Role of postnatal androgens in sexual differentiation of the lordosis-inhibiting effect of central injections of cholecystokinin

The neuropeptide cholecystokinin (CCK) inhibits lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMN) of female rats and has no effect when infused into the VMN of male rats. To test whether this sex difference develops under the control of perinatal steroids, male rats were castrated or given sham surgeries within 3 h of birth and female rats were injected with either 0 or 100 micrograms testosterone propionate on postnatal day 5. As adults, these rats were castrated as necessary, implanted with unilateral cannulae directed at the VMN, and tested for their ability to display female sexual behavior and to respond to CCK. Neonatal castration of males prevented defeminization of this response. When treated with 5 micrograms estradiol benzoate (EB), neonatally castrated males showed both lordosis behavior and a profound inhibition of that behavior after infusions of CCK. Neonatally castrated males did not display lordosis behavior when treated with 2 micrograms EB. Control males showed no lordosis behavior and, therefore, no response to CCK. Both doses of EB induced lordosis behavior in neonatally androgenized females. Significantly, these neonatally androgenized females were less responsive to CCK's inhibition of lordosis and were also anovulatory. These results imply that androgens alter the development of CCK responsive circuits as well as defeminize cyclic gonadotropin release. Levels of 125I-sCCK-8 binding in the VMN were correlated closely with an individual's ability to respond to sCCK-8. In summary, the inhibition of female sexual behavior caused by exogenously administered CCK in normal adult female rats appears to be controlled at least partially by levels of CCK receptors in the VMN and to differentiate under the control of perinatally present testosterone.     

Glutamate AMPA/kainate receptors, not GABA(A) receptors, mediate estradiol-induced sex differences in the hypothalamus

Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.     

Recurrent hypoglycemia reduces the glucose sensitivity of glucose-inhibited neurons in the ventromedial hypothalamus nucleus

Recurrent hypoglycemia blunts the brain's ability to sense and respond to subsequent hypoglycemic episodes. Glucose-sensing neurons in the ventromedial hypothalamus nucleus (VMN) are well situated to play a role in hypoglycemia detection. VMN glucose-inhibited (GI) neurons, which decrease their firing rate as extracellular glucose increases, are extremely sensitive to decreased extracellular glucose. We hypothesize that recurrent hypoglycemia decreases the glucose sensitivity of VMN GI neurons. To test our hypothesis, 14- to 21-day-old Sprague-Dawley rats were subcutaneously injected with regular human insulin (4 U/kg) or saline (control) for three consecutive days. Blood glucose levels 1 h after insulin injection on day 3 were significantly lower than on day 1, reflecting an impaired ability to counteract hypoglycemia. On day 4, the glucose sensitivity of VMN GI neurons was measured using conventional whole cell current-clamp recording. After recurrent insulin-induced hypoglycemia, VMN GI neurons only responded to a glucose decrease from 2.5 to 0.1, but not 0.5, mM. Additionally, lactate supplementation also decreased glucose sensitivity of VMN GI neurons. Thus our findings suggest that decreases in glucose sensitivity of VMN GI neurons may contribute to the impairments in central glucose-sensing mechanisms after recurrent hypoglycemia.     

Manipulation of neonatal gonadal steroid milieu and leptin secretion in later life in male and female rats

The mechanism underlying the gender-based difference in circulating leptin levels (females>males) is still uncertain, because the difference persists even after adjustment for fat mass and sex steroid concentrations. In this study, we tested the possibility that the neonatal sex steroid milieu, which is critical for the sexual differentiation of the brain, may permanently affect leptin secretion in rats of both sexes. Male rats were neonatally castrated (NC), and females were neonatally androgenized (NA) by testosterone (T). Two subsets of the NC males were given T on postnatal day 1 or 29. Appropriate controls for all these groups were prepared. The animals were sacrificed on postnatal day 57, and at this age, the percent body fat was similar among all the male and female groups. NC males had a two-fold, significantly higher level of leptin than intact males. This hyperleptinemia induced by NC was prevented by T when it was given neonatally, but not on the day 29. By contrast, NA for females was without effect on leptin titers in later life. These results suggest that neonatal T in male rats may, at least in part, mediate the sex-related difference in leptin secretion that becomes apparent in later life. However, as intact females still had significantly higher leptin titers than NC males, it is very likely that additional factors may also be responsible for the sexually dimorphic leptin secretion in rats.     

Effects of ventromedial nucleus lesions on estradiol induced ovulation in the rat

Small bilateral stereotaxic lesions were made in the hypothalamic ventromedial nucleus (SVMN) to determine: (i) whether estrogen would restore early receptivity in unreceptive SVMN lesioned female rats and (ii) whether SVMN lesions would suppress estrogen induced ovulation in the rat. SVMN lesions were shown to completely suppress spontaneous early receptivity and seriously impair estrous receptivity in 5-day cyclic female Wistar rats. A loss of early receptivity in response to 10 μg estradiol benzoate (EB) was also observed in SVMN lesioned females, in comparison to unoperated, sham VMN and dorsomedial nucleus (DMN) lesioned animals. Isolated SVMN lesioned females exhibited a weak ovulatory response to 10 μg EB, but, where shown to be unreceptive prior to estrogen injection, they never ovulated. On the contrary, ovulation occured in about 50% of cases in isolated unoperated and in sham VMN and DMN lesioned females following estrogen administration. The mechanisms whereby EB brought about precocious ovulation in 5-day cyclic female rats were therefore concluded to be dependent on VMN functional integrity and thereby on the degree of early sexual receptivity in the rat.     

Hypothalamic Monoaminergic Activity in 11-Week-Old Cold-Exposed Female Lean (Fa/Fa) and Obese (fa/fa) Zucker Rats

We previously reported that serotonergic activity was reduced in the ventromedial hypothalamic nucleus (VMN) of obese vs. lean male Zucker rats. To verify that this reduction was associated with genotype rather than gender, we measured monoamines and their major metabolites in hypothalamic nuclei of ll-week-old female lean (Fa/Fb) and obese (fa/fb) Zucker rats. In addition, since the thermic response to cold is reported to differ between lean and obese rats, some rats were also exposed to 9° or 22° C for 2h to determine if cold exposure altered hypothalamic monoaminergic activity. As in males, levels of 5-hydroxyindoleacetic acid [5-HIAA; major metabolite of serotonin (5-HT)] and the ratio of 5-HIANS-HT were lower in the VMN of obese vs. lean females (P = 0.008, 0.001, respectively). S-HIANS-HT was also reduced in the paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the obese compared to the lean females. Cold exposure significantly stimulated brown fat mitochondria1 GDP binding in lean but not obese rats. Similarly, levels of norepinephrine, dopamine (DA), 5-HIAA, and 5-HT in the PVN, and 5-HIAA in the SCN increased in cold-exposed lean but not obese rats. In contrast, VMN and preoptic 3,4-dihydroxyphenylacetic acid (DOPAC; major metabolite of DA) increased in the cold-exposed obese but not lean animals. We conclude that: (1) the blunted peripheral response to cold in obese vs. lean Zucker rats is accompanied by altered hypothalamic monoaminergic activity, the physiological role of which needs further evaluation; and 2) depressed VMN serotonergic activity is associated with the obese genotype (fa/fa) rather than gender and as such may contribute to the reduced sympathetic and enhanced parasympathetic outflow from the VMN .     

Influence of androgen in the neonatal period on ejaculatory and postejaculatory behavior in the rat

The objective of the present report was to investigate the influence of androgen in the neonatal period on the development of ejaculatory and postejaculatory behavior. At birth, male rats were either castrated (neonatally castrated males), implanted with a Silastic tube of the aromatase inhibitor androsta-1,4,6-triene-3,17-dione for the first 10 days (ATD males), or left untreated (normal males). Female rats were either injected with 0.5 mg testosterone propionate (TP) on Days 1 (day of birth) and 2 (androgenized females) or left untreated (normal females). All gonadally intact animals were castrated at 60 days of age. Following TP administration, all animals were tested for ejaculatory and postejaculatory behavior under both shock and nonshock conditions. All animals were capable of showing the intromission pattern; however, the ejaculatory pattern was exhibited regularly only by those animals exposed to androgen at birth (normal males, androgenized females, and ATD males). The normal males required fewer intromissions to achieve ejaculation than the other two groups exhibiting this reflex. This result is discussed in terms of peripheral genital stimulation deficits and the differentiation of neural tissue responsible for masculine copulatory behavior. Androgenized females and ATD males displayed a refractory period, characterized by 22-kHz vocalizations, equal to or longer than that found in normal males. These results indicate that defeminization is not necessary for the display of normal ejaculatory and postejaculatory behavior.     

Long-term ovariectomy and hormone-induced sexual behavior, progestin receptors, and hypothalamic morphology in female rats

Long-term ovariectomy reduces the ability of estradiol and progesterone treatment to induce sexual receptivity in female rats. Previous researchers suggested that this effect may be due to a decreased induction of neural progestin receptors by estradiol in the long-term ovariectomized rats. The present study was designed to replicate and extend this finding, and to search for neuroanatomical correlates by measuring the volume of the ventromedial nucleus (VMN) of the hypothalamus, a putative site of action of estradiol and progesterone for the induction of female sexual behavior. Long-term ovariectomy (5 to 6 weeks) as compared to short-term ovariectomy (1 week) reduced the ability of estradiol-17 beta and progesterone treatment to induce sexually receptive and proceptive behaviors. Consistent with previous reports, our data show that the reduced levels of cytosol progestin receptors after long-term ovariectomy and estradiol treatment are related to a reduced ability of estradiol to induce the receptors. Long-term ovariectomy did not affect the concentration of cytosol progestin receptors in the preoptic area, suggesting a neuroanatomical specificity to this effect. Contrary to our predictions, long-term ovariectomy did not affect the volume of the VMN. In fact, estradiol treatment, while blocking the effect of long-term ovariectomy on sexual behavior, decreased the volume of the VMN. Therefore, the measurement of the volume of the VMN is not a good predictor of the responsiveness to steroid hormone induction of sexual behavior.     

Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus

We have studied the effects on neuropeptide Y (NPY) concentration in six hypothalamic nuclei, viz. medial preoptic area (MPOA), paraventricular nucleus (PVN), median eminence (ME), arcuate nucleus (ARC), ventromedial nucleus (VMN) and dorsomedial nucleus (DMN) of food deprivation (FD) for 2, 3, or 4 days or FD for 4 days followed by one day ad lib food intake (FI) in male rats. Hypothalamic nuclei were microdissected by the technique of Palkovits and processed for measurement of NPY immunoreactivity by RIA. NPY-like immunoreactivity in the ME, VMN and DMN was unaffected by FD or FI, but the remaining three nuclei--the ARC, MPOA and NPY--displayed a different pattern of changes in NPY levels in response to either FD or FD followed by FI. In the ARC, NPY levels rose significantly at day 3 and 4 after FD and remained elevated even after one day of FI. In the MPOA, while FD for 4 days had no effect, NPY concentration increased significantly in response to FI. In contrast, in the PVN, a site implicated in the control of feeding behavior, the NPY response to FD and FI was markedly different. FD elicited a gradual, time-related increase in NPY levels to reach highest concentration on day 4 and thereafter, following one day of FI, NPY levels fell dramatically to the range found in control satiated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maternal Deprivation Exacerbates the Response to a High Fat Diet in a Sexually Dimorphic Manner

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including affectation of metabolism. Indeed, MD for 24 hours during the neonatal period reduces body weight throughout life when the animals are maintained on a normal diet. However, little information is available regarding how this early stress affects the response to increased metabolic challenges during postnatal life. We hypothesized that MD modifies the response to a high fat diet (HFD) and that this response differs between males and females. To address this question, both male and female Wistar rats were maternally deprived for 24 hours starting on the morning of postnatal day (PND) 9. Upon weaning on PND22 half of each group received a control diet (CD) and the other half HFD. MD rats of both sexes had significantly reduced accumulated food intake and weight gain compared to controls when raised on the CD. In contrast, when maintained on a HFD energy intake and weight gain did not differ between control and MD rats of either sex. However, high fat intake induced hyperleptinemia in MD rats as early as PND35, but not until PND85 in control males and control females did not become hyperleptinemic on the HFD even at PND102. High fat intake stimulated hypothalamic inflammatory markers in both male and female rats that had been exposed to MD, but not in controls. Reduced insulin sensitivity was observed only in MD males on the HFD. These results indicate that MD modifies the metabolic response to HFD intake, with this response being different between males and females. Thus, the development of obesity and secondary complications in response to high fat intake depends on numerous factors.     

Hormone-dependent regulation of GABAA receptor gamma subunit mRNAs in sexually dimorphic regions of the rat brain.

Transmission mediated by gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) and the ventromedial nucleus (VMN) of the hypothalamus is known to play critical, but contrasting, roles in regulating steroid-dependent sexual behaviours in rats. Previous studies have demonstrated a striking dichotomy in receptor composition between the two regions with regard to gamma, but not alpha or beta, subunit expression. To test if gonadal steroids regulate the expression of the gamma subunit genes within the mPOA and the VMN, in situ hybridization analysis for messenger RNAs encoding the gamma 1, gamma 2Short (gamma 2S) and gamma 2Long (gamma 2L) subunits was done in gonadectomized male and female rats and in gonadally intact females over the oestrous cycle. No significant differences in the expression of the gamma subunit mRNAs were observed in gonadectomized male versus female rats. Significant effects of gonadal state in female rats were observed for gamma 1 mRNA levels in the mPOA and gamma 2L levels in the VMN. These data demonstrate that gonadal hormones exert activational control of expression of GABAA receptor gamma subunit mRNAs and suggest that differences in receptor structure may contribute to the functional modulation of female sexual behaviours mediated by GABAergic transmission in these regions.     

A contributory role for midbrain progesterone in the facilitation of female sexual behavior in rats

Progesterone (P) facilitation of sexual receptivity in rodents has been achieved by intracranial administration to the ventral hypothalamus; the preoptic area; and midbrain areas such as central gray, mesencephalic reticular formation, and ventral tegmental nucleus. In our laboratory, by far the most effective site in rats has been the ventromedial nucleus of the hypothalamus (VMN). However, several reports of sensitivity to P in the midbrain of rats and other rodent species led us to investigate whether stimulation of the ventral midbrain of female rats might contribute to facilitation of sexual receptivity. Ovariectomized Long-Evans rats received one cannula aimed at the VMN, and another aimed at the contralateral ventral mesencephalon. P in both cannulae, following a priming dose of estradiol, caused significantly higher lordosis quotients (LQ) than blank tubes. Controls with bilateral cannulae in the VMN responded when both tubes were filled with P, but did not respond to unilateral VMN P stimulation. P in the VMN and contralateral anterior preoptic area did not result in a greater degree of receptivity than did the empty tubes. These studies indicate that although progesterone stimulation in the midbrain alone is not sufficient to facilitate receptivity in female rats with our methods, the midbrain may play an auxiliary role. P implants in the midbrain appear to facilitate receptivity in the case of VMN implant treatments that are subthreshold for stimulating lordosis. The results are discussed in light of similar studies in other rodent species, and in the context that more than one brain site may be important in the natural stimulation of sexual receptivity by gonadal hormones.     

Influence of age, sex and hypoxia on plasma dopamine-beta-hydroxylase activity in the rat

Using rats (Wistar strain) of our own breed, we studied dopamine-beta-hydroxylase (E.C. 1.14.17.1) (DBH) activity in the plasma of animals of different ages (in correlation to sex) under normal conditions and after exposure to altitude hypoxia (corresponding to 7000 or 9000 m and lasting 20 min). The enzyme was determined by the method of Kato et al. (1974). We found that the given plasma enzyme activity was significantly higher in females than in males, throughout the whole life-span. In addition, we found that minimum activity was reached on about the 14th and 21st day of postnatal life and again on the 40th day, while maximum activity was recorded at the ages of 5, 30 and 35 days and in adult rats. In adult animals (males and females), exposure to altitude hypoxia was followed by a statistically significant increase in plasma DBH activity, which was much more pronounced in females than in males. In males, 240 min after terminating hypoxia plasma DBH activity had returned to normal, but in females it was still significantly raised; after 48 h, plasma DBH activity in females was identical to the activity before exposure to hypoxia. In rats aged 5 and 35 days, hypoxia evoked a fluctuating response. A decrease in activity immediately after terminating hypoxia was followed at 60 min by a return to normal, but at 240 min there was again a significant decrease. In 21-day-old rats, hypoxia did not induce any significant change in plasma DBH activity (the initial activity level in this group was very low).     

Estrogenic Regulation of Histamine Receptor Subtype H1 Expression in the Ventromedial Nucleus of the Hypothalamus in Female Rats

Female sexual behavior is controlled by central estrogenic action in the ventromedial nucleus of the hypothalamus (VMN). This region plays a pivotal role in facilitating sex-related behavior in response to estrogen stimulation via neural activation by several neurotransmitters, including histamine, which participates in this mechanism through its strong neural potentiating action. However, the mechanism through which estrogen signaling is linked to the histamine system in the VMN is unclear. This study was undertaken to investigate the relationship between estrogen and histamine receptor subtype H1 (H1R), which is a potent subtype among histamine receptors in the brain. We show localization of H1R exclusively in the ventrolateral subregion of the female VMN (vl VMN), and not in the dorsomedial subregion. In the vl VMN, abundantly expressed H1R were mostly colocalized with estrogen receptor α. Intriguingly, H1R mRNA levels in the vl VMN were significantly elevated in ovariectomized female rats treated with estrogen benzoate. These data suggest that estrogen can amplify histamine signaling by enhancing H1R expression in the vl VMN. This enhancement of histamine signaling might be functionally important for allowing neural excitation in response to estrogen stimulation of the neural circuit and may serve as an accelerator of female sexual arousal.     

Nonresponsiveness of the thyroid gland to goitrogen in the early neonatal period in the rat: light- and electron-microscopic observations.

The thyroid response of fetal and neonatal rats to propylthiouracil (PTU) as a goitrogen was studied with observation of the thyroid glands by light and electron microscopy. On day 19 of gestation and on days 1, 3, 5 and 8 after birth, fetal and neonatal rats were given a subcutaneous injection of PTU and were autopsied 2 days later. PTU induced conspicuous goiters in fetal rats but did not in neonatal rats aged up to day 5 after birth. Beyond that age, PTU again induced goiters. Histologically, the follicular cell height in goitrous thyroid glands was significantly increased. Ultrastructurally, follicular cells in goitrous thyroid glands often had colloid droplets and lysosomes. It seems that nonresponsiveness of the thyroid glands in early neonatal rats to goitrogen is due to a temporary decline of the pituitary activity of thyrotropin secretion. About 5 days or more after birth, the pituitary-thyroid system begins to operate again in response to goitrogen.     

Ovarian hormones after postnatal day 20 reduce neuron number in the rat primary visual cortex

Previous work from our lab has documented a sex difference in neuron number in the binocular region of the adult rat primary visual cortex (Oc1B), with males having 19% more neurons than females. In the present study, the role of developmental steroid hormones in the formation of this difference was explored. Male and female rats underwent neonatal hormone manipulation (female + testosterone or dihydrotestosterone; male + flutamide) followed by gonadectomy on postnatal day 20. Animals that did not undergo hormone manipulation were either gonadectomized or sham operated at day 20. Neuron number was quantified in the monocular (Oc1M) and binocular (Oc1B) subfields of the adult rat primary visual cortex using the optical disector technique. As adults, day 20 gonadectomized females, as well as females + testosterone and females + dihydrotestosterone, had significantly more neurons than intact females. There was no difference in neuron number between postnatal day 20 gonadectomized males, males + flutamide, and intact males. Also, intact males had significantly more neurons than intact females in both in Oc1M and Oc1B. It appears that ovarian steroids after day 20 are the primary cause of the lower number of neurons in the primary visual cortex of the female rat.     

Influence of neonatally administered gonadotropin on the sexual function of adult rats]

Male and female rats were daily injected with 10 IU HCG plus 10 IU FSH from the 1st to 14th day of life in order to investigate the influence of neonatal gonadotrophin administration on the sex-specific differentiation of the brain. When adult, the males showed hypogonadism associated with approximately normal sexual activity. In the females, precocious puberty, indicated by premature vaginal opening and spontaneous estrus, occurred. Furthermore, bisexuality with a tendency towards more male behavioural patterns was observed, but no impairment of ovarian cyclicity. Thus, hypergonadotrophic hypergonadism during the hypothalamic differentiation phase gave rise to bisexual behaviour in adult female rats associated with normal ovarian cycles. The question of a direct or indirect influence of gonadotrophins on the sex-specific brain differentiation is discussed.     

Effects of morphine and naloxone administered to pregnant rats on the adrenal glands and testes of the offspring]

Relative testis and adrenal weights, testosterone and corticosterone levels in the blood were determined in 9-, 16-day and 2-month-old rats born to females injected with morphine (10 mg/kg/day), naloxone (10 mg/kg/day) or saline throughout the 15-18 days of gestation. Opioid receptors agonist morphine caused a long-lasting inhibition of the testes and activation of the adrenals. Saline injections to the females, that are known to be a stressor for them, also inhibited the testis of the neonatal offsprings. The block of the opioid receptors by the naloxone prevented the effect of prenatal stress on the testis, but inverted the negative correlation between the testes and the adrenals, that can be observed in the normal development.     

Effects of morphine on electrical activity of the emotion-producing zones in the hypothalamus of adult and aged rats

In the experiments performed on adult and aged rats, the effect of morphine on the electrical activity, recorded from the emotion-producing zones of the hypothalamus, the ventromedial nucleus (VMN), and the lateral hypothalamic area (LHA), was studied. In thein vitro experiments, an age-dependent reduction of background impulse activity (BIA) was found in the VMN single neurons, but not in the LHA neurons. Morphine reduced BIA in most of the VMN neurons, but enhanced it in the LHA neurons of adult rats, and enhanced BIA in the neurons of both structures of the aged rats. The inhibitory effect of morphine on the VMN and LHA neurons and its excitatory effect on the LHA neurons decreased with age. In thein vivo experiments, an age-dependent reduction of the background field electrical activity (background electrogram, BEG) was found in the neurons of both emotion-producing zones. Morphine reduced the BEG magnitude in the VMN and LHA more effectively in the aged rats than in the adult rats. The results allow us to suggest that both the opiate regulation of hypothalamic functions and formation of an opiate dependence in the adult rats essentially differ from those in the aged rats.Neirofiziologiya/Neurophysiology, Vol. 27, No. 2, pp. 126–133, March–April, 1995.     

Long-term perspectives of thyroxine administration in neonatal rats.

Newborn pups were injected with normal saline (group A) and exogenous thyroxine (group B). Elevated T4 and decreased TSH levels from day 7 in group B continued until day 35. T4 and TSH were in normal range by day 42 and were similar to group A. Weight gain was significantly lower in group B. On day 45, half hourly injections (subcutaneous) of TRH were given to half of group A and group B each. Remaining halves were injected with saline. TSH response to TRH was significantly decreased in group B rats. Thus, neonatal hyperthyroidism results in (1) permanent decrease in pituitary reserve of TSH secretion and (2) a permanent imprinting regarding growth and thyroidal development and thus, neonatal period is critical for thyroidal development.