Journal of Comparative and Physiological Psychology 1979, Vol. 93, No. 2, 360-367

Effects of Lesions of the Amygdala, Preoptic Area, and Hypothalamus on Estradiol-Induced Activity in the Female Rat James M. King University of Texas at Arlington Lesions of the anterior hypothalamic nucleus and the medial preoptic area sharply attenuated enhancement of wheel running by estradiol benzoate in ovariectomized female rats. Lesions of the corticomedial amygdala had no effect on this behavior. The hormonal effects on activity were largely independent of any changes in body weight. Results of this first experiment indicated that the anterior hypothalamic and medial preoptic areas are critically involved in the induction of activity by estradiol. However, this experiment provided no support for suggestions that the corticomedial amygdala inhibits those structures that mediate the estrogenic induction of activity. In the second experiment, food deprivation was used to stimulate activity. Results of this experiment suggested that the reduction in the ability of estradiol to induce activity following anterior hypothalamic and medial preoptic lesions does not reflect a general inability to become more active. In the female rat, elevated estrogen levels are associated with reduced food intake and body weight (King & Cox, 1973), enhanced sexual receptivity (Pfaff, 1970), and increased locomotor activity (Finger, 1969). It has been reported that estradiol implanted in or near the medial preoptic area (MP) enhanced locomotor activity in ovariectomized rats (Wade & Zucker, 1970), whereas an implant in the anterior hypothalamic area (AH) was without effect on activity (Wade & Zucker, 1970). Colvin and Sawyer (1969) noted that wheel running in ovariectomized rats was stimulated not only by estrogen implants in and around the AH but also by implants in posterior hypothalamic areas. Colvin and Sawyer obtained equivocal results with their MP placements, however. This report is based on a dissertation submitted to the graduate faculty of the University of Texas at Arlington in partial fulfillment of the requirements for the PhD degree. The assistance of the members of the dissertation committee (Verne C. Cox, Ira H. Bernstein, D. H. Whitmore, James Miller, and Garvin McCain) is gratefully acknowledged. The author also wishes to thank M. A. Short, J. Stone, and K. Adams for assisting with portions of the surgery and histology. Requests for reprints should be sent to James M. King, who is now at the Neuropsychology Branch, Biomedical Laboratory, DRDAR-CLL-MN, E3100, Edgewood Area, Aberdeen Proving Ground, Maryland 21010.

As vaginal cornification was not observed in these studies, the hormone likely remained in the immediate area of the implant (Davidson, Smith, Rodgers, & Bloch, 1968). Kennedy (1964) reported two studies that addressed the issue of AH involvement in wheel running induced by estradiol. In the first study, rats with AH lesions responded normally to the hormone, but in the second, many were refractory to its effects on activity. Further, Hitt (1967) found that neither MP nor AH lesions altered the periodicity of estrous activity cycles, although the overall activity level was reduced following these lesions. Thus, it is not clear from the available data that either the AH or the MP is critically involved in the induction of locomotor activity by estrogens. Mabry and Campbell (1975) suggested that the corticomedial amygdala (CM) may be involved in mediating estradiol enhancement of activity. These investigators suggested that the CM may inhibit more medially situated hypothalamic and preoptic structures which exert excitatory influences on activity. The finding that hyperactivity is induced by CM lesions, as reported by Mabry and Campbell, is by no means universal. Some investigators have found that lesions of the amygdala have no effect on activity (Cole, 1975), and others have re-

Copyright 1979 by the American Psychological Association, Inc.

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ported decrements in activity (Mclntyre & Stein, 1973) following such lesions. Thus, there is little agreement regarding the effects of amygdaloid lesions on activity. The effects of CM lesions on estradiol-induced activity have not been investigated. Autoradiographic studies have suggested that the CM, along with the AH and MP, is an estrogen target tissue (Pfaff & Keiner, 1973). Experiment 1 was designed to explore the affects of lesions of the AH, MP, and CM on the induction of wheel-running activity by estradiol. Experiment 1 Method Subjects. The subjects were 56 experimentally naive female albino rats (Holtzman) which weighed 257.0 ± 1.5 (mean ± SE) g at the start of the experiment. They were housed in individual cages with Purina Laboratory Chow and water continuously available. A reversed 16:8 hr dark/light cycle was maintained in the animal colony; temperature was automatically maintained at 21 °C. This experiment was run in two replications. Rats were assigned to one of four lesion conditions (AH, MP, CM, or sham) and to one of two hormone conditions (estradiol benzoate or oil). Procedure. Following a 2-wk adaptation period, the rats were given access to Wahmann LC-34 activity wheels for 1-hr per day during the dark portion of the light/dark cycle. Each rat was run in the same wheel throughout the experiment. The effectiveness of this activity-measuring procedure has been demonstrated with both endogenous (Hitt, Gerall, & Giantonio, 1968) and exogenous (King & Cox, 1976) estrogens. The experimental room was illuminated with a 25-W red light during data collection periods. All rats were weighed at the conclusion of each day's activity testing. Data on baseline activity and body weight were collected for 15 days prior to surgery. The rats were anesthetized with an ip injection of 2.4 ml/kg Chloropent (Fort Dodge) supplemented with Fluothane (halothane; Ayerst) when required. All rats were bilaterally ovariectomized and received scalp incisions and bilateral trephinations. Rats in the lesion groups received stereotaxically placed bilateral electrolytic lesions. With the skull level between lambda and bregma, the AP and lateral coordinates were determined relative to bregma, and the depth coordinate was relative to the skull surface. The coordinates in millimeters for lesions of the MP, AH, and CM, respectively were AP -.3, .6 Right, .4 Left, D -8.0; AP -1.2, .6 Right, .4 Left, D -8.5; and AP -2.0, L ± 3.5, D —9.0. Current parameters were 1 mA for 15 sec for AH and MP lesions and 1 mA for 30 sec for CM lesions. The cathode lead was placed at the anus. In the sham groups, a lesion probe was lowered 3-4 mm below the surface of the skull, but no current was passed. Incisions were closed with sutures and wound clips. Rats

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were given two im ,1-ml injections of Combiotic (Pfizer Laboratories) and were returned to their cages for a 15-day recovery period. Daily sc .1-ml injections of 3.0 Mg of estradiol benzoate (EB) or oil were begun on Day 3 following surgery and were continued for the duration of the experiment. This dosage has been shown to be effective in influencing body weight (King & Cox, 1973) and activity (King & Cox, 1976) in rats. Following the recovery period, data on activity and body weight were collected for 15 days. Activity and body weight scores for each subject were converted to percentages of their means for the last 5 baseline days and averaged into 5-day blocks. The data were analyzed by analyses of variance. Histology. At the conclusion of the experiment, all rats with lesions were anesthetized with Chloropent and perfused intracardially with .9% saline followed by 10% formalin. The brains were removed and fixed in 10% formalin. Alternate 60-^m frozen sections were stained with cresyl violet and were examined to determine the extent and location of the lesions, which were evaluated relative to the appropriate plates in Konig and Klippel (1963). The lesions were evaluated by two independent judges without knowledge of the body weight or activity data obtained from the rats.

Results Histology. Drawings of representative AH, MP, and CM lesions are shown in Figure 1. Except as noted below, only those subjects that sustained complete bilateral damage to the intended target at the center of its anterior posterior extent, the middle plates for each lesion in Figure 1, were included in the data analysis. Thus, these lesions are illustrative of the extent of the damage to neural tissue when the stated criteria were met. The MP lesions extended rostrally to the level at which the anterior commissure becomes continuous, while their posterior boundary was defined by the appearance of the suprachiasmatic nucleus. The AH lesions began at this level and ended anterodorsally to the rostral tip of the ventromedial hypothalamic nucleus. The AH and MP lesions extended laterally and dorsoventrally throughout the entire extent of the nuclei involved, but they did not systematically damage more laterally or dorsally placed structures. The CM lesions arose at the anterior boundaries of the amygdaloid complex and extended caudally throughout the extent of the CM nuclei to the level, of the posterior tip of the ventromedial hypothalamic nucleus. The medial boundary of the CM lesions was defined by

Figure 1. Drawings of serial sections along the course of representative corticomedial amygdala (left), medial preoptic (center), and anterior hypothalamic nucleus (right) lesions. (The cross-hatched areas represent the extent of the lesions at each level. Drawings are based on plates from Konig and Klippel, 1963. The number at the lower right-hand corner of each drawing is the anterior-posterior coordinate of the corresponding plate.)

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the optic tracts. Damage was generally confined to the medial, basomedial, and corticomedial amygdaloid nuclei. One rat was included in the AH-Oil group in which, unilaterally, 20% of this nucleus had been spared, and three rats that had sustained additional, though moderate, damage to the basolateral amygdaloid nuclei were included in the CM-Oil group. These four rats could not be distinguished behaviorally from the other rats in their groups. As more than seven rats with acceptable damage to the intended target were available in all but the AH-Oil group, groups of seven subjects were constituted to maximize the similarity among baseline activity means and ranges. Activity. The mean baseline activity was 399.2 (± 24.0) turns per hour. There were no significant differences among groups in baseline activity. The postoperative activity percentage data are contained in Figure 2. As indicated by the significant hormone effect, F(l, 48) = 49.32, p < .001, EB treatment 200 ,-

enhanced activity, compared with Oil administration. The EB induction of activity was dramatically attenuated following AH and MP lesions. This is reflected in the lesion effect, F(3, 48) = 19.43, p < .001, and Hormone X Lesion interaction, F(3, 48) = 8.33, p < .001. The CM lesions had no consistent effect on the EB induction of activity. Further analysis revealed that MP and AH lesions significantly reduced activity in both the EB, F(3, 24) = 14.51, p < .001, and the Oil, F(3,24) = 7.69, p < .001, treatment conditions. Thus, MP and AH lesions enhanced the postovariectomy decline in and essentially eliminated the EB enhancement of activity. Body weight. There were no significant differences among groups in baseline body weight. • The body weight percentage data are presented in Figure 3. The EB treatment significantly suppressed body weight in all lesion conditions, F(l, 48) = 183.51, p < .001. Although this effect was less marked

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Figure 2. Effects of lesions of the corticomedial amygdala, medial preoptic area, and anterior hypothalamic nucleus on estradiol benzoate (EB) induced activity. (The data presented are 5-day block means ± SE. Note the differences in scale between the upper and lower portions of the figure.)

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Figure 3. Effects of lesions of the corticomedial amygdala, medial preoptic area, and anterior hypothalamic nucleus on the modulation of body weight by estradiol benzoate (EB). (The data presented are 5-day block means ± SE.)

in the AH and MP lesion conditions, as a considerable reduction in activity which shown by the lesion effect, F(3,48) = 2.93, p can be reversed by treatment with estrogen < .05, and Hormone X Lesion interaction, (e.g., Mook, Kenney, Roberts, Nussbaum, & F(3, 48) = 7.62, p < .001, it was nonetheless Rodier, 1972). Indeed, this phenomenon substantial even in these groups. Three was observed in the sham and CM groups MP-EB rats, one AH-EB rat, and one MP- but not in the AH and MP groups. Oil rat lost weight following surgery. These The results of the present experiment rats were offered vanilla Carnation Slender suggest that estradiol may act on both the and a .25% saccharin plus 3.0% glucose so- MP and the AH to produce increased locolution, in addition to their regular diet, for motor activity. The inability of EB to enup to 5 days, ending on the seventh day after hance activity following MP and AH lesions surgery. During postoperative testing, these could be due to general debilitation, alrats could not be distinguished from the though there are data that argue against others in their respective groups. such an interpretation. First, in the present experiment, MP and AH rats maintained Discussion nearly normal levels of body weight and were Ovariectomy has been reported to produce only slightly less active than rats in the

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sham-Oil group. Second, direct observation of rats with lesions of the MP and AH suggested no obvious debilitation. Third, Powers and Valenstein (1972) found that MP lesions facilitated female sexual behavior. Finally, Kennedy (1964) found that rostral hypothalamic lesions did not interfere with activity induced by food deprivation, although the site of Kennedy's lesions was not well defined. An alternative explanation, which cannot be so readily excluded in the present case, proposes that MP lesions, AH lesions, or both interfere with a general activity system that mediates increased locomotor activity in response to a variety of stimuli, rather than disrupting a system, or systems, specific to the induction of activity by estrogens. Experiment 2 was designed to address this question.

Results and Discussion

Histology. The lesions obtained in the present experiment met the criteria described above. Drawings of representative AH and MP lesions are shown in Figure 1. Groups of five rats each were constituted as outlined in Experiment 1. Activity and body weight. The mean baseline and deprivation activity values in turns per hour for the AH, MP, and sham groups are shown in Table 1. These are postovariectomy activity data. The significant lesion effect, F(2, 12) = 4.34, p < .05, indicates that animals in the AH and MP groups were less active than sham animals. This is consistent both with results obtained by Hitt (1967) and with those of Experiment 1 in that AH and MP lesions enhanced the postovariectomy reduction in activity. Food deprivation stimulated activity, F(l, 12) = Experiment 2 19.63, p < .01, and did so to a similar extent in all groups, as indicated by the nonsignifFood deprivation has been found to pro- icant Lesion X Deprivation interaction, F(2, duce a marked hyperactivity in rats (e.g., 12) < 1. The activity percentage data are Jakubczak, 1973). The present experiment presented in Figure 4. Four days of food examined the effects of this activity-induc- deprivation significantly enhanced activity, ing stimulus on the wheel running of ovari- F(3, 36) = 4.03, p < .025; and the nonsignifectomized female rats following MP, AH, or icant lesion effect, F(2,12) < 1, and Lesion sham lesions. X Days interaction, F(6, 36) = 1.38, suggest that this effect was of similar magnitude in all groups. In addition, there were no sigMethod nificant effects of lesion condition on body weight in this experiment. Subjects. The subjects were 15 experimentally naive Thus, AH, MP, and sham rats all refemale albino rats (Holtzman) which weighed 282 (± sponded to food deprivation with equivalent 6.0) g at the start of the experiment. The rats were housed in individual cages with Purina Laboratory increases in activity and decreases in body Chow and water continuously available except as noted weight. Animals with MP lesions tended to below. Food was not available in the activity wheels. display a greater increment in activity in The environmental conditions and activity testing response to short periods of deprivation than procedures are described in Experiment 1. Procedure. The AH, MP, and sham lesions and ovariectomies were performed as described above. Rats were allowed to adapt to the light/dark cycle during an 8-day period following surgery. During a subsequent 8-day baseline phase, the rats were given 1-hr access daily to activity wheels. Following this baseline period, total food deprivation was begun for all rats. Activity testing was terminated after 4 days of food deprivation, as rats in all groups displayed the hunched posture and hypothermia characteristic of severely deprived rats. At this point, all rats with lesions were killed and their brains were collected as in Experiment 1. The activity and body weight data were converted to percentages of each subject's mean activity or body weight for the last four baseline days. The data were analyzed by analyses of variance.

Table 1 Effects of Food Deprivation on WheelRunning Activity (Turns/Hour) Group

Baseline phase

Deprivation phase

Sham MP AH

249.7 ± 68.4 84.2 ± 15.0 128.0 ± 9.5

482.4 ± 101.8 201.1 ± 84.8 306.2 ± 58.9

Nate. Data are 4-day block means ± SE. MP = medial preoptic area lesions; AH = anterior hypothalamic nucleus le-

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Figure 4. Effects of lesions of the medial preoptic area and anterior hypothalamic nucleus on fooddeprivation-induced activity and body weight loss. (The data presented are daily means ± SE.)

did animals in the sham and AH lesion conditions, and the MP rats did not show the same positive relation between duration of food deprivation and activity level that was apparent in the other groups. The results of this experiment indicate that ovariectomized rats with MP and AH lesions are capable both of displaying substantial increments in activity and of sustaining these activity increments. This suggests that the inability of estradiol to induce increases in activity following AH and MP lesions reflects the loss of critical sites of action for this hormone. General Discussion The results of the first experiment revealed that both MP and AH lesions attenuated the effects of estradiol on activity. The CM lesions failed to interfere with this effect. Experiment 2 demonstrates that neither MP nor AH lesions blocked induction of activity by food deprivation. Following MP or AH lesions, rats were, if anything, relatively more reactive to food deprivation than were sham subjects. The present data support Wade and Zucker's (1970) finding regarding the role of the MP in estradiol-induced activity and, consistent with the results of Colvin and Sawyer (1969) and Pfaff and Keiner (1973), suggest that the AH is also an estrogen target tissue. Since

some MP efferents pass caudally through the AH (Rodgers & Schwartz, 1976), the effects of AH lesions may be due to deefferentation of the MP. Colvin and Sawyer also observed that application of EB to sites in the posterior hypothalamus/rostral midbrain stimulated activity in ovariectomized rats. These sites are distributed in much the same pattern as are the estrogen-concentrating neurons in this region (Pfaff & Keiner, 1973). The present results suggest that the action of EB on these sites may be sufficient to produce an enhancement of activity only in the presence of an intact MP or AH. Contrary to the suggestion made by Mabry and Campbell (1975), the results of Experiment 1 indicate that estradiol has a normal effect on activity following CM lesions. Any of a number of variables, including sex of subjects, activity measuring device, and size of lesions, could account for this discrepancy. Although autoradiographic studies have indicated that the CM is an estrogen target tissue (Pfaff & Keiner, 1973), lesions of this structure do not interfere with the effects of estradiol on food intake and body weight (Cox & King, 1974) or activity. Some estrogen target tissues, such as the CM, may be involved in the neuroendocrine control of the estrous cycle. Doty (1974) proposed that the increased activity that precedes estrus reflects sexual motivation on the part of the female rat.

LESIONS AND ESTRADIOL ACTIVITY

The MP lesions have been found both to facilitate (Powers & Valenstein, 1972) and to inhibit (Short, 1978) female sexual behavior, and AH lesions have either depressed (Singer, 1968) or failed to affect (Short, 1978) these responses. However, neither MP nor AH lesions disrupted soliciting, another behavior that may indicate sexual motivation in the female rat (Short, 1978). Thus, locomotor activity and motivational components of female sexual behavior may be differentially affected by MP and AH lesions, and increased activity may be a primary behavioral effect of estrogens. References Cole, S. 0. Attenuation of the effects of amphetamine on the activity of rats following amygdala lesions. Bulletin of the Psychonomic Society, 1975, 5, 447_449. Colvin, G. B., & Sawyer, C. H. Induction of running activity by intracerebral implants of estrogen in ovariectomized rats. Neuroendocrinology, 1969,4, 309-320. Cox, V. C., & King, J. M. The effects of estradiol on food intake and weight in ovariectomized rats with amygdaloid lesions. Physiological Psychology, 1974, 2, 371-373. Davidson, J. M., Smith, E. R., Rodgers, C. H., & Bloch, G. J. Relative thresholds of behavioral and somatic responses to estrogen. Physiology and Behavior, 1968, 3, 227-229. Doty, R. L. A cry for the liberation of the female rodent: Courtship and copulation in rodentia. Psychological Bulletin, 1974,81, 159-172. Finger, F. W. Estrous and general activity in the rat. Journal of Comparative and Physiological Psychology, 1969, 68, 461-466. Hitt, J. C. The effects of forebrain and hypothalamic lesions on estrous activity cycles in the albino rat (Doctoral dissertation, Tulane University, 1966). Dissertation Abstracts International, 1967, 27, 3687B. (University Microfilms No. 67-4458) Hitt, J. C., Gerall, A. A., & Giantonio, G. W. Detection of estrous activity cycle by 1-hour samples of running activity. Psychonomic Science, 1968,10, 159-160. Jakubczak, L. F. Frequency, duration, and speed of wheel running of rats as a function of age and starvation. Animal Learning and Behavior, 1973, 1, 13-16. Kennedy, G. C. Hypothalamic control of the endocrine and behavioral changes associated with oestrous in

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the rat. Journal of Physiology, 1964, 772, 383392. King, J. M., & Cox, V. C. The effects of estrogens on food intake and body weight following ventromedial hypothalamic lesions. Physiological Psychology, 1973,1, 261-264. King, J. M., & Cox, V. C. Measurement of estradiolinduced activity with brief time samples. Bulletin of the Psychonomic Society, 1976, 8, 47-48. Konig, J. F. R., & Klippel, R. A. The rat brain: A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore: Williams & Wilkins, 1963. Mabry, P. D., & Campbell, B. A. Food-deprivationinduced arousal: Mediation by hypothalamus and amygdala. Journal of Comparative and Physiological Psychology, 1975,89, 19-38. Mclntyre, M., & Stein, D. G. Differential effects of one-versus-two-stage amygdaloid lesions on activity, exploratory, and avoidance behavior in the albino rat. Behavioral Biology, 1973,9, 451-465. Mook, D. G., Kenney, N. J., Roberts, S., Nussbaum, A. I. & Rodier, W. I. Ovarian-adrenal interactions in regulation of body weight by female rats. Journal of Comparative and Physiological Psychology, 1972, 87, 198-211. Pfaff, D. W. Nature of sex hormone effects on rat sex behavior: Specificity of effects and individual patterns of response. Journal of Comparative and Physiological Psychology, 1970, 73, 349-358. Pfaff, D. W., & Keiner, M. Atlas of estradiol-concentrating cells in the central nervous system of the female rat. Journal of Comparative Neurology, 1973, 757, 121-157. Powers, B., & Valenstein, E. S. Sexual receptivity: Facilitation by medial preoptic lesions in female rats. Science, 1972, 775, 1003-1005. Rodgers, C. H., & Schwartz, N. B. Differentiation between neural and hormonal control of sexual behavior and gonadotrophin secretion in the female rat. Endocrinology, 1976,98, 778-786. Short, M. A. An examination of the roles of the medial preoptic nucleus and the anterior hypothalamic nucleus in the mediation of sexual behavior in the female rat (Doctoral dissertation, The University of Texas at Arlington, 1977). Dissertation Abstracts International, 1978, 38, 3440B. (University Microfilms No. 77-27,791) Singer, J. Hypothalamic control of male and female sexual behavior in female rats. Journal of Comparative and Physiological Psychology, 1968, 66, 738-742. Wade, G. N., & Zucker, I. Modulation of food intake and locomotor activity in female rats by diencephalic hormone implants. Journal of Comparative and Physiological Psychology, 1970, 72, 328-336. Received May 17,1978 •