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Cholecystokinin stimulates and inhibits lordosis behavior in female rats
Physiology&Behavior, Vol. 39, pp. 217-224. Copyright©PergamonJournals Ltd., 1987. Printedin the U.S.A.
0031-9384/87$3.00 + .00
Cholecystokinin Stimulates and Inhibits Lordosis Behavior in Female Rats I GEORGE J. BLOCH, ALEX M. BABCOCK, ROGER A. GORSKI AND PAUL E MICEVYCH Department o f Anatomy and the Laboratory o f Neuroendocrinology, Brain Research Institute UCLA School o f Medicine, Los Angeles, CA 90024 R e c e i v e d 3 July 1986 BLOCH, G. J., A. M. BABCOCK, R. A. GORSKI AND P. E MICEVYCH. Cholecystokinin stimulates and inhibits lordosis behavior in female rats. PHYSIOL BEHAV 39(2) 217-224, 198_7.--Recently,IP CCK-8 has been shown to inhibit lordosis in sexually experienced, estradiol benzoate (EB) and progesterone (P) primed rats. However, receptivity is influenced by prior sexual experience and/or exposure to sex steriods, as well as the steroid dosage administered before testing. Thus, we examined the effect of CCK-8 (3/~g/kg; IP) on lordosis in rats with different degrees of receptivity. Three weeks after ovariectomy, females were treated with EB followed 48 hr later with P, or with EB alone. CCK-8 significantly facilitated lordosis in rats given 5 tzg EB. Following a 5 week nonexperimental period, animals were more receptive and CCK-8 significantly inhibited lordorsis in the 5 or 10/zg EB groups. In a separate experiment, rats were ovariectomized, adrenalectomized, and treated with EB alone. As in the first experiment, CCK-8 facilitated and inhibited lordosis. CCK-8's effects were highly dependent on the female's receptivity, facilitating lordosis when receptivity was low and inhibiting lordosis when receptivity was high (but not maximal). In conclusion, IP CCK-8 modulates lordosis behavior independent of P, but its effects depend on the female's degree of receptivity. Lordosis behavior
Cholecystokinin
Estrogen
Progesterone
S E V E R A L findings suggest that the brain-gut octapeptide cholecystokinin (CCK-8) may be important in the regulation of reproductive processes. The distribution of CCK-8 levels in the medial preoptic area, mediobasal hypothalamus, and posterior pituitary is sexually differentiated [18, 28, 37], and there are sex differences in the apparent number of CCK-8 binding sites within the ventromedial nucleus of the hypothalamus [2]. In addition, 125I-CCK-8 binding sites in the ventromedial nucleus of the female are depressed on estrus or following ovariectomy and estrogen replacement (submitted for publication). Depending on the steroid environment and the site of central administration, CCK-8 has also been reported to inhibit [43] and to stimulate [25] the release of LH. Recently we have demonstrated that CCK-8 inhibits the stimulated release of L H R H in vitro from male, ovariectomized, and ovariectomized estrogen-primed female rat hypothalami [27]. Mendelson and Gorzaika [26] recently reported that 3 /zg/kg CCK-8 administered intraperitoneally (IP) inhibited lordosis behavior in ovariectomized, sexually experienced rats injected with estradiol benzoate (EB) and progesterone (P), and they suggested that this inhibitory effect was due to an interaction between CCK-8 and P, but not EB. It is known that prior administration of sex steroids and/or prior experience with mating "sensitizes" females so that in subsequent mating tests they are more sexually responsive to a given dose of steroids than before [5, 19, 20, 45]. In addition,
Adrenalectomy
the effects of some peptides (i.e., aMSH and ACTH 4-10) on lordosis behavior may depend on the state of receptivity of the female [46]. Because of these concerns, we undertook to study the effects of CCK on lordosis behavior in detail, and have observed the effects of CCK-8 in ovariectomized and EB-treated females showing various degrees of receptivity.
G E N E R A L METHOD
Animals and Surgery Animals were 81 100-day-old female Long-Evans rats obtained from Charles River, Wilmington, MA (Experiment 1) or Simonsen, Gilroy, CA (Experiments 2 and 3). These rats, housed 3 to 4 to a cage, were given ad lib access to Purina lab chow and water in a room maintained on a partially reversed light schedule (lights on from 1:30 a.m. to 3:30 p.m.). All rats were ovariectomized approximately one week after arrival using ether anesthesia, and were given their first mating test three weeks later.
Drug Procedures Fifty-three hours before each mating test (11:30 a.m.), rats were given a subcutaneous (SC) injection of EB dissolved in 0.1 ml sesame oil or 0.1 ml oil, followed 48 hr later
1Supported by NIH grants NS 21220 (P.E M.) and HD 01182 (R.A.G.). Preliminary reports were given at the Society for Neuroscience and the Endocrine Society in 1986.
217
BLOCH ET AL.
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The purpose of this experiment was to assess the effect of CCK-8 on the lordosis behavior of female rats demonstrating various degrees of receptivity.
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FIG. 1. Effect of IP CCK-8 on lordosis behavior: first test. Rats were primed with EB alone (5/0, 10/0 groups), or followed 48 hr later with P (5/50, 10/50 groups), then given CCK-8 (dark bars) or saline (clear bars) 10 minutes before the mating test. Number of rats," *p<0.05 vs. saline, independent t-test.
Three weeks after ovariectomy, rats were injected with EB alone or with P 48 hr later as follows: 0/0 group, each given an injection of oil followed 48 hr later with oil (n=8); 5/0 group, each given 5/zg EB, then oil (n= 12); 10/0 group, each given 10/~g EB, then oil (n= 12); 5/50 group, each given 5/xg EB, then 50/xg P (n= 12); 10/50 group, each given 10 tzg EB, then 50 /zg P (n=8). Half the animals in each group received 3/xg/kg CCK-8 10 minutes before the mating test; the others received vehicle (saline). Mean LQ scores of saline and CCK-8 injected rats were analyzed separately for each steroid group using a two-tailed Student's t-test.
RESULTS AND DISCUSSION
with an SC injection of 50 t~g P in the same volume of oil (see specific experiments). Ten minutes before each mating test (4:30 p.m.), rats were given an IP injection of 3/xg/kg suiphated CCK-8 (Bachem, Tustin, CA) or vehicle (saline). A fresh CCK-8 solution was prepared from a lyophilized aliquot immediately before each testing session.
Behavioral Testing Mating tests were the same in all experiments. Test females were placed in a Plexiglas arena illuminated with a 25 watt red bulb. A Long-Evans male with proven sexual vigor, adapted to the mating arena for at least 10 rain, was permitted to mount the test female 10 times. A lordosis quotient (LQ) was computed by dividing the number of lordotic responses by the number of mounts and multiplying by 100. A male's behavior was counted as a mount only if he clasped the female with his forelegs, " p a l p a t e d " the female by rapidly moving his forelegs along her flanks, and executed vigorous pelvic thrusting movements which resulted in contact between the pelvic region of the male and female. Because both positions--flattening and arching of the b a c k - allow the male to achieve intromission, a female' s behavior was scored as a lordosis when she displayed either flattening or arching of the back and a raising of the head.
Radioimmunoassay In Experiments 3 and 4, plasma corticosterone values were obtained by radioimmunoassay. Total corticosterone was measured in unextracted medium using antiserum from Radioassay System laboratories, Inc. The tracer steroid was 1,2,6,7 3H(N)-corticosterone. Plasma samples were heatdenatured, and dextran-coated charcoal was used for phase separation. The antibody crossreacts with progesterone (0.29%), deoxycorticosterone (6.1%), 20a-dihydroprogesterone (0.08%), and 11-deoxycortisol (0.03%). The sensitivity of the assay was 30 pg/ml plasma, and the intra- and interassay variations were 9.9% and 11.8%, respectively.
In the 5/0 group, CCK-8 significantly increased lordosis behavior as compared to saline treated controls (54.3_+ 19.0 vs. 11.7_+8.3, p<0.05, Fig. 1). There were no significant effects of CCK-8 in the 10/0, 5/50, and 10/50 groups although the 10/0 group showed a trend toward CCK-8-induced facilitation as well (see Fig. 1). No behavior was observed in the 0/0 group. The results of this experiment demonstrate that IP CCK-8 can facilitate lordosis behavior and that this facilitation is dependent on the administration of EB but not P. Although mechanisms responsible are presently unknown, the CCK-8-induced facilitation appears to be associated with low levels of receptivity since it was observed only in groups where control L Q ' s were low.
Experiment lb In order to further test the effects of CCK-8 in rats with varying degress of receptivity, we retested the animals used in Experiment l a following a 5 week nonexperimental period. Since Gerall and Dunlap [19] had reported that females remaining in the home cage for three weeks before injection with EB and P for a second time show levels of receptivity as low as the first time, we reasoned that this 5 week nonexperimental period between test 1 (Experiment la) and test 2 would allow the females to be retested in a state of minimal receptivity. During subsequent mating tests, we expected to see an increase in receptivity as has been reported [5, 19, 45], allowing us to assess the effects of CCK-8 as receptivity increased. Thus, we hypothesized that this experiment would allow us to replicate the observation of CCK-8-induced facilitation of lordosis behavior during test 2, and to observe the reported inhibition of lordosis behavior [26] during subsequent tests.
METHOD
In a crossover design with respect to the administration of CCK-8 or saline, the animals in the 5/0, 10/0, 5/50, and 10/50 groups of Experiment l a were tested 5 weeks later and every
C H O L E C Y S T O K I N I N A N D LORDOSIS B E H A V I O R
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FIG. 2. Effect of IP CCK-8 on lordosis behavior: tests 2 through 5. Rats were primed with EB alone (5/0, 10/0 groups), or followed 48 hr later with P (5/50, 10/50 groups), then given either CCK-8 (dark triangles, individual mating tests; striped bars, mean value of two tests) or saline (clear squares and bars) in a crossover design l0 minutes before the mating test, *p<0.05, **p <0.01, matched pair t-test.
10-12 days thereafter (tests 2 through 5) and a few animals in the 10/0, 5/50, and 10/50 groups 15 days after test 5 (test 6). Statistical evaluation was performed by comparing the mean saline LQ value to the mean CCK-8 value for each animal over tests 2 through 5 using a two-tailed matched-pair t-test. RESULTS AND DISCUSSION The mean lordosis quotients with standard errors of the 5/0, 10/0, 5/50, and 10/50 groups during tests 2 through 5 are shown in Fig. 2. There was a significant inhibition of lordosis behavior with CCK-8 as compared to saline controls in the 5/0 and 10/0 groups. There was no significant inhibition in the 5/50 and 10/50 groups over tests 2 through 5. As reported by others, the control (sahne injected) animals showed a marked increase in receptivity subsequent to their first steroid injection and mating experierrce. To our knowledge, this is the first demonstration that this increase in receptivity occurs as late as 5 weeks after the first test. In addition, CCK-8 failed to inhibit lordosis behavior as control females became maximally receptive (LQ of 95-100). This suggests that the effects o f CCK-8 might depend on the degree of receptivity of the female. Indeed, when the effects of
IP CCK-8 were correlated with the degree of receptivity of the saline-injected controls for each mating test conducted in Experiments 1 and 2 (tests 1 through 6), there was a significant negative correlation ( r = - . 8 0 7 , p < 0 . 0 0 1 ) . Because there was no effect of CCK-8 in those mating tests where the saline controls were maximally receptive, we recalculated the correlation and plotted the regression line without these tests, as illustrated in Fig. 3. Although the data in Fig. 3 suggest that the correlation might not be significant without the data points obtained when the controls showed low L Q ' s , a significant negative correlation was still evident without these two data points ( r = - .55, p<0.05). It is important to note that the present relation between receptivity and the effects of CCK-8 cannot be explained as a regression toward a mean value, a nonspecific effect that may occur when subjects are preselected into low- and high-receptive groups prior to testing.
EXPERIMENT 2 The purpose of this experiment was to assess the effect of CCK-8 on the lordosis behavior of adrenalectomized rats demonstrating various degrees of receptivity.
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Experiment 2a Because CCK-8 facilitated lordosis behavior only in animals treated with EB alone, this suggests that P may not be necessary for the facilitation. Adrenal P can, however, contribute to an increase in lordosis behavior in estrogentreated rats [15,17] even though lordosis behavior can be produced in adrenalectomized rats injected with estrogen alone [12,13]. Moreover, peripheral administration of CCK-8 has been shown to increase adrenal corticosterone secretion in viva and basal ACTH output from hemipituitaries in vitro [23, 33, 36]. The purpose of this experiment, therefore, was to test the hypothesis that the facilitation of lordosis behavior observed in Experiment la was independent of any CCK-8 stimulated release of adrenal P.
METHOD Two weeks after ovariectomy, 17 rats were adrenalectomized under ether anesthesia. These animals were then maintained on 0.9% saline for the duration of the experiment. One week after adrenalectomy, all animals were mjected with 5 /~g EB, 53 hr before the mating test. Ten minutes before behavioral testing, animals were given 3 p,g/kg CCK-8 (n=9) or saline (n=8). The following afternoon, 1-2 hr before lights off, animals were moved from the animal room into the laboratory, and 0.5 ml blood was taken from the jugular vein of each animal within 3 minutes after the onset of ether anesthesia. Plasma was frozen and a radioimmunoassay for
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FIG. 4. LQ of adrenalectomized rats primed with 5 /~g EB, then given CCK-8 (dark bars and squares) or saline (clear bars and squares) l0 minutes before the mating test. In Experiment 2a, the bars represent the mean values of the I I animals qualifying as adrenalectomized during test 1. In Experiment 2b, the squares represent individual mating tests (number of animals), and the bars represent the values of the 10 animals tested in a crossover design in tests 2 and 3. **p <0.01, independentt-test; *p <0.05, matched-pair t-test.
corticosterone levels was subsequently performed to assure completeness of adrenalectomy. Mean LQ scores of saline and CCK-8 injected rats were analyzed using a two-tailed Student's t-test.
RESULTS AND DISCUSSION AS in Experiment la, CCK-8 facilitated lordosis behavior during the first mating test. After CCK-8 or saline, the mean LQ's with standard errors of the adrenalectomized females were 92.8+4.3 and 5 1 . 3+- 10.9 respectively (Fig. 4). One can therefore conclude that adrenal P is not required for the observed facilitation with CCK-8. Plasma levels of corticosterone are shown in Table 1. Although corticosterone values that are not higher than 10 ng/ml have been used as a criterion for defining adrenalectomy in rats [1], there are a number of reasons to assume that our cut-off point of 12.1 ng/ml was not too high: (1) Moving the animals from their home cages into the laboratory as in the present experiment would result in levels of corticosterone that are at least 100 ng/ml in rats with intact adrenal glands (M. Dallman, personal communication); (2) The cut-off point of 10 ng/ml is based on morning samples, whereas the values in the present study were obtained from samples taken in the afternoon, a time when corticosterone is reported to be approximately 7 times higher in normal animals [24]; and (3) It is likely that under the stress of having moved the animals 45 to 105 minutes before taking blood, the animals with intact adrenals would respond to ether with high levels of corticosterone even within the 2-3 minutes before blood was taken [34]. Thus, CCK-8 caused a significant facilitation of lordosis
C H O L E C Y S T O K I N I N AND LORDOSIS BEHAVIOR
221
TABLE 1 LEVELS OF CORTICOSTERONEIN THE PLASMAOF ADRENALECTOMIZEDRATS: ng/ml -+ SE (NUMBEROF ANIMALS)
Adrenalectomized Functional Adrenal Tissue
1st Test: 9 Days After Adrenalectomy*
2nd Test: 19 Days After Adrenaiectomy*
3rd Test: 31 Days After Adrenaiectomy*
0.84 _+ 0.69 (11)*
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2.96 _+ 1.54(10)§
21.85 _+ 1.87 (6)
45.84 _+ 3.79 (5)
60.13 _ 10.77 (6)
*Blood taken the following afternoon. *Nine out of 11 animals had plasma levels of corticosterone that were below the sensitivity of the assay, and these were assigned a value of 0.03 ng/ml (see the General Method section). The other 2 animals had levels of 7.6 and 1.4 ng/ml. *Eight out of 11 animals had plasma levels below the sensitivity of the assay; the others had levels of 10.0, 2.9, and 2.9 ng/ml. §Seven out of 10 animals had plasma levels below the sensitivity of the assay; the others had levels of 12.1, 10.4, and 6.9 ng/ml.
behavior in EB-treated ovariectomized, adrenalectomized females, and therefore increased levels of adrenal progesterone resulting from activation of the hypothalamopituitary-adrenal axis by CCK-8 is not required for this effect.
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Experiment 2b Rats injected with EB alone showed a significant CCK-8induced inhibition (Experiment lb). This could mean that P is not required for the inhibition, an interpretation that does not agree with the suggestion of Mendelson and Gorzalka [26]. It is possible, however, that progesterone--in this case, of adrenal origin--is necessary. To test this hypothesis, the animals used in the previous experiment were retested in order to determine whether adrenal P is required for the inhibitory effect of CCK-8.
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METHOD In a crossover design with respect to the administration of CCK-8 or saline, the ovariectomized, adrenalectomized rats from Experiment 2a were retested 10 days later (test 2) and 22 days later (test 3), 53 hr after the injection of 5/zg EB. As in Experiment 2a, blood samples were taken from each animal the afternoon after a mating test, and plasma corticosterone levels were measured to assure completeness of adrenalectomy. Mean saline LQ values were compared to mean CCK-8 values using a two-tailed matched-pair t-test.
RESULTS AND DISCUSSION As shown in Fig. 4, CCK-8 significantly inhibited lordosis behavior in the 10 animals considered to be adrenalectomized during tests 2 and 3 (see Table 1). The LQ's of the animals were lower in test 3 than in test 2. Whether this decline in receptivity was related to the long period of complete adrenalectomy or some other factor is not known, but the inhibition with CCK-8 was apparent in each of these tests (/7<0.025 for test 2, p<0.01 for test 3, independent t-test) as
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well as for each animal tested in the crossover design (see Fig. 4). One can therefore conclude that adrenal progesterone is not necessary for the observed inhibition with CCK-8.
EXPERIMENT 3 The injection of 5/zg EB resulted in a much lower LQ in the saline-injected controls of Experiment la than in Exper-
222
BLOCH E T A L .
iment 2a (mean LQ's of 11.7 vs. 51.3, respectivley). CCK-8 facilitated lordosis behavior in both experiments, suggesting that low levels of receptivity were not, per se, an important factor in determining the effects of CCK-8. There were two important differences, however, in the animals used for the two experiments: (1) The animals used in Experiment la were ovariectomized while those used in Experiment 2a were ovariectomized as well as adrenalectomized; and (2) Rats used in Experiment la were obtained from Charles River while those used in Experiment 2a were obtained from Simonsen. With regard to the first difference, rats that are both ovariectomized and adrenalectomized appear to be behaviorally more sensitive to estrogen stimulation than are ovariectomized rats with intact adrenal glands [12,13]. In order to determine whether rats from Simonsen were behaviorally more sensitive to estrogen stimulation than Charles River rats, and to further study the relation between receptivity and the effects of CCK-8 in Simonsen rats, a group of Simonsen rats was ovariectomized, then injected with EB and given mating tests with CCK-8 or saline.
METHOD Twelve Long-Evans rats (Simonsen) were given mating tests 21 (test 1) and 32 (test 2) days after ovariectomy, 53 hr after the administration of 10/xg EB. In a crossover design, animals were given 3 p.g/kg CCK-8 IP (n=6) or saline (n=6) 10 minutes before the mating tests. Mean LQ's of saline and CCK-8 injected rats were analyzed using a Student's t-test for test 1, and a matched-pair t-test for tests 1 and 2. RESULTS AND DISCUSSION There was no effect of CCK-8 on lordosis behavior during the first mating test: LQ's of 68.3-+14.9 (saline) and 33.3-+18.2 (CCK-8). Similarly, when the LQ's were compared over the two mating tests, there were no significant differences between the groups: LQ's of 61.7___9.4 (saline) and 50_+13.7 (CCK-8). Regarding the first test, when the LQ's of the saline-injected animals of this experiment were compared to the LQ's of the l0 /zg EB, saline-injected animals of Experiment la (Charles River), Simonsen females were behaviorally more receptive than the Charles River females (LQ's of 68.3+-14.9 vs. 16.7_+16.7, p<0.025, Student's t-test). When the effects of IP CCK-8 were correlated with the degree of receptivity of the saline-injected controls for each mating test conducted in Experiments 2 and 3, there was a significant negative correlation ( r = - . 9 8 1 , p<0.02, Fig. 5). This indicates that the general pattern of facilitation and inhibition observed in Long-Evans rats obtained from Charles River (Fig. 3) was also apparent in those obtained from Simonsen, although " l o w " levels of receptivity were higher in Simonsen than Charles River rats.
G E N E R A L DISCUSSION The major finding of the present study is that CCK-8 facilitates lordosis behavior in rats showing low levels of receptivity while it inhibits lordosis behavior in rats showing high but not maximal levels of receptivity. The facilitatory and inhibitory effects were observable after administration of EB, and they were independent of P. This latter finding
contradicts Mendelson and Gorzalka [26], who proposed that CCK-8 inhibits lordosis through a direct interaction with P. Our results indicate that the degree of receptivity, rather than a specific action of P, is the important factor, and it is noteworthy that Mendelson and Gorzalka's data are consistent with this interpretation. In addition, these authors did not observe a facilitation of lordosis after administering CCK-8. Although they used a different strain (SpragueDawley rats), our data indicate that the females used by Mendelson and Gorzalka were already too receptive at the time of testing, perhaps because the rats had already received mating tests and injections of EB and P prior to the experiment. Many substances are known to stimulate lordosis behavior when administered to estrogen-primed animals. These include peptides (e.g., LHRH, substance P, ctMSH, oxytocin), steroids (estrogen, progesterone), and a variety of neurotransmitters, and this has led to speculation that a nonspecific mechanism may be involved [44]. A number of substances affect lordosis behavior in opposite ways. Injection of the peptides aMSH or ACTH 4-10 has been reported to facilitate or inhibit lordosis behavior depending on whether the rats are showing low or high (but not maximal) levels of receptivity [42,46]. Although the animals in these studies were preselected on the basis of their receptivity, making interpretation more difficult than in the present study, these results are very similar to those observed with CCK-8. The authors suggested that aMSH and ACTH 4-10 might be influencing lordosis by stimulating the animal's level of arousal, increasing behavioral efficiency when minimally receptive, decreasing behavioral efficiency when highly receptive. It is unlikely, however, that the CCK-8 effect is through a similar mechanism, since CCK-8 decreases rather than increases levels of arousal as measured by exploratory behavior [8-10]. Other substances known to facilitate or inhibit lordosis behavior include various dopamine and serotonin agonists, and serotonin antagonists [11,22]. One way to explain the neurotransmitter effects is to assume that low doses of the substance stimulate (inhibit) presynaptic receptors that inhibit endogenous transmission, while high doses stimulate (inhibit) post-synaptic receptors [11]. A similar hypothesis holds that different degrees of receptivity induced by different steroid regimens (different doses of estrogen, or estrogen alone vs. estrogen plus progesterone) will have different effects on neurotransmitter activity and thereby affect lordosis behavior differentially [22]. It is unlikely, however, that the dual effect of CCK-8 on lordosis can be explained by either of these mechanisms, since the effect is still apparent when the dose of steroid and the dose of CCK-8 are held constant, only the receptivity of the animal having changed. The facilitatory and inhibitory effects of CCK-8 cannot be explained by the known correlation between female receptivity and the concentration of estrogen-induced hypothalamic progestin receptors [29,31]. The concentration of progestin receptors should have been equally low 3 weeks after ovariectomy (test l, Experiment la) and 5 weeks later (test 2, Experiment lb) [7], yet levels of receptivity of controls were much higher in test 2. Similarly, there should be no differences in the concentration of estrogen receptors between the two tests [7,30]. In this context, one should note that it is unlikely that the single injection of 5 or l0/xg EB before test l produced significant plasma levels of estrogen for more than the first few days of the 5 week period. Our maximum dose (10/xg) has been reported to produce peak
C H O L E C Y S T O K I N I N AND LORDOSIS BEHAVIOR plasma estradiol levels within 32 hr after injection, followed by a rapid fall in the next 10 hr and a continued decline that would result in no detectable estrogen by 3-5 days after injection (calculated from [21,41]). The present finding, that IP CCK-8 can produce opposite effects depending on the degree of receptivity of the female, is of considerable importance. Depending on the hormonal priming procedures, degree of prior sexual experience, as well as the strain [16] and even supplier of rats, there may be differences in observed effects of various experimental treatments on lordosis behavior. Indeed, while Sakuma and Pfaff [35] reported that electrical stimulation of the mesencephalic central gray of rats primed with a single injection of 5 /~g EB resulted in a facilitation of lordosis behavior, Arendash and Gorski [3], using very similar stimulation parameters in the same strain, found that electrical stimulation of this same area of the brain inhibited lordosis in rats injected with 2/~g EB/day for 3 days, then 500/~g P. Although there are other possible explanations for this discrepancy, one possibility is the difference in receptivity of the rats between the two studies: before electrical stimulation, the control LQ's in Arendash and Gorski's study were approximately 90, while in Sakuma and Pfaff's study they were approximately 35. The finding that a chemical can stimulate and inhibit a reproductive process is, of course, not unique to sexual behavior. Intraventricular injection of CCK-8 has been reported to inhibit LH secretion in ovariectomized rats [43], while injection in the medial preoptic area of ovariectomized, estrogen-treated rats has been reported to increase LH secretion [25]. Also, intraventricular injection of angiotensin II has been reported to inhibit LH secretion in ovariectomized rats, but to stimulate LH secretion in ovariectomized rats pretreated with both estrogen and progesterone [39]. In addition to testosterone's effect of in-
223 hibiting LH secretion in male rats, acute effects of castration or the effects of low doses of testosterone include stimulation of LH secretion [6,38]. Furthermore, it is well known that depending on the time of administration, P can either facilitate or inhibit lordosis behavior and gonadotropin secretion in female rats [14,29]. Important to the present study, however, is the finding that opposing effects can be demonstrated without changing the steroids administered, the dosage used, or the timing of administration. In males, we have not observed any effects of IP CCK-8 on male copulatory behavior, either before or after castration [4]. In addition, we have observed no effects of CCK-8 on lordosis behavior in castrated males injected with 5/~g EB/day for 3 days prior to testing [4]. This suggests a sex difference in the effects of CCK-8 on lordosis behavior, a possibility that we are currently investigating. Peripheral injection of CCK-8 may influence the ventromedial nucleus of the hypothalamus because 3Hcaerulein, a decapeptide with structural and physiological homologies to CCK-8, has been shown to accumulate in this nucleus after peripheral injection [40]. The ventromedial nucleus appears to be especially important in regulating estrogen-stimulated lordosis behavior, while the medial preoptic area may be involved in inhibiting lordosis behavior [32]. Because CCK-8 binding sites in the ventromedial nucleus are depressed by EB following ovariectomy (submitted for publication), the behaviorally stimulating effect of estrogen may, in part, be due to a disinhibition mediated through a down regulation of CCK-8 binding sites within this area of the brain. The tissue content of CCK-8 varies differentially in certain regions of the brain during the estrus cycle [28,37], so it is possible that CCK-8 is exerting opposing effects on lordosis behavior through specific actions in discrete regions of the brain that are differentially regulated by estrogen.
REFERENCES 1. Akana, S. F., C. S. Cascio, J. Shinsako and M. F. Dallman. Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am J Physio1249: R527-R532, 1985. 2. Akesson, T. R., P. W. Mantyh, C. R. Mantyh, D. W. Matt and P. E. Micevych. Sex differences in 125 I-CCK-octapeptide binding sites in the ventromedial nucleus. Neuroendocrinology, in press. 3. Arendash, G. W. and R. A. Gorski. Suppression of lordotic responsiveness in the female rat during mesencephalic electrical stimulation. Pharmacol Biochem Behav 19: 351-357, 1983. 4. Babcock, A. M., D. Bloom, G. J. Bioch and P. E. Micevych. CCK does not effect sexual behavior in male rats. Soc Neurosci Abstr 12: 674, 1986. 5. Beach, F. A. and R. K. Orndoff. Variation in the responsiveness of female rats to ovarian hormones as a function of preceding hormonal deprivation. Horm Behav 5: 201-205, 1974. 6. Bloch, G. J., J. F. Masken, C. L. Kragt and W. F. Ganong. Effect of testosterone on plasma LH in male rats of various ages. Endocrinology 94: 947-951, 1974. 7. Clark, C. R., N. J. MacLusky, B. Parsons and F. Naftolin. Effects of estrogen deprivation on brain estrogen and progestin receptor levels and the activation of female sexual behavior. Horm Behav 15: 289-298, 1981. 8. Crawley, J. N. Cholecystokinin accelerates the rate of habituation to a novel environment. Pharmacol Biochem Behav 20: 23-27, 1984.
9. Crawley, J. N., S. E. Hays, T. L. O'Donahue, S. M. Paul and F. K. Goodwin. Neuropeptide modulation of social and exploratory behaviors in laboratory rodents. Peptides 2: Suppl l, 123129, 1981. 10. Crawley, J. N., S. E. Hays, S. M. Paul and F. K. Goodwin. Cholecystokinin reduces exploratory behavior in mice. Physiol Behav 27: 407-411, 1981. 11. Crowley, W. R. and F. P. Zemlan. The neurochemical control of mating behavior. In: Neuroendocrinology of Reproduction, edited by N. T. Adler. New York: Plenum Press, 1981, pp. 451-484. 12. Davidson, J. M., C. H. Rogers, E. R. Smith and G. J. Bloch. Stimulation of female sex behavior in adrenalectomized rats with estrogen alone. Endocrinology 82: 193-195, 1968. 13. Eriksson, H. and P. Sodersten. A failure to facilitate lordosis behavior in adrenalectomized and gonadectomized estrogenprimed rats with monoamine-synthesisinhibitors. Horm Behav 4: 89-97, 1973. 14. Feder, H. H. and B. L. Marrone. Progesterone: its role in the central nervous system as a facilitator and inhibitor of sexual behavior and gonadotropin release. Ann N Y Acad Sci 286: 331-354, 1977. 15. Feder, H. H. and K. B. Ruf. Stimulation of progesterone release and estrous behavior by ACTH in ovariectomized rodents. Endocrinology 84. 171-174, 1969. 16. Fitten, L. J., J. Shryne and R. A. Gorski. Behavioral responsiveness to estrogen in female rats: influence of strain and of aging. Soc Neurosci Abstr 11: 951, 1985.
BLOCH ET AL.
224 17. Franck, J. E. Short latency induction of lordosis by ether in estrogen-primed female rats. Physiol Behav 19: 245-247, 1977. 18. Frankfurt, M., R. A. Siegel, I. Sim and W. Wuttke. Cholecystokinin and substance P concentrations in discrete areas of the rat brain: sex differences. Brain Res 358: 53-58, 1985. 19. Gerall, A. A. and J. L. Dunlap. The effect of experience and hormones on the initial receptivity in female and male rats. Physiol Behav 10: 851-854, 1973. 20. Gerall, A. A., J. L. Dunlap and S. E. Hendricks. Effect of ovarian secretions on female behavioral potentiality in the rat. J Comp Physiol Psychol 82: 449-465, 1972. 21. Henderson, S. R., C. Baker and G. Fink. Oestradiol-17 and pituitary responsiveness to luteinizing hormone releasing factor in the rat: a study using rectangular pulse oestradiol-17 monitored by non-chromatographic radioimmunoassay. J Endocrinol 73: 441-453, 1977. 22. Hunter, A. J., D. R. Hole and C. A. Wilson. Studies into the dual effects of serotonergic pharmacological agents on female sexual behavior in the rat: preliminary evidence that endogenous 5HT is stimulatory. Pharmacol Biochem Behav 22: 5-13, 1985. 23. Itoh, S., R. Hirota and G. Katsuura. Effect of cholecystokinin octapeptide and vasoactive intestinal polypeptide on adrenocortical secretion in the rat. Jpn J Physiol 32: 553-560, 1982. 24. Kaneko, M., T. Hiroshige, J. Shinsako and M. F. Dallman. Diurnal changes in amplification of hormone rhythms in the adrenocortical system. Am J Physiol 239: R309-R316, 1980. 25. Kimura, F., R. Hashimoto and M. Kawakami. The stimulatory effect of cholecystokinin implanted in the medial preoptic area on luteinizing hormone secretion in the ovariectomized estrogen-primed rat. Endocrinol Jpn 30: 305-309, 1983. 26. Mendelson, S. D. and B. B. Gorzalka. Cholecystokininoctapeptide produces inhibition of lordosis in the female rat. Pharmaol Biochem Behav 21: 755-759, 1984. 27. Micevych, P. E., A. M. Babcock, D. W. Matt and J. K. H. Lu. Cholecystokinin inhibits k+ stimulated LHRH release from male and female rat hypothalami in vitro. Soc Neurosci Abstr 12: 1415, 1986. 28. Micevych, P. E., V. W. Go and D. W. Matt. Effect of gonadal steroid hormones on levels of cholecystokinin in the hypothalamus. Sot" Neurosci Abstr 11: 1228, 1985. 29. Moguilewsky, M. and J.-P. Raynaud. The relevance of hypothalamic and hypophyseal progestin receptor regulation in the induction and inhibition of sexual behavior in the female rat. Endocrinology 105: 516-522, 1979. 30. Parsons, B., N. J. MacLusky, M. S. Krieger, B. S. McEwen and D. W. Pfaff. The effects of long-term estrogen exposure on the induction of sexual behavior and measurements of brain estrogen and progestin receptors in the female rat. Horm Behav 13: 301-313, 1979.
31. Parsons, B., B. S. McEwen and D. W. Pfaff. A discontinuous schedule of estradiol treatment is sufficient to activate progesterone-facilitated feminine sexual behavior and to increase cytosoi receptors for progestins in the hypothalamus of the rat. Endocrinology 110: 613-619, 1982. 32. Pfaff, D. W. Estrogen and Brain Stimulation. New York: Springer-Verlag, 1980. 33. Porter, J. R. and L. D. Sander. The effect of cholecystokinin octapeptide on pituitary-adrenal hormone secretion. Regul Pept 2" 245-252, 1981. 34. Sakellaris, P. C. and J. Vernikos-Danellis. Increased rate of response of pituitary-adrenal system in rats adapted to chronic stress. Endocrinology 97: 597-602, 1975. 35. Sakuma, Y. and D. W. Pfaff. Facilitation of female reproductive behavior from mesencephalic gray in the rat. A m J Physiol 237: R278-R284, 1979. 36. Sander, L. D. and J. R. Porter. Influence of cholecystokinin on hypothalamic-stalk median-eminence-extract stimulation of ACTH output from isolated pituitary cells. Life Sci 31: 11031110, 1982. 37. Siegel, R. A., M. Frankfurt, U. Pahnke and W. Wuttke. Cholecystokinin in discrete rat brain regions as a function of sex and estrous cycle. Ann N Y A c a d Sci 448: 651-653, 1985. 38. Sodersten, P., P. Eneroth and A. Pettersson. Episodic secretion of luteinizing hormone and androgen in male rats. J Endocrinol 97: 145-153, 1983. 39. Steele, M. K., R. V. Gallo and W. F. Ganong. Stimulatory or inhibitory effects of angiotensin II upon LH secretion in ovariectomized rats: a function of gonadal steroids. Neuroendocrinology 40: 210-216, 1985. 40. Stern, J. J., C. A. Cudillo and J. Kruper. Ventromedial hypothalamus and short-term feeding suppression by caerulein in male rats. J Comp Physiol Psychol 90: 484-490, 1976. 41. Tapper, C. M., F. Greig and K. Brown-Grant. Effects of steroid hormones on gonadotrophin secretion in female rats after ovariectomy during the oestrous cycle. J Endocrinol 62: 511525, 1974. 42. Thody, A. J., C. A. Wilson and D. Everard. Facilitation and inhibition of sexual receptivity in the female rat by MSH. Physiol Behav 22: 447-450, 1979. 43. Vijayan, E., W. K. Samson and S. M. McCann. In vivo and in vitro effects of cholecystokinin on gonadotropin, prolactin, growth hormone and thyrotropin release in the rat. Brain Res 172: 295-302, 1979. 44. Whalen, R. E. and A. H. Lauber. Progesterone substitutes: cGMP mediation. Neurosci Biobehav Rev 10: 47-53, 1986. 45. Whalen, R. E. and K. Nakayama. Induction of estrous behavior: facilitation by repeated hormone treatments. J Endocrinol 33: 525-526, 1965. 46. Wilson, C. A., A. J. Thody and D. Everard. Effect of various ACTH analogs on lordosis behavior in the female rat. Horm Behav 13: 293-300, 1979.
NOTE ADDED IN PROOF Akesson, T., P. Mantyh, C. Mantyh, D. Matt and P. Micevych. Estrous Cyclicity of l~5I-Cholecystokinin Octapeptide Binding in the Ventromedial Hypothalamic Nucleus: Evidence for down modulation by estrogen. Neuroendocrinology, 1987.
0031-9384/87$3.00 + .00
Cholecystokinin Stimulates and Inhibits Lordosis Behavior in Female Rats I GEORGE J. BLOCH, ALEX M. BABCOCK, ROGER A. GORSKI AND PAUL E MICEVYCH Department o f Anatomy and the Laboratory o f Neuroendocrinology, Brain Research Institute UCLA School o f Medicine, Los Angeles, CA 90024 R e c e i v e d 3 July 1986 BLOCH, G. J., A. M. BABCOCK, R. A. GORSKI AND P. E MICEVYCH. Cholecystokinin stimulates and inhibits lordosis behavior in female rats. PHYSIOL BEHAV 39(2) 217-224, 198_7.--Recently,IP CCK-8 has been shown to inhibit lordosis in sexually experienced, estradiol benzoate (EB) and progesterone (P) primed rats. However, receptivity is influenced by prior sexual experience and/or exposure to sex steriods, as well as the steroid dosage administered before testing. Thus, we examined the effect of CCK-8 (3/~g/kg; IP) on lordosis in rats with different degrees of receptivity. Three weeks after ovariectomy, females were treated with EB followed 48 hr later with P, or with EB alone. CCK-8 significantly facilitated lordosis in rats given 5 tzg EB. Following a 5 week nonexperimental period, animals were more receptive and CCK-8 significantly inhibited lordorsis in the 5 or 10/zg EB groups. In a separate experiment, rats were ovariectomized, adrenalectomized, and treated with EB alone. As in the first experiment, CCK-8 facilitated and inhibited lordosis. CCK-8's effects were highly dependent on the female's receptivity, facilitating lordosis when receptivity was low and inhibiting lordosis when receptivity was high (but not maximal). In conclusion, IP CCK-8 modulates lordosis behavior independent of P, but its effects depend on the female's degree of receptivity. Lordosis behavior
Cholecystokinin
Estrogen
Progesterone
S E V E R A L findings suggest that the brain-gut octapeptide cholecystokinin (CCK-8) may be important in the regulation of reproductive processes. The distribution of CCK-8 levels in the medial preoptic area, mediobasal hypothalamus, and posterior pituitary is sexually differentiated [18, 28, 37], and there are sex differences in the apparent number of CCK-8 binding sites within the ventromedial nucleus of the hypothalamus [2]. In addition, 125I-CCK-8 binding sites in the ventromedial nucleus of the female are depressed on estrus or following ovariectomy and estrogen replacement (submitted for publication). Depending on the steroid environment and the site of central administration, CCK-8 has also been reported to inhibit [43] and to stimulate [25] the release of LH. Recently we have demonstrated that CCK-8 inhibits the stimulated release of L H R H in vitro from male, ovariectomized, and ovariectomized estrogen-primed female rat hypothalami [27]. Mendelson and Gorzaika [26] recently reported that 3 /zg/kg CCK-8 administered intraperitoneally (IP) inhibited lordosis behavior in ovariectomized, sexually experienced rats injected with estradiol benzoate (EB) and progesterone (P), and they suggested that this inhibitory effect was due to an interaction between CCK-8 and P, but not EB. It is known that prior administration of sex steroids and/or prior experience with mating "sensitizes" females so that in subsequent mating tests they are more sexually responsive to a given dose of steroids than before [5, 19, 20, 45]. In addition,
Adrenalectomy
the effects of some peptides (i.e., aMSH and ACTH 4-10) on lordosis behavior may depend on the state of receptivity of the female [46]. Because of these concerns, we undertook to study the effects of CCK on lordosis behavior in detail, and have observed the effects of CCK-8 in ovariectomized and EB-treated females showing various degrees of receptivity.
G E N E R A L METHOD
Animals and Surgery Animals were 81 100-day-old female Long-Evans rats obtained from Charles River, Wilmington, MA (Experiment 1) or Simonsen, Gilroy, CA (Experiments 2 and 3). These rats, housed 3 to 4 to a cage, were given ad lib access to Purina lab chow and water in a room maintained on a partially reversed light schedule (lights on from 1:30 a.m. to 3:30 p.m.). All rats were ovariectomized approximately one week after arrival using ether anesthesia, and were given their first mating test three weeks later.
Drug Procedures Fifty-three hours before each mating test (11:30 a.m.), rats were given a subcutaneous (SC) injection of EB dissolved in 0.1 ml sesame oil or 0.1 ml oil, followed 48 hr later
1Supported by NIH grants NS 21220 (P.E M.) and HD 01182 (R.A.G.). Preliminary reports were given at the Society for Neuroscience and the Endocrine Society in 1986.
217
BLOCH ET AL.
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EXPERIMENT I
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The purpose of this experiment was to assess the effect of CCK-8 on the lordosis behavior of female rats demonstrating various degrees of receptivity.
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FIG. 1. Effect of IP CCK-8 on lordosis behavior: first test. Rats were primed with EB alone (5/0, 10/0 groups), or followed 48 hr later with P (5/50, 10/50 groups), then given CCK-8 (dark bars) or saline (clear bars) 10 minutes before the mating test. Number of rats," *p<0.05 vs. saline, independent t-test.
Three weeks after ovariectomy, rats were injected with EB alone or with P 48 hr later as follows: 0/0 group, each given an injection of oil followed 48 hr later with oil (n=8); 5/0 group, each given 5/zg EB, then oil (n= 12); 10/0 group, each given 10/~g EB, then oil (n= 12); 5/50 group, each given 5/xg EB, then 50/xg P (n= 12); 10/50 group, each given 10 tzg EB, then 50 /zg P (n=8). Half the animals in each group received 3/xg/kg CCK-8 10 minutes before the mating test; the others received vehicle (saline). Mean LQ scores of saline and CCK-8 injected rats were analyzed separately for each steroid group using a two-tailed Student's t-test.
RESULTS AND DISCUSSION
with an SC injection of 50 t~g P in the same volume of oil (see specific experiments). Ten minutes before each mating test (4:30 p.m.), rats were given an IP injection of 3/xg/kg suiphated CCK-8 (Bachem, Tustin, CA) or vehicle (saline). A fresh CCK-8 solution was prepared from a lyophilized aliquot immediately before each testing session.
Behavioral Testing Mating tests were the same in all experiments. Test females were placed in a Plexiglas arena illuminated with a 25 watt red bulb. A Long-Evans male with proven sexual vigor, adapted to the mating arena for at least 10 rain, was permitted to mount the test female 10 times. A lordosis quotient (LQ) was computed by dividing the number of lordotic responses by the number of mounts and multiplying by 100. A male's behavior was counted as a mount only if he clasped the female with his forelegs, " p a l p a t e d " the female by rapidly moving his forelegs along her flanks, and executed vigorous pelvic thrusting movements which resulted in contact between the pelvic region of the male and female. Because both positions--flattening and arching of the b a c k - allow the male to achieve intromission, a female' s behavior was scored as a lordosis when she displayed either flattening or arching of the back and a raising of the head.
Radioimmunoassay In Experiments 3 and 4, plasma corticosterone values were obtained by radioimmunoassay. Total corticosterone was measured in unextracted medium using antiserum from Radioassay System laboratories, Inc. The tracer steroid was 1,2,6,7 3H(N)-corticosterone. Plasma samples were heatdenatured, and dextran-coated charcoal was used for phase separation. The antibody crossreacts with progesterone (0.29%), deoxycorticosterone (6.1%), 20a-dihydroprogesterone (0.08%), and 11-deoxycortisol (0.03%). The sensitivity of the assay was 30 pg/ml plasma, and the intra- and interassay variations were 9.9% and 11.8%, respectively.
In the 5/0 group, CCK-8 significantly increased lordosis behavior as compared to saline treated controls (54.3_+ 19.0 vs. 11.7_+8.3, p<0.05, Fig. 1). There were no significant effects of CCK-8 in the 10/0, 5/50, and 10/50 groups although the 10/0 group showed a trend toward CCK-8-induced facilitation as well (see Fig. 1). No behavior was observed in the 0/0 group. The results of this experiment demonstrate that IP CCK-8 can facilitate lordosis behavior and that this facilitation is dependent on the administration of EB but not P. Although mechanisms responsible are presently unknown, the CCK-8-induced facilitation appears to be associated with low levels of receptivity since it was observed only in groups where control L Q ' s were low.
Experiment lb In order to further test the effects of CCK-8 in rats with varying degress of receptivity, we retested the animals used in Experiment l a following a 5 week nonexperimental period. Since Gerall and Dunlap [19] had reported that females remaining in the home cage for three weeks before injection with EB and P for a second time show levels of receptivity as low as the first time, we reasoned that this 5 week nonexperimental period between test 1 (Experiment la) and test 2 would allow the females to be retested in a state of minimal receptivity. During subsequent mating tests, we expected to see an increase in receptivity as has been reported [5, 19, 45], allowing us to assess the effects of CCK-8 as receptivity increased. Thus, we hypothesized that this experiment would allow us to replicate the observation of CCK-8-induced facilitation of lordosis behavior during test 2, and to observe the reported inhibition of lordosis behavior [26] during subsequent tests.
METHOD
In a crossover design with respect to the administration of CCK-8 or saline, the animals in the 5/0, 10/0, 5/50, and 10/50 groups of Experiment l a were tested 5 weeks later and every
C H O L E C Y S T O K I N I N A N D LORDOSIS B E H A V I O R
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FIG. 2. Effect of IP CCK-8 on lordosis behavior: tests 2 through 5. Rats were primed with EB alone (5/0, 10/0 groups), or followed 48 hr later with P (5/50, 10/50 groups), then given either CCK-8 (dark triangles, individual mating tests; striped bars, mean value of two tests) or saline (clear squares and bars) in a crossover design l0 minutes before the mating test, *p<0.05, **p <0.01, matched pair t-test.
10-12 days thereafter (tests 2 through 5) and a few animals in the 10/0, 5/50, and 10/50 groups 15 days after test 5 (test 6). Statistical evaluation was performed by comparing the mean saline LQ value to the mean CCK-8 value for each animal over tests 2 through 5 using a two-tailed matched-pair t-test. RESULTS AND DISCUSSION The mean lordosis quotients with standard errors of the 5/0, 10/0, 5/50, and 10/50 groups during tests 2 through 5 are shown in Fig. 2. There was a significant inhibition of lordosis behavior with CCK-8 as compared to saline controls in the 5/0 and 10/0 groups. There was no significant inhibition in the 5/50 and 10/50 groups over tests 2 through 5. As reported by others, the control (sahne injected) animals showed a marked increase in receptivity subsequent to their first steroid injection and mating experierrce. To our knowledge, this is the first demonstration that this increase in receptivity occurs as late as 5 weeks after the first test. In addition, CCK-8 failed to inhibit lordosis behavior as control females became maximally receptive (LQ of 95-100). This suggests that the effects o f CCK-8 might depend on the degree of receptivity of the female. Indeed, when the effects of
IP CCK-8 were correlated with the degree of receptivity of the saline-injected controls for each mating test conducted in Experiments 1 and 2 (tests 1 through 6), there was a significant negative correlation ( r = - . 8 0 7 , p < 0 . 0 0 1 ) . Because there was no effect of CCK-8 in those mating tests where the saline controls were maximally receptive, we recalculated the correlation and plotted the regression line without these tests, as illustrated in Fig. 3. Although the data in Fig. 3 suggest that the correlation might not be significant without the data points obtained when the controls showed low L Q ' s , a significant negative correlation was still evident without these two data points ( r = - .55, p<0.05). It is important to note that the present relation between receptivity and the effects of CCK-8 cannot be explained as a regression toward a mean value, a nonspecific effect that may occur when subjects are preselected into low- and high-receptive groups prior to testing.
EXPERIMENT 2 The purpose of this experiment was to assess the effect of CCK-8 on the lordosis behavior of adrenalectomized rats demonstrating various degrees of receptivity.
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BLOCH ET A L . 364 [~ 5 / 0 group
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Receptivity (Saline LQ)
FIG. 3. Percent change in LQ after IP CCK-8 plotted against receptivity of saline-injected controls for the mating tests conducted in Experiments 1 and 2. The percent change with CCK-8 was calculated as the mean LQ of CCK-8-injected rats (LQccr.) minus the mean LQ of the saline controls (LQsAL) divided by LQsAL × 100: (LQccK - LQsg~fl~QskL) × 100. Each square represents a separate calculation based on the results of a separate mating test. The values in the boxed area are tests where the controls were maximally receptive (LQ's of 95-100); these values were not included when calculating the regression line of this figure (r = -.903, p <0.001). The numbers associated with each square indicate the mating test (1 through 6).
Experiment 2a Because CCK-8 facilitated lordosis behavior only in animals treated with EB alone, this suggests that P may not be necessary for the facilitation. Adrenal P can, however, contribute to an increase in lordosis behavior in estrogentreated rats [15,17] even though lordosis behavior can be produced in adrenalectomized rats injected with estrogen alone [12,13]. Moreover, peripheral administration of CCK-8 has been shown to increase adrenal corticosterone secretion in viva and basal ACTH output from hemipituitaries in vitro [23, 33, 36]. The purpose of this experiment, therefore, was to test the hypothesis that the facilitation of lordosis behavior observed in Experiment la was independent of any CCK-8 stimulated release of adrenal P.
METHOD Two weeks after ovariectomy, 17 rats were adrenalectomized under ether anesthesia. These animals were then maintained on 0.9% saline for the duration of the experiment. One week after adrenalectomy, all animals were mjected with 5 /~g EB, 53 hr before the mating test. Ten minutes before behavioral testing, animals were given 3 p,g/kg CCK-8 (n=9) or saline (n=8). The following afternoon, 1-2 hr before lights off, animals were moved from the animal room into the laboratory, and 0.5 ml blood was taken from the jugular vein of each animal within 3 minutes after the onset of ether anesthesia. Plasma was frozen and a radioimmunoassay for
TEST ! EXPERIMENT 2o
/
FIG. 4. LQ of adrenalectomized rats primed with 5 /~g EB, then given CCK-8 (dark bars and squares) or saline (clear bars and squares) l0 minutes before the mating test. In Experiment 2a, the bars represent the mean values of the I I animals qualifying as adrenalectomized during test 1. In Experiment 2b, the squares represent individual mating tests (number of animals), and the bars represent the values of the 10 animals tested in a crossover design in tests 2 and 3. **p <0.01, independentt-test; *p <0.05, matched-pair t-test.
corticosterone levels was subsequently performed to assure completeness of adrenalectomy. Mean LQ scores of saline and CCK-8 injected rats were analyzed using a two-tailed Student's t-test.
RESULTS AND DISCUSSION AS in Experiment la, CCK-8 facilitated lordosis behavior during the first mating test. After CCK-8 or saline, the mean LQ's with standard errors of the adrenalectomized females were 92.8+4.3 and 5 1 . 3+- 10.9 respectively (Fig. 4). One can therefore conclude that adrenal P is not required for the observed facilitation with CCK-8. Plasma levels of corticosterone are shown in Table 1. Although corticosterone values that are not higher than 10 ng/ml have been used as a criterion for defining adrenalectomy in rats [1], there are a number of reasons to assume that our cut-off point of 12.1 ng/ml was not too high: (1) Moving the animals from their home cages into the laboratory as in the present experiment would result in levels of corticosterone that are at least 100 ng/ml in rats with intact adrenal glands (M. Dallman, personal communication); (2) The cut-off point of 10 ng/ml is based on morning samples, whereas the values in the present study were obtained from samples taken in the afternoon, a time when corticosterone is reported to be approximately 7 times higher in normal animals [24]; and (3) It is likely that under the stress of having moved the animals 45 to 105 minutes before taking blood, the animals with intact adrenals would respond to ether with high levels of corticosterone even within the 2-3 minutes before blood was taken [34]. Thus, CCK-8 caused a significant facilitation of lordosis
C H O L E C Y S T O K I N I N AND LORDOSIS BEHAVIOR
221
TABLE 1 LEVELS OF CORTICOSTERONEIN THE PLASMAOF ADRENALECTOMIZEDRATS: ng/ml -+ SE (NUMBEROF ANIMALS)
Adrenalectomized Functional Adrenal Tissue
1st Test: 9 Days After Adrenalectomy*
2nd Test: 19 Days After Adrenaiectomy*
3rd Test: 31 Days After Adrenaiectomy*
0.84 _+ 0.69 (11)*
1.46 + 0.92 (11)$
2.96 _+ 1.54(10)§
21.85 _+ 1.87 (6)
45.84 _+ 3.79 (5)
60.13 _ 10.77 (6)
*Blood taken the following afternoon. *Nine out of 11 animals had plasma levels of corticosterone that were below the sensitivity of the assay, and these were assigned a value of 0.03 ng/ml (see the General Method section). The other 2 animals had levels of 7.6 and 1.4 ng/ml. *Eight out of 11 animals had plasma levels below the sensitivity of the assay; the others had levels of 10.0, 2.9, and 2.9 ng/ml. §Seven out of 10 animals had plasma levels below the sensitivity of the assay; the others had levels of 12.1, 10.4, and 6.9 ng/ml.
behavior in EB-treated ovariectomized, adrenalectomized females, and therefore increased levels of adrenal progesterone resulting from activation of the hypothalamopituitary-adrenal axis by CCK-8 is not required for this effect.
81- I~:' r'1 5/0 group, odranolecfomlzed 5:3- ~
• I0/0 group
Experiment 2b Rats injected with EB alone showed a significant CCK-8induced inhibition (Experiment lb). This could mean that P is not required for the inhibition, an interpretation that does not agree with the suggestion of Mendelson and Gorzalka [26]. It is possible, however, that progesterone--in this case, of adrenal origin--is necessary. To test this hypothesis, the animals used in the previous experiment were retested in order to determine whether adrenal P is required for the inhibitory effect of CCK-8.
'e'°e°'2_-5-3-C3,°0°gec..2_si.2Oo,ter
r.o.°,'r-02 l.,,'°"°°' '
-59-
-87-
METHOD In a crossover design with respect to the administration of CCK-8 or saline, the ovariectomized, adrenalectomized rats from Experiment 2a were retested 10 days later (test 2) and 22 days later (test 3), 53 hr after the injection of 5/zg EB. As in Experiment 2a, blood samples were taken from each animal the afternoon after a mating test, and plasma corticosterone levels were measured to assure completeness of adrenalectomy. Mean saline LQ values were compared to mean CCK-8 values using a two-tailed matched-pair t-test.
RESULTS AND DISCUSSION As shown in Fig. 4, CCK-8 significantly inhibited lordosis behavior in the 10 animals considered to be adrenalectomized during tests 2 and 3 (see Table 1). The LQ's of the animals were lower in test 3 than in test 2. Whether this decline in receptivity was related to the long period of complete adrenalectomy or some other factor is not known, but the inhibition with CCK-8 was apparent in each of these tests (/7<0.025 for test 2, p<0.01 for test 3, independent t-test) as
t'l 3
5',.3
s~.9
60',5
65'.~
6~.7
74',3
I I I I I I
78:9 ~__~
Receptivity {Saline LQ)
FIG. 5. Percent change in LQ after IP CCK-8 plotted against receptivity of saline-injected controls for the mating tests conducted in Simonsen rats in Experiments 2 and 3 (see legend to Fig. 3 for more details). There was a significant negative correlation when the maximally-receptive test was excluded (r=-.981, p<0.02). The numbers associated with each square indicate the mating test (1 through 3).
well as for each animal tested in the crossover design (see Fig. 4). One can therefore conclude that adrenal progesterone is not necessary for the observed inhibition with CCK-8.
EXPERIMENT 3 The injection of 5/zg EB resulted in a much lower LQ in the saline-injected controls of Experiment la than in Exper-
222
BLOCH E T A L .
iment 2a (mean LQ's of 11.7 vs. 51.3, respectivley). CCK-8 facilitated lordosis behavior in both experiments, suggesting that low levels of receptivity were not, per se, an important factor in determining the effects of CCK-8. There were two important differences, however, in the animals used for the two experiments: (1) The animals used in Experiment la were ovariectomized while those used in Experiment 2a were ovariectomized as well as adrenalectomized; and (2) Rats used in Experiment la were obtained from Charles River while those used in Experiment 2a were obtained from Simonsen. With regard to the first difference, rats that are both ovariectomized and adrenalectomized appear to be behaviorally more sensitive to estrogen stimulation than are ovariectomized rats with intact adrenal glands [12,13]. In order to determine whether rats from Simonsen were behaviorally more sensitive to estrogen stimulation than Charles River rats, and to further study the relation between receptivity and the effects of CCK-8 in Simonsen rats, a group of Simonsen rats was ovariectomized, then injected with EB and given mating tests with CCK-8 or saline.
METHOD Twelve Long-Evans rats (Simonsen) were given mating tests 21 (test 1) and 32 (test 2) days after ovariectomy, 53 hr after the administration of 10/xg EB. In a crossover design, animals were given 3 p.g/kg CCK-8 IP (n=6) or saline (n=6) 10 minutes before the mating tests. Mean LQ's of saline and CCK-8 injected rats were analyzed using a Student's t-test for test 1, and a matched-pair t-test for tests 1 and 2. RESULTS AND DISCUSSION There was no effect of CCK-8 on lordosis behavior during the first mating test: LQ's of 68.3-+14.9 (saline) and 33.3-+18.2 (CCK-8). Similarly, when the LQ's were compared over the two mating tests, there were no significant differences between the groups: LQ's of 61.7___9.4 (saline) and 50_+13.7 (CCK-8). Regarding the first test, when the LQ's of the saline-injected animals of this experiment were compared to the LQ's of the l0 /zg EB, saline-injected animals of Experiment la (Charles River), Simonsen females were behaviorally more receptive than the Charles River females (LQ's of 68.3+-14.9 vs. 16.7_+16.7, p<0.025, Student's t-test). When the effects of IP CCK-8 were correlated with the degree of receptivity of the saline-injected controls for each mating test conducted in Experiments 2 and 3, there was a significant negative correlation ( r = - . 9 8 1 , p<0.02, Fig. 5). This indicates that the general pattern of facilitation and inhibition observed in Long-Evans rats obtained from Charles River (Fig. 3) was also apparent in those obtained from Simonsen, although " l o w " levels of receptivity were higher in Simonsen than Charles River rats.
G E N E R A L DISCUSSION The major finding of the present study is that CCK-8 facilitates lordosis behavior in rats showing low levels of receptivity while it inhibits lordosis behavior in rats showing high but not maximal levels of receptivity. The facilitatory and inhibitory effects were observable after administration of EB, and they were independent of P. This latter finding
contradicts Mendelson and Gorzalka [26], who proposed that CCK-8 inhibits lordosis through a direct interaction with P. Our results indicate that the degree of receptivity, rather than a specific action of P, is the important factor, and it is noteworthy that Mendelson and Gorzalka's data are consistent with this interpretation. In addition, these authors did not observe a facilitation of lordosis after administering CCK-8. Although they used a different strain (SpragueDawley rats), our data indicate that the females used by Mendelson and Gorzalka were already too receptive at the time of testing, perhaps because the rats had already received mating tests and injections of EB and P prior to the experiment. Many substances are known to stimulate lordosis behavior when administered to estrogen-primed animals. These include peptides (e.g., LHRH, substance P, ctMSH, oxytocin), steroids (estrogen, progesterone), and a variety of neurotransmitters, and this has led to speculation that a nonspecific mechanism may be involved [44]. A number of substances affect lordosis behavior in opposite ways. Injection of the peptides aMSH or ACTH 4-10 has been reported to facilitate or inhibit lordosis behavior depending on whether the rats are showing low or high (but not maximal) levels of receptivity [42,46]. Although the animals in these studies were preselected on the basis of their receptivity, making interpretation more difficult than in the present study, these results are very similar to those observed with CCK-8. The authors suggested that aMSH and ACTH 4-10 might be influencing lordosis by stimulating the animal's level of arousal, increasing behavioral efficiency when minimally receptive, decreasing behavioral efficiency when highly receptive. It is unlikely, however, that the CCK-8 effect is through a similar mechanism, since CCK-8 decreases rather than increases levels of arousal as measured by exploratory behavior [8-10]. Other substances known to facilitate or inhibit lordosis behavior include various dopamine and serotonin agonists, and serotonin antagonists [11,22]. One way to explain the neurotransmitter effects is to assume that low doses of the substance stimulate (inhibit) presynaptic receptors that inhibit endogenous transmission, while high doses stimulate (inhibit) post-synaptic receptors [11]. A similar hypothesis holds that different degrees of receptivity induced by different steroid regimens (different doses of estrogen, or estrogen alone vs. estrogen plus progesterone) will have different effects on neurotransmitter activity and thereby affect lordosis behavior differentially [22]. It is unlikely, however, that the dual effect of CCK-8 on lordosis can be explained by either of these mechanisms, since the effect is still apparent when the dose of steroid and the dose of CCK-8 are held constant, only the receptivity of the animal having changed. The facilitatory and inhibitory effects of CCK-8 cannot be explained by the known correlation between female receptivity and the concentration of estrogen-induced hypothalamic progestin receptors [29,31]. The concentration of progestin receptors should have been equally low 3 weeks after ovariectomy (test l, Experiment la) and 5 weeks later (test 2, Experiment lb) [7], yet levels of receptivity of controls were much higher in test 2. Similarly, there should be no differences in the concentration of estrogen receptors between the two tests [7,30]. In this context, one should note that it is unlikely that the single injection of 5 or l0/xg EB before test l produced significant plasma levels of estrogen for more than the first few days of the 5 week period. Our maximum dose (10/xg) has been reported to produce peak
C H O L E C Y S T O K I N I N AND LORDOSIS BEHAVIOR plasma estradiol levels within 32 hr after injection, followed by a rapid fall in the next 10 hr and a continued decline that would result in no detectable estrogen by 3-5 days after injection (calculated from [21,41]). The present finding, that IP CCK-8 can produce opposite effects depending on the degree of receptivity of the female, is of considerable importance. Depending on the hormonal priming procedures, degree of prior sexual experience, as well as the strain [16] and even supplier of rats, there may be differences in observed effects of various experimental treatments on lordosis behavior. Indeed, while Sakuma and Pfaff [35] reported that electrical stimulation of the mesencephalic central gray of rats primed with a single injection of 5 /~g EB resulted in a facilitation of lordosis behavior, Arendash and Gorski [3], using very similar stimulation parameters in the same strain, found that electrical stimulation of this same area of the brain inhibited lordosis in rats injected with 2/~g EB/day for 3 days, then 500/~g P. Although there are other possible explanations for this discrepancy, one possibility is the difference in receptivity of the rats between the two studies: before electrical stimulation, the control LQ's in Arendash and Gorski's study were approximately 90, while in Sakuma and Pfaff's study they were approximately 35. The finding that a chemical can stimulate and inhibit a reproductive process is, of course, not unique to sexual behavior. Intraventricular injection of CCK-8 has been reported to inhibit LH secretion in ovariectomized rats [43], while injection in the medial preoptic area of ovariectomized, estrogen-treated rats has been reported to increase LH secretion [25]. Also, intraventricular injection of angiotensin II has been reported to inhibit LH secretion in ovariectomized rats, but to stimulate LH secretion in ovariectomized rats pretreated with both estrogen and progesterone [39]. In addition to testosterone's effect of in-
223 hibiting LH secretion in male rats, acute effects of castration or the effects of low doses of testosterone include stimulation of LH secretion [6,38]. Furthermore, it is well known that depending on the time of administration, P can either facilitate or inhibit lordosis behavior and gonadotropin secretion in female rats [14,29]. Important to the present study, however, is the finding that opposing effects can be demonstrated without changing the steroids administered, the dosage used, or the timing of administration. In males, we have not observed any effects of IP CCK-8 on male copulatory behavior, either before or after castration [4]. In addition, we have observed no effects of CCK-8 on lordosis behavior in castrated males injected with 5/~g EB/day for 3 days prior to testing [4]. This suggests a sex difference in the effects of CCK-8 on lordosis behavior, a possibility that we are currently investigating. Peripheral injection of CCK-8 may influence the ventromedial nucleus of the hypothalamus because 3Hcaerulein, a decapeptide with structural and physiological homologies to CCK-8, has been shown to accumulate in this nucleus after peripheral injection [40]. The ventromedial nucleus appears to be especially important in regulating estrogen-stimulated lordosis behavior, while the medial preoptic area may be involved in inhibiting lordosis behavior [32]. Because CCK-8 binding sites in the ventromedial nucleus are depressed by EB following ovariectomy (submitted for publication), the behaviorally stimulating effect of estrogen may, in part, be due to a disinhibition mediated through a down regulation of CCK-8 binding sites within this area of the brain. The tissue content of CCK-8 varies differentially in certain regions of the brain during the estrus cycle [28,37], so it is possible that CCK-8 is exerting opposing effects on lordosis behavior through specific actions in discrete regions of the brain that are differentially regulated by estrogen.
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NOTE ADDED IN PROOF Akesson, T., P. Mantyh, C. Mantyh, D. Matt and P. Micevych. Estrous Cyclicity of l~5I-Cholecystokinin Octapeptide Binding in the Ventromedial Hypothalamic Nucleus: Evidence for down modulation by estrogen. Neuroendocrinology, 1987.