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Effects of hippocampal lesions on the copulatory behavior of male rats
Phystology and Behavior. Vol 3, pp 651-656. Pergamon Press, 1968. Printed in Great Brttam
Effects of Hippocampal Lesions on the Copulatory Behavior of Male Rats D O N A L D A. D E W S B U R Y , E D W A R D D. G O O D M A N , P A T R I C I A J. S A L I S A N D B R A D F O R D N. B U N N E L L
Department of Psychology and Center for Neurobiological Sciences, University of Florida (Received 8 February 1968) P. J. SALISAND B. N. BUNNELL. Effects of hippocampal lesions on the copulatory (5) 651-656, 1968. Performance on two preoperative and two postoperative copulation tests was compared for male rats given total hippocampal, dorsal hippocampal, neocortical, and no lesions. Eight rats were in each group. Tests were continued to a satiety criterion. Statistically significant increases in Mount Latency and Intromission Latency were found m the rats with total hippocampal lesions. There were no other significant differences. DEWSBURY,
D. A., E. D.
GOODMAN,
behavior of male rats. PHYSIOL.BEHAV.3
Hippocampal lesions
Copulatton
Mount latency
Intromission latency
15 min, it is possible that major lesion effects could have occurred and not been detected with the measures they used. In the present study tests were continued to a satiety criterion wtth latency data reported and data grouped according to copulatory series following the general method of Beach and Jordan [1 ]. In addition, the hippocampal lesions made in both earlier studies spared the ventral ~k-½of the hippocampal formation. In the present study two hippocampectomized groups were used: (1) one with dorsal hippocampal lesions and; (2) one in which an attempt was made to ablate the entire hippocampal formation.
THE ROLEOF THE hippocampal formation in the control of the copulatory behavior of male rats is not clear. The two lesion studies in the literature, those of Kim [7] and Kimble, Rogers, and Hendrickson [8] leave questions regarding both the power of the behavioral measures used and the extent of the hippocampal lesions. Copulatory acts in male rats are usually divided into three categories: mounts, where there is no vaginal penetration, intromissions, with vaginal penetrations lasting approximately 0.3 sec [12] and ejaculations. The mounts and intromissions usually occur in series which are terminated by ejaculations; successive series are separated from each other by postejaculatory refractory periods. Rats typically reach a criterion of satiety, such as 30 min without an intromission, after about seven ejaculations [1]. It may take several hr for the animal to satiate. Kim [7] did not distinguish mounts from intromtssions and ejaculations but lumped them into a single measure, "mounting acts." He found that the number of "mounting acts" in 15 min tests increased following dorsal hippocampal lesions. The "mounting acts" measure is a complex one and could be affected by changes in such factors as efficiency in achieving vaginal penetration, latency to initiate mounting, number of intromissions required to produce ejaculation, and duration of post-ejaculatory refractory periods. Kimble et aL [8], who also used 15 min tests, compared the copulatory behavior of sexually naive hippocampectomized and unoperated male rats. They scored mounts, intromissions, and ejaculations and reported that their lesioned rats were indistinguishable from the normals. As no latency measures were reported, data were not grouped according to copulatory series, and tests were continued for only
METHOD
Subjects The subjects for this experiment were 32 male Long-Evans rats purchased from Simonson Laboratories, Gilroy, California. They were 170 days of age at the beginning of preoperative testing and had been given 2-5 copulation tests to a satiety criterion. A total of 102 female rats of the same strain and 90-170 days of age at the beginning of testing were used as partners in the mating tests.
Apparatus All tests were conducted in four plexiglass observation cages which were 32 in. in dia. and 26 in. high. Cages were placed on wooden bases which were covered with commercially produced litter. All copulatory events were recorded manually on an Esterline-Angus event recorder.
Whis research was supported by grants GB-6590 and GB-4604 from the National Science Foundation. ~The hormones used were Progynon brand of estradiol benzoate and Proluton brand of progesterone supplied through the courtesy of Dr. Preston L. Perlman of the Schering Corporation, Bloomfield, New Jersey. 651
652
DEWSBURY, GOODMAN, SAL1S AND BUNNELL
Surgical Procedure Surgery was performed under sodmm pentobarbital anesthesia (60 mg/kg) with atropine sulphate (0.12 mg) used to reduce respiratory complications. All lesions were made by aspiration following a procedure similar to that described by Kim [7]. The sham operations were identical to the hippocampal and neocortical lesions except that no tissue was removed. A dissecting microscope was used for the hippocampal ablations.
Testing Procedure The rats were maintained on a reversed light-dark cycle with the lights coming on at 12:00 midnight and going off at 12:00 noon. Four males were tested simultaneously in adjacent cages. Five min after the males were placed an the test cages the females were introduced and tests begun. The occurrence of mounts, intromissions, and ejaculations was determined behaviorally [2] and recorded. If there were no intromisslons in the 15 rain period beginning a test, the test was discontinued and the male retested later in the week. If copulation was initiated the test was continued to the satiety criterion, 30 min with no intromissions. In a few cases where widelyspaced intromissions continued over extended periods of time, tests were terminated 2 hr after the occurrence of the last ejaculation• The following measures were taken: Ejaculation Frequency (EF), the total number of ejaculations occurring in a test; Mount Latency (ML), the time from the introduction of the female to the first mount with clasping by the male; Intromission Latency (IL), the time from the introduction of the female to the first vaginal penetration; Ejaculation Latency (EL), the latency from the first intromission to the ejaculation in each series; Intromission Frequency (IF), the number of intromissions in each ejaculatory series; Mean InterIntromission Interval (MIII), the mean interval separating
successive intromlssions within an ejaculatory series; and Post-Ejaculatory Interval (PEt), the interval from each ejaculation to the next intromission. The two preoperative tests were given in the afternoon four weeks apart with one observer recording the data. On the basis of preoperative data the animals were divided into four groups which were matched as closely as possible on mean EFs and first series ELs. These were to become the four groups: total hippocampal, dorsal hippocampal, cortical control, and control. The control group was further divided into sham operated and normal subgroups. As the results showed no differences between these subgroups, their data have been combined and treated as a single group. For the purposes of scheduling postoperative tests the 32 animals were divided into four squads of eight with two ammals from each group in each squad. Squads were begun on a schedule of les~ons and postoperative tests one week apart from each other. The operations were performed one to four weeks after the last preoperative test. Each squad received its first postoperative test 17-20 days after surgery. The second postoperative test followed two weeks later. Postoperative tests were conducted in the evening with two observers present for each test. The four rats tested simultaneously included one from each group with test cage and observer varied across groups• Due to d~fficulties with the females used in two of the tests (one with a dorsal hippocampal male and one with a cortical male) these tests could not be completed. As a result the postoperative tests for these two animals occurred two weeks after those of the other rats. Females were brought into behavioral estrus with exogenous intramuscular injections of 0.l mg estradiol benzoate 72 hr before testing and 1.0 mg progesterone 3 hr before testing. Prior to the experimental tests the females were placed briefly with non-experimental males to insure their receptivity. Females which did not show behavioral estrus were not used for testing.
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FIG. 1. Median Mount Latency and Intromission Latency as a funcUon of test and lesion group.
HIPPOCAMPAL LESIONS AND COPULATORY BEHAVIOR RESULTS
Behavior Median MLs and ILs for each test are presented in Fig. 1. A small but clear postoperative increase is evident in both ML and IL in the total hippocampal group relative to the three other groups. The significance of this difference was tested with a Kruskal-WaUis one-way analysis of variance [14]. Shifts from mean preoperative score to mean postoperative score were used in the analyses. There was a significant effect of lesion group on both measures (For ML: H = 14.2, df= 3, p < 0 . 0 1 ; F o r IL: H = 10.3, d f ~ 3, p < 0 . 0 2 ) . When the total hippocampal group was then compared with the control group using a Mann-Whitney U test (Siegel, 1956), significant differences were found on both measures (For M L ; U = 11, p < 0 . 0 1 4 ; F o r l L : U = 8, p < 0 . 0 0 5 ) . All groups showed slight increases in ejaculation frequency postoperatively, but there was no significant effect of lesion group (H = 0.5, df = 3, p > 0.05). Data from successive series were grouped and analyzed in the manner of Beach and Jordan [1 ]. N o apparent differences were seen. A summary of these data is presented in Fig. 2. Shown in this figure are scores on EL, M i l l , IF, and PEI for the first four and last two ejaculatory series. Each score
653 represents the percent postoperative shift in the measure, determined by subtracting the median preoperative scores from the median postoperative scores and dividing the difference by the median preoperative scores for each group. There are few systematic differences. On visual inspection the increases in the last series for the dorsal hippocampal group appeared appreciable. Therefore these data were analyzed with the Kruskal-Wallis one-way analysis of variance as used above. None of the measures showed statistically significant differences as a function of lesion group in the 1st series (HEL = 3.4, HMIII ~- 1.9, H]F = 0.3, HpEI = 1.4; df= 3; p > 0.05). In order to compare these data w~th those of Kim the number of "mounting acts" in the first 15 min of each test was determined by summing the number of mounts, intromissions, and ejaculations. All four groups showed increases in the mean frequency of the mounting act on postoperative trials (total hippocampal q- 5.6, dorsal hippocampal + 8.0, neocortical + 7.4, and control + 0.7). The Kruskal-Wallis one-way analysis of variance on the difference scores (postoperative mounting acts--preoperative mounting acts) revealed no significant difference as a function of lesion group (H = 3.8, df = 3, p > 0.05).
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FIG. 2. Per cent change in Ejaculation Latency, Intromission Frequency, Post-Ejaculatory Interval, and Mean lnter-lntromission Interval for the first four and last two series as a function of lesion group. Percentage Change z (Mdn. Postoperative score--Mdn. Preoperative score)/Mdn. Preoperative score.
Upon completion of testing all operated animals were sacrificed, perfused with saline and 10 per cent formalin, and their brains embedded in celloidin. Sections were cut at 30 micra and every seventh section was mounted and stained with cresyl violet. Tracings of a representative lesion from each of the three operated groups are presented in Fig. 3. In the total hippocampal group the anterior margin of the lesion generally invaded the dorsal hippocampal commisure and, in a few cases, cut the fornix superior and damaged the fornix; in one animal the lesion extended forward into the dorsal posterior portion of the lateral septal nucleus unilaterally. In three animals small medial fragments of the anterior tip of the dorsal hippocampus remained on one side; for the most part, the entire dorsal hippocampus and dentate gyrus, overlying corpus callosum, cingulum, and median cerebral cortex were destroyed. In all animals the ventral portion of the hippocampus was left intact, at least unilaterally, for a distance of about 0.75 mm from its anterior tip; thus there was no involvement of the amygdala. The remainder of the hippocampal formation was almost completely destroyed as were parts of the subiculum and entorhinal cortex in the posterior cerebral hemispheres. Portions of the visual cortex were spared in all animals. The anterior portion of the lesion spared the fimbria and the stria terminalis as well as the stria medullaris; slight unilateral damage to the nucleus anterior dorsalis, and all but one animal had superficial damage to the nucleus lateralis of the thalamus; in these cases, the damage was usually unilateral, or, if it occurred bilaterally, occurred at different levels along the dorsal edge of the nucleus. The posterior part of the lesion severed the fimbria in all animals and produced unilateral damage to the pretectal, posterior lateral thalamic, and dorsal lateral geniculate bodies in two animals. In all, between 80 and 90 per cent of the hippocampal formation was removed in the animals of this group and this was accompanied by removal of 18-30 per cent of total neocortex. The lesions of the dorsal hippocampal group were similar
654
DEWSBURY, GOODMAN, SALIS AND BUNNELL CORTICAL
CONTROL
DORSAL HIPPOCAMPAL
TOTAL HIPPOCAMPAL
Z:A2' FIG. 3. Representatwe lesions for one ammal from each of the three operated groups: Control (neocort]cal), Dorsal Hippocarnpal, and Total H]ppocampal. The lesions (stippled areas) have been reconstructed on plates traced from Konig and Khppel [10]. The levels chosen reading from top to bottom, are 5660, 4110, 2790, 1610 micra anterior to the frontal zero plane of Konig and Khppel.
to those of the total hippocampal in terms of anterior margin and the locus of extrahippocampal destruction. In addition, one animal had unilateral, and one bilateral superficial damage to the superior colliculus. The maximum posterior extent of the dorsal lesions was at approximately 1.5 mm anterior to the frontal zero plane as defined by Konig and Klippel [10], whde the depth of the posterior portion of the dorsal lesions was at, or slightly above the K f n i g and Klippel horizontal zero plane. There was considerable variation in the size of these lesions. In all, between 45-60 per cent of the hippocampus was spared in these animals. I n most animals, the neocortical damage in the dorsal group was similar to that of the total hippocampal group; in a few cases however, there was more sparing of posterior neocortex than in the total hippocampal animals (see Fig. 3). Neocortical destruc-
t]on in the dorsal hippocampal group amounted to 15-25 per cent of total neocortex. The cortical control group had between 20-30 per cent of total neocortex removed. In general, these lesions were somewhat more anteriorly placed than was the neocort~cal destruction m the two hlppocampal groups. Their anterior margin was approximately 6.5 mm ahead of the frontal zero plane of K6nig and Klippel wh]le the posterior limit was about 1.5 mm anterior to this plane. These lesions removed the corpus callosum over the dorsal hippocampus, destroyed the cingulum, and, in all but one case, produced slight damage to the alveus. In one animal the h~ppocampal pyramids received moderate damage bilaterally. Medially, the cortical lesions spared a port]on of the cingulate cortex. Fortunately, the les~ons of the dorsal hippocampal group
HIPPOCAMPAL LESIONS AND COPULATORY BEHAVIOR
655
were similar to those of the total hippocampal group in terms of amount of destruction of the median cortex and, in view of the behavioral results obtained, it did not appear necessary to run additional cortical control animals in which median cortex was completely removed.
campus and septum remained functional in our dorsal hippocampal group. We are currently examining the effects of septal lesions on copulatory behavior in the male rat. Kimble et al. [8] reported that their large bilateral dorsal hippocampal lesions destroyed the fimbria or, at best, left only fragments of it. This suggests that the effects we obtained may have been due to the destruction of a posteroventral hippocampal-allocortical, or hlppocampal-neocortical system. A third alternative, that a mass action effect in operating cannot be discounted by our data, but the studies of Jarrard and Kimura, cited above, which suggest that there is a unique effect of posteroventral hippocampal lesions on other behaviors, lead us to believe that a mass factor is not critical. There was a nonsignificant increase in EL and M I I I in our dorsal hippocampal group in the last series before the satiety criterion was reached. The high interanimal variation in this group led us to look for a correlation between scores on the last series and extrahippocampal brain damage. N o such relationship was found and we have no explanation for this trend. Larsson [11] found that male rats continued to copulate despite extensive lesions of the posterior neocortex involving the entire occipital cortex and portions of parietal and temporal cortex. Increases in IL, MIII, and PEI did appear. These changes were attributed to deficits in the ability of the male to locate and maintain contact with the female and were interpreted as being secondary to visual deficits. Neocortical lesions in the present study never exceeded 30 per cent of total neocortex and some sparing of visual cortex invariably occurred. In view of the differences in size and location of the lesions in our neocortical group as compared with the posterior lesion group in the Larsson study, it is not surprising that we did not observe either the qualitative or the quantitative changes he obtained. Since the completion of this work, Bermant, Glickman, and Davidson [3] have published an additional study of the effects of hippocampal lesions on the copulatory behavior of rats. Bermant et al. report that rats with dorsal hippocampal lesions showed progressively decreasing MIIIs and PEIs during their three postoperative tests. In a second experiment, the behavior of control rats was compared with that of rats with combined dorsal and ventral hippocampal lesions. N o significant differences attributable to the lesions were reported. The data from the present study have been reanalyzed in a search for the effects reported by Bermant et al. Data from M I I I and PEI in the first four series were examined in two ways: (1). Postoperative test 1 vs. postoperative test 2, and (2). Mean preoperative vs. Postoperative test 2. The 16 appropriate Kruskal-Wallis one-way analyses of variance revealed no significant differences as a function of lesion group. However, there was a consistent trend in the direction suggested by the data of Bermant et aL In all 8 comparisions of shifts in PEI, the dorsal hippocampal group showed the largest shift in the direction of faster performance in the second postoperative test. The dorsal hippocampal group showed the largest mean decrease in five of the eight comparisons of M I I I in the second postoperative test and the lowest summed ranks in 7 of 8 comparisons. Thus, the differences between the present data and those of Bermant et al. may be of degree rather than kind. The few differences between the present data and those of Bermant, et aL might easily result from differences in the
DISCUSSION
F r o m the present data it would appear that the hippocampus is of little importance in the mediation of the normal mating pattern of the sexually experienced male rat. The only statistically significant effects of total hippocampal lesions were increases in ML and IL. Thus, the effect of near total removal of the hippocampus was simply to increase the latency of the first copulation by the male. Once the behavior was initiated it was carried out through the normal number of copulatory series in a manner which did not appear to be qualitatively different from that of the control animals. There were no indications that hippocampectomized animals had any difficulty in orienting toward and maintaining contact with the female. Resection of the dorsal hippocampus with lesions corresponding roughly to the size and location of those made by Kim [7] and Kimble et al. [8] failed to produce significant changes in any measures taken. The same was true for the neocortical lesions. The delay in the initiation of the sexual response in totally hippocampectomized animals may be a reflection of decreased "distractability" [4]. This argument assumes that an animal is engaged in some compelling behavioral sequence (e.g. exploring the test cage, grooming, etc.) and the introduction of the female represents a novel stimulus to which the experimental animal is less attentive. It is more likely, in view of our failure to observe any obvious qualitative differences in the behavior of the different groups during the 5 min pretest adaptation periods, that the effects are more specifically related to the latency of arousal of the copulatory response. The question of the anatomical region whose removal produces this deficit remains. In analyzing the projections of the four hippocampal fields, Raisman, Cowan, and Powell [13] have said that the hippocampal field CA1 projects primarily to postcommisural fornix while fields CA3 and CA4 project via fimbria and precommisural fornix to septum and the nucleus of the diagonal band. Jarrard [5], taking note of this, pointed out that in the rat most of CA3 and CA4 lie in the caudal third of the hippocampus. Jarrard cites evidence from his laboratory which indicates that the behavioral effects of posteroventral hippocampal lesions differ from the effects of ablation of the dorsal hippocampus. In an earlier study Kimura [9] found that posterior hippocampal lesions interfered with passive avoidance, but more anterior lesions did not. Kaada, Rasmussen, Kveim [6] found no differences between anterior and posterior lesions on a passive avoidance task, but it is not clear from their description whether K a a d a et al. actually invaded the posteroventral hippocampus with their "posterior" hippocampal lesions. Because it is difficult to remove that portion of the fimbria which lies between thalamus and caudate nucleus without damaging these structures, this region was largely intact in most of our animals. Such sparing may also have been present in many of Kim's [7] animals. This being the case, it is possible that pathways between posteroventral hippo-
656
DEWSBURY, GOODMAN, SALIS AND BUNNELL
lesions and/or testing procedures. The electrolytic lesions in the later study appear to be smaller, particularly in the case of the amount of ventral h~ppocampus and neocortex ablated. The duration of each test, spacing of the tests, and the amount of postoperative recovery time differed in the two experiments. The third postoperative test of Bermant
et al. occurred slightly sooner after the operations than our second test. The differences shown by the Bermant, et al. dorsal hippocampal group are most striking on the third postoperative test. We ran only two postoperative tests and it is possible that the number of such tests may be a significant factor in the demonstration of a dorsal hippocampal effect.
REFERENCES 1. Beach, F. A. and L. Jordan. Sexual exhaustion and recovery in the male rat. Q. Jl exp. Psychol. 7: 121-133, 1956. 2. Bermant, G. Rat sex behavior: photographic analysis of the intromission response. Psychonom. Sci. 2: 65-66, 1965. 3. Bermant, G., S. E. Glickman and J. M. Davidson. Effects of limbic lesions on copulatory behavior of male rats. J. comp. physiol. Psychol. 65: 118-125, 1968. 4. lsaacson, R. L. Hippocampus. Paper presented at symposium on "The Limbic-midbram system and behavior" at meetings of the American Psychological Association in New York, N.Y., September 4, 1966. 5. Jarrard, L. E. On the importance of &fferent hippocampal lesions for behavioral investigators. Ammon's Horn. Spring, 15-21, 1967. 6. Kaada, B. R., E. W. Rasmussen and O. Kvenn. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic, and insular lesions in rats. J. comp. phystol. Psychol. 55: 661-670, 1962. 7. Klm, C. Sexual activity of male rats following ablation of hippocampus. J. comp. physiol. Psychol. 53: 553-557, 1960.
8. Kimble, D. P., L. Rogers and C. W. Hendrickson. Hippocampal lesions &srupt maternal, not sexual, behavior m the albino rat. J. comp. physiol. Psychol. 63: 401--407, 1967. 9. Kimura, D. Effects of selective hippocampal damage on avoidance behavior in the rat. Can. J. Psychol. 12" 213-218, 1958. 10. Konig, F. R. and Renate A. Klippel. The rat brain" A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore: Williams and Wilkins, 1963. 11. Larsson, K. Mating behavior in male rats after cerebral cortex ablation. II. Effects of lesions in the frontal lobes compared to lesions m the posterior half of the hemispheres. J. exp. Zool. 155: 203-214, 1964. 12. Pierce, J. T. and R. L. Nutall. Duration of sexual contacts in the rat. J. comp. physiol. Psychol. 54: 585-587, 1961. 13. Raisman, G., W. M. Cowan and T. P. S. Powell. An experimental analysis of the efferent projection of the hippocampus. Brain, 89: 83-108, 1966. 14. Siegel, S. Non-parametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956.
Effects of Hippocampal Lesions on the Copulatory Behavior of Male Rats D O N A L D A. D E W S B U R Y , E D W A R D D. G O O D M A N , P A T R I C I A J. S A L I S A N D B R A D F O R D N. B U N N E L L
Department of Psychology and Center for Neurobiological Sciences, University of Florida (Received 8 February 1968) P. J. SALISAND B. N. BUNNELL. Effects of hippocampal lesions on the copulatory (5) 651-656, 1968. Performance on two preoperative and two postoperative copulation tests was compared for male rats given total hippocampal, dorsal hippocampal, neocortical, and no lesions. Eight rats were in each group. Tests were continued to a satiety criterion. Statistically significant increases in Mount Latency and Intromission Latency were found m the rats with total hippocampal lesions. There were no other significant differences. DEWSBURY,
D. A., E. D.
GOODMAN,
behavior of male rats. PHYSIOL.BEHAV.3
Hippocampal lesions
Copulatton
Mount latency
Intromission latency
15 min, it is possible that major lesion effects could have occurred and not been detected with the measures they used. In the present study tests were continued to a satiety criterion wtth latency data reported and data grouped according to copulatory series following the general method of Beach and Jordan [1 ]. In addition, the hippocampal lesions made in both earlier studies spared the ventral ~k-½of the hippocampal formation. In the present study two hippocampectomized groups were used: (1) one with dorsal hippocampal lesions and; (2) one in which an attempt was made to ablate the entire hippocampal formation.
THE ROLEOF THE hippocampal formation in the control of the copulatory behavior of male rats is not clear. The two lesion studies in the literature, those of Kim [7] and Kimble, Rogers, and Hendrickson [8] leave questions regarding both the power of the behavioral measures used and the extent of the hippocampal lesions. Copulatory acts in male rats are usually divided into three categories: mounts, where there is no vaginal penetration, intromissions, with vaginal penetrations lasting approximately 0.3 sec [12] and ejaculations. The mounts and intromissions usually occur in series which are terminated by ejaculations; successive series are separated from each other by postejaculatory refractory periods. Rats typically reach a criterion of satiety, such as 30 min without an intromission, after about seven ejaculations [1]. It may take several hr for the animal to satiate. Kim [7] did not distinguish mounts from intromtssions and ejaculations but lumped them into a single measure, "mounting acts." He found that the number of "mounting acts" in 15 min tests increased following dorsal hippocampal lesions. The "mounting acts" measure is a complex one and could be affected by changes in such factors as efficiency in achieving vaginal penetration, latency to initiate mounting, number of intromissions required to produce ejaculation, and duration of post-ejaculatory refractory periods. Kimble et aL [8], who also used 15 min tests, compared the copulatory behavior of sexually naive hippocampectomized and unoperated male rats. They scored mounts, intromissions, and ejaculations and reported that their lesioned rats were indistinguishable from the normals. As no latency measures were reported, data were not grouped according to copulatory series, and tests were continued for only
METHOD
Subjects The subjects for this experiment were 32 male Long-Evans rats purchased from Simonson Laboratories, Gilroy, California. They were 170 days of age at the beginning of preoperative testing and had been given 2-5 copulation tests to a satiety criterion. A total of 102 female rats of the same strain and 90-170 days of age at the beginning of testing were used as partners in the mating tests.
Apparatus All tests were conducted in four plexiglass observation cages which were 32 in. in dia. and 26 in. high. Cages were placed on wooden bases which were covered with commercially produced litter. All copulatory events were recorded manually on an Esterline-Angus event recorder.
Whis research was supported by grants GB-6590 and GB-4604 from the National Science Foundation. ~The hormones used were Progynon brand of estradiol benzoate and Proluton brand of progesterone supplied through the courtesy of Dr. Preston L. Perlman of the Schering Corporation, Bloomfield, New Jersey. 651
652
DEWSBURY, GOODMAN, SAL1S AND BUNNELL
Surgical Procedure Surgery was performed under sodmm pentobarbital anesthesia (60 mg/kg) with atropine sulphate (0.12 mg) used to reduce respiratory complications. All lesions were made by aspiration following a procedure similar to that described by Kim [7]. The sham operations were identical to the hippocampal and neocortical lesions except that no tissue was removed. A dissecting microscope was used for the hippocampal ablations.
Testing Procedure The rats were maintained on a reversed light-dark cycle with the lights coming on at 12:00 midnight and going off at 12:00 noon. Four males were tested simultaneously in adjacent cages. Five min after the males were placed an the test cages the females were introduced and tests begun. The occurrence of mounts, intromissions, and ejaculations was determined behaviorally [2] and recorded. If there were no intromisslons in the 15 rain period beginning a test, the test was discontinued and the male retested later in the week. If copulation was initiated the test was continued to the satiety criterion, 30 min with no intromissions. In a few cases where widelyspaced intromissions continued over extended periods of time, tests were terminated 2 hr after the occurrence of the last ejaculation• The following measures were taken: Ejaculation Frequency (EF), the total number of ejaculations occurring in a test; Mount Latency (ML), the time from the introduction of the female to the first mount with clasping by the male; Intromission Latency (IL), the time from the introduction of the female to the first vaginal penetration; Ejaculation Latency (EL), the latency from the first intromission to the ejaculation in each series; Intromission Frequency (IF), the number of intromissions in each ejaculatory series; Mean InterIntromission Interval (MIII), the mean interval separating
successive intromlssions within an ejaculatory series; and Post-Ejaculatory Interval (PEt), the interval from each ejaculation to the next intromission. The two preoperative tests were given in the afternoon four weeks apart with one observer recording the data. On the basis of preoperative data the animals were divided into four groups which were matched as closely as possible on mean EFs and first series ELs. These were to become the four groups: total hippocampal, dorsal hippocampal, cortical control, and control. The control group was further divided into sham operated and normal subgroups. As the results showed no differences between these subgroups, their data have been combined and treated as a single group. For the purposes of scheduling postoperative tests the 32 animals were divided into four squads of eight with two ammals from each group in each squad. Squads were begun on a schedule of les~ons and postoperative tests one week apart from each other. The operations were performed one to four weeks after the last preoperative test. Each squad received its first postoperative test 17-20 days after surgery. The second postoperative test followed two weeks later. Postoperative tests were conducted in the evening with two observers present for each test. The four rats tested simultaneously included one from each group with test cage and observer varied across groups• Due to d~fficulties with the females used in two of the tests (one with a dorsal hippocampal male and one with a cortical male) these tests could not be completed. As a result the postoperative tests for these two animals occurred two weeks after those of the other rats. Females were brought into behavioral estrus with exogenous intramuscular injections of 0.l mg estradiol benzoate 72 hr before testing and 1.0 mg progesterone 3 hr before testing. Prior to the experimental tests the females were placed briefly with non-experimental males to insure their receptivity. Females which did not show behavioral estrus were not used for testing.
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FIG. 1. Median Mount Latency and Intromission Latency as a funcUon of test and lesion group.
HIPPOCAMPAL LESIONS AND COPULATORY BEHAVIOR RESULTS
Behavior Median MLs and ILs for each test are presented in Fig. 1. A small but clear postoperative increase is evident in both ML and IL in the total hippocampal group relative to the three other groups. The significance of this difference was tested with a Kruskal-WaUis one-way analysis of variance [14]. Shifts from mean preoperative score to mean postoperative score were used in the analyses. There was a significant effect of lesion group on both measures (For ML: H = 14.2, df= 3, p < 0 . 0 1 ; F o r IL: H = 10.3, d f ~ 3, p < 0 . 0 2 ) . When the total hippocampal group was then compared with the control group using a Mann-Whitney U test (Siegel, 1956), significant differences were found on both measures (For M L ; U = 11, p < 0 . 0 1 4 ; F o r l L : U = 8, p < 0 . 0 0 5 ) . All groups showed slight increases in ejaculation frequency postoperatively, but there was no significant effect of lesion group (H = 0.5, df = 3, p > 0.05). Data from successive series were grouped and analyzed in the manner of Beach and Jordan [1 ]. N o apparent differences were seen. A summary of these data is presented in Fig. 2. Shown in this figure are scores on EL, M i l l , IF, and PEI for the first four and last two ejaculatory series. Each score
653 represents the percent postoperative shift in the measure, determined by subtracting the median preoperative scores from the median postoperative scores and dividing the difference by the median preoperative scores for each group. There are few systematic differences. On visual inspection the increases in the last series for the dorsal hippocampal group appeared appreciable. Therefore these data were analyzed with the Kruskal-Wallis one-way analysis of variance as used above. None of the measures showed statistically significant differences as a function of lesion group in the 1st series (HEL = 3.4, HMIII ~- 1.9, H]F = 0.3, HpEI = 1.4; df= 3; p > 0.05). In order to compare these data w~th those of Kim the number of "mounting acts" in the first 15 min of each test was determined by summing the number of mounts, intromissions, and ejaculations. All four groups showed increases in the mean frequency of the mounting act on postoperative trials (total hippocampal q- 5.6, dorsal hippocampal + 8.0, neocortical + 7.4, and control + 0.7). The Kruskal-Wallis one-way analysis of variance on the difference scores (postoperative mounting acts--preoperative mounting acts) revealed no significant difference as a function of lesion group (H = 3.8, df = 3, p > 0.05).
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FIG. 2. Per cent change in Ejaculation Latency, Intromission Frequency, Post-Ejaculatory Interval, and Mean lnter-lntromission Interval for the first four and last two series as a function of lesion group. Percentage Change z (Mdn. Postoperative score--Mdn. Preoperative score)/Mdn. Preoperative score.
Upon completion of testing all operated animals were sacrificed, perfused with saline and 10 per cent formalin, and their brains embedded in celloidin. Sections were cut at 30 micra and every seventh section was mounted and stained with cresyl violet. Tracings of a representative lesion from each of the three operated groups are presented in Fig. 3. In the total hippocampal group the anterior margin of the lesion generally invaded the dorsal hippocampal commisure and, in a few cases, cut the fornix superior and damaged the fornix; in one animal the lesion extended forward into the dorsal posterior portion of the lateral septal nucleus unilaterally. In three animals small medial fragments of the anterior tip of the dorsal hippocampus remained on one side; for the most part, the entire dorsal hippocampus and dentate gyrus, overlying corpus callosum, cingulum, and median cerebral cortex were destroyed. In all animals the ventral portion of the hippocampus was left intact, at least unilaterally, for a distance of about 0.75 mm from its anterior tip; thus there was no involvement of the amygdala. The remainder of the hippocampal formation was almost completely destroyed as were parts of the subiculum and entorhinal cortex in the posterior cerebral hemispheres. Portions of the visual cortex were spared in all animals. The anterior portion of the lesion spared the fimbria and the stria terminalis as well as the stria medullaris; slight unilateral damage to the nucleus anterior dorsalis, and all but one animal had superficial damage to the nucleus lateralis of the thalamus; in these cases, the damage was usually unilateral, or, if it occurred bilaterally, occurred at different levels along the dorsal edge of the nucleus. The posterior part of the lesion severed the fimbria in all animals and produced unilateral damage to the pretectal, posterior lateral thalamic, and dorsal lateral geniculate bodies in two animals. In all, between 80 and 90 per cent of the hippocampal formation was removed in the animals of this group and this was accompanied by removal of 18-30 per cent of total neocortex. The lesions of the dorsal hippocampal group were similar
654
DEWSBURY, GOODMAN, SALIS AND BUNNELL CORTICAL
CONTROL
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Z:A2' FIG. 3. Representatwe lesions for one ammal from each of the three operated groups: Control (neocort]cal), Dorsal Hippocarnpal, and Total H]ppocampal. The lesions (stippled areas) have been reconstructed on plates traced from Konig and Khppel [10]. The levels chosen reading from top to bottom, are 5660, 4110, 2790, 1610 micra anterior to the frontal zero plane of Konig and Khppel.
to those of the total hippocampal in terms of anterior margin and the locus of extrahippocampal destruction. In addition, one animal had unilateral, and one bilateral superficial damage to the superior colliculus. The maximum posterior extent of the dorsal lesions was at approximately 1.5 mm anterior to the frontal zero plane as defined by Konig and Klippel [10], whde the depth of the posterior portion of the dorsal lesions was at, or slightly above the K f n i g and Klippel horizontal zero plane. There was considerable variation in the size of these lesions. In all, between 45-60 per cent of the hippocampus was spared in these animals. I n most animals, the neocortical damage in the dorsal group was similar to that of the total hippocampal group; in a few cases however, there was more sparing of posterior neocortex than in the total hippocampal animals (see Fig. 3). Neocortical destruc-
t]on in the dorsal hippocampal group amounted to 15-25 per cent of total neocortex. The cortical control group had between 20-30 per cent of total neocortex removed. In general, these lesions were somewhat more anteriorly placed than was the neocort~cal destruction m the two hlppocampal groups. Their anterior margin was approximately 6.5 mm ahead of the frontal zero plane of K6nig and Klippel wh]le the posterior limit was about 1.5 mm anterior to this plane. These lesions removed the corpus callosum over the dorsal hippocampus, destroyed the cingulum, and, in all but one case, produced slight damage to the alveus. In one animal the h~ppocampal pyramids received moderate damage bilaterally. Medially, the cortical lesions spared a port]on of the cingulate cortex. Fortunately, the les~ons of the dorsal hippocampal group
HIPPOCAMPAL LESIONS AND COPULATORY BEHAVIOR
655
were similar to those of the total hippocampal group in terms of amount of destruction of the median cortex and, in view of the behavioral results obtained, it did not appear necessary to run additional cortical control animals in which median cortex was completely removed.
campus and septum remained functional in our dorsal hippocampal group. We are currently examining the effects of septal lesions on copulatory behavior in the male rat. Kimble et al. [8] reported that their large bilateral dorsal hippocampal lesions destroyed the fimbria or, at best, left only fragments of it. This suggests that the effects we obtained may have been due to the destruction of a posteroventral hippocampal-allocortical, or hlppocampal-neocortical system. A third alternative, that a mass action effect in operating cannot be discounted by our data, but the studies of Jarrard and Kimura, cited above, which suggest that there is a unique effect of posteroventral hippocampal lesions on other behaviors, lead us to believe that a mass factor is not critical. There was a nonsignificant increase in EL and M I I I in our dorsal hippocampal group in the last series before the satiety criterion was reached. The high interanimal variation in this group led us to look for a correlation between scores on the last series and extrahippocampal brain damage. N o such relationship was found and we have no explanation for this trend. Larsson [11] found that male rats continued to copulate despite extensive lesions of the posterior neocortex involving the entire occipital cortex and portions of parietal and temporal cortex. Increases in IL, MIII, and PEI did appear. These changes were attributed to deficits in the ability of the male to locate and maintain contact with the female and were interpreted as being secondary to visual deficits. Neocortical lesions in the present study never exceeded 30 per cent of total neocortex and some sparing of visual cortex invariably occurred. In view of the differences in size and location of the lesions in our neocortical group as compared with the posterior lesion group in the Larsson study, it is not surprising that we did not observe either the qualitative or the quantitative changes he obtained. Since the completion of this work, Bermant, Glickman, and Davidson [3] have published an additional study of the effects of hippocampal lesions on the copulatory behavior of rats. Bermant et al. report that rats with dorsal hippocampal lesions showed progressively decreasing MIIIs and PEIs during their three postoperative tests. In a second experiment, the behavior of control rats was compared with that of rats with combined dorsal and ventral hippocampal lesions. N o significant differences attributable to the lesions were reported. The data from the present study have been reanalyzed in a search for the effects reported by Bermant et al. Data from M I I I and PEI in the first four series were examined in two ways: (1). Postoperative test 1 vs. postoperative test 2, and (2). Mean preoperative vs. Postoperative test 2. The 16 appropriate Kruskal-Wallis one-way analyses of variance revealed no significant differences as a function of lesion group. However, there was a consistent trend in the direction suggested by the data of Bermant et aL In all 8 comparisions of shifts in PEI, the dorsal hippocampal group showed the largest shift in the direction of faster performance in the second postoperative test. The dorsal hippocampal group showed the largest mean decrease in five of the eight comparisons of M I I I in the second postoperative test and the lowest summed ranks in 7 of 8 comparisons. Thus, the differences between the present data and those of Bermant et al. may be of degree rather than kind. The few differences between the present data and those of Bermant, et aL might easily result from differences in the
DISCUSSION
F r o m the present data it would appear that the hippocampus is of little importance in the mediation of the normal mating pattern of the sexually experienced male rat. The only statistically significant effects of total hippocampal lesions were increases in ML and IL. Thus, the effect of near total removal of the hippocampus was simply to increase the latency of the first copulation by the male. Once the behavior was initiated it was carried out through the normal number of copulatory series in a manner which did not appear to be qualitatively different from that of the control animals. There were no indications that hippocampectomized animals had any difficulty in orienting toward and maintaining contact with the female. Resection of the dorsal hippocampus with lesions corresponding roughly to the size and location of those made by Kim [7] and Kimble et al. [8] failed to produce significant changes in any measures taken. The same was true for the neocortical lesions. The delay in the initiation of the sexual response in totally hippocampectomized animals may be a reflection of decreased "distractability" [4]. This argument assumes that an animal is engaged in some compelling behavioral sequence (e.g. exploring the test cage, grooming, etc.) and the introduction of the female represents a novel stimulus to which the experimental animal is less attentive. It is more likely, in view of our failure to observe any obvious qualitative differences in the behavior of the different groups during the 5 min pretest adaptation periods, that the effects are more specifically related to the latency of arousal of the copulatory response. The question of the anatomical region whose removal produces this deficit remains. In analyzing the projections of the four hippocampal fields, Raisman, Cowan, and Powell [13] have said that the hippocampal field CA1 projects primarily to postcommisural fornix while fields CA3 and CA4 project via fimbria and precommisural fornix to septum and the nucleus of the diagonal band. Jarrard [5], taking note of this, pointed out that in the rat most of CA3 and CA4 lie in the caudal third of the hippocampus. Jarrard cites evidence from his laboratory which indicates that the behavioral effects of posteroventral hippocampal lesions differ from the effects of ablation of the dorsal hippocampus. In an earlier study Kimura [9] found that posterior hippocampal lesions interfered with passive avoidance, but more anterior lesions did not. Kaada, Rasmussen, Kveim [6] found no differences between anterior and posterior lesions on a passive avoidance task, but it is not clear from their description whether K a a d a et al. actually invaded the posteroventral hippocampus with their "posterior" hippocampal lesions. Because it is difficult to remove that portion of the fimbria which lies between thalamus and caudate nucleus without damaging these structures, this region was largely intact in most of our animals. Such sparing may also have been present in many of Kim's [7] animals. This being the case, it is possible that pathways between posteroventral hippo-
656
DEWSBURY, GOODMAN, SALIS AND BUNNELL
lesions and/or testing procedures. The electrolytic lesions in the later study appear to be smaller, particularly in the case of the amount of ventral h~ppocampus and neocortex ablated. The duration of each test, spacing of the tests, and the amount of postoperative recovery time differed in the two experiments. The third postoperative test of Bermant
et al. occurred slightly sooner after the operations than our second test. The differences shown by the Bermant, et al. dorsal hippocampal group are most striking on the third postoperative test. We ran only two postoperative tests and it is possible that the number of such tests may be a significant factor in the demonstration of a dorsal hippocampal effect.
REFERENCES 1. Beach, F. A. and L. Jordan. Sexual exhaustion and recovery in the male rat. Q. Jl exp. Psychol. 7: 121-133, 1956. 2. Bermant, G. Rat sex behavior: photographic analysis of the intromission response. Psychonom. Sci. 2: 65-66, 1965. 3. Bermant, G., S. E. Glickman and J. M. Davidson. Effects of limbic lesions on copulatory behavior of male rats. J. comp. physiol. Psychol. 65: 118-125, 1968. 4. lsaacson, R. L. Hippocampus. Paper presented at symposium on "The Limbic-midbram system and behavior" at meetings of the American Psychological Association in New York, N.Y., September 4, 1966. 5. Jarrard, L. E. On the importance of &fferent hippocampal lesions for behavioral investigators. Ammon's Horn. Spring, 15-21, 1967. 6. Kaada, B. R., E. W. Rasmussen and O. Kvenn. Impaired acquisition of passive avoidance behavior by subcallosal, septal, hypothalamic, and insular lesions in rats. J. comp. phystol. Psychol. 55: 661-670, 1962. 7. Klm, C. Sexual activity of male rats following ablation of hippocampus. J. comp. physiol. Psychol. 53: 553-557, 1960.
8. Kimble, D. P., L. Rogers and C. W. Hendrickson. Hippocampal lesions &srupt maternal, not sexual, behavior m the albino rat. J. comp. physiol. Psychol. 63: 401--407, 1967. 9. Kimura, D. Effects of selective hippocampal damage on avoidance behavior in the rat. Can. J. Psychol. 12" 213-218, 1958. 10. Konig, F. R. and Renate A. Klippel. The rat brain" A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore: Williams and Wilkins, 1963. 11. Larsson, K. Mating behavior in male rats after cerebral cortex ablation. II. Effects of lesions in the frontal lobes compared to lesions m the posterior half of the hemispheres. J. exp. Zool. 155: 203-214, 1964. 12. Pierce, J. T. and R. L. Nutall. Duration of sexual contacts in the rat. J. comp. physiol. Psychol. 54: 585-587, 1961. 13. Raisman, G., W. M. Cowan and T. P. S. Powell. An experimental analysis of the efferent projection of the hippocampus. Brain, 89: 83-108, 1966. 14. Siegel, S. Non-parametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956.