lorens, 1973; Lorens et al., 1975; Olton, 1973; Srebro & Lorens, 1975), latent inhibition (Ackil, Mellgren, Halgren, & Frommer, 1969; Asin, Wirtshafter, & Kent, 1978), and spontaneous alternation (Asin, Wirtshafter, & Kent, 1977; Dalland, 1970; Geyer et al., 1976b). X-ray induced agenesis of the dentate gyrus granule cell layer or electrolytic lesion of the cingulate gyrus impairs both the acquisition and extinction of a runway task (Bruner, Haggbloom, & Gazzara, 1974; Glass, Ison, & Thomas, 1969), a pattern of deficits similar to those reported here. Furthermore, like median raphe-lesioned animals, rats with lesions of the hippocampus display impaired acquisition and extinction when trained under long, but not short, intertrial intervals (Jarrard, Isaacson, & Wickelgren, 1964; Means, Woodruff, & Isaacson, 1972). Since the median
254 raphe appears tures, further destruction of median raphe parallels.
ASIN, WIRTSHAFTER, AND KENT to h a v e a b u n d a n t a n a t o m i c a l c o n n e c t i o n s w i t h l i m b i c s t r u c comparisons of the behavioral changes seen following the t h e s e a r e a s m a y p r o v e f r u i t f u l in e l u c i d a t i n g t h e r o l e o f t h e in b e h a v i o r . W e a r e c u r r e n t l y i n v e s t i g a t i n g s u c h p o s s i b l e
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