Reid L. Norman

Consequences of restraint stress on natural killer cell activity, behavior, and hormone levels in rhesus macaques (Macaca mulatta)

Three experiments were performed to determine the effect of stress on the neuroendocrine-immune system in nonhuman primates. In Experiment 1 the diurnal variation in cell and hormone levels was determined. The percentages of neutrophils, monocytes, and eosinophils fluctuated throughout the 24-hr period, while white blood cell (WBC), neutrophil-to-lymphocyte ratio (N:L), hemoglobin (Hgb), natural killer cell cytotoxicity (NK activity) and beta-endorphin levels did not. Experiment 2 investigated the effects of ketamine and restraint on behavior. Scratching was increased in control monkeys and animals receiving ketamine, whereas passivity was increased in chair-restrained animals. In Experiment 3, eight adult male rhesus monkeys were restrained in primate chairs at 0600h. Behavior was filmed for 3 hr and blood samples were collected at 0700, 0800, and 0900. Whole blood was analyzed for total WBC and percentage of each leukocyte type. NK activity was also measured. Plasma levels of cortisol and beta-endorphin were determined and behavior was quantitated from video-records. WBC and the percentage of neutrophils increased during the restraint period, while the percent lymphocytes and monocytes decreased. NK activity also decreased over time after restraint whereas plasma cortisol and beta-endorphin levels increased significantly. Although after the 3 hr of restraint stress, changes were found in hormone levels, behavior, and NK activity, there were no significant correlations between the parameters measured. Thus, our results indicate that there is not a common neuroendocrine response or single neuroendocrine mediator that results in predictable behavioral changes and immune suppression following stress.

Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E1 into pentobarbital-blocked rats

Abstract Five to 20 μg of prostaglandin (PG) E1 infused into the third ventricle of 38 rats on the afternoon of vaginal proestrus reversed the pentobarbital (Pb) blockade of ovulation in 25. Plasma levels of luteinizing hormone (LH) were higher in the PGE1-infused females than in the Pb-blocked controls, but the peak concentrations were only half as high and lasted only half as long as comparable values in normal proestrous females. PGE1 was a more potent stimulator of ovulation in Pb-blocked females than either PGE2 or PGF2α. If infused into the pituitary or injected subcutaneously, however, PGE1 (10 μg) failed to induce ovulation in Pb-blocked animals. The data indicate that PGE1 stimulates LH release and ovulation in the Pb-treated rat by activating a neurally-controlled gonadotropin-releasing mechanism.

Intracerebroventricular porcine corticotropin-releasing hormone and cortisol effects on pig immune measures and behavior

The effects of intracerebroventricular (icv) administration of porcine corticotropin-releasing hormone (pCRH) and cortisol on the immune system and behavior were examined in domestic pigs. In Experiment 1, 50 micrograms of pCRH in 200 microliters of saline or 200 microliters of vehicle was administered i.c.v. at 0600 h. Blood samples were obtained at 0600 (prior to injection), 0700, and 0800 h. Plasma cortisol concentrations were higher at 1 and 2 h after pCRH than after saline. Generally, pCRH failed to effect NK cytotoxicity or lymphocyte proliferation in response to phytohemagluttin (PHA). However, 1 h postinjection, pigs administered pCRH i.c.v. had marginally lower NK activity than control pigs. Pigs injected with pCRH had substantially lower neutrophil chemotaxis (CHTX) than the control pigs at 1 and 2 h postinjection. As blood cortisol concentration increased, neutrophil CHTX decreased. Pigs injected i.c.v. with pCRH had higher neutrophil numbers and neutrophil:lymphocyte ratios than control pigs. Percentage of lymphocytes was higher among control than treated pigs. Central pCRH increased overall activity, particularly walking, standing, licking, rooting, and increased activity-related sequences (e.g., sit, walk and stand, walk), but reduced complex oral/nasal sequences (e.g., root, lick). In Experiment 2, pigs were injected i.c.v. with 10 micrograms of cortisol in 200 microliters of saline or with vehicle at 0600 h. Administration of cortisol failed to effect NK cytotoxicity, lymphocyte proliferation, CHTX, or leukocyte distribution. Pigs given cortisol had no apparent change in behavior. These data indicate leukocyte distribution and specific neutrophil function in pigs were significantly modulated by stress-related hormones of the hypothalamic-pituitary-adrenal axis and complexity of behavioral sequences (pigs repeating certain behavioral sequences) associated with increased activity was reduced. Oral/nasal stereotypies (as seen among confined sows) were not elevated among pigs given i.c.v. pCRH. CRH given by i.c.v. administration may serve as a better model for acute rather than chronic stress.

Short-term leptin infusion does not affect circulating levels of LH, testosterone or cortisol in food-restricted pubertal male rhesus macaques.

Although the adipocyte protein leptin has been implicated in the control of reproductive function in rodents, its role in primate reproductive physiology is poorly understood. Because primates in puberty show nighttime LH secretion and there is considerable evidence that the fertile state requires adequate nutrition, we reasoned that animals on the verge of reproductive competence would respond to leptin infusions by secreting LH. Food restriction reduces circulating leptin levels and slows or stops the GnRH pulse generator. Therefore, we examined the endocrine effects of leptin infusions in food‐restricted male pubertal primates during the night when they normally secrete LH. In addition, we investigated the effect of leptin on in vitro testosterone production by Leydig cells.

Neuroendocrine Consequences of Fasting in Adult Male Macaques: Effects of Recombinant Rhesus Macaque Leptin Infusion

Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 µg/h) into fasted adult male rhesus macaques (6–9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42–57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 ± 1.4 ng/ml control vs. 2.7 ± 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 ± 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 ± 1.0 ng/ml) and the fasted + rrhLep (2.85 ± 1.0 ng/ml) groups compared to controls (16.3 ± 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 ± 0.95 pulses/15 h control vs. 12.8 ± 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 ± 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 ± 0.37 ng/ml in control animals, 0.7 ± 0.2 ng/ml in fasted animals and 10.1 ± 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque.

Exposure to ovarian steroids elicits a female pattern of plasma cortisol levels in castrated male macaques

Our recent observations (1) that there is a difference in circadian patterns of plasma cortisol levels between male and female macaques and (2) that after gonadectomy these differences in the patterns and in the levels of cortisol were reduced prompted us to investigate how 17 beta-estradiol (E2) and progesterone affect cortisol secretion in orchidectomized male rhesus macaques. Five male macaques, castrated as adults, were implanted subcutaneously with segments of silastic tubing filled with E2 and with progesterone in a manner such that the levels and the sequence of these hormones mimicked those that occur during the menstrual cycle of intact female macaques. Since previous studies had shown that the difference in cortisol patterns was due to higher levels in females during the day, these studies were conducted from 0800 to 2000 hours. Blood samples were collected in an adjacent room at 15-minute intervals. Separate trials were conducted 2 weeks after E2 was implanted and levels were 110 +/- 14 pg/ml and again 2 weeks later after progesterone was implanted and E2 levels were 59 +/- 15 pg/ml; progesterone levels averaged 4.0 +/- 0.65 ng/ml. Mean plasma concentrations of cortisol (microgram/100 ml) for the 12-hour period were three-fold higher in orchidectomized males treated with E2 (17.2) and with E2 + progesterone (18.0) than in intact males (4.9). Levels in males treated with ovarian steroids were double that (8.5 micrograms/100 ml) observed for intact females.(ABSTRACT TRUNCATED AT 250 WORDS)

17β-Hydroxysteroid dehydrogenase activity in the pituitary gland and neural tissue of rhesus monkeys

Ovariectomized rhesus monkeys were treated with estradiol-17β (E2) for 24 days; E2 for 24 days and progesterone (P) for the last 12 days; or P alone for 12 days. Parts of the brain (hypothalamus, preoptic area, amygdala, frontal cortex, and cerebellum) and anterior pituitary gland were analyzed for activity of the 17β-hydroxy steroid dehydrogenase (17β-HSDH) by quantification of the amount of [14C]-estrone (14C-E1) or [3H]-androstenedione ([3h]-a) formed from [14C]-E2 or [3H]-testosterone ([3H]-T) by minced tissue in a 2-h incubation period at pH 7.4. No significant effect of treatment was observed. Therefore, the between treatment data were pooled and analyzed together. The anterior pituitaries, both in the amount of [14C]-E1 formed from [14C]-E2 and in the amount of [3H]-A formed from [3H]-t, had a significantly higher level of enzyme activity (P < 0.001) than any of the neural tissue analyzed. The difference between the activity of the 17β-HSDHs in anterior pituitary and brain tissue was confirmed after we incubated the 800 g supernatant of these tissues and used radioimmunoassayable E1 as an end point of 17β-HSDH activity. With this technique, the activity was lost after freezing, was greater at pH 8.0 than at 7.0, and was linear at protein concentrations from 0.3 to 1.0 mg/ml of incubation medium. The fact that this enzyme is localized with such intensity within the anterior pituitary may indicate that it participates in E2 action at the pituitary level in this species.

Progesterone Suppression of the Estradiol Receptor in the Reproductive Tract of Macaques, Cats, and Hamsters

Few biological phenomena are so well described and yet so poorly understood as the periodic morphological changes that occur in the mammalian reproductive tract during estrous or menstrual cycles.

Dopaminergic and opioid compounds. Effects on prolactin and LH release after electrical stimulation of the hypothalamus in ovariectomized rhesus monkeys.

In ovariectomized rhesus monkeys ( Macaco mulatto ) electrical stimulation (ES) of the medial basal hypothalamus (MBH) for 30 min with stimulus parameters that caused no overt behavio

Effects of bilateral amygdaloid lesions on gonadal and pituitary hormones in serum and on sexual behavior in female rhesus monkeys

Bilateral lesions that destroyed the amygdaloid nuclear complex failed to alter either serum concentrations of luteinizing hormone (LH), estradiol (E2), progesterone, and testosterone or sexual behavior in female rhesus monkeys. In 3 of 5 females ovulation occurred in the postoperative cycle, and in 4 of 5 females exogenous E2 treatment released a surge of LH whose pattern was indistinguishable from the normal preovulatory surge. A postcastration rise in serum LH also occurred in the 5 lesioned monkeys, and E2 again induced an LH release. The data suggest that the amygdala does not play a major role in regulation of LH or ovarian steroid secretion during the menstrual cycle. Moreover amygdaloid lesions in female monkeys do not alter their sexual behavior.