Richard P. Michael

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.

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.

Androgen Receptors and Estrogen Receptors Are Colocalized in Male Rat Hypothalamic and Limbic Neurons that Express Fos Immunoreactivity Induced by Mating

Conversion of testosterone into estradiol is important for male rat sexual behavior, and both steroids probably contribute to mating. The distributions of neurons containing androgen receptors (AR) and estrogen receptors (ER) overlap, and many AR-immunoreactive (AR-ir) neurons express Fos immunoreactivity (Fos-ir) induced by mating. Because mating-induced Fos-ir in the male rat occurs mainly in AR-ir neurons, and because both steroids are important for mating, we hypothesized that (i) AR-ir and ER-ir are colocalized and that (ii) some of these neurons are activated during mating. We examined, in adjacent sections from the medial preoptic area (MPN) through the central tegmental field (CTF), the expression of ER-ir in: (i) AR-ir-containing neurons, and (ii) Fos-ir-expressive neurons. PG21 anti-AR, OA-11-824 anti-c-fos, H222 or 1D5 anti-ER primary antibodies were visualized, respectively, with cyanine-conjugated, fluorescein- or cyanine-conjugated, and fluorescein-conjugated secondary antibodies in male rats which were killed 1 h after ejaculating with a receptive female. In MPN, bed nucleus of the stria terminalis (BNST), and medial amygdala (MEA), 80–90% of ER-ir labeling occurred in AR-ir-positive neurons but only about 30% of AR-ir neurons were ER-ir-positive. No ER-ir was found in the CTF. This suggests the presence of three types of brain neurons sensitive to gonadal steroid hormones: neurons sensitive to androgens only, neurons sensitive to both androgens and estrogens, and neurons sensitive to estrogens only. About 50% of ER-ir labeling occurred in cells expressing mating-induced Fos-ir but only about 30% of Fos-ir neurons were ER-ir-positive. These findings suggest that, in the MPN, at least two different neuronal populations are activated during mating: the first contains AR-ir only and the second contains AR-ir and ER-ir. In the BNST and MEA, at least three hormonally sensitive populations are activated during mating: the two described above plus a third population which expresses ER-ir only.

Observations Upon the Sexual Behaviour of the Domestic Cat (Felis Catus L.) Under Laboratory Conditions

Detailed descriptions are given, based upon upwards of one thousand standardized Mating Tests, of the Patterns of Sexual Behaviour shown by the adult female cat. The changes observed in these patterns are described in terms of a Behaviour Cycle. The characteristics of the four distinct stages of this Cycle have been determined by studying the behaviour of thirty animals in response both to the coital activity of the male cat and to artificial sexual stimulation. The conspicuous external signs of the Cycle, together with the highly stereotyped pattern of Mating Behaviour which constitutes a sharp end-point for quantitative study, greatly facilitate the further investigation of sexual behaviour in this form. Nothing resembling oestrous or pro-oestrous behaviour was seen either in immature animals or in spayed adult females. Only behaviourally oestrous animals can be mated, and mating was never observed after removal of the ovaries during four hundred mating tests conducted with thirty animals over a period of fourteen months. It is concluded that the state of Sexual Receptivity in the female cat is strictly dependant upon the presence of ovarian hormone. Correlations are made between the behavioural patterns and the condition of the genital tract as revealed by vaginal smears; a highly significant association exists between the degree of vaginal cornification and the behavioural state at every stage of the oestrous cycle. Oestrous behaviour and mating continue throughout the period of vaginal oestrus. Mating behaviour in association with an anoestrous vaginal epithelium is a very rare occurrence in this form. The full organisation and development of the sexual reflexes in the female feline which result in the adoption of the full oestrous posture when stimulated artificially by vaginal probing is not necessarily associated with a state of sexual receptivity. It follows that the only certain criterion of Receptivity is a positive mating test conducted with an active male. Reasons have been given for putting forward the view that a central mechanism exists, underlying the development of the state of Receptivity, which requires a higher level of ovarian hormone for its activation than that required for the organisation of the segmental, postural reflexes. Attention has been drawn to the territory characteristics of the male as well as to spontaneously occurring distortions of sexual activity. Many of the aberrant patterns of sexual behaviour, which have been described in the literature as developing in brain-damaged animals, have been found to occur in unoperated animals simply as a result of laboratory conditioning and training.

Intracerebral Infusion of an Aromatase Inhibitor, Sexual Behavior and Brain Estrogen Receptor-Like Immunoreactivity in Intact Male Rats

Copulatory behavior was studied in five groups of sexually experienced, gonadally intact male rats in which: (i) the nonsteroidal aromatase inhibitor Fadrozole (CIBA-Geigy CGS 16949A) was delivered bilaterally (0.756 microgram in 6.0 microliters saline in 24 h to each side) into the medial preoptic area (POM) together with normal saline given s.c. via osmostic minipumps (n = 10); (ii) normal saline was delivered bilaterally into POM together with the same dose of Fadrozole s.c. (n = 9); (iii) Fadrozole was delivered bilaterally into the lateral preoptic area together with normal saline given s.c. (n = 6); (iv) Fadrozole was delivered bilaterally into the cerebral cortex together with normal saline given s.c. (n = 6), and (v) unoperated controls (n = 14). Mounting and ejaculation were significantly decreased in rats receiving Fadrozole in POM compared with the behavior of rats in the other 4 groups. Few differences occurred between rats in the latter 4 groups, all of which continued to mate. The H222 and ER-715 anti-estrogen receptor (ER) antibodies were used to examine the distribution of ER immunoreactive (ERir) neurons in hypothalamic and limbic sites in gonadectomized controls and in some of the rats in groups i, ii and v. Since labeling of ERir neurons in rat brain with the H222 anti-ER antibody is reported to be inhibited by estrogen, it was used here to identify regions (with staining) where the aromatization of testosterone (T) into estradiol (E2) had been suppressed. Intense H222 ERir nuclear neuronal labeling was confined to the POM of males receiving Fadrozole in POM, and significantly more labeled neurons were found in the POM of these rats than in the POM of rats treated with saline in POM. In contrast, the ER-715 antibody, which is reported to stain neurons independently of hormonal status, labeled neuronal nuclei in hypothalamic and limbic regions of all groups, demonstrating the presence of ER. These findings show that conversion of T into E2 in the POM of the male rat is important for male rat copulatory behavior and that H222 ERir nuclear neuronal labeling can be used to identify the neurons in POM that were affected by Fadrozole.

Fos Induced by Mating or Noncontact Sociosexual Interaction Is Colocalized with Androgen Receptors in Neurons within the Forebrain, Midbrain, and Lumbosacral Spinal Cord of Male Rats☆

This study was designed to determine the extent to which Fos immunoreactivity (induced either by mating or noncontact sociosexual interaction) and androgen receptor (AR) immunoreactivity are colocalized in brain and spinal cord of male rats. Some males (Mated) were allowed to mate to ejaculation; others (Social Controls) were placed with females but physical contact was prevented by a wire mesh screen; remaining males (Isolated) were placed alone in the test jar for the duration of the test period. After testing, brains and spinal cords were examined for AR and Fos immunoreactivity (ir). PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In both brain and spinal cord, the number of Fos-ir neurons varied according to group: Mated males > Social Controls > Isolated males. Fos was highly localized in subsets of AR-ir neurons within the medial preoptic nucleus, bed nucleus of the stria terminalis, dorsomedial nucleus of the amygdala, and central tegmental field. Fos was also localized in subsets of AR-ir neurons within the L5, L6, and S1 segments of the spinal cord. Spinal cord concentrations of AR-ir and Fos-ir neurons were greatest in Lamina X, and the vast majority of Fos-ir neurons in the dorsal part of Lamina X were also AR-ir. Thus, in both brain and spinal cord, androgen-sensitive neurons are active during mating, and transmission of sexually relevant information from cord to brain is probably accomplished via hormone-sensitive spinal neurons.

Androgen receptor immunoreactivity and mating-induced Fos expression in forebrain and midbrain structures in the male rat

The distribution of androgen receptor immunoreactive-neurons, mapped with the PG21 anti-androgen receptor antibody, was compared in male rat brains with the distribution of Fos-immunoreactive neurons induced by mating. In gonadally intact, but not in castrated male rats, substantial numbers of androgen receptor-containing neurons were present in a variety of forebrain and midbrain regions. The PG21 antibody apparently had a higher affinity for occupied than for non-occupied androgen receptors. Androgen receptor-immunoreactive regions included the medial preoptic area and other forebrain areas previously identified as containing androgen receptors, the dorsal and ventral periaqueductal gray, and a midbrain region that included the lateral part of the central tegmental field, part of the caudal zona incerta, the subparafascicular nucleus of the thalamus and the peripeduncular nucleus. Fos-expressive neurons were essentially absent in non-mated males but were present in the brains of rats which mated to ejaculation. All brain regions in which androgen receptor-immunoreactive neurons were counted also expressed Fos immunoreactivity after mating, and there was considerable overlap between the distributions of androgen receptor- and Fos-immunoreactive neurons. In a second experiment, we used immunofluorescent techniques to document the intraneuronal co-localization of Fos with androgen receptor immunoreactivity in the medial preoptic area, medial amygdala, and central tegmental field. In these regions mating-induced Fos immunofluorescence was exclusively localized in androgen receptor-immunofluorescent neurons. However, not all androgen receptor neurons were Fos expressive, suggesting that only some androgen-sensitive neurons were activated during mating. These results are consonant with the view that hormone actions on forebrain and midbrain structures influence the neuronal activity correlated with mating.

Changes in response rates and reinforcement thresholds for intracranial self-stimulation during morphine withdrawal

Rats were implanted with stimulating electrodes in the medial forebrain bundle-lateral hypothalamus and were trained in an auto-titration brain self-stimulation paradigm. When response rates and reinforcement thresholds were stable, the animals were implanted with subcutaneous osmotic minipumps (Alzet, 2ML1) which continually delivered morphine (1.2 mg/kg/hr as the base, n = 16) or saline (10.0 microliter/hr, n = 11). After one week the pumps were removed, and the animals were again tested in the auto-titration paradigm following the daily administration of either saline (spontaneous withdrawal) or 1.0 mg/kg naloxone (precipitated withdrawal). During the eight-day withdrawal phase there was a decrease in the rate of lever-pressing for the morphine dependent animals and this was greatest on the first day. The magnitude of the decrease was greater in the precipitated withdrawal group than in the spontaneous withdrawal group and an increase in the reinforcement threshold occurred only with precipitated withdrawal. Animals in both groups lost weight when measured each morning, but the precipitated group showed greater weight loss during the day. In addition, animals in the precipitated withdrawal group had diarrhea and showed a higher incidence of withdrawal signs than both the non-dependent (control) and spontaneous withdrawal groups. These experiments provide a detailed account of opiate withdrawal following the continuous subcutaneous infusion of a small dose of morphine for one week.

Acute effects of neuroleptics on brain self-stimulation thresholds in rats

The acute effects of 5 neuroleptic drugs were tested in rats implanted with stimulating electrodes in the medial forebrain bundle and trained in a brain selfstimulation threshold procedure. Haloperidol (0.01–0.10 mg/kg) and loxapine (0.03–0.56 mg/kg) produced increases in reinforcement thresholds accompanied by reductions in response rates. Chlorpromazine (0.10–3.0 mg/kg) did not significantly alter reinforcement thresholds, but did produce dose-dependent reductions in response rates. Pimozide (0.1–1.75 mg/kg) was similar to chlorpromazine and significantly increased the reinforcement threshold only at the highest dose, although a graded decrease in response rates occurred over a wide dose-range. Clozapine (0.1–1.75 mg/kg) increased reinforcement thresholds without producing any significant changes in response rates, but when 3.0 mg/kg was administered, a marked disruption of behavior occurred. The results suggested that a distinction can be made between the effects of neuroleptics on motor behavior and on central reinforcement thresholds, and this may help in the interpretation of the relation between the chemical and clinical potency of antipsychotic drugs.

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.