Human vaginal secretions: volatile fatty acid content.

Vaginal samples (682) were collected by a tampon method from 50 healthy young women. Samples were analyzed by gas chromatography. The volatile aliphatic acids increased during the late follicular phase of the menstrual cycle and declined progressively during the luteal phase. Women on oral contraceptives had lower amounts of volatile acids and did not show any rhythmic changes in acid content during the menstrual cycle. These same substances possess sex-attractant properties in other primate species.

Pheromones: isolation of male sex attractants from a female primate.

Fractionation of vaginal secretions from rhesus monkeys by partitioning and chromatographic procedures, combined with behavioral studies, demonstrates that short-chain aliphatic acids are responsible for stimulating the sexual behavior of males. Injection of estradiol into ovariectomized females increases the concentration of volatile acids in secretions which will then sexually stimulate these male primates.

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).

The nuclear accumulation of [3H]testosterone and [3H]estradiol in the brain of the female primate: evidence for the aromatization hypothesis.

To identify the metabolites of estradiol (E2) and testosterone (T) in nuclei obtained from the female primate brain and, hence, to investigate the mechanism of their actions on behavior, 9 ovariectomized adult rhesus monkeys were studied. Two of these females were injected with 5.5 mCi [3H]T, and 30 min later, samples of 14 brain areas, pituitary gland, and peripheral tissues were removed and homogenized. Purified cell nuclei and a crude cytosol fraction were prepared, extracted with ether, and fractionated by HPLC to identify steroid metabolites. In nuclei from the hypothalamus, preoptic area, and amygdala, [3H]E2 formed locally was the major form of radioactivity. In nuclei from the clitoris, [3H]dihydrotestosterone was the major form of radioactivity, and in nuclei in all other brain samples and in the pituitary gland and uterus, [3H]T predominated. Two females (controls) were pretreated for 5 days with oil sc, injected with 1 mCi [3H]E2, and killed 60 min later. In these females, elevated nuclear concentrations of [3H]E2 were found in the hypothalamus, preoptic area, amygdala, pituitary gland, and uterus. Similar results were obtained in 2 females that were pretreated for 5 days with 2 mg/day dihydrotestosterone propionate, sc, and then injected with 1 mCi [3H]E2. In 3 females that were pretreated for 5 days with 2 mg/day T propionate, sc, and then injected with 1 mCi [3H]E2, levels of [3H]E2 were reduced by 100% (P less than 0.01) in nuclei from preoptic area and amygdala compared with control values and by 78% (P less than 0.05) in nuclei from the hypothalamus. There were no comparable reductions in steroid levels in cerebral cortex, pituitary gland, or uterus. This is the first direct evidence in the brain of a female primate that the actions of T and E2 involve the same receptor systems.

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.

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.

Localization of the synthetic progestin 3H-ORG 2058 in neurons of the primate brain: evidence for the site of action of progestins on behavior.

The location of neurons that concentrate progestin in the brains of female cynomolgus monkeys was mapped by autoradiography using the specific synthetic progestin receptor ligand 3H-ORG 2058. Three females were ovariectomized and treated with estrogen (20 micrograms estradiol benzoate daily for 7 days), and one of them was also pretreated with progesterone. Each received an i.v. injection of 1 mCi 3H-ORG 2058 and was killed 1 hour later. Thaw-mount autoradiograms revealed intense accumulation of radioactivity in the nuclei of many neurons in the mediobasal hypothalamus, particularly in the ventromedial nucleus (n.), arcuate n., and premammillary n. Neuronal labeling was also observed frequently in the medial preoptic n., and occasionally in the anterior hypothalamic area, paraventricular n., and organum vasculosum of the lamina terminalis. In the pituitary gland, about 5% of cells in the pars distalis were intensely labeled. In the female pretreated with progesterone, however, labeling was almost completely blocked. Analysis of samples by high-performance liquid chromatography demonstrated that the radioactivity extracted from brain and pituitary gland cell nuclei was almost entirely unmetabolized 3H-ORG 2058. The nuclear concentration of progestin was much greater in the pituitary gland than in the brain, and was greater in the hypothalamus than in any other brain area. These results revealed well-localized groups of progestin-concentrating neurons in the primate brain which presumably mediate the effects of progesterone on both gonadotropin secretion and female sexual behavior.

Behavior of rhesus monkeys during 28-day cycles of estrogen treatment.

Sixteen ovariectomized rhesus monkeys were paired, each with two males, during thirty-two 28-day periods during which they received one or more of the following schedules of daily injections: (a) different mixtures of estradiol benzoate, progesterone, and testosterone propionate (artificial menstrual cycles), (b) the doses of estradiol benzoate given in artificial cycles but without the other hormones (cyclic estradiol regimens), and (c) appropriate control treatments (625 one-hour behavioral tests). Hormone doses were adjusted to give plasma levels in the physiological range (335 blood samples). Measures of male sexual activity during artificial cycles and during cyclic estradiol regimens showed significant cyclic changes, but these changes were indistinguishable from each other. In four females studied in an operant conditioning paradigm, there was a midcycle dip in the times taken to obtain access to males during the cyclic estradiol regimens. The data indicated that the estrogen state of the female plays a preponderant role in determining the cyclic changes in the behavioral interactions of the pair independently of the effects of other ovarian hormones.

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.

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.

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.

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)

Estrogen binding and the actions of testosterone in the brain of the male rhesus monkey.

Autoradiography and high performance liquid chromatography (HPLC) were used to determine where metabolites of testosterone interact with estrogen binding sites in the brain of the male primate. Three days after castration, animals received a subcutaneous injection of either estradiol benzoate (EB, 200 micrograms/kg, n = 4) or oil vehicle (controls, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an intravenous bolus. At 60 min, brains were rapidly removed, left halves were used for autoradiography and right halves were dissected into 14 samples for HPLC of nuclear and supernatant fractions. In control males, labeled neurons were observed in preoptic area, hypothalamus and amygdala. In EB-pretreated males, the number of labeled neurons was reduced by 35% in the anterior hypothalamus and ventromedial nucleus, and by 65% in the cortical and accessory basal amygdaloid nuclei, but was not significantly reduced in other brain regions. In hypothalamus, preoptic area and amygdala, EB-pretreatment reduced nuclear concentrations of [3H]estradiol to 37-55% of control levels, but reduced neither the nuclear concentrations of [3H]testosterone nor the supernatant concentrations of [3H]estradiol and [3H]testosterone. The data suggest that the actions of testosterone in regions such as the arcuate nucleus and lateral septal nucleus primarily involve unchanged testosterone or dihydrotestosterone, while in regions such as the amygdala, aromatization and interaction with estrogen receptors is involved also.

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.

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.

Brain self-stimulation, locomotor activity and tissue concentrations of ethanol in male rats.

These studies were aimed at correlating the effects of ethanol on operant behavior and on locomotor activity with its distribution in selected tissues in the body. One group of male rats was trained on a continuous reinforcement schedule for intracranial self-stimulation (ICSS) with electrodes in the lateral hypothalamus. Another group was studied in a locomotor activity apparatus, and both groups were given ethanol intraperitoneally over the dose-range 0.3-1.7 g/kg. Urine was collected 15 min and 60 min after ethanol administration and samples of blood, brain, heart, lung, liver, muscle and testis were obtained at both time points. Depressions of ICSS and of locomotor activity occurred, and these changes in behavior were correlated with increasing concentrations of ethanol in blood, urine and tissue. Thus, the disrupting effects of ethanol on behavior which occurred shortly after its acute administration were closely linked to its concentrations throughout the body.

Pre-optic and hypothalamic neurons accumulate [3H]medroxyprogesterone acetate in male cynomolgus monkeys.

Medroxyprogesterone acetate (MPA) is a synthetic progestin that is reported to be effective in the treatment of paraphilic behavior, including paraphilic aggression, in men. The mechanisms and sites of action for its behavioral effects are not known. Thaw-mount autoradiography was used to help identify sites in the brain at which MPA may act in a male primate. Two adult, castrated male cynomolgus monkeys were administered [3H]MPA and killed one hour later. Radioactivity was concentrated in the nuclei of many neurons in the medial preoptic nucleus (n.), anterior hypothalamic area, ventromedial hypothalamic n., and arcuate n. Virtually no labeled cells were observed in the bed n. of the stria terminalis, lateral septal n., or amygdala. Analysis by high performance liquid chromatography of brain samples from the same animals demonstrated that 84% of the extractable radioactivity in cell nuclei from the hypothalamus and preoptic area was in the form of unmetabolized [3H]MPA. The localization of MPA-concentrating neurons in regions of the brain known to be implicated in regulating both sexual behavior and pituitary function suggests that, among other sites of action, MPA may act directly upon the brain.

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.

Characterization by high performance liquid chromatography of nuclear metabolites of testosterone in the brains of male rhesus monkeys.

Testosterone (T) restores the potency of castrated male rhesus monkeys, and our autoradiographic data have demonstrated that 3H-T or its metabolites concentrate in cell nuclei in the corticomedial amygdala, bed nucleus of stria terminalis, preoptic area, and hypothalamus. In rat, 3H-estradiol (3H-E2) is a major nuclear metabolite of 3H-T in areas of the limbic system, but comparable data are lacking for the primate. We have therefore developed an improved technique using high performance liquid chromatography for investigating metabolites of 3H-T that accumulate in cell nuclei in small amounts of tissue obtained from the brain of the rhesus monkey. Two castrated male rhesus monkeys were injected with 5 mCi of 3H-T and were killed 30 min later. In amygdala, preoptic area-bed nucleus of stria terminalis, and hypothalamus, 48-70% of the nuclear radioactivity was in the form of 3H-E2 (Type I tissues). In six other brain areas and in pituitary, 35-85% of the nuclear radioactivity was in the form of 3H-T (Type II tissues), whereas in genital tract tissues, 86-99% of the nuclear radioactivity was in the form of 3'-dihydrotestosterone (3H-DHT) (Type III tissues). In plasma and in supernatants from both Type I and Type II tissues, the proportions of 3H-T were high, and 3H-E2 did not exceed 10% of the total extractable radioactivity. These data suggest that, as in rodents, some of the central actions of T in primates may be mediated by estrogen target neurons.

Testosterone and its metabolites in male cynomolgus monkeys (Macaca fascicularis): behavior and biochemistry.

To extend our previous study on the behavioral effects of testosterone propionate (TP) and dihydrotestosterone propionate (DHTP) to a dose-range producing supra-physiological plasma androgen levels, 4 castrated cynomolgus monkeys were tested with the same 4 females during successive 4-week treatment periods while receiving 800 micrograms, 1.6 mg, 3.2 mg, 6.4 mg and 12.8 mg of TP or DHTP SC/day in counterbalanced order (16 pairs, 828 1-hr tests). Both androgens increased male sexual activity, but DHTP was less effective than TP in increasing the numbers of ejaculations per test and failed to restore ejaculations to intact levels. Giving androgen-treated males single injections of 50 micrograms and 100 micrograms estradiol benzoate (EB) was without any additional effect on behavior (16 pairs, 256 tests). To examine hormonal effects in the brain, castrated males were given either 3H-T or 3H-DHT, and tissues were examined by high performance liquid chromatography (hplc). After 3H-T, 3H-E2 and unchanged 3H-T were the major forms of radioactivity in nuclei from hypothalamus, preoptic area and amygdala. After 3H-DHT, unchanged 3H-DHT predominated. The lower behavioral effectiveness of DHT could not be ascribed to its failure to enter the brain. The data suggested a role for unchanged T in the regulation of ejaculatory behavior in a male primate.