Rats selectively-bred for behavior related to affective disorders: proclivity for intake of alcohol and drugs of abuse, and measures of brain monoamines.

Several lines of rats potentially useful for studying affective disorders have been developed in our laboratory though selective breeding for behavioral characteristics. The propensity of these lines to consume alcohol and other drugs of abuse (amphetamine and cocaine) was examined. Also, measurement of the concentration of brain monoamines - norepinephrine, dopamine, and serotonin - as well as estimation of their metabolism by measurement of the major extracellular metabolites of these monoamines was carried out to examine possible relationships of brain chemistry to the behavioral characteristics shown by these lines, as well as to their propensity for drug usage. The lines of rats are: Swim Low-active (SwLo) and Swim High-active (SwHi), which show either very low (SwLo) or very high (SwHi) amounts of motor activity in a swim test; Swim-test Susceptible (Susceptible or SUS) and Swim-test Resistant (Resistant or RES), which are highly susceptible (SUS) or highly resistant (RES) to having their swim-test activity depressed by being exposed to a stressful condition prior to the swim test; and Hyperactive (HYPER), which show spontaneous nocturnal hyperactivity compared to non-selectively bred (i.e., normal) rats as well as both extreme hyperactivity and behavioral depression after being exposed to a stressful condition. Regarding alcohol and drug usage, SUS rats readily consume alcohol while all other lines including non-selected, normal rats do not, and SwLo rats show a strong tendency to consume amphetamine and cocaine. Marked differences in brain monoamines were found between the various lines and normal rats, with salient differences seen in norepinephrine, particularly in the hippocampus, and in dopamine in forebrain regions (striatum and nucleus accumbens).

Depletion of brain norepinephrine does not reduce spontaneous ambulatory activity of rats in the home cage.

6-Hydroxydopamine (6-OHDA) lesions of brain noradrenergic neurons and terminals were made in rats to assess the importance of forebrain norepinephrine (NE) for mediating circadian patterns of spontaneous ambulatory activity that rats show in the home cage. 6-OHDA was injected intracranially into the fibers of the ascending noradrenergic dorsal and ventral bundle pathways or infused into the lateral ventricle or both. Rats living in a 12/12 h light/dark cycle exhibit a marked increase in ambulatory activity during the dark period in comparison to the light period and a 'W-shaped' pattern of activity during the 12 h of the dark phase. Results showed that near-total depletion of brain NE did not impair the capacity to generate normal patterns of spontaneous ambulatory activity that occur in the home cage. In the animals that sustained the most complete NE lesions, the amounts of activity generated at times of peak activity were exaggerated in comparison to the control animals, which is consistent with the possibility that NE in the brain exerts a moderating influence on behavior.

Rats selectively bred for high and low swim-test activity show differential responses to dopaminergic drugs.

RATIONALE: Selective breeding of Sprague-Dawley rats has been used to generate a line of animals with very low swim-test activity (SwLo) in an attempt to model certain characteristics of depression. For comparison with the SwLo animals, a line bred for high swim-test activity (SwHi) and a non-selectively bred line (SwNS) have been generated. Previous studies using these lines suggested an inverse relationship between dopamine (DA) function in the brain and inactivity in the swim test. OBJECTIVES: The current experiments investigated the possibility that SwLo and SwHi rats show differences in central DA processes, as suggested by responsiveness to DA agonists. RESULTS: The increase in ambulation produced by d-amphetamine (0.25-1.0 mg/kg) was largest in SwHi rats and smallest in SwLo rats, with SwNS rats showing an intermediate response. Amphetamine levels in plasma and brain tissue were similar in SwHi and SwLo rats, indicating that pharmacokinetic differences were not responsible for the behavioral differences. Repeated amphetamine administration produced enhancement in the ambulation-increasing effects of this drug (i.e., sensitization), with significant enhancement seen in all three lines. Apomorphine in doses that stimulate postsynaptic receptors (0.25-4.0 mg/kg) produced mainly increased sniffing behaviors in SwHi and SwNS rats and oral behaviors in SwLo rats, suggesting that the lines differ in proportions of D1, D2, and D3 postsynaptic receptors. CONCLUSIONS: The findings suggest that DA function differs in lines of rats selectively bred for differences in swim behavior, a feature that may make these lines useful for studying certain depressive symptoms that might be related to DA function.

Galanin: a significant role in depression?

This paper describes a hypothesis that attempts to account for how changes in noradrenergic systems in the brain can affect depression-related behaviors and symptoms. It is hypothesized that increased activity of the locus coeruleus (LC) neurons, the principal norepinephrine (NE)-containing cells in the brain, causes release of galanin (GAL) in the ventral tegmentum (VTA) from LC axon terminals in which GAL is colocalized with NE. It is proposed that GAL release in VTA inhibits the activity of dopaminergic cell bodies in this region whose axons project to forebrain, thereby resulting in two of the principal symptoms seen in depression, decreased motor activation and decreased appreciation of pleasurable stimuli (anhedonia). The genesis of this hypothesis, which derives from studies using an animal model of depression, is described as well as recent data consistent with the hypothesis. The formulation proposed suggests that GAL antagonists may be of therapeutic benefit in the treatment of depression.

Behavioral responses to Depo-Provera, Fadrozole, and estradiol in castrated, testosterone-treated cynomolgus monkeys (Macaca fascicularis): the involvement of progestin receptors.

Sexual motivation and behavior decreased in male cynomolgus monkeys given either Depo-Provera (medroxyprogesterone acetate, MPA), which reduces androgen uptake by brain, or the nonsteroidal aromatase inhibitor, Fadrozole, which virtually eliminates the conversion of testosterone (T) to estradiol (E2) in brain. This suggested that both unchanged T and E2 are important for the control of male primate sexual behavior, but combined treatment with MPA and Fadrozole did not have the anticipated summatory effects in intact males: the behavioral decrements when MPA-treated males were given Fadrozole were about half those observed when Fadrozole was given alone. The present study tested the hypothesis that Fadrozole suppressed the behavioral effects of MPA by preventing the induction by E2 of progestin receptors in the brain to which MPA binds. Eight castrated, T-treated males were each tested with an estrogenized female i) during baseline, ii) during MPA treatment, iii) during treatment with MPA and Fadrozole together, and iv) with E2 treatment added to condition (iii) (256 1-h behavior tests). All dosages were those used in previous studies. Sexual motivation, as reflected in mounting attempts and mounting attempt latencies, was further diminished by E2 treatment in males receiving both MPA and Fadrozole, but ejaculatory activity was not changed. Immunocytochemistry demonstrated that the distributions of progestin and androgen receptors were little affected by MPA treatment, and that progestin receptor immunoreactivity was almost completely abolished in the brains of males receiving both MPA and Fadrozole but present in those receiving additional E2 treatment, findings that supported the hypothesis.

Effects of the nonsteroidal aromatase inhibitor, fadrozole, on the sexual behavior of male cynomolgus monkeys (Macaca fascicularis).

In many vertebrates, castration and hormone replacement and, more recently, the use of aromatase inhibitors, have shown that male sexual activity is mediated by the aromatization of testosterone (T) to estradiol (E2). In macaques, however, the systemic administration of E2, either alone or in combination with androgen, failed either to maintain or to restore the sexual activity of castrated males. The present study examines the effects of administering the nonsteroidal aromatase inhibitor, Fadrozole, either alone or combined with E2, to castrated, T-treated male cynomolgus monkeys at a dose of 0.25 mg/kg/day. This dose inhibited by over 98% the conversion of T to E2 and the subsequent accumulation of the latter in hypothalamic cell nuclei. Castrated males bearing sc Silastic impants of T were each tested with an ovariectomized, E2-treated female partner before, during, and after being given minipumps delivering either Fadrozole or water (240 1-hr tests). Within 2 weeks, Fadrozole significantly reduced ejaculatory activity and male sexual motivation in the absence of changes in plasma T levels, which remained in the upper range for intact males. Additional estradiol treatment produced small but significant increases in ejaculations by three of the six males only, and measures of male sexual motivation remained unchanged (120 tests). The present results, which stand in contrast to our previous findings in macaques, support the view that aromatization of T is important for ejaculatory activity and sexual motivation in a male primate. They also suggest that exogenous E2, which reaches the brain from the systemic circulation, does not fully duplicate the behavioral effects of E2 produced locally in the brain by the aromatization of T.

Effects of the nonsteroidal aromatase inhibitor, Fadrozole, on sexual behavior in male rats.

The new nonsteroidal aromatase inhibitor, Fadrozole (CGS 16949A, CIBA-Geigy Corp.), was tested for its ability (i) to inhibit the conversion of testosterone (T) to estradiol (E2) in brain and (ii) to suppress male sexual activity. Sprague-Dawley rats were castrated and immediately given sc Silastic T-implants and osmotic minipumps delivering 2.5 mg/kg/day Fadrozole (N = 4), 0.25 mg/kg/day Fadrozole (N = 4), or water (N = 4 controls). T-implants were removed after 6 days and, 3 days later, 3H-T (1 microCi/g) was given as an iv bolus. No 3H-E2 was detected in hypothalamic or amygdaloid nuclear pellets from Fadrozole-treated males but this metabolite predominated in controls. However, nuclear concentrations of 3H-T and [3H]dihydrotestosterone were similar in all groups. In another group of males (N = 18), brain aromatase activity was reduced by more than 96% at the 0.25 mg/kg dose level. Additional castrated, T-implanted males received minipumps delivering 0.25 mg/kg/day Fadrozole (six males) or water (six behaviorally matched controls) and were tested weekly with receptive females. After 2 weeks, ejaculations were reduced by 77% compared with controls (P less than 0.01) and, after 4 weeks, intromissions were also significantly reduced (P less than 0.05) but less so (48%). Radioenzymatic estimates of plasma aromatase inhibitor levels remained elevated throughout Fadrozole treatment. These males were then given Silastic E2 implants: intromissions increased significantly in 1 week (P less than 0.01), but ejaculations remained below control values. Results supported the view that aromatization is important for sexual behavior in male rats and suggested that Fadrozole has utility for studying the mechanisms by which testosterone affects behavior.

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey.

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.

Developmental changes in the uptake of testosterone by the primate brain.

During the neonatal period in male macaques, the testis produces adult-like levels of plasma testosterone (T), but the function of this in development is not understood. To investigate the interaction of T with the neonatal brain, 4 male and 5 female cynomolgus monkeys were gonadectomized 2-5 days after birth, and were injected subcutaneously 3 days later with 500 microCi [3H]-testosterone ([3H]-T). 60 min later, brains and other tissue samples were removed. Purified nuclear pellets were prepared by centrifugation through 2 M sucrose, extracted into ether and analyzed by high-performance liquid chromatography. The aromatized metabolite, [3H]-estradiol [( 3H]-E2), was found only in the hypothalamus (HYP) and amygdala (AMG). In HYP, [3H]-E2 represented 55 +/- 3% of the radioactivity in males and 53 +/- 3% in females. In AMG, [3H]-E2 represented 40 +/- 9% of the radioactivity in males and 47 +/- 3% in females. Concentrations of unchanged [3H]-T were higher than those of [3H]-dihydrotestosterone [( 3H]-DHT). Both androgens were present in nuclear pellets from all 8 brain regions studied, and concentrations were significantly higher in females than in males (p less than 0.005). [3H]-T was also the main form of radioactivity in nuclear pellets from pituitary gland, adrenal gland, uterus and liver, but very high levels of [3H]-DHT were found in seminal vesicles, prostate and penis. Comparisons were made with previous results from orchidectomized fetuses at 122 days gestation and from fully adult male castrates, and the largest developmental changes occurred in the AMG where concentrations of [3H]-E2 were 20-fold higher in adults than in fetuses, and most of this increase took place after the neonatal stage. Nuclear concentrations of [3H]-T also increased markedly during development in most brain regions except the cerebellar cortex where they declined.

The interaction of testosterone with the brain of the orchidectomized primate fetus.

At certain times during gestation, the testes of the fetal macaque produce plasma levels of testosterone (T) that are similar to those of adults. It is thought that testosterone acts on the brain via estrogen and androgen receptors to organize the development of sexually dimorphic neural structures that underlie sex differences in behavior. To test the proposition that there are male-female differences in the occupation of steroid receptor binding sites during fetal development in the cynomolgus macaque, we have compared the uptake of [3H]T and its metabolites in: (1) 5 intact males (plasma T 571.2 +/- 215.5 ng/100 ml); (2) 5 intact females (33.8 +/- 25.2 ng/100 ml); (3) in 5 males orchidectomized in utero (14.6 +/- 5.7 ng/100 ml). About 1 week after fetal gonadectomy or sham-operation, all fetuses were given 500 microCi [3H]T s.c. and were then delivered 60 min later by Cesarean section. Brains were removed and dissected into blocks containing the hypothalamus and preoptic area, amygdala, hippocampus, and midbrain. Samples of cerebral and cerebellar cortex were also obtained. Purified nuclear pellets were prepared by centrifugation through 2 M sucrose and were extracted into ether and analyzed by high performance liquid chromatography. Hypothalamic nuclear concentrations of [3H]E2 in intact males (847 +/- 195 dpm per mg DNA) were significantly lower than those in sham-operated females (2147 +/- 542 dpm per mg DNA) (P less than 0.05), but those in orchidectomized males (2233 +/- 345 dpm per mg DNA) were similar to concentrations in females.(ABSTRACT TRUNCATED AT 250 WORDS)

Medroxyprogesterone acetate, aggression, and sexual behavior in male cynomolgus monkeys (Macaca fascicularis).

Medroxyprogesterone acetate (MPA) is used clinically to treat male sex offenders, but there are conflicting reports about its effects on aggression. To investigate these matters in a nonhuman primate, four intact male cynomolgus monkeys were studied in a testing paradigm that involved the presence of a caged, aggression-arousing stimulus male either immediately before or during a pair-test with an ovariectomized, untreated female partner. After two 4-week periods of pretreatment baseline, males received weekly injections of 40 mg MPA either alone (two 4-week treatment periods) or in combination with testosterone replacement with sc implants (one period) and additional daily injections of 2 mg testosterone propionate (two periods). MPA was then withdrawn while testosterone replacement continued (three periods). The testing paradigm was effective in maintaining aggression, especially male-male aggression, for many months. Male-male aggression increased with MPA treatment, and increased further with testosterone replacement, whereas male-female aggression tended to change in the opposite direction. As in earlier studies, MPA decreased both plasma testosterone and male sexual activity, but restoring plasma testosterone levels in treated males failed to restore their sexual activity. MPA therefore has behavioral effects that are not mediated primarily by its suppression of circulating androgens.

Medroxyprogesterone acetate and the nuclear uptake of testosterone and its metabolites by brain, pituitary gland and genital tract in male cynomolgus monkeys.

The synthetic progestin, medroxyprogesterone acetate (MPA), is used to treat male sex offenders, and it is also suppresses sexual activity in male monkeys. To examine the possibility that MPA may act as an anti-androgen in the primate brain, 4 intact male cynomolgus monkeys were given MPA (40 mg i.m.) once a week for 16 weeks, while 4 control males received i.m. injections of vehicle. All males were then castrated and 3 days later were given 3 mCi [3H]testosterone ([3H]T) i.v.; 1 h after injection males were killed, and radioactivity in nuclear pellets obtained from the hypothalamus (HYP), preoptic area (POA), amygdala (AMG), septum, pituitary gland and genital tract was analyzed by HPLC. Concentrations of [3H]T and [3H]dihydrotestosterone in nuclear pellets were 65-96% lower in MPA-treated males than in controls (P less than 0.001), but the aromatized metabolite, [3H]estradiol, which was the major form of radioactivity present in nuclear pellets from HYP, POA and AMG, was unchanged. There were no differences in concentrations of [3H]T in supernatants from the tissues of MPA-treated and control males. Because the reduced nuclear uptake of androgen in brain occurred in males whose androgen-dependent behavior had been suppressed by MPA treatments, it is proposed that MPA may have anti-androgenic effects at the level of the cell nucleus in brain regions that control behavior.

Uptake of medroxyprogesterone acetate by progestin and androgen target neurons in the brain and pituitary gland of male cynomolgus monkeys.

Medroxyprogesterone acetate (MPA), a synthetic progestin that reduces plasma testosterone levels, has been used in the treatment of male sex offenders. It also reduces the sexual activity of male macaques. To investigate its sites and mechanisms of action, 11 adult male cynomolgus monkeys were castrated and 7 and 21 h later were pretreated with 20 mg progesterone s.c. (Prog, n = 3), or 5 mg dihydrotestosterone propionate s.c. (DHTP, n = 3) or oil vehicle (controls, n = 5). Twenty-four hours after castration, all males were injected i.v. with 5 mCi [3H]-MPA, and killed after 60 min. Left halves of the brains were processed for thaw-mount autoradiography to identify the neurons accumulating radioactivity, and right halves were analyzed by high performance liquid chromatography (HPLC) to measure the uptake of [3H]-MPA in nuclear fractions. In males pretreated with oil, there were labeled neurons in the ventromedial nucleus (n.), arcuate n., medial preoptic n. and anterior hypothalamic area. In progesterone-pretreated males, labeling was reduced by 84-100% compared with controls (p less than 0.001), but in DHTP-pretreated males there was no effect, and labeling was not significantly different from control levels. Nuclear concentrations of [3H]-MPA measured by HPLC in controls were highest in the hypothalamus, amygdala, preoptic area and pituitary gland. Pretreatments with progesterone reduced the nuclear concentrations of [3H]-MPA in hypothalamus, preoptic area and pituitary gland by 82-95% compared with controls (p less than 0.05), but DHTP pretreatments had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The uptake of tritiated diethylstilbestrol by the brain, pituitary gland, and genital tract of the fetal macaque: a combined chromatographic and autoradiographic study.

To examine the possible sites of action of the synthetic estrogen diethylstilbestrol (DES) in the developing primate, [3H]DES (250 mu Ci, iv, or 500 mu Ci, sc) was administered directly into two rhesus and nine cynomolgus macaque fetuses at about 122 days gestation (range, 121-124 days). The location of cells accumulating radioactivity 60 min later was examined by autoradiography in two males and two females. In females, labeled neurons were observed in the hypothalamus, preoptic area, and amygdala, but not in the cerebral cortex. In one male a similar pattern of uptake was observed, but percentages of labeled neurons were lower, and in the other male very little labeling was observed in any region. The chemical identity of the radioactivity in cell nuclei was determined by high performance liquid chromatography in three males and four females. Concentrations of radioactivity in nuclear pellets were highest in the hypothalamus and lowest in the cerebral cortex. This regional variation was highly significant (P less than 0.001), but there was no significant difference between nuclear concentrations of radioactivity in males and females. In supernatant fractions, concentrations of radioactivity showed no significant variation between brain regions and after 60 min, 52-67% of the extracted radioactivity was no longer in the form of [3H]DES. Nuclear levels of radioactivity in pituitary glands and genital tracts of both male and female fetuses were 2-5 times higher than those in hypothalamus. The results demonstrated a direct interaction between DES and cell nuclei from specific regions of the brain, pituitary gland, and genital tract at this stage of gestation in a primate.

Comparisons of the nuclear uptake of [3H]-testosterone and its metabolites by the brains of male and female macaque fetuses at 122 days of gestation.

Testosterone secreted by the testis of the macaque fetus is thought to influence certain aspects of the brain's subsequent development which may be responsible for the ontogeny of sexually dimorphic patterns of behavior. To compare the interactions between testosterone and the receptors for androgens and estrogens in brain cell nuclei in the two sexes, 7 intact female fetuses and 5 intact male fetuses were injected in utero at about 120 days of gestation with [3H]-testosterone (250 microCi i.v. or 500 microCi s.c.). One hour later, fetuses were delivered by cesarean section, and samples of brain and peripheral tissues were homogenized and separated into purified nuclear and supernatant fractions. Fractions were analyzed by high performance liquid chromatography to measure levels of [3H]-testosterone and its metabolites. Concentrations of radioactivity extracted from cell nuclei were significantly higher in the hypothalamus-preoptic area than in other brain areas (p less than 0.001); [3H]-estradiol represented 65.0 +/- 5.7% of this radioactivity and nuclear concentrations of this metabolite were 73% lower in males than in females (p less than 0.001). Nuclear concentrations of [3H]-testosterone in the pituitary gland (68.9 +/- 8.8% of extracted radioactivity) were 48% lower in males than in females (p less than 0.001). There was no evidence of a sex difference in the tissue uptake of radioactive steroids from blood, but in males, levels of endogenous plasma testosterone (599.8 +/- 208.2 ng/100 ml) were significantly higher than in females (37.7 +/- 28.5 ng/100 ml; p less than 0.01), and the specific activity of [3H]-testosterone in blood was consequently lower in males than in females.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol administration and the sexual activity of castrated male rhesus monkeys (Macaca mulatta).

To examine whether estradiol might be effective in maintaining sexual behavior after castration or after testosterone withdrawal, we have observed male rhesus monkeys during daily 1-hr tests alternately with each of two ovariectomized, estradiol-treated females (four males, four females, eight male-female pairs, 798 tests). Estradiol (2-5 micrograms/kg sc/day) or vehicle was administered in counterbalanced order immediately after castration and again immediately after withdrawal of testosterone propionate treatments (800 micrograms and 1.6 mg sc/day). There were no significant differences in behavior during vehicle and estradiol treatments to indicate that estradiol helped to maintain male sexual activity. Instead, estradiol treatment tended to interfere with the capacity to intromit. This supported the results of other studies, namely, that the systemic administration of estradiol does not enhance the sexual behavior of castrated male macaques, and raises questions about the role of both aromatization and estrogen receptors in the male primate brain.

Sites in the male primate brain at which testosterone acts as an androgen.

Quantitative autoradiographic analysis was used to identify regions in the brain of the male primate where androgen binding sites may be involved in the actions of testosterone. Three days after castration, adult male rhesus monkeys received a subcutaneous injection of either dihydrotestosterone propionate (DHTP, 20 mg, n = 6), testosterone propionate (TP, 100 mg, n = 2), or oil vehicle (control males, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an i.v. bolus. At 60 min, brains were rapidly removed and the left halves were used for autoradiography. In control males, highest percentages of labeled neurons (20-84% using a rigorous Poisson criterion) were observed in the ventromedial, arcuate and premammillary nuclei (n.) of the hypothalamus, medial preoptic n., bed n. of stria terminalis, intercalated mammillary n., lateral septal n. and the medial, cortical and accessory basal n. of the amygdala. Pretreatment with DHTP eliminated labeling in androgen target tissues of the genital tract, and reduced the percentages of labeled neurons to 4-22% of control values in the arcuate, lateral septal, premammillary and intercalated mammillary n., indicating that in these regions testosterone acted predominantly at androgen binding sites. However, in the medial preoptic n., the ventromedial hypothalamic n. and the accessory basal amygdaloid n., DHTP pretreatment resulted in much less blocking which, together with other data, suggested that in these sites, testosterone's actions involved aromatization and interaction with estrogen-binding sites.

Pretreatments with 5 alpha-dihydrotestosterone and the uptake of testosterone by cell nuclei in the brains of male rhesus monkeys.

An in vivo competition method was used in adult male rhesus monkeys to determine if testosterone binds to high affinity binding agents, notably androgen receptors, in brain cell nuclei. Castrated males received 5 alpha-dihydrotestosterone propionate (DHTP, 20 mg, N = 6), testosterone propionate (TP, 100 mg, N = 3) or oil vehicle (controls, N = 6) followed 3 h later by 5 mCi [3H]testosterone [( 3H]T) as an intravenous bolus. Brain and peripheral tissue samples were removed after 60 min, homogenized and separated into supernatant and purified nuclear fractions. Radioactive metabolites of [3H]T [( 3H]estradiol, [3H]DHT) and unchanged [3H]T were identified by high performance liquid chromatography (HPLC). Androgen pretreatments reduced the nuclear uptake of [3H]T by 67-98% in hypothalamus (HYP), preoptic area (POA) and pituitary gland (PIT). This blockade was presumed to be due to prior occupation of nuclear androgen receptors by unlabeled androgens because pretreatments had no effects on levels of [3H]T in supernatants. Since [3H]T was the major radioactive androgen present in brain cell nuclei, results strongly suggested that the principal nuclear androgen receptor ligand in HYP, POA and PIT was unchanged [3H]T rather than [3H]DHT as occurs in the genital tract. In the amygdala the situation was quite different. Here, nuclear concentrations of [3H]T were reduced by 67% following TP pretreatment but were not changed following DHTP pretreatment, indicating a different uptake mechanism in this region that could have particular relevance for testosterone's central actions on behavior.

Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone.

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.

The uptake of [3H]testosterone and its metabolites by the brain and pituitary gland of the fetal macaque.

Testosterone is secreted by the fetal testis during gestation, and this is thought to influence certain aspects of the brain's subsequent development. To study this action at the neuronal level, nine macaque fetuses were injected with 250 microCi [3H]testosterone via the umbilical vein at about 120 days gestation. After 60 min, samples of brain and peripheral tissue were studied by autoradiography or HPLC. Purified nuclear pellets were prepared, and radioactivity in ether extracts was fractionated by HPLC and identified by coelution with internal standard steroids. Concentrations of radioactivity were significantly higher (P less than 0.05) in the hypothalamus-preoptic area than in amygdala, hippocampus, midbrain, and cerebral and cerebellar cortexes, and most of the radioactivity (75%) in the hypothalamus-preoptic area coeluted with 17 beta-estradiol. Radioactivity coeluting with 17 beta-estradiol was also detected in nuclear fractions from amygdala (44%). In contrast, 80% of the radioactivity extracted from pituitary gland nuclei coeluted with testosterone. Most of the neurons labeled in autoradiograms were located in the hypothalamus and preoptic area, fewer were found in the amygdala, and labeling in the frontal or motor cortex did not exceed chance levels. Results suggested that aromatization and, consequently, estrogen receptors play a role in the effects of testosterone on the hypothalamus and amygdala of the primate fetus at this stage of development.