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Neonatal ACTH and corticosterone alter hypothalamic monoamine innervation and reproductive parameters in the female rat
Peptides.Vol. 14, lap. 379-384, 1993
0196-9781/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the USA.
Neonatal ACIH and Corticosterone Alter Hypothalamic Monoamine Innervation and Reproductive Parameters in the Female Rat STEPHEN
E. A L V E S , H O M A Y O O N
M. A K B A R I ,
EFRAIN
C. A Z M I T I A
AND
FLEUR
L. S T R A N D ~
Department of Biology and the Center for Neural Science, New York University, Washington Square, New York, N Y 10003 R e c e i v e d 10 M a r c h 1992 ALVES, S. E., H. M. AKBARI, E. C. AZMITIA AND F. L. STRAND. Neonatal ACTH and corticosterone alter hypothalamic monoamine innervation and reproductive parameters in the female rat. PEPTIDES 14(2) 379-384, 1993.--Female SpragueDawley rat pups were injected SC with either ACTH(1-24) (0.5 mg/kg) or saline vehicle once daily from postnatal day 1 (day of birth) to day 7. Plasma corticosterone (CORT) levels were recorded via radioimmunoassay (RIA) on day 4 to measure adrenal response to these treatments. Hypothalamic 5-HT and DA fiber densities were assessed using high-atfinity specific 3H-5-HT and 3H-DA uptake at days 7, 25, and at adulthood (80-90 days). Animals were checked daily for vaginal opening (starting on day 30) as a sign of sexual maturation and later tested for sexual behavior as virgins (60-70 days of age). Plasma estradiol and progesterone levels were measured via RIA. Plasma CORT levels were greatly increased among ACTH-treated animals during the treatment period. The 5-HT uptake was significantly increased in ACTH-treated animals at day 7 (p < 0.01 ) and at adulthood (p < 0.02) compared to controls. The DA uptake was significantly higher among ACTH-treated animals at day 7 (p < 0.01). The sexual maturation of ACTH-treated animals was delayed when compared to control animals (p < 0.01). The ACTH-treated animals displayed slight deficits in female sexual behavior compared to control animals (p < 0.05). No significant changes in plasma sex steroid levels were found. Based on this study, we suggest that monoamine innervation into the developing female hypothalamus is susceptible to early postnatal manipulation with ACTH and CORT. and that the resulting changes in these monoamine fiber densities may be responsible for the observed deficits in reproductive maturation and behavior. ACTH Hypothalamic monoamines Sexual behavior
Serotonin
Dopamine
T H E monoaminergic innervation of the hypothalamus is believed to play an important regulatory role over reproductive behavior and function in the female rat ( 12,13,18,19,25-27,3 i ). The activity of these neurons within discrete hypothalamic nuclei, mediated in part by the gonadal steroids, dictates the expression of sexual behavior (the lordosis response) upon the appropriate stimulus or the eventual release of gonadotropins from the pituitary. The development of this neurocircuitry in the female direction (a cyclic hypothalamus) begins during the last week of gestation and continues through the first week of postnatal life. Manipulation of these neurotransmitter systems during this critical time can permanently alter the future expression of sexual behavior in adult female rats (20,21). Increasing serotonin (5-HT) levels by the administration of its precursor, /-tryptophan, during the first 7 days of postnatal life results in a decreased capacity for lordosis behavior among adult female rats (21 ). While sex steroids dominate the organization of developing neural pathways essential for male or female sexual behavior,
Female reproductive maturation
Lordosis
either perinatal stress (9,17,42) or perinatal administration of the stress-evoked peptide hormone adrenocorticotropic hormone (ACTH) (36) seriously diminishes subsequent male sexual behavior. A stress-induced rise of plasma corticosteroid levels can influence the maturation of developing central m o n o a m i n e systems (11). Adrenocorticotropic hormone, a 39 a m i n o acid pituitary peptide, is the primary endogenous releaser of corticosteroids from the adrenal cortex during a stress response. In addition to this classical role, this peptide hormone and its fragments [ACTH(4-10), A C T H ( 4 - 9 ) [Org 2766], A C T H ( I - 1 3 ) [aMSH], and A C T H ( I - 2 4 ) ] have direct neurotrophic properties, affecting the development and regeneration of both central and peripheral neurons (7,38). The central 5-HT system appears to be intimately associated with this pituitary-adrenal endocrine system, as these neurons are affected by both the A C T H peptides (6, I0) and the adrenal steroids (4,40). In addition, recent evidence suggests that dopamine neurons are also sensitive to ACTH/corticosteroid modulation (34,43). Both of these monoamines are believed to
i Requests for reprints should be addressed to Dr. Fleur L. Strand, Biology Department, Room 1009 Main Bldg., New York University, Washington Square, New York, NY 10003. 379
380
ALVES ET AL.
be involved in the control of reproductive processes in the female rat. The developing 5-HT system appears to be particularly sensitive to changes in plasma corticosteroid concentration, and it has been suggested that behavioral alterations seen in offspring from stressed mothers may be due to changes in this monoamine system (11). Prenatal administration of ACTH(1-24) (which has the full corticosteroidogenic properties of the whole peptide) during the last week of gestation alters the sexual differentiation of the brain in male rats, decreasing the expression of male sexual behavior among these animals at adulthood (36). This decrease in sexual behavior is correlated with an increase in 5-HT in the medial preoptic area (MPOA), the primary regulatory site for male sexual behavior in the rat. Another study in our laboratory has shown that early postnatal administration of ACTH accelerates the developing 5-HT system within the brainstem and hippocampus of neonatal and juvenile rats (22). The current study investigates whether exposure to elevated levels of the pituitary-adrenal stress hormones, ACTH and corticosterone (CORT), during the first postnatal week would affect the development of 5-HT or DA systems within the hypothalamus of female rats, and the subsequent reproductive capacity of these animals at adulthood. METHOD Pregnant Sprague-Dawley rats were ordered from Hilltop Laboratories, housed individually, and water and Purina rat chow were provided ad lib. They were kept in a 12L:12D photoperiod with lights offat 1200. On the day of birth (day 1), the pups were weighed and each litter culled to five females and three males to maintain the same sex ratio.
Treatment Female rat pups were injected from day 1 to day 7 with either ACTH(1-24) (0.5 mg/kg), donated by Organon Inc., or 0.9% saline, once daily. The ACTH(1-24) was dissolved in saline and administered subcutaneously (SC) in a volume of 0.1 ml/10 g per injection. On postnatal day 22, males and females were separated from the dam and housed in groups of the same sex with two to three animals per cage.
Corticosterone Radioimmunoassay Two dosages of ACTH(1-24) were used for this study. On postnatal day 4, 45 rain following the injection of ACTH(1-24) (0.5 mg/kg), ACTH(I-24) (0.1 mg/kg), or saline vehicle, five pups from each treatment group were decapitated; trunk blood was collected in EDTA and pooled. The blood was centrifuged with a Beckman T J-6 refrigerated centrifuge at 5000 × g for 25 min. The plasma was placed in vials and stored at - 7 0 ° C until the assay was performed. Plasma levels of corticosterone were determined using a Coat-A-Count rat corticosterone radioimmunoassay from Diagnostic Products Corporation with a sensitivity of up to 5.7 ng/ml. Determination of hormonal levels was interpolated from a standard curve prepared in triplicate using calibrators and quality control serum pools. The data reduction was calculated by linear regression and logit-log representation with the aid of a computer program. The size of the aliquot required was 50 vl, and all samples were run in triplicate.
Measurement of Neurotransmitter Fiber Densities Hypothalamic synaptosomal high-aflinity specific uptake was measured to assess hypothalamic neurotransmitter fiber density. Specific 5-HT and DA uptake was measured at various stages
during development: day 7 (neonatal), day 25 (juvenile), and adult (80-90 days, diestrus). High-affinity uptake. Following the technique of Azmitia et al. (5), animals were rapidly decapitated under sodium pentobarbital anesthesia and the hypothalamus was dissected out, weighed, and placed in ice-cold 0.32 M sucrose (10X volume). The tissue was homogenized with a glass Teflon homogenizer and centrifuged at 300 × g for 10 min; supernatant (SI) was saved, the pellet was resuspended in 2 ml 0.32 M sucrose, and centrifuged at 300 × g for 10 min. The resulting supernatant ($2) was saved and the pellet was discarded. Both St and $2 were combined and centrifuged at 12,500 × g for l0 min. The supernatant was discarded and the pellet was resuspended in 10X v/w Krebs-Ringer (K-R) (Sigma) with 10 s M pargyline (P), pH 7.4. Tissue (40 vl) was incubated for 5 min at 37°C with either 140 /~1 K-R and P or 120 /~1 K-R and P, and 20 ul of fluoxetine or nomifensine (5 × 10-5 M final concentration) for determination of nonspecific uptake of 5-HT and DA, respectively: 20 ul of SH-5HT (5 × 10_8 Mfinal concentration) or 3HDA (1 × l0 7 31 final concentration) were added to each well and tissue was incubated for an additional 10 min. A total of eight wells/condition was performed (four total uptake and four nonspecific uptake). Tissue was harvested onto nitrocellulose filters (Skatron, Inc.) with 0.1 M phosphate-buffered saline using a Titertek cell harvester. Filters were placed in counting vials with 3 ml of Liquiscint and counted in a Beckman counter (efficiency 0.38). Protein content of each sample was calculated by the method of Lowry. et al. (24).
Reproductive Parameters Maturation. Beginning on postnatal day 30, female offspring were checked daily for vaginal opening as an indication of reproductive maturation (onset of puberty). Female sexual behavior. Between 60 and 70 days of age, the animals were tested for female sexual behavior as intact virgins. One week prior to testing, vaginal smears were recorded daily, 2 h before the onset of darkness. Animals in late proestrus (basal bodies being the predominant cell type in the smear) were tested for sexual behavior 2 h after the onset of darkness. Each female was placed in a rectangular aquarium under red lighting, containing a sexually experienced, sterile male (castrated males were used to prevent impregnation of the females). Under a combination ketamine-xylazine anesthesia, these males had been castrated and 4 cm of silastic tubing, filled with testosterone propionate [a dosage that is effective in producing male sexual behavior among castrates (Luine, personal communication)], was implanted subcutaneously in the dorsal neck region. The lordosis quotient (LQ), a quantitative measure of female sexual behavior, was determined by rotating the female between three males until she had been mounted a total of 10 times. The LQ for each animal was then calculated by dividing the total number of lordotic responses by 10 mounts and multiplying this value by 100. The lordosis quality score (LQS) is a measure of the intensity of the lordosis response. Each lordotic response was scored on a scale from 1 to 3, with 3 representing the most intense response. A score of 3 was given to a lordotic response in which the rat maximally arched its back and thrust its head upward, to nearly a 90 ° angle to the cage floor. A score of 2 was given to a lordotic response where the rat arched its back readily upon being mounted, and thrust its head upward to roughly a 60 ° angle. A score of l was given to a rat who flattened her back but did not arch it and did not thrust the head upward, but held her nose directly outward, level with the straight line of her back. A score of 0 was given to an animal that failed to display a receptive pose to the mounting male. A 0 score was not included
N E O N A T A L A C T H A N D C O R T IN T H E F E M A L E R A T
381
150-
TABLE l PLASMA CORTICOSTERONE LEVELS ON POSTNATAL DAY 4 FOLLOWING DAILY SC INJECTIONS WITH SALINE OR ACTH(l-24) Plasma Corticosterone (ng/ml)
Treatment
140 130.J 0 z
0 (D
Saline ACTH (0.1 mg/kg/day) ACTH (0.5 mg/kg/day)
n.m. 331.8 + 12.8 586.9 + 31.4
Plasma was collected 45 rain following SC injection with either saline or one of the two dosages of ACTH on postnatal day 4. Each value is the mean _+ SE of pooled samples from five animals per group (n.m. = not measurable).
to calculate the LQS, as this is a measure of the intensity of the lordosis response. A score of 0 represents failure to lordose and this is demonstrated in the LQ. The mean LQS was then calculated for each animal.
Measurement of Plasma Ovarian Steroid Hormone Levels (Radioimmunoassay) Plasma estradiol and progesterone levels were measured in adults during late proestrus, 2 h before the onset of darkness. Blood was collected via cardiac puncture with a 10-ml syringe filled with 1 ml of EDTA. The blood was centrifuged in a Beckman T J-6 refrigerated centrifuge at 5000 × g for 25 min. The plasma was placed in separate vials and stored at - 7 0 ° C until the day the assays were performed. Plasma hormone levels were determined using Coat-a-Count estradiol and progesterone radioimmunoassay (RIA) kits (sample size of 100/zl) from Diagnostics Products Corporation. The data reduction was calculated by the conventional RIA techniques of linear regression and logit-log representation with its calibrators and the aid of a computer program. The estradiol assay has a sensitivity of 8 pg/ml and the progesterone assay has a sensitivity of 0.05 ng/ml. All samples were assayed in duplicate.
Statistical Analysis Data were analyzed by the Student's t-test. Significance was determined at p < 0.05. RESULTS
Plasma Corticosterone The ACTH-treated animals showed a dose-dependent increase in plasma C O R T levels at postnatal day 4. Saline-treated animals had unrecordable levels of the stress hormone, suggesting that the stress of injection alone was not sufficient to elevate levels of endogenous A C T H and thus C O R T in the control animals (Table l).
120 -
1101009080-
706050
* p( 0.02 ** p( 0.01
f---I Saline ACTH 1 - 2 4 Treatment: ~ 7 Days F~I 25 Days i Adult
FIG. I. Changes in high-al~nity specific serotonin (5-HT) uptake (percent of control) in the hypothalamus of 7 day, 25 day, and adult (80-90 day) rats following daily ACTH(1-24) injections (SC, 0.5 mg/kg) during the first week of postnatal life. Values for saline-treated animals (control) at each age were normalized to 100% and are represented by one bar (error bar is mean of the errors over the three time points, n = 4 per subgroup).
was 18% higher among ACTH-treated animals compared to controls. Specific DA uptake was also significantly higher in A C T H treated animals at day 7 (p < 0.0 l) compared to saline animals (Fig. 2). However, at day 25 there was no difference between DA uptake values of ACTH-treated or control animals. At adulthood, although the mean specific DA uptake value was 24% above the control mean, this difference was not statistically significant because of the variability among the animals of the A C T H treatment group.
Reproductive Parameters The ACTH-treated animals had a delayed onset of puberty, as measured by the day of vaginal opening. The mean day of vaginal opening among saline-treated animals (n = 20) was 34.7 + 0.8. A m o n g ACTH-treated animals (n = 21), this value was 38.0 _+ 0.3 days postnatal (p < 0.01).
150 140,
130.
120. 110. 100
90 80 70 60
Neurotransmitter Fiber Density Assessed by Reuptake Measure The ACTH-treated animals had significantly elevated specific 5-HT uptake in the hypothalamus on day 7 (p < 0.01) and at adulthood (p < 0.02) compared to saline control animals (Fig. 1), suggesting increased fiber outgrowth at this stage in development. The mean specific 5-HT uptake at day 7 was 25% above control. By 25 days postnatal (juvenile stage), there was no significant difference in specific uptake of 5-HT between the two groups. However, at adulthood, the mean specific 5-HT uptake
50 * p( 0.01
12~ Saline ACTH 1 - 2 4 Treatment: i
z o uoy8 Adult
FIG. 2. Changes in high-at~nity specific dopamine (DA) uptake (percent of control) in the hypothalamus of 7 day, 25 day, and adult (80-90 day) rats following daily ACTH(I-24) injections (SC, 0.5 mg/kg) during the first week of postnatal life. Values for saline-treated animals (control) at each age were normalized to 100% and are represented by one bar (error bar is mean of the errors over the three time points, n = 4 per subgroup).
382
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ACTH Treatment
Saline Treatment
Saline Treatment
(*p
59%
.4
(*p(O.05)
53%
., ,i
6%
~
tntUiiiUiiWiilmh!' iJ+
-----
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29%
35%
41%
35% ~] 90%
12%
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29%
BB 100%
ACTH Treatment
[D 80%
[ ~ 70% or Less
BB3.0-2.4
~2.3-
1.7
:-:~ 1.6- 1.0
FIG. 3. Percent distribution of lordosis quotients among saline-and ACTH(1-24) (0.5 mg/kg/day)-treated animals during the first week postnatal (n = 17 per group) tested as intact virgins at 60-70 days of age. The percent of ACTH-treated animals achieving an LQ of 90-100 is only 42%, whereas about 94% of the control animals achieved scores within this range (p < 0.05).
FIG. 4. Percent distribution of mean lordotic quality scores of salineand ACTH(I-24) (0.5 mg/kg/day)-treatedanimals during the first week postnatal (n = 17 per group) tested as virginsat 60-70 days of age. The percent of saline-treated females achieving LQSs in the highest range (2.4-3.0) is 53%, a level achieved by only 12% of the treated rats (p < 0.05 ).
Animals treated with ACTH displayed decreased female sexual behavior, having lower LQs and LQSs compared to salinetreated animals when tested as young intact virgins. However, this treatment did not eradicate the expression of the lordosis response among any animals, the lowest score recorded being 40. The ACTH-treated animals had a mean LQ of 78.2 +_ 3.74 and mean LQS of 1.80 _+ 0.11 compared to 95.3 + 1.50 and 2.35 _+ 0.07 for saline-treated animals, respectively (n = 17 per group, p < 0.05). The percent distribution of these scores among each treatment group is expressed in Figs. 3 and 4.
uptake. Beginning at puberty (days 30-40), a great deal of neuronal reorganization and maturation occurs, and in fact the greatest increase in synaptic formation within the hypothalamus and other brain regions of the rat occurs around the fifth week of postnatal life (1,28,32). During this ensuing synaptogenesis that occurs at puberty, the outgrowth and/or synaptic maturation of these neuronal systems in ACTH-treated animals once again surpasses the growth among saline-treated control animals. The result is an increase in hypothalamic 5-HT innervation, and a trend toward increased DA fiber density, in adult ACTH-treated animals. The final adult innervation pattern of 5-HT neurons is not reached until 3 months postnatal (3,5), and the plasticity of these immature serotonergic neurons is shown by their susceptibility to ACTH/CORT modulation during both early development and puberty. Thus, changes occurring during the pubertal period are evident at the adulthood time point (80-90 days of age), when these neurons are mature. The DA neurons appear to mature at an earlier time; however, the first few weeks of postnatal life are also a vulnerable time for their functional maturation (8). Whether these neural changes during this peripubescent period were responsible for the delayed vaginal opening observed among these animals is not certain. The attainment of reproductive maturation in female rats (the first estrous cycle and vaginal opening) is dependent upon a luteinizing hormone (LH) surge on the morning before the first estrus (33). Luteinizing
Plasma Ovarian Steroid Levels No statistically significant differences were found in plasma estradiol or progesterone levels in adult ACTH- or saline-treated animals during late proestrus, 2 h before the onset of darkness (Table 2). DISCUSSION These results suggest that early postnatal exposure to ACTH(1-24) and CORT affects both hypothalamic monoamine innervation and reproductive parameters in the female rat. Daily administration of ACTH(1-24) during the first week of postnatal life, which in turn dramatically elevates endogenous plasma CORT levels, accelerates the maturation of the 5-HT and DA systems that innervate the hypothalamus of female rats, as measured by synaptosomal high-affinity specific uptake. While there is a linear increase in neurotransmitter uptake as neurons mature in normal, untreated animals (22), this process is accelerated in ACTH-treated animals, as evidenced by the increase in both specific 5-HT and DA uptake at day 7. By day 25, no differences exist in the uptake of either monoamine between ACTH-treated animals and controls. However, at adulthood (days 80-90), increases in specific 5-HT and DA uptake are once again observed. Thus, this treatment appears eventually to produce a permanent increase in hypothalamic monoamine innervation, particularly of 5-HT fibers, where a statistically significant increase was also found at adulthood. At 25 days of age, rats are in their juvenile stage and have not yet reached reproductive maturity. During this stage, both hypothalamic DA and 5-HT neurological systems appear to have reached a similar level of development in the control and ACTHtreated animals, with no evident differences in neurotransmitter
TABLE 2 PLASMA ESTRADIOLAND PROGESTERONELEVELS OF ADULT RATS DURING LATE PROESTRUSFOLLOWINGPOSTNATAL EXPOSURE TO ACTH(I-24) Treatment
PlasmaEstradiol (Ig/ml)
Plasma Progesterone (ng/ml)
Saline ACTH
104.0_+ 8.8 85.1 _+ 12.9
47.2 +_ 12.9 33.3-+ 11.6
Saline or ACTH(1-24) (0.5 mg/kg, SC) was administered daily from day I-7 postnatal. Plasma was collectedduring late pro~trus, 2 h before the onset of darkness in adult animals. Values represent the mean _+SE (n = 4 per treatment group). None of the values of the ACTH-treated rats is significantlydifferent from saline controls.
NEONATAL ACTH AND CORT IN THE FEMALE RAT
383
hormone release is under control ofgonadotropin-releasing hormone (GnRH) neurons within the MPOA-medial basal hypothalamus (MBH), which have monoaminergic connections. It has been suggested that the loss of an inhibitory DA tone on GnRH release just prior to puberty may be responsible for the elevated GnRH and, in turn, LH release, which ultimately leads to reproductive maturation (23,33). Perhaps the increased DA innervation of the hypothalamus among ACTH-treated animals may have contributed to a delay in this process. Our results indicate that, like the male brain, sexual differentiation of the female rat brain is susceptible to perinatal manipulation with ACTH( 1-24)/CORT, although the decreases observed in the lordosis response among females were not as dramatic as the permanent decreases in mounting behavior among prenatally treated males (36). The effect of this treatment among females was demonstrable in the delayed reproductive maturation and the decreased female sexual behavior observed among ACTH-treated animals as young adults, compared to control animals. Although some ACTH-treated animals performed as well as control animals, the initial mating stimulus evoked lower mean LQs and LQSs among ACTH-treated animals. A study by Stylianpoulou et al. (39) demonstrated that the administration of deoxycorticosterone (DOCA), an endogenous adrenal steroid, to female rat pups on postnatal days 3 and 5 significantly decreased the expression of female sexual behavior among these animals at adulthood. In addition to this apparent defeminization, these animals also appeared to have been at least partially masculinized by this treatment, as they displayed increased heterotypic or male sexual behavior following ovariectomy and testosterone replacement, compared to control animals (39). As in our study with ACTH and CORT, DOCA treatment did not eradicate the lordosis response in any animals. Treatment with the male sex steroid testosterone during the first week postnatal nearly annihilates this female sexual reflex (39). These data suggest that the stress hormones, although not potent enough to fully determine brain sexual differentiation, can somewhat alter the process by disrupting some of the neurocircuitry responsible for sexual behaviors, both masculine and feminine. Serotonin has been studied extensively for its putative role as an endogenous modulator of reproductive behavior and function in the rat. Findings suggest that this indolamine has a tonic inhibitory effect over female reproductive behavior within the ventromedial (VMN) and arcuate (ARC) nuclei of the hypothalamus and the midbrain central gray (MCG), and a possible stimulatory role in the MPOA (16,19,35), although lesioning of the MPOA facilitates the lordosis response among both male and female rats (37). From pharmacological studies investigating the mechanism of action of 5-HT on female sexual behavior, it appears that 5-HT~A receptor binding suppresses lordosis behavior in hormonally primed, ovariectomized rats (2,29) and, as shown more recently, among intact, normally cycling rats (41 ). In contrast, 5-HTIB receptor activation appears to facilitate female sexual behavior (30). The effective neural locations of these 5-HT receptor-mediated actions on lordosis have not been identified. However, an increase in 5-HT activity in the medial basal hypothalamus (MBH) is inhibitory to female sexual behavior (15) and infusion of 8-hydroxy-2-(di-n-propyl-
amino)tetralin (8-OH-DPAT), a specific 5-HTtA agonist, directly into the VMN results in a rapid, dose-dependent suppression of lordosis behavior (41 ). These data suggest that 5-HT may exert its inhibitory effect on lordosis by activation of 5-HT~A receptors within these hypothalamic regions. Dopamine appears to induce its inhibitory effect on lordosis behavior via D2 receptor activation (14). Treatment with quinpirole, a selective D2 receptor agonist, results in an inhibition of lordosis among female rats. Administration of haloperidol, a dopaminergic D2 antagonist, blocks the inhibitory effect of DA on this behavior (14). These data support the idea that this monoamine system also plays an inhibitory role in the lordosis response, probably through D2 receptor binding. Although there was a trend toward a decrease in both estradiol and progesterone among ACTH-treated animals, plasma levels of these female sex steroids were not significantly different between animals from either treatment group (Table 2). These hormones function synergistically to alter hypothalamic neurotransmitter activity to ultimately induce receptivity among female rats. The ability of the hypothalamus to respond to these hormones in this manner is determined during development when the brain is sexually differentiated. Exposure to elevated levels of ACTH and CORT during this critical time in development may disrupt this neurocircuitry necessary for maximal response to these hormones. As mentioned previously, stress hormone treatment did not eradicate the expression of receptivity; however, about 30% of the ACTH-treated animals performed receptivity scores below those observed among salinetreated animals (Figs. 3 and 4). In addition, rejection behavior, where the female kicks and attempts to repel the mounting male, accompanied by distress sounds, was observed among many of the stress hormone-treated animals, whereas this behavior was rarely seen in control animals. In general, the animals that showed this aggressive behavior were those with low receptivity scores. However, if the male was successful in mounting, very often these females would display lordosis scores within normal range. As mentioned previously, alterations in neurotransmitter systems during development can have long-term effects on the subsequent sexual behavior of the adult animal (20,21). We observed an increase in hypothalamic 5-HT and DA innervation among ACTH(l-24)-treated animals at 7 days of age. At adulthood, these animals maintained a significant increase in 5-HT fiber density, and a trend toward increased DA fiber density. The increases in these monoamine systems during development and at adulthood may be, at least in part, responsible for the delayed reproductive maturation and/or the decrease in female sexual behavior observed among ACTH-treated animals. Since ACTH(1-24) administration caused a simultaneous elevation in plasma CORT levels, we must assume that both of these stress hormones are involved in the neural and behavioral responses we are reporting. ACKNOWLEDGEMENTS We wish to thank Tommy S. Lee for his help in manuscript preparation, and Organon Inc. for supplyingthe ACTH(1-24). This work was supported by the Council for Tobacco Research.
REFERENCES
1. Aghajanian,G. K.; Bloom, F. E. The formation ofsynaptic junctions in developing rat brain: A quantitative electron microscopic study. Brain Res. 6:716-727; 1967. 2. Ahlenius, S. N.: Fernandez-Guasti, A.; Hjorth, S.; Larsson, K.
Suppression of lordosis behavior by the putative 5-HT receptor agonist 8-OH-DPATin the rat. Eur. J. Pharmacol. 124:361-363; 1986. 3. Azmitia, E. C. The serotonin producing neurons of the dorsal and median raphe nuclei. In: Iverson, L. L.; Iverson, S. D.; Snyder,
384
4. 5.
6. 7.
8.
9. 10.
11. 12. 13. 14.
15. 16.
17. 18.
19.
20.
21.
22.
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S. H., eds. The handbook of psychopharmacology. New York: Plenum Press; 1978:233-314. Azmitia, E. C.; McEwen, B. S. Adrenalconical influence on rat brain tryptophanhydroxylase activity. Brain Res. 78:291-302; 1974. Azmitia, E. C.; Brennan, M.: Quartermain, D. Adult development of the hippocampal-serotonin system of C57BL/UN mice: Analysis of high-affinity uptake of [3H]5-HT in slices and synaptosomes. Neurochem. Int. 5:39-44: 1983. Azmitia, E. C.; de Kloet, E. ACTH neuropeptide stimulation of serotonergic neuronal maturation in tissue culture: Modulation by hippocampal cells. Prog. Brain Res. 72:311-318; 1987. Biir, P. R. D.; Sbrama, L. H.; Gispen, W. H. Neurotrophic effects of ACTH/MSH-Iike peptides in the peripheral nervous system. In: de Weld, D., ed. Neuropeptides: Basics and perspectives. New York: Elsevier: 1990:175-211. Cuomo, V.; Cortese, I.; Cagiano, R.; Renna, G.; Principi, L.; Mocchetti, I.; Brunello, N.; Racagni, G. Neurochemical and behavioral changes produced by developmental exposure to psychotropic drugs influencing noradrenergic and dopaminergic transmission in the rat. In: Caiagli, F.; Giacobini, E.; Paoletti, R., eds. Developmental neuroscience: Physiological, pharmacological and clinical aspects. New York: Elsevier; 1984:167-174. Dahloff, L. G.; Hard, E.; Larsson, K. Influence of maternal stress on offspring sexual behavior. Anita. Behav. 25:958-963; 1977. Davila-Garcia, M. I.; Hlibczuk, V.; Akbari, H. M.; Alves, S. E.; Azmitia, E. C. Postnatal effects of prenatal administration of neuropeptides on 3H-5-HT uptake by serotonergic neurons. Soc. Neurosci. Abstr. 14:416; 1988. Endroczi, E. Stress and adaptation. Budapest: Akademiai Kiado: 1991. Everitt, B. J.; Fuxe, K.: Hokfelt, T. Inhibitory role ofdopamine and 5-hydroxytryptamine in the sexual behavior of female rats. Eur. J. Pharmacol. 29:187-191; 1974. Everitt, B. J.; Fuxe, K.; Hokfelt, T.; Johnsson, G. Role of monoamines in the control by hormones of sexual receptivity in the female rat. J. Comp. Physiol. Psychol. 89:556-572; 1975. Fernfindez-Guasti, A.; Ahlenius, S.; Hjonh, S.; Larsson, K. Separation ofdopaminergic and serotonergic inhibitory mechanisms in the mediation of estrogen-induced lordosis behavior in the rat. Pharmacol. Biochem. Behav. 27:93-98; 1987. Foreman, M. M.; Moss, R. k. Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat. Horm. Behav. 10:97-106: 1978. Frankfurt, M.; Rennet, K.; Azmitia, E. C.; Luine, V. Intrahypothalamic 5,7-dihydroxytryptamine: Temporal analysis of effect on 5hydroxytryptamine content in brain nuclei and on facilitated lordosis behavior. Brain Res. 340:127-133; 1985. GOtz, F.; Drrner, G. Homosexual behavior in prenatally stressed male rats after castration and oestrogen treatment in adulthood. Endokrinologie 76(1):115-117; 1980. Hansen, S.; Stanfield, E. J.; Everitt, B. J. The role of ventral bundle noradrenergic neurons in sensory components of sexual behavior and coitus-induced pseudopregnancy. Nature. 286:152-154: 1980. James, M. D.; Hole, D. R.; Wilson, C. A. Differential involvement of 5-bydroxytryptamine (5-HT) in specific hypothalamic areas in the mediation of steroid-induced changes in gonadotrophin release and sexual behavior in female rats. Neuroendocrinology 49:561569; 1989. Jarzab, B.; Dohler, K. D. Serotonergic influences on sexual differentiation of the rat brain. In: De Vries, G. J.; de Bruin, J. P. C.; Uylings, H. B. M.; Comer, M. A., eds. Progress in brain research. vol. 61. Amsterdam: Elsevier Science Publishers; 1984:119. Jarzab, B.; Sickmoller, P. M.; Kokocinska, D.; Kaminski, M.; Gubala, E.; Achtlik, W.; Wagiet, J.; Dohler, K. D. Influence of neurotransmitters on sexual differentiation of the brain. In: Gupta, D.; Wollmann, H. A.; Ranke, M. B., eds. Neuroendocrinology: New frontiers. Tubingen: Brain Research Promotion; 1990:87-96. King, J. A.; Davila-Garcia, M.; Azmitia, E. C.; Strand, F. L. Differential effects of prenatal and postnatal ACTH or nicotine exposure
23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33. 34.
35. 36. 37. 38. 39.
40. 41.
42. 43.
on 5-HT high affinity uptake in the neonatal rat brain. Int. J. De~. Neurosci. 9(3):281-286:1991. Lamberts, R.: Wuttke, W. Puberty of tkmale rats may in part be explained by decreased hypothatamic dopamine receptor sensitivit.~. Brain Res. 215:375-381; 1981. Lowry, O.: Rosebrough, N.; Farr, A.: Randall, R. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275: 1951. kuine, V. N.; Fischettc, C. T. Inhibition of lordosis behavior by intrahypothalamic implants of pargyline. Neuroendocrinology 34: 237-244: 1982. kuine, V. N.: Frankfurt, M.; Rainbow, T. C.: Biegon, A.; Azmitia, E. C. Intrahypothalamic 5,7-dihydroxytryptamine facilitates feminine sexual behavior and decreases [3H]imipramine binding and 5HT uptake. Brain Res. 264:344-348; 1983. Luine, V. N.; Thorton. J. E.: Frankfurt, M.; MacLusky, N. J. Effects of hypothalamic scrotonin depletion on lordosis behavior and gonadal hormone receptors. Brain Res. 426:47-54; 1987. Matsumoto, A,; Arai. Y. Developmental changes in synaptic formation in the hypothalamus arcuate nucleus of female rats. Cell Tissue Res. 169:143-146:1976. Mendelson, S. D.: Gorsalka, B. B. 5-HT~A receptors: Differential involvement in female and male sexual behavior in the rat. Physiol. Behav. 37:345-351: 1986. Mendelson, S. D.: Gorsalka, B. B. Differential effects of 5-HT~B agonists on female and male sexual behavior in the rat. Soc. Neurosei. Abstr. 14:372: 1988. Meyerson, B. J. Central nervous monoamines and hormone-induced estrus behavior in the spayed rat. Acta Physiol. Seand. 63(Suppl. 241 ):3-32: 1964. Miller, L. D. P.: Aoki, A. Stereological analysis of the hypothalamic ventromedial nucleus II. Hormone-induced changes in the synaptogenic pattern. Dev. Brain Res. 61:189-196; 1991. Ojeda. S. R.: Urbanski, H. F. Puberty in the rat. in: Knobil, E.; Neill, J., eds. The physiology of reproduction, vol. II. New York: Raven Press, Ltd.: 1988:1699-1737. Parvez, H.; Casimiri, V.: Kiuchi, Y.; Nevo, I.; Elisebetb, P.; Castro, L.: Hobel, C. J.; Parvez, S. Contribution of pituitary-adrenal hormones in maturation of catecholamine regulating enzymes during pre and postnatal life. In: Gupta, D.; Wollmann, H. A.; Ranke, M. B., eds. Neuroendocrinology: New frontiers. Ttibingen, Germany: Brain Research Promotion; 1990:104. Renner, K. J.: Krey. L. C.: kuine, V. N. Effect of progesterone on monoamine turnover in the brain of the estrogen-primed rat. Brain Res. Bull. 19:105-202: 1987. Segarra, A. C.: Luine, V. N.: Strand, F. L. Sexual behavior of male rats is differentially affected by timing of perinatal ACTH administration. Physiol. Behav. 50:689-697; 1991. Singer. J. Hypothalamic control ofmale and female sexual behavior in female rats. J. Comp. Physiol. Psychol. 66:738-742: 1968. Strand, F. k.: Rose. K. J.: Zuccarelli, L. A.: Kume, J.: Alves, S. E.; Antonawich, F. J.; Garrett, k. Y. Neuropeptide hormones as neurotrophic factors. Physiol. Rev. 71:1017-1046; 1991. Stylianopoulou, F.; Fameli, M.: Brountzos, E.; Contopoulos, A. N. Neonatal neural organizing effects of exogenous corticosteroids on sexual differentiation of the brain in the female rat. Horm. Behav. 17:332-341: 1983. Sze, P. Y.; Neckers, L.; Towle. A. C. Glucocorticoids as a regulatory factor for brain tryptophan hydroxylase. J. Neurochem. 26:169173: 1976. Uphouse, L.; Montanez, S.: Richards-Hill, R.; Caldarola-Pastuszka, M.; Droge, M. Effects of the 5-HT~Aagonist, 8-OH-DPAT, on sexual behaviors of the proestrus rat. Pharmacol. Biochem. Behav. 39:635640; 199 I. Ward, I. L. Prenatal stress feminizes and demasculinizes the behavior of males. Science 143:212-218:1972. Wolterink, G.: Van Zanten, E.: Kamsteeg, H.; Radhakishun, F. S.: Van Ree, J. M. Functional recovery after destruction ofdopamine systems in the nucleus accumbens of rats.ll. Facilitation by the ACTH-(4-9) analog ORG 2766. Brain Res. 507:101-108; 1990.
0196-9781/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the USA.
Neonatal ACIH and Corticosterone Alter Hypothalamic Monoamine Innervation and Reproductive Parameters in the Female Rat STEPHEN
E. A L V E S , H O M A Y O O N
M. A K B A R I ,
EFRAIN
C. A Z M I T I A
AND
FLEUR
L. S T R A N D ~
Department of Biology and the Center for Neural Science, New York University, Washington Square, New York, N Y 10003 R e c e i v e d 10 M a r c h 1992 ALVES, S. E., H. M. AKBARI, E. C. AZMITIA AND F. L. STRAND. Neonatal ACTH and corticosterone alter hypothalamic monoamine innervation and reproductive parameters in the female rat. PEPTIDES 14(2) 379-384, 1993.--Female SpragueDawley rat pups were injected SC with either ACTH(1-24) (0.5 mg/kg) or saline vehicle once daily from postnatal day 1 (day of birth) to day 7. Plasma corticosterone (CORT) levels were recorded via radioimmunoassay (RIA) on day 4 to measure adrenal response to these treatments. Hypothalamic 5-HT and DA fiber densities were assessed using high-atfinity specific 3H-5-HT and 3H-DA uptake at days 7, 25, and at adulthood (80-90 days). Animals were checked daily for vaginal opening (starting on day 30) as a sign of sexual maturation and later tested for sexual behavior as virgins (60-70 days of age). Plasma estradiol and progesterone levels were measured via RIA. Plasma CORT levels were greatly increased among ACTH-treated animals during the treatment period. The 5-HT uptake was significantly increased in ACTH-treated animals at day 7 (p < 0.01 ) and at adulthood (p < 0.02) compared to controls. The DA uptake was significantly higher among ACTH-treated animals at day 7 (p < 0.01). The sexual maturation of ACTH-treated animals was delayed when compared to control animals (p < 0.01). The ACTH-treated animals displayed slight deficits in female sexual behavior compared to control animals (p < 0.05). No significant changes in plasma sex steroid levels were found. Based on this study, we suggest that monoamine innervation into the developing female hypothalamus is susceptible to early postnatal manipulation with ACTH and CORT. and that the resulting changes in these monoamine fiber densities may be responsible for the observed deficits in reproductive maturation and behavior. ACTH Hypothalamic monoamines Sexual behavior
Serotonin
Dopamine
T H E monoaminergic innervation of the hypothalamus is believed to play an important regulatory role over reproductive behavior and function in the female rat ( 12,13,18,19,25-27,3 i ). The activity of these neurons within discrete hypothalamic nuclei, mediated in part by the gonadal steroids, dictates the expression of sexual behavior (the lordosis response) upon the appropriate stimulus or the eventual release of gonadotropins from the pituitary. The development of this neurocircuitry in the female direction (a cyclic hypothalamus) begins during the last week of gestation and continues through the first week of postnatal life. Manipulation of these neurotransmitter systems during this critical time can permanently alter the future expression of sexual behavior in adult female rats (20,21). Increasing serotonin (5-HT) levels by the administration of its precursor, /-tryptophan, during the first 7 days of postnatal life results in a decreased capacity for lordosis behavior among adult female rats (21 ). While sex steroids dominate the organization of developing neural pathways essential for male or female sexual behavior,
Female reproductive maturation
Lordosis
either perinatal stress (9,17,42) or perinatal administration of the stress-evoked peptide hormone adrenocorticotropic hormone (ACTH) (36) seriously diminishes subsequent male sexual behavior. A stress-induced rise of plasma corticosteroid levels can influence the maturation of developing central m o n o a m i n e systems (11). Adrenocorticotropic hormone, a 39 a m i n o acid pituitary peptide, is the primary endogenous releaser of corticosteroids from the adrenal cortex during a stress response. In addition to this classical role, this peptide hormone and its fragments [ACTH(4-10), A C T H ( 4 - 9 ) [Org 2766], A C T H ( I - 1 3 ) [aMSH], and A C T H ( I - 2 4 ) ] have direct neurotrophic properties, affecting the development and regeneration of both central and peripheral neurons (7,38). The central 5-HT system appears to be intimately associated with this pituitary-adrenal endocrine system, as these neurons are affected by both the A C T H peptides (6, I0) and the adrenal steroids (4,40). In addition, recent evidence suggests that dopamine neurons are also sensitive to ACTH/corticosteroid modulation (34,43). Both of these monoamines are believed to
i Requests for reprints should be addressed to Dr. Fleur L. Strand, Biology Department, Room 1009 Main Bldg., New York University, Washington Square, New York, NY 10003. 379
380
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be involved in the control of reproductive processes in the female rat. The developing 5-HT system appears to be particularly sensitive to changes in plasma corticosteroid concentration, and it has been suggested that behavioral alterations seen in offspring from stressed mothers may be due to changes in this monoamine system (11). Prenatal administration of ACTH(1-24) (which has the full corticosteroidogenic properties of the whole peptide) during the last week of gestation alters the sexual differentiation of the brain in male rats, decreasing the expression of male sexual behavior among these animals at adulthood (36). This decrease in sexual behavior is correlated with an increase in 5-HT in the medial preoptic area (MPOA), the primary regulatory site for male sexual behavior in the rat. Another study in our laboratory has shown that early postnatal administration of ACTH accelerates the developing 5-HT system within the brainstem and hippocampus of neonatal and juvenile rats (22). The current study investigates whether exposure to elevated levels of the pituitary-adrenal stress hormones, ACTH and corticosterone (CORT), during the first postnatal week would affect the development of 5-HT or DA systems within the hypothalamus of female rats, and the subsequent reproductive capacity of these animals at adulthood. METHOD Pregnant Sprague-Dawley rats were ordered from Hilltop Laboratories, housed individually, and water and Purina rat chow were provided ad lib. They were kept in a 12L:12D photoperiod with lights offat 1200. On the day of birth (day 1), the pups were weighed and each litter culled to five females and three males to maintain the same sex ratio.
Treatment Female rat pups were injected from day 1 to day 7 with either ACTH(1-24) (0.5 mg/kg), donated by Organon Inc., or 0.9% saline, once daily. The ACTH(1-24) was dissolved in saline and administered subcutaneously (SC) in a volume of 0.1 ml/10 g per injection. On postnatal day 22, males and females were separated from the dam and housed in groups of the same sex with two to three animals per cage.
Corticosterone Radioimmunoassay Two dosages of ACTH(1-24) were used for this study. On postnatal day 4, 45 rain following the injection of ACTH(1-24) (0.5 mg/kg), ACTH(I-24) (0.1 mg/kg), or saline vehicle, five pups from each treatment group were decapitated; trunk blood was collected in EDTA and pooled. The blood was centrifuged with a Beckman T J-6 refrigerated centrifuge at 5000 × g for 25 min. The plasma was placed in vials and stored at - 7 0 ° C until the assay was performed. Plasma levels of corticosterone were determined using a Coat-A-Count rat corticosterone radioimmunoassay from Diagnostic Products Corporation with a sensitivity of up to 5.7 ng/ml. Determination of hormonal levels was interpolated from a standard curve prepared in triplicate using calibrators and quality control serum pools. The data reduction was calculated by linear regression and logit-log representation with the aid of a computer program. The size of the aliquot required was 50 vl, and all samples were run in triplicate.
Measurement of Neurotransmitter Fiber Densities Hypothalamic synaptosomal high-aflinity specific uptake was measured to assess hypothalamic neurotransmitter fiber density. Specific 5-HT and DA uptake was measured at various stages
during development: day 7 (neonatal), day 25 (juvenile), and adult (80-90 days, diestrus). High-affinity uptake. Following the technique of Azmitia et al. (5), animals were rapidly decapitated under sodium pentobarbital anesthesia and the hypothalamus was dissected out, weighed, and placed in ice-cold 0.32 M sucrose (10X volume). The tissue was homogenized with a glass Teflon homogenizer and centrifuged at 300 × g for 10 min; supernatant (SI) was saved, the pellet was resuspended in 2 ml 0.32 M sucrose, and centrifuged at 300 × g for 10 min. The resulting supernatant ($2) was saved and the pellet was discarded. Both St and $2 were combined and centrifuged at 12,500 × g for l0 min. The supernatant was discarded and the pellet was resuspended in 10X v/w Krebs-Ringer (K-R) (Sigma) with 10 s M pargyline (P), pH 7.4. Tissue (40 vl) was incubated for 5 min at 37°C with either 140 /~1 K-R and P or 120 /~1 K-R and P, and 20 ul of fluoxetine or nomifensine (5 × 10-5 M final concentration) for determination of nonspecific uptake of 5-HT and DA, respectively: 20 ul of SH-5HT (5 × 10_8 Mfinal concentration) or 3HDA (1 × l0 7 31 final concentration) were added to each well and tissue was incubated for an additional 10 min. A total of eight wells/condition was performed (four total uptake and four nonspecific uptake). Tissue was harvested onto nitrocellulose filters (Skatron, Inc.) with 0.1 M phosphate-buffered saline using a Titertek cell harvester. Filters were placed in counting vials with 3 ml of Liquiscint and counted in a Beckman counter (efficiency 0.38). Protein content of each sample was calculated by the method of Lowry. et al. (24).
Reproductive Parameters Maturation. Beginning on postnatal day 30, female offspring were checked daily for vaginal opening as an indication of reproductive maturation (onset of puberty). Female sexual behavior. Between 60 and 70 days of age, the animals were tested for female sexual behavior as intact virgins. One week prior to testing, vaginal smears were recorded daily, 2 h before the onset of darkness. Animals in late proestrus (basal bodies being the predominant cell type in the smear) were tested for sexual behavior 2 h after the onset of darkness. Each female was placed in a rectangular aquarium under red lighting, containing a sexually experienced, sterile male (castrated males were used to prevent impregnation of the females). Under a combination ketamine-xylazine anesthesia, these males had been castrated and 4 cm of silastic tubing, filled with testosterone propionate [a dosage that is effective in producing male sexual behavior among castrates (Luine, personal communication)], was implanted subcutaneously in the dorsal neck region. The lordosis quotient (LQ), a quantitative measure of female sexual behavior, was determined by rotating the female between three males until she had been mounted a total of 10 times. The LQ for each animal was then calculated by dividing the total number of lordotic responses by 10 mounts and multiplying this value by 100. The lordosis quality score (LQS) is a measure of the intensity of the lordosis response. Each lordotic response was scored on a scale from 1 to 3, with 3 representing the most intense response. A score of 3 was given to a lordotic response in which the rat maximally arched its back and thrust its head upward, to nearly a 90 ° angle to the cage floor. A score of 2 was given to a lordotic response where the rat arched its back readily upon being mounted, and thrust its head upward to roughly a 60 ° angle. A score of l was given to a rat who flattened her back but did not arch it and did not thrust the head upward, but held her nose directly outward, level with the straight line of her back. A score of 0 was given to an animal that failed to display a receptive pose to the mounting male. A 0 score was not included
N E O N A T A L A C T H A N D C O R T IN T H E F E M A L E R A T
381
150-
TABLE l PLASMA CORTICOSTERONE LEVELS ON POSTNATAL DAY 4 FOLLOWING DAILY SC INJECTIONS WITH SALINE OR ACTH(l-24) Plasma Corticosterone (ng/ml)
Treatment
140 130.J 0 z
0 (D
Saline ACTH (0.1 mg/kg/day) ACTH (0.5 mg/kg/day)
n.m. 331.8 + 12.8 586.9 + 31.4
Plasma was collected 45 rain following SC injection with either saline or one of the two dosages of ACTH on postnatal day 4. Each value is the mean _+ SE of pooled samples from five animals per group (n.m. = not measurable).
to calculate the LQS, as this is a measure of the intensity of the lordosis response. A score of 0 represents failure to lordose and this is demonstrated in the LQ. The mean LQS was then calculated for each animal.
Measurement of Plasma Ovarian Steroid Hormone Levels (Radioimmunoassay) Plasma estradiol and progesterone levels were measured in adults during late proestrus, 2 h before the onset of darkness. Blood was collected via cardiac puncture with a 10-ml syringe filled with 1 ml of EDTA. The blood was centrifuged in a Beckman T J-6 refrigerated centrifuge at 5000 × g for 25 min. The plasma was placed in separate vials and stored at - 7 0 ° C until the day the assays were performed. Plasma hormone levels were determined using Coat-a-Count estradiol and progesterone radioimmunoassay (RIA) kits (sample size of 100/zl) from Diagnostics Products Corporation. The data reduction was calculated by the conventional RIA techniques of linear regression and logit-log representation with its calibrators and the aid of a computer program. The estradiol assay has a sensitivity of 8 pg/ml and the progesterone assay has a sensitivity of 0.05 ng/ml. All samples were assayed in duplicate.
Statistical Analysis Data were analyzed by the Student's t-test. Significance was determined at p < 0.05. RESULTS
Plasma Corticosterone The ACTH-treated animals showed a dose-dependent increase in plasma C O R T levels at postnatal day 4. Saline-treated animals had unrecordable levels of the stress hormone, suggesting that the stress of injection alone was not sufficient to elevate levels of endogenous A C T H and thus C O R T in the control animals (Table l).
120 -
1101009080-
706050
* p( 0.02 ** p( 0.01
f---I Saline ACTH 1 - 2 4 Treatment: ~ 7 Days F~I 25 Days i Adult
FIG. I. Changes in high-al~nity specific serotonin (5-HT) uptake (percent of control) in the hypothalamus of 7 day, 25 day, and adult (80-90 day) rats following daily ACTH(1-24) injections (SC, 0.5 mg/kg) during the first week of postnatal life. Values for saline-treated animals (control) at each age were normalized to 100% and are represented by one bar (error bar is mean of the errors over the three time points, n = 4 per subgroup).
was 18% higher among ACTH-treated animals compared to controls. Specific DA uptake was also significantly higher in A C T H treated animals at day 7 (p < 0.0 l) compared to saline animals (Fig. 2). However, at day 25 there was no difference between DA uptake values of ACTH-treated or control animals. At adulthood, although the mean specific DA uptake value was 24% above the control mean, this difference was not statistically significant because of the variability among the animals of the A C T H treatment group.
Reproductive Parameters The ACTH-treated animals had a delayed onset of puberty, as measured by the day of vaginal opening. The mean day of vaginal opening among saline-treated animals (n = 20) was 34.7 + 0.8. A m o n g ACTH-treated animals (n = 21), this value was 38.0 _+ 0.3 days postnatal (p < 0.01).
150 140,
130.
120. 110. 100
90 80 70 60
Neurotransmitter Fiber Density Assessed by Reuptake Measure The ACTH-treated animals had significantly elevated specific 5-HT uptake in the hypothalamus on day 7 (p < 0.01) and at adulthood (p < 0.02) compared to saline control animals (Fig. 1), suggesting increased fiber outgrowth at this stage in development. The mean specific 5-HT uptake at day 7 was 25% above control. By 25 days postnatal (juvenile stage), there was no significant difference in specific uptake of 5-HT between the two groups. However, at adulthood, the mean specific 5-HT uptake
50 * p( 0.01
12~ Saline ACTH 1 - 2 4 Treatment: i
z o uoy8 Adult
FIG. 2. Changes in high-at~nity specific dopamine (DA) uptake (percent of control) in the hypothalamus of 7 day, 25 day, and adult (80-90 day) rats following daily ACTH(I-24) injections (SC, 0.5 mg/kg) during the first week of postnatal life. Values for saline-treated animals (control) at each age were normalized to 100% and are represented by one bar (error bar is mean of the errors over the three time points, n = 4 per subgroup).
382
.,~[.VES ET .~1,.
ACTH Treatment
Saline Treatment
Saline Treatment
(*p
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35%
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29%
BB 100%
ACTH Treatment
[D 80%
[ ~ 70% or Less
BB3.0-2.4
~2.3-
1.7
:-:~ 1.6- 1.0
FIG. 3. Percent distribution of lordosis quotients among saline-and ACTH(1-24) (0.5 mg/kg/day)-treated animals during the first week postnatal (n = 17 per group) tested as intact virgins at 60-70 days of age. The percent of ACTH-treated animals achieving an LQ of 90-100 is only 42%, whereas about 94% of the control animals achieved scores within this range (p < 0.05).
FIG. 4. Percent distribution of mean lordotic quality scores of salineand ACTH(I-24) (0.5 mg/kg/day)-treatedanimals during the first week postnatal (n = 17 per group) tested as virginsat 60-70 days of age. The percent of saline-treated females achieving LQSs in the highest range (2.4-3.0) is 53%, a level achieved by only 12% of the treated rats (p < 0.05 ).
Animals treated with ACTH displayed decreased female sexual behavior, having lower LQs and LQSs compared to salinetreated animals when tested as young intact virgins. However, this treatment did not eradicate the expression of the lordosis response among any animals, the lowest score recorded being 40. The ACTH-treated animals had a mean LQ of 78.2 +_ 3.74 and mean LQS of 1.80 _+ 0.11 compared to 95.3 + 1.50 and 2.35 _+ 0.07 for saline-treated animals, respectively (n = 17 per group, p < 0.05). The percent distribution of these scores among each treatment group is expressed in Figs. 3 and 4.
uptake. Beginning at puberty (days 30-40), a great deal of neuronal reorganization and maturation occurs, and in fact the greatest increase in synaptic formation within the hypothalamus and other brain regions of the rat occurs around the fifth week of postnatal life (1,28,32). During this ensuing synaptogenesis that occurs at puberty, the outgrowth and/or synaptic maturation of these neuronal systems in ACTH-treated animals once again surpasses the growth among saline-treated control animals. The result is an increase in hypothalamic 5-HT innervation, and a trend toward increased DA fiber density, in adult ACTH-treated animals. The final adult innervation pattern of 5-HT neurons is not reached until 3 months postnatal (3,5), and the plasticity of these immature serotonergic neurons is shown by their susceptibility to ACTH/CORT modulation during both early development and puberty. Thus, changes occurring during the pubertal period are evident at the adulthood time point (80-90 days of age), when these neurons are mature. The DA neurons appear to mature at an earlier time; however, the first few weeks of postnatal life are also a vulnerable time for their functional maturation (8). Whether these neural changes during this peripubescent period were responsible for the delayed vaginal opening observed among these animals is not certain. The attainment of reproductive maturation in female rats (the first estrous cycle and vaginal opening) is dependent upon a luteinizing hormone (LH) surge on the morning before the first estrus (33). Luteinizing
Plasma Ovarian Steroid Levels No statistically significant differences were found in plasma estradiol or progesterone levels in adult ACTH- or saline-treated animals during late proestrus, 2 h before the onset of darkness (Table 2). DISCUSSION These results suggest that early postnatal exposure to ACTH(1-24) and CORT affects both hypothalamic monoamine innervation and reproductive parameters in the female rat. Daily administration of ACTH(1-24) during the first week of postnatal life, which in turn dramatically elevates endogenous plasma CORT levels, accelerates the maturation of the 5-HT and DA systems that innervate the hypothalamus of female rats, as measured by synaptosomal high-affinity specific uptake. While there is a linear increase in neurotransmitter uptake as neurons mature in normal, untreated animals (22), this process is accelerated in ACTH-treated animals, as evidenced by the increase in both specific 5-HT and DA uptake at day 7. By day 25, no differences exist in the uptake of either monoamine between ACTH-treated animals and controls. However, at adulthood (days 80-90), increases in specific 5-HT and DA uptake are once again observed. Thus, this treatment appears eventually to produce a permanent increase in hypothalamic monoamine innervation, particularly of 5-HT fibers, where a statistically significant increase was also found at adulthood. At 25 days of age, rats are in their juvenile stage and have not yet reached reproductive maturity. During this stage, both hypothalamic DA and 5-HT neurological systems appear to have reached a similar level of development in the control and ACTHtreated animals, with no evident differences in neurotransmitter
TABLE 2 PLASMA ESTRADIOLAND PROGESTERONELEVELS OF ADULT RATS DURING LATE PROESTRUSFOLLOWINGPOSTNATAL EXPOSURE TO ACTH(I-24) Treatment
PlasmaEstradiol (Ig/ml)
Plasma Progesterone (ng/ml)
Saline ACTH
104.0_+ 8.8 85.1 _+ 12.9
47.2 +_ 12.9 33.3-+ 11.6
Saline or ACTH(1-24) (0.5 mg/kg, SC) was administered daily from day I-7 postnatal. Plasma was collectedduring late pro~trus, 2 h before the onset of darkness in adult animals. Values represent the mean _+SE (n = 4 per treatment group). None of the values of the ACTH-treated rats is significantlydifferent from saline controls.
NEONATAL ACTH AND CORT IN THE FEMALE RAT
383
hormone release is under control ofgonadotropin-releasing hormone (GnRH) neurons within the MPOA-medial basal hypothalamus (MBH), which have monoaminergic connections. It has been suggested that the loss of an inhibitory DA tone on GnRH release just prior to puberty may be responsible for the elevated GnRH and, in turn, LH release, which ultimately leads to reproductive maturation (23,33). Perhaps the increased DA innervation of the hypothalamus among ACTH-treated animals may have contributed to a delay in this process. Our results indicate that, like the male brain, sexual differentiation of the female rat brain is susceptible to perinatal manipulation with ACTH( 1-24)/CORT, although the decreases observed in the lordosis response among females were not as dramatic as the permanent decreases in mounting behavior among prenatally treated males (36). The effect of this treatment among females was demonstrable in the delayed reproductive maturation and the decreased female sexual behavior observed among ACTH-treated animals as young adults, compared to control animals. Although some ACTH-treated animals performed as well as control animals, the initial mating stimulus evoked lower mean LQs and LQSs among ACTH-treated animals. A study by Stylianpoulou et al. (39) demonstrated that the administration of deoxycorticosterone (DOCA), an endogenous adrenal steroid, to female rat pups on postnatal days 3 and 5 significantly decreased the expression of female sexual behavior among these animals at adulthood. In addition to this apparent defeminization, these animals also appeared to have been at least partially masculinized by this treatment, as they displayed increased heterotypic or male sexual behavior following ovariectomy and testosterone replacement, compared to control animals (39). As in our study with ACTH and CORT, DOCA treatment did not eradicate the lordosis response in any animals. Treatment with the male sex steroid testosterone during the first week postnatal nearly annihilates this female sexual reflex (39). These data suggest that the stress hormones, although not potent enough to fully determine brain sexual differentiation, can somewhat alter the process by disrupting some of the neurocircuitry responsible for sexual behaviors, both masculine and feminine. Serotonin has been studied extensively for its putative role as an endogenous modulator of reproductive behavior and function in the rat. Findings suggest that this indolamine has a tonic inhibitory effect over female reproductive behavior within the ventromedial (VMN) and arcuate (ARC) nuclei of the hypothalamus and the midbrain central gray (MCG), and a possible stimulatory role in the MPOA (16,19,35), although lesioning of the MPOA facilitates the lordosis response among both male and female rats (37). From pharmacological studies investigating the mechanism of action of 5-HT on female sexual behavior, it appears that 5-HT~A receptor binding suppresses lordosis behavior in hormonally primed, ovariectomized rats (2,29) and, as shown more recently, among intact, normally cycling rats (41 ). In contrast, 5-HTIB receptor activation appears to facilitate female sexual behavior (30). The effective neural locations of these 5-HT receptor-mediated actions on lordosis have not been identified. However, an increase in 5-HT activity in the medial basal hypothalamus (MBH) is inhibitory to female sexual behavior (15) and infusion of 8-hydroxy-2-(di-n-propyl-
amino)tetralin (8-OH-DPAT), a specific 5-HTtA agonist, directly into the VMN results in a rapid, dose-dependent suppression of lordosis behavior (41 ). These data suggest that 5-HT may exert its inhibitory effect on lordosis by activation of 5-HT~A receptors within these hypothalamic regions. Dopamine appears to induce its inhibitory effect on lordosis behavior via D2 receptor activation (14). Treatment with quinpirole, a selective D2 receptor agonist, results in an inhibition of lordosis among female rats. Administration of haloperidol, a dopaminergic D2 antagonist, blocks the inhibitory effect of DA on this behavior (14). These data support the idea that this monoamine system also plays an inhibitory role in the lordosis response, probably through D2 receptor binding. Although there was a trend toward a decrease in both estradiol and progesterone among ACTH-treated animals, plasma levels of these female sex steroids were not significantly different between animals from either treatment group (Table 2). These hormones function synergistically to alter hypothalamic neurotransmitter activity to ultimately induce receptivity among female rats. The ability of the hypothalamus to respond to these hormones in this manner is determined during development when the brain is sexually differentiated. Exposure to elevated levels of ACTH and CORT during this critical time in development may disrupt this neurocircuitry necessary for maximal response to these hormones. As mentioned previously, stress hormone treatment did not eradicate the expression of receptivity; however, about 30% of the ACTH-treated animals performed receptivity scores below those observed among salinetreated animals (Figs. 3 and 4). In addition, rejection behavior, where the female kicks and attempts to repel the mounting male, accompanied by distress sounds, was observed among many of the stress hormone-treated animals, whereas this behavior was rarely seen in control animals. In general, the animals that showed this aggressive behavior were those with low receptivity scores. However, if the male was successful in mounting, very often these females would display lordosis scores within normal range. As mentioned previously, alterations in neurotransmitter systems during development can have long-term effects on the subsequent sexual behavior of the adult animal (20,21). We observed an increase in hypothalamic 5-HT and DA innervation among ACTH(l-24)-treated animals at 7 days of age. At adulthood, these animals maintained a significant increase in 5-HT fiber density, and a trend toward increased DA fiber density. The increases in these monoamine systems during development and at adulthood may be, at least in part, responsible for the delayed reproductive maturation and/or the decrease in female sexual behavior observed among ACTH-treated animals. Since ACTH(1-24) administration caused a simultaneous elevation in plasma CORT levels, we must assume that both of these stress hormones are involved in the neural and behavioral responses we are reporting. ACKNOWLEDGEMENTS We wish to thank Tommy S. Lee for his help in manuscript preparation, and Organon Inc. for supplyingthe ACTH(1-24). This work was supported by the Council for Tobacco Research.
REFERENCES
1. Aghajanian,G. K.; Bloom, F. E. The formation ofsynaptic junctions in developing rat brain: A quantitative electron microscopic study. Brain Res. 6:716-727; 1967. 2. Ahlenius, S. N.: Fernandez-Guasti, A.; Hjorth, S.; Larsson, K.
Suppression of lordosis behavior by the putative 5-HT receptor agonist 8-OH-DPATin the rat. Eur. J. Pharmacol. 124:361-363; 1986. 3. Azmitia, E. C. The serotonin producing neurons of the dorsal and median raphe nuclei. In: Iverson, L. L.; Iverson, S. D.; Snyder,
384
4. 5.
6. 7.
8.
9. 10.
11. 12. 13. 14.
15. 16.
17. 18.
19.
20.
21.
22.
ALVES ET AL.
S. H., eds. The handbook of psychopharmacology. New York: Plenum Press; 1978:233-314. Azmitia, E. C.; McEwen, B. S. Adrenalconical influence on rat brain tryptophanhydroxylase activity. Brain Res. 78:291-302; 1974. Azmitia, E. C.; Brennan, M.: Quartermain, D. Adult development of the hippocampal-serotonin system of C57BL/UN mice: Analysis of high-affinity uptake of [3H]5-HT in slices and synaptosomes. Neurochem. Int. 5:39-44: 1983. Azmitia, E. C.; de Kloet, E. ACTH neuropeptide stimulation of serotonergic neuronal maturation in tissue culture: Modulation by hippocampal cells. Prog. Brain Res. 72:311-318; 1987. Biir, P. R. D.; Sbrama, L. H.; Gispen, W. H. Neurotrophic effects of ACTH/MSH-Iike peptides in the peripheral nervous system. In: de Weld, D., ed. Neuropeptides: Basics and perspectives. New York: Elsevier: 1990:175-211. Cuomo, V.; Cortese, I.; Cagiano, R.; Renna, G.; Principi, L.; Mocchetti, I.; Brunello, N.; Racagni, G. Neurochemical and behavioral changes produced by developmental exposure to psychotropic drugs influencing noradrenergic and dopaminergic transmission in the rat. In: Caiagli, F.; Giacobini, E.; Paoletti, R., eds. Developmental neuroscience: Physiological, pharmacological and clinical aspects. New York: Elsevier; 1984:167-174. Dahloff, L. G.; Hard, E.; Larsson, K. Influence of maternal stress on offspring sexual behavior. Anita. Behav. 25:958-963; 1977. Davila-Garcia, M. I.; Hlibczuk, V.; Akbari, H. M.; Alves, S. E.; Azmitia, E. C. Postnatal effects of prenatal administration of neuropeptides on 3H-5-HT uptake by serotonergic neurons. Soc. Neurosci. Abstr. 14:416; 1988. Endroczi, E. Stress and adaptation. Budapest: Akademiai Kiado: 1991. Everitt, B. J.; Fuxe, K.: Hokfelt, T. Inhibitory role ofdopamine and 5-hydroxytryptamine in the sexual behavior of female rats. Eur. J. Pharmacol. 29:187-191; 1974. Everitt, B. J.; Fuxe, K.; Hokfelt, T.; Johnsson, G. Role of monoamines in the control by hormones of sexual receptivity in the female rat. J. Comp. Physiol. Psychol. 89:556-572; 1975. Fernfindez-Guasti, A.; Ahlenius, S.; Hjonh, S.; Larsson, K. Separation ofdopaminergic and serotonergic inhibitory mechanisms in the mediation of estrogen-induced lordosis behavior in the rat. Pharmacol. Biochem. Behav. 27:93-98; 1987. Foreman, M. M.; Moss, R. k. Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat. Horm. Behav. 10:97-106: 1978. Frankfurt, M.; Rennet, K.; Azmitia, E. C.; Luine, V. Intrahypothalamic 5,7-dihydroxytryptamine: Temporal analysis of effect on 5hydroxytryptamine content in brain nuclei and on facilitated lordosis behavior. Brain Res. 340:127-133; 1985. GOtz, F.; Drrner, G. Homosexual behavior in prenatally stressed male rats after castration and oestrogen treatment in adulthood. Endokrinologie 76(1):115-117; 1980. Hansen, S.; Stanfield, E. J.; Everitt, B. J. The role of ventral bundle noradrenergic neurons in sensory components of sexual behavior and coitus-induced pseudopregnancy. Nature. 286:152-154: 1980. James, M. D.; Hole, D. R.; Wilson, C. A. Differential involvement of 5-bydroxytryptamine (5-HT) in specific hypothalamic areas in the mediation of steroid-induced changes in gonadotrophin release and sexual behavior in female rats. Neuroendocrinology 49:561569; 1989. Jarzab, B.; Dohler, K. D. Serotonergic influences on sexual differentiation of the rat brain. In: De Vries, G. J.; de Bruin, J. P. C.; Uylings, H. B. M.; Comer, M. A., eds. Progress in brain research. vol. 61. Amsterdam: Elsevier Science Publishers; 1984:119. Jarzab, B.; Sickmoller, P. M.; Kokocinska, D.; Kaminski, M.; Gubala, E.; Achtlik, W.; Wagiet, J.; Dohler, K. D. Influence of neurotransmitters on sexual differentiation of the brain. In: Gupta, D.; Wollmann, H. A.; Ranke, M. B., eds. Neuroendocrinology: New frontiers. Tubingen: Brain Research Promotion; 1990:87-96. King, J. A.; Davila-Garcia, M.; Azmitia, E. C.; Strand, F. L. Differential effects of prenatal and postnatal ACTH or nicotine exposure
23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33. 34.
35. 36. 37. 38. 39.
40. 41.
42. 43.
on 5-HT high affinity uptake in the neonatal rat brain. Int. J. De~. Neurosci. 9(3):281-286:1991. Lamberts, R.: Wuttke, W. Puberty of tkmale rats may in part be explained by decreased hypothatamic dopamine receptor sensitivit.~. Brain Res. 215:375-381; 1981. Lowry, O.: Rosebrough, N.; Farr, A.: Randall, R. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275: 1951. kuine, V. N.; Fischettc, C. T. Inhibition of lordosis behavior by intrahypothalamic implants of pargyline. Neuroendocrinology 34: 237-244: 1982. kuine, V. N.: Frankfurt, M.; Rainbow, T. C.: Biegon, A.; Azmitia, E. C. Intrahypothalamic 5,7-dihydroxytryptamine facilitates feminine sexual behavior and decreases [3H]imipramine binding and 5HT uptake. Brain Res. 264:344-348; 1983. Luine, V. N.; Thorton. J. E.: Frankfurt, M.; MacLusky, N. J. Effects of hypothalamic scrotonin depletion on lordosis behavior and gonadal hormone receptors. Brain Res. 426:47-54; 1987. Matsumoto, A,; Arai. Y. Developmental changes in synaptic formation in the hypothalamus arcuate nucleus of female rats. Cell Tissue Res. 169:143-146:1976. Mendelson, S. D.: Gorsalka, B. B. 5-HT~A receptors: Differential involvement in female and male sexual behavior in the rat. Physiol. Behav. 37:345-351: 1986. Mendelson, S. D.: Gorsalka, B. B. Differential effects of 5-HT~B agonists on female and male sexual behavior in the rat. Soc. Neurosei. Abstr. 14:372: 1988. Meyerson, B. J. Central nervous monoamines and hormone-induced estrus behavior in the spayed rat. Acta Physiol. Seand. 63(Suppl. 241 ):3-32: 1964. Miller, L. D. P.: Aoki, A. Stereological analysis of the hypothalamic ventromedial nucleus II. Hormone-induced changes in the synaptogenic pattern. Dev. Brain Res. 61:189-196; 1991. Ojeda. S. R.: Urbanski, H. F. Puberty in the rat. in: Knobil, E.; Neill, J., eds. The physiology of reproduction, vol. II. New York: Raven Press, Ltd.: 1988:1699-1737. Parvez, H.; Casimiri, V.: Kiuchi, Y.; Nevo, I.; Elisebetb, P.; Castro, L.: Hobel, C. J.; Parvez, S. Contribution of pituitary-adrenal hormones in maturation of catecholamine regulating enzymes during pre and postnatal life. In: Gupta, D.; Wollmann, H. A.; Ranke, M. B., eds. Neuroendocrinology: New frontiers. Ttibingen, Germany: Brain Research Promotion; 1990:104. Renner, K. J.: Krey. L. C.: kuine, V. N. Effect of progesterone on monoamine turnover in the brain of the estrogen-primed rat. Brain Res. Bull. 19:105-202: 1987. Segarra, A. C.: Luine, V. N.: Strand, F. L. Sexual behavior of male rats is differentially affected by timing of perinatal ACTH administration. Physiol. Behav. 50:689-697; 1991. Singer. J. Hypothalamic control ofmale and female sexual behavior in female rats. J. Comp. Physiol. Psychol. 66:738-742: 1968. Strand, F. k.: Rose. K. J.: Zuccarelli, L. A.: Kume, J.: Alves, S. E.; Antonawich, F. J.; Garrett, k. Y. Neuropeptide hormones as neurotrophic factors. Physiol. Rev. 71:1017-1046; 1991. Stylianopoulou, F.; Fameli, M.: Brountzos, E.; Contopoulos, A. N. Neonatal neural organizing effects of exogenous corticosteroids on sexual differentiation of the brain in the female rat. Horm. Behav. 17:332-341: 1983. Sze, P. Y.; Neckers, L.; Towle. A. C. Glucocorticoids as a regulatory factor for brain tryptophan hydroxylase. J. Neurochem. 26:169173: 1976. Uphouse, L.; Montanez, S.: Richards-Hill, R.; Caldarola-Pastuszka, M.; Droge, M. Effects of the 5-HT~Aagonist, 8-OH-DPAT, on sexual behaviors of the proestrus rat. Pharmacol. Biochem. Behav. 39:635640; 199 I. Ward, I. L. Prenatal stress feminizes and demasculinizes the behavior of males. Science 143:212-218:1972. Wolterink, G.: Van Zanten, E.: Kamsteeg, H.; Radhakishun, F. S.: Van Ree, J. M. Functional recovery after destruction ofdopamine systems in the nucleus accumbens of rats.ll. Facilitation by the ACTH-(4-9) analog ORG 2766. Brain Res. 507:101-108; 1990.