Effects of morphine on luteinizing hormone secretion and catecholamine turnover in the hypothalamus of estrogen-treated rats

This study examined the effects of morphine sulfate and naloxone alone or in combination on phasic luteinizing hormone (LH) secretion in estrogen-treated ovariectomized rats. Thereafter, the effects of morphine on initial concentrations, rate constants and rates of turnovers of norepinephrine and dopamine were evaluated in untreated or morphine-injected, estrogen-primed rats. Morphine, when given at 12.30 h, completely suppressed the spontaneous LH surges which occur in steroid-treated rats. The opiate antagonist, naloxone, (12.15 h) markedly amplified and advanced the time of LH release and a combination of morphine and naloxone produced peak afternoon LH values which were intermediate between those obtained in controls and in rats receiving only naloxone. Norepinephrine (NE) and dopamine (DA) turnover were calculated from data obtained in groups of rats sacrificed 0,45 or 90 min after administering 300 mg/kg b. wt. i.p. ofα-methyl-p-tyrosine (α-MPT) at 10.00 or 15.00 h. In these experiments, the medial preoptic nucleus (MPN) and the median eminence (ME) were microdissected and analyzed for changes in NE and DA concentrations by a radioenzymatic procedure. In estrogen-treated rats, NE rate constants and turnover significantly increased at 15.00 vs 10.00 h in MPN and ME concomitant with increases in serum LH. Morphine blocked both increases in rate constants and turnover in the MPN and ME and also significantly reduced initial concentrations of NE in the MPN. None of the DA parameters measured in MPN or ME changed in estrogen-treated controls between morning and afternoon. Further, while morphine did not affect DA turnover in the MPN, DA turnover declined in the ME. These data add to accumulating evidence which demonstrates an important modulatory role for hypothalamic opiate neurons in regulating catecholamine activity and gonadotropin secretion.     

Effects of p-chlorophenylalanine on hypothalamic indoleamine levels and the associated changes which occur in catecholamine dynamics and LH surges in estrogen-treated ovariectomized rats

Recent studies have demonstrated that the serotonergic and noradrenergic systems are functionally and anatomically linked and both systems have been implicated as contributors to the regulation of the phasic release of LH. Consequently, perturbations within the serotonergic system could secondarily affect noradrenergic system activity and result in a loss of phasic LHRH secretion. In the present studies we examined the effects of p-chlorophenylalanine (PCPA) on LH surges and the associated changes which occur in hypothalamic serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations. We also evaluated the effects of this drug on norepinephrine (NE) and dopamine (DA) initial concentrations, rate constants and turnover rates in the medial preoptic area (MPN), suprachiasmatic nuclei (SCN), paraventricular nuclei (PVN) and median eminence (ME). Seven days after ovariectomy, rats received estradiol (E2) capsules (day 0) and on day 1 some animals also received PCPA (250 mg/kg b. wt., i.p.) while the remainder served as controls. LH surges occurred in control animals but not in PCPA-treated rats on days 2, 3 and 4. PCPA produced a significant decline in 5-HT and 5-HIAA concentrations in all microdissected hypothalamic regions at 09.00 and 15.00 h on day 2. In control rats, there were no significant changes in initial concentrations of NE in the MPN, PVN and ME between 09.00 and 15.00 h with the exception of the SCN where a slight decline had occurred by 15.00 h. NE rate constants and turnover rates increased during the afternoon in controls in the MPN, SCN and ME and declined in the PVN concomitant with LH surges. PCPA had variable effects in suppressing NE initial concentrations depending upon the hypothalamic area studied and the time of day. More importantly, the drug abolished the diurnal rhythm in rate constants observed in controls and consequently, neither the MPN, SCN nor ME showed any increase in NE turnover rates in the afternoon of day 2. In contrast, a significant decline in rate constants and turnover rates occurred in the PVN of both control and PCPA-treated rats during the afternoon of day 2. DA initial concentrations declined in controls between 09.00 and 15.00 h in the MPN and ME but not in the SCN or PVN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of pulsatile luteinizing hormone secretion but not luteinizing hormone surge in leptin resistant obese Zucker rats

The adipose tissue-derived hormone leptin may be a primary mediator linking nutritional status and reproduction. The present study used the leptin-resistant obese female Zucker rat to investigate whether leptin signalling is required for normal pulsatile luteinizing hormone (LH) secretion and/or generation of the LH surge. For the pulsatile LH secretion study, an indwelling atrial catheter was implanted and a low dose of oestrogen given as a subcutaneous implant to lean and obese ovariectomized (OVX) Zucker rats. One week following OVX, blood samples were collected every 10 min for 3 h during the morning. Plasma LH concentrations were measured by radioimmunoassay. For the LH surge study, lean and obese OVX rats were given a high dose of oestrogen as a subcutaneous implant. Two days later, rats were given progesterone at 09.00 h to induce a proestrus-like LH surge. Blood samples were collected from an indwelling atrial catheter throughout that and the following day and plasma LH concentrations were measured by radioimmunoassay. LH pulse amplitude and mean LH secretion were profoundly attenuated in obese Zucker rats compared with lean littermates, whereas LH pulse frequency was not significantly different between phenotypes. The opioid receptor antagonist naloxone did not affect the pattern of pulsatile LH secretion in obese rats, suggesting that leptin does not exert its facilitatory effects on LH secretion through an opioidergic pathway. Both lean and obese rats showed characteristic steroid-induced LH surges. It therefore appears that a leptin signal is required for generation of a normal pattern of pulsatile LH secretion, but is not a necessary component of the steroid-induced LH surge.     

Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels Increase in A1 but not Locus Ceruleus Noradrenergic Neurons in Proestrous Rats but not in Diestrous or Androgen-Sterilized Animals

Norepinephrine (NE) turnovers (an index of secretion) increase in the hypothalamus of proestrous rats concomitant with luteinizing hormone surges, whereas, neither of these events are observed in diestrous nor in androgen-sterilized rats. Increased hypothalamic NE release may occur as a consequence of the withdrawal of local inhibitory γ-aminobutyric acid and opiate controls on specific presynaptic NE terminals and/or as a result of an increase in activity within noradrenergic neurons. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of NE and our earlier studies revealed that increases in TH mRNA in A1 and locus ceruleus (LC) neurons can serve as an index of increased activity within these cells. In the present study, we evaluated whether TH message levels change in A1 and LC neurons prior to and during the hours when luteinizing hormone surges and increased NE turnovers are observed. As controls, TH mRNA levels in A1 and LC neurons were evaluated at the same hours of day in diestrous day 2 and in androgen-sterilized rats. In situ hybridization histochemistry and quantitative image analysis methods were used to measure changes in TH mRNA levels. Luteinizing hormone surges in proestrous rats began at 1500 h, peaked between 1600 and 1700 h and declined, thereafter, to 2000 h. In contrast, plasma luteinizing hormone remained basal throughout the day in diestrous and androgen-sterilized rats. While A1 neuronal TH mRNA levels did not differ in the three groups of rats during the morning (0930 to 1030 h), these message levels were significantly elevated in proestrous rats during the afternoon (1645 to 1715 h) and remained high at 2000 to 2030 h. In contrast, no changes in TH mRNA levels were observed in A1 neurons throughout the afternoon in diestrous animals or androgen-sterilized rats. TH mRNA levels in the LC did not differ in the three groups of rats and they remained unchanged throughout the afternoon hours we examined. From these observations we conclude that concomitant with afternoon proestrous luteinizing hormone surges and the accompanying increase in hypothalamic NE secretion, there is an increase in activity within A1 but not LC neurons. These data suggest that the proestrous increase in hypothalamic NE turnover we previously observed is not due solely to withdrawal of local inhibitory controls of presynaptic NE release but it also involves an increase in activity within A1 but not LC neurons.     

A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat

Orexins are novel hypothalamic peptides which stimulate food intake. In view of the well-known tight connection between the nutritional state and the reproductive function, in this study we examined a possible role of orexin-A in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in ovariectomized rats. Experiments were performed on both normally-fed and 3-day-fasted rats. Although fasting led to abolition of both LH and PRL surges, intracerebroventricular administration of orexin-A (0.3 and 3.0 nmol) resulted in a dose-dependent recovery of the hormonal surges. In addition, anti-orexin-A antisera given to normally-fed rats completely abrogated the surges of both hormones. These results demonstrate for the first time a significant participation of orexin-A in the preovulatory LH and PRL surges in the rat.     

Neonatal exposure to estradiol prevents the expression of ovarian hormone-induced luteinizing hormone and prolactin surges in adulthood but not antecedent changes in neuropeptide Y or adrenergic transmitter activity: Implications for sexual differentiation of gonadotropin secretion

Sex differences in adult patterns of mating behavior and gonadotropin secretion in rats are determined in part by the presence or absence of gonadal steroids during a perinatal critical period. For example, male rats and female rats exposed neonatally to androgen do not exhibit LH surge patterns when treated appropriately with ovarian hormones in adulthood, and there is evidence that this may be due to a failure of ovarian hormones to activate the hypothalamic neuronal systems that stimulate LH secretion in such animals. Because considerable evidence suggests that estradiol formed centrally from testosterone is responsible for the permanent defeminization of mating behavior and gonadotropin secretion, the present studies compared normal females with normal males and with females treated neonatally with estradiol on the ability of ovarian hormones to induce several important neurochemical changes antecedent to the LH surge, including changes in neuropeptide Y (NPY) and LH-releasing hormone (LHRH) concentrations in the median eminence, as well as changes in turnover rates for catecholamine transmitters in the medial basal hypothalamus and medial preoptic area. Normal ovariectomized female rats responded to sequential treatment with estradiol followed by progesterone with afternoon LH and prolactin (PRL) surges, and with sequential accumulation followed by decline in concentrations of LHRH and NPY in the median eminence prior to the LH surge. In addition, administration of progesterone increased the turnover rates of norepinephrine (NE) and epinephrine (EPI) in the arcuate-median eminence region of normal females. Gonadectomized male rats receiving the same ovarian hormone treatment failed to exhibit LH or PRL surges and displayed none of the changes in neurotransmitter turnover or peptide concentrations characteristically seen in the normal female. Unexpectedly however, when females that were treated with estradiol benzoate on days 1–3 postpartum were ovariectomized and treated with ovarian hormones in adulthood, they showed the same accumulation/decline in median eminence NPY concentrations and the same activation of NE and EPI turnover in the arcuate-median eminence region as normal females, even though they showed no LH or PRL surges or changes in median eminence LHRH concentrations. These results suggest that estradiol may not mediate all of the defeminizing actions of androgen exerted during the early neonatal period, and particularly those actions that result in a lack of responsiveness in central noradrenergic, adrenergic and NPY systems in adulthood. However, an action of neonatal estradiol may result in uncoupling of the LHRH neurosecretory system from normal excitatory neurochemical influences.     

Luteinizing hormone and luteinizing hormone-releasing hormone secretion is under locus coeruleus control in female rats

It has been suggested that norepinephrine (NE) from the locus coeruleus (LC) plays an important role in triggering the preovulatory surge of gonadotropins. This work intended to study the role of LC in luteinizing hormone (LH) secretion during the estrous cycle and in ovariectomized rats treated with estradiol and progesterone (OVXE(2)P) and to correlate it with LH releasing hormone (LHRH) content in the medial preoptic area (MPOA) and median eminence (ME). Female rats on each day of the estrous cycle and OVXE(2)P were submitted to jugular cannulation and LC electrolytic lesion or sham-operation, at 09:00 h. Blood samples were collected hourly from 11:00 to 18:00 h, when animals were decapitated and their brains removed to analyze LC lesion and punch out the MPOA and ME. Plasma LH levels and LHRH content of MPOA and ME were determined by radioimmunoassay. During metestrus, diestrus and estrus, LC lesion did not modify either LH plasma concentrations or LHRH content, but completely abolished the preovulatory LH surge during proestrus and the surge of OVXE(2)P. These blockades were accompanied by an increased content of LHRH in the MPOA and ME. The results suggest that: (1). LC does not participate in the control of basal LH secretion but its activation is essential to trigger spontaneous or induced LH surges, and (2). the increased content of LHRH in the MPOA and ME may be due to a decreased NE input to these areas. Thus, LC activation may be required for depolarization of LHRH neurons and consequent LH surges.     

Locus ceruleus lesions block pulsatile LH release in ovariectomized rats

Luteinizing hormone (LH) secretion during the reproductive cycle and in ovariectomized (OVX) rats is pulsatile and this pattern of secretion is determined by intermittent discharges of LH-releasing hormone (LHRH) into the hypophysial portal vessels. LHRH secretion is probably controlled by prior pulsatile norepinephrine (NE) release. The locus ceruleus (LC) is an important source of NE to the LHRH neurons. We have shown previously that LC lesions block the preovulatory LH surge and ovulation and also cause a decrease in plasma LH concentrations in OVX rats. The possible role of the LC in regulating pulsatile LH release has not been explored. Therefore, the aim of this work was to investigate, in OVX rats, the effects of LC lesions on pulsatile LH secretion. LC lesions were produced in adult female rats three weeks after OVX. On the next morning, the jugular vein was catheterized and, on the afternoon of the same day, blood samples (0.3 ml) were withdrawn every 5 min, during 90 min, from conscious freely moving rats. Plasma LH was measured by radioimmunoassay. LC lesions greatly suppressed pulsatile LH secretion by decreasing both LH pulse frequency and amplitude. The basal as well as total secretion of LH were also decreased. This inhibitory effect of the lesions was observed only when at least 50% of the nucleus was destroyed. Data from sham-operated animals as well as those with less than 50% destruction of the LC did not differ from those of the control rats without brain lesions. Since LC lesions induce a decrease in NE content in the preoptic area and median eminence, the inhibition of pulsatile LH release in ovariectomized rats with LC lesions occurs presumably as result of decreased pulsatile NE release into these areas of the brain that decreases both the frequency and the amount of LHRH released per pulse.     

Morphine but not Naloxone Enhances Luteinizing Hormone-Releasing Hormone Neuronal Responsiveness to Norepinephrine

Some axon terminals of hypothalamic opiate neurons directly synapse on luteinizing hormone-releasing hormone (LHRH) neurons. To determine whether such synaptic connections affect LHRH neuronal activity, we have examined the profiles and concentrations of LH released in response to intracerebroventricular (icv) norepinephrine (NE, 45 μg) infusions alone or following medial preoptic area (MPOA) electrochemical stimulation (ECS) in estrogen-treated ovariectomized rats. Similar studies were performed in rats treated with naloxone (5 mg/kg ip) or morphine (20 mg/kg sc) given 15 min prior to MPOA-ECS or 30 min prior to icv NE. Naloxone neither augmented nor suppressed the LH response obtained with NE alone. MPOA-ECS evoked a significant increase in plasma LH. When the preoptic area was stimulated (0 min) and NE was infused at 30 min, a significant amplification of LH release occurred. Prior treatment of rats (-15 min) with naloxone had no effect on LH responses elicited by either preoptic stimulation alone or combined with icv NE. In the second study, morphine was given sc and had no effect on basal plasma LH levels. However, when morphine was given (-15 min) and icv NE infusions were made (30 min), the rise in plasma LH induced by NE was significantly enhanced. Preoptic ECS (0 min) evoked a rise in plasma LH and this response was also enhanced by morphine pretreatment. The major effect on LH release occurred when sc morphine injections (-15 min) were combined with MPOA-ECS (0 min) followed by icv NE (30 min). In these rats, a remarkable and highly significant release of LH occurred which reached peak levels even greater than those observed during spontaneous LH surges (2,392 versus 16 to 1,800 ng/ml). Since morphine has profound effects on the serotonergic system, in the third series of studies, morphine was infused into the dorsal raphe nucleus (DRN) and LH responses to MPOA-ECS or icv NE alone or following combined ECS + NE were examined. DRN morphine did not affect basal LH release but it produced a rapid and highly significant rise in plasma prolactin. When DRN morphine was given (-15 min) and NE was infused icv (30 min), there was marked amplification in LH release compared to those values observed after only NE. However, there were no appreciable differences in LH values obtained after sc versus DRN morphine injections in response to NE. Similarly, the amplification of LH release which occurred following DRN morphine (-15 min) + MPOA-ECS (0 min) was not different from that obtained after sc morphine. In the final group of rats, DRN morphine was given (-15 min), the preoptic area was stimulated (0 min) and NE was infused (30 min). Following this treatment, plasma LH release was also markedly enhanced and did not differ appreciably (except at 60 and 120 min) from the levels of LH released after sc morphine. Prolactin concentrations rose slowly after icv NE to reach peak levels 75 min after treatment. Combinations of morphine + MPOA-ECS without or with NE neither augmented nor suppressed the high prolactin concentrations achieved after only DRN morphine infusions. We conclude from these data that: 1) those opiate neuronal terminals which synapse directly on LHRH neurons do not affect LHRH neuronal responsiveness to either NE, to MPOA-ECS or to combined preoptic stimulation+ NE, and 2) morphine has profound effects on LHRH neuronal responsiveness to both NE, to MPOA-ECS and, in particular, to combined ECS + NE. Since amplification of LH release occurs after treatment of rats with morphine either by sc injections or DRN infusions, the augmented LH and prolactin responses observed are most likely due to the morphine-induced release of serotonin and not to direct morphine effects on LHRH neurons.     

Noradrenaline release in the medial preoptic area during the rat oestrous cycle: temporal relationship with plasma secretory surges of prolactin and luteinising hormone

During the rat oestrous cycle, the afternoon of pro-oestrous is characterised by preovulatory surges of luteinising hormone (LH) and prolactin. On the afternoon of oestrous, a secretory surge of prolactin has also been reported. Because the medial preoptic area (MPOA) is known to regulate prolactin and LH secretory surges and noradrenaline has been demonstrated to stimulate these hormones release, we evaluated whether noradrenaline release in the MPOA was temporally associated with plasma prolactin and LH surges in cycling rats. During the 4 days of oestrous cycle, noradrenaline concentrations were determined in microdialysates from the MPOA, collected at 30-min intervals from 10.30 h to 19.00 h. Plasma prolactin and LH levels were measured in blood samples withdrawn hourly from 14.00 h to 19.00 h on pro-oestrous and from 13.00 h to 18.00 h on the other days of the cycle. On the afternoons of both pro-oestrous and oestrous, noradrenaline levels increased at 14.00 h and remained elevated until 16.30 h. Conversely, they were low and constant throughout metoestrous and dioestrous. Correlating with noradrenaline release in the MPOA, plasma prolactin surges occurred during the afternoons of both pro-oestrous and oestrous. On pro-oestrous, the afternoon LH surge was also preceded by the increase in MPOA noradrenaline whereas, during oestrous, LH secretion was low and unaltered. A temporal association between noradrenaline release and prolactin secretion suggests that noradrenergic neurotransmission in the MPOA regulates prolactin surges in female rats. Moreover, our data also suggest that MPOA noradrenaline requires specific conditions to physiologically regulate LH secretion, which seems to occur during the afternoon of pro-oestrous.     

Sex differences in the responses of hypothalamic luteinizing hormone-releasing hormone and catecholamine systems to ovarian hormones and naloxone: implications for sexual differentiation of luteinizing hormone secretion in rats

Normal male rats, or female rats exposed neonatally to androgens or estrogens, do not respond in adulthood to ovarian hormone treatments that stimulate preovulatory-like surges of luteinizing hormone (LH) or mating behavior in normal females. As an attempt to understand the neurochemical basis for this insensitivity, the present studies tested whether sex differences also exist with respect to several important neural events that are antecedent to and essential for the appearance of an LH surge induced by ovarian hormone treatment. Administration of estradiol via Silastic capsules to adult, gonadectomized rats resulted in a suppression of LH release that was equivalent in males and females, but only the estrogen-primed females responded to injections of progesterone with an LH surge. Similarly, in estrogen-primed females but not males, progesterone induced a presurge sequential accumulation and decline of LH-releasing hormone (LH-RH) concentrations in the median eminence and increased the turnover rates of norepinephrine (NE) and epinephrine (E) in the medial basal hypothalamus during the time of LH-RH accumulation. Ovarian hormones may activate NE and E release in females by removing a tonic inhibition over catecholamine release exerted by endogenous opioids. In order to test whether direct antagonism of opiate mechanisms would produce equivalent neuroendocrine or neurochemical responses in males and females, additional studies tested the effects of the opiate receptor blocker naloxone on LH release and on activity of catecholamines in the medial basal hypothalamus. In contrast to females, estrogen-primed male rats did not display either an increase in serum LH or an enhancement of the alpha-methyltyrosine-induced decline of NE or E after treatment with naloxone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intravenous injections of nicotine decrease the pulsatile secretion of LH by inhibiting the gonadotropin-releasing hormone (GnRH) pulse generator activity in female rats.

Whether nicotine inhibits the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator to suppress pulsatile LH secretion, and whether this suppression of LH secretion by nicotine is mediated by opioid neurons, were studied in ovariectomized rats by examining changes in LH secretion and the multiunit activity (MUA) of the medial basal hypothalamus. Intravenous (i.v.) injection of nicotine (nicotine bitartrate, 100 micrograms) significantly increased the interval between characteristic increases (volleys) in MUA and LH pulses. This inhibitory effect of nicotine on the GnRH pulse generator activity was not blocked by the prior injection of an opiate receptor antagonist naloxone (naloxone hydrochlolide, 2 mg/kg bw), which was effective in significantly decreasing the interval between MUA volleys. The results suggest that nicotine alters the activity of the GnRH pulse generator, and that cholinergic neurons appear to be directly involved in suppressing pulsatile secretion of LH.     

Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges

Studies were undertaken to determine whether nitric oxide (NO) is involved in induction of the prolactin surge on proestrus and in that induced by ovarian steroids in ovariectomized (ovx) rats, by using inhibitors of NO synthase, the enzyme that generates NO. Two week-ovariectomized rats were treated either with estradiol benzoate (EB, 30 μg/rat, s.c.) alone, or with EB and 2 days later with progesterone (P, 2 mg/rat, s.c.) to evoke a prolactin surge in the afternoon. Injections of the NO synthase inhibitor methyl ester (40 mg/kg, s.c.) at 10.00, 12.00, and 14.00 h completely suppressed the steroid-induced prolactin surge in the afternoon. Similarly, another NO synthase inhibitor, (100 mg/kg, s.c.), injected at 1000, 1200, and 1400 h on proestures suppressed prolactin and luteinizing hormone (LH) surges, but failed to alter the daily increase of corticosterone. These studies confirm our earlier report and show that NO is involved in the complex processes that initiate the afternoon prolactin and LH surges of proestrus.     

Hypothalamic monoamines and their catabolites in relation to the estradiol-induced luteinizing hormone surge

Monoamines and non-conjugated catabolites (serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), norepinephrine (NE), and dopamine (DA] were measured in the medial basal hypothalamus (MBH) and preoptic area (POA) of ovariectomized (OVX) and OVX estradiol (E2)-treated rats using high-performance liquid chromatography with electrochemical detection. These E2 treatments were sufficient to induce an LH surge. The use of MHPG/NE ratios as estimates of NE release was validated in the rat hypothalamus by the major decreases of MHPG after injection of the alpha 2-adrenergic agonist, clonidine, and by MHPG increases after the alpha 2-antagonist, yohimbine. The ratio, MHPG/NE, decreased between morning and afternoon in the MBH but not in the POA; there were no differences between OVX and E2-treated rats. Previous studies using a variety of methods indicate that NE turnover increases during LH surges. The present data suggest that unconjugated MHPG is not a sensitive measure of NE release in the rat hypothalamus, but can detect the large changes produced by stimulating or inhibiting the alpha 2-adrenergic autoreceptor. The ratios of DOPAC/DA and 5-HIAA/5-HT in the MBH decreased consistently between morning and afternoon in OVX rats, with or without E2 treatment. This suggests that the release of DA and 5-HT decreases during the day regardless of steroidal milieu.     

Role of the locus coeruleus in the prolactin secretion of female rats

Since locus coeruleus (LC) lesion blocks preovulatory prolactin surge, the aim of this study was to determine if this lesion would also block prolactin surges induced by steroids in ovariectomized rats and would modify basal prolactin secretion. To determine the time of the steroid-induced prolactin surges, ovariectomized rats treated with estradiol (OVE) or estradiol and progesterone (OVEP) were cannulated at 08:00 h and blood samples were collected hourly between 14:00 and 18:00 h. Ovariectomized rats treated with oil (OV-Oil) were used as control. Prolactin peaked at 16:00 h in OVE rats and at 15:00 h in OVEP. In a second experiment, male rats, cycling rats, OVE, OVEP, and OV-Oil groups were cannulated at 08:00 h, followed by LC lesion or sham-surgery. Blood samples were withdrawn at times of basal and peak prolactin levels. LC lesion blocked afternoon prolactin surges of OVE, OVEP and proestrus rats. However, the low levels observed at 16:00 h in OV-Oil, diestrus and male rats as well as at 11:00 h in OVE, OVEP, estrus, and proestrus rats were not modified by LC lesion. The high prolactin levels observed on estrus afternoon were dramatically reduced by LC lesion. Data suggest that LC neurons are important for steroid-induced prolactin surge genesis, but not for prolactin basal secretion.     

Plasma LH levels in the long term ovariectomized rat after anterolateral deafferentation of the medial basal hypothalamus and lesions in the medial preoptic area

This study was designed to investigate the effect of anterolateral hypothalamic deafferentation (ALHD) and medial preoptic area (MPOA) lesions on plasma LH levels in the long term ovariectomized rat. The deafferentations were carried out with a Halasz-Pupp knife (radius of 1.5 mm and height of 2.0 mm) and the MPOA lesions with a platinum electrode. Sham treated and an intact group served as controls. Blood samples were obtained from the jugular vein under light ether anesthesia before and at 1, 2, 4 and 6 weeks after brain surgery. After the sixth week sample all rats were treated with 50 μg of estradiol benzoate (EB) and two days later blood samples were collected during the morning and afternoon. Hypothalamic deafferentation resulted in a more significant (p<0.01) drop in plasma LH levels in one half of the group (ALHD-1) than in the other half (p<0.05) (ALHD-2) when compared to the controls. Treatment of the controls with EB resulted in a significant (p<0.01) depression of LH levels in the morning and an LH surge during the afternoon. EB also resulted in a suppression (p<0.01) of LH levels during the morning in all of the ALHD rats; however, only the ALHD-1 group had an LH surge during the afternoon following EB. Plasma LH levels in the ALHD-2 remained suppressed during the afternoon after EB treatment. Lesions in the MPOA had no effect on plasma LH levels at 1 to 6 weeks when compared to controls. Treatment of the MPOA lesion group with EB resulted in a significant (p<0.01) drop in plasma LH levels during the morning as well as the afternoon. These data suggest that the fibers that are critical for the control of tonic and phasic LH secretion enter the medial basal hypothalamus laterally and that the deafferentations carried out here were selective in interrupting fibers involved with tonic LH secretion in some rats and those involved with the phasic secretion in others. These data also suggest that the MOPA components involved with tonic LH secretion are separate from those controlling phasic LH secretion.     

Lipopolysaccharide inhibits luteinizing hormone release through interaction with opioid and excitatory amino acid inputs to gonadotropin-releasing hormone neurones in female rats: possible evidence for a common mechanism involved in infection and immobilization stress

Acute immobilization stress suppresses naloxone- and N-methyl-d-aspartate (NMDA)-induced, but not gonadotropin-releasing hormone (GnRH)-induced, luteinizing hormone (LH) release in ovariectomized oestrogen-primed rats. To explore whether a common mechanism may underlie inhibition of gonadotropin secretion by various stressors, we examined in the present study the effect of lipopolysaccharide (LPS) on LH release induced by progesterone, GnRH, naloxone and NMDA. The effect of LPS on Fos expression in GnRH neurones was also examined in association with its effect on steroid-induced LH release. Injection of progesterone (1 mg/rat) at noon induced an LH surge in the afternoon in ovariectomized rats pretreated with oestradiol benzoate. In these rats, the majority of hypothalamic GnRH neurones expressed Fos in the evening. Intravenous (i.v.) administration of LPS (10 micro g/rat) inhibited steroid-induced LH release and also reduced the Fos expression in GnRH neurones. In separate experiments, an i.v. injection of GnRH (50 ng/kg), naloxone (10 mg/kg) or NMDA (20 mg/kg) significantly elevated serum LH concentrations within 10 min. Pretreatment with LPS, which did not affect basal LH release or GnRH-induced LH release, inhibited naloxone-induced and NMDA-induced LH release. These results show that LPS has a suprapituitary site(s) of action to suppress the activity of GnRH neurones in female rats, and suggest that LPS affects the opioid, as well as the excitatory amino acidergic regulation of GnRH neurones. The similarity of effects of LPS and immobilization stress further suggests that a common mechanism is involved in inhibition of GnRH neurones by different stressors.     

The release of pituitary LH and sprouting of LH-releasing hormone (LHRH)-containing neurons after anterior hypothalamic deafferentation

The chronology of changes in plasma luteinizing hormone (LH) and the distribution of immunoreactive neuronal processes containing LH releasing hormone (LHRH-ir) were studied in the female rat after surgical interruption of anterior neural connections of the mediobasal hypothalamus (MBH). Spontaneous LH surges on the afternoon of proestrus and LH release after estradiol benzoate (EB) followed 48 h later by progesterone (P) administration were studied in ovariectomized (OVX) rats. The maximum increase in plasma concentrations (delta maxLH) after EBP was calculated for each rat at several intervals over 140 days. Control animals given EBP at monthly intervals after OVX had comparably large delta maxLH surges during the first few months of study. However, a gradual decline in control delta maxLH followed becoming significant 3 months after the start of the experiment. In contrast, frontal cuts (FC), which interrupted anterior MBH connections, produced an abrupt decrease in delta maxLH surges after EBP to 11% of preoperative levels. However, during subsequent EBP trials, there was a gradual improvement in LH surges to about 50% of preoperative levels over 100 days. In some cases, individual improvement became equal to preoperative LH surge levels, in others there was no recovery. Examination of LHRH-ir nerve fiber growth responses after FC suggested that sprouting by these peptide-containing neuronal processes may have contributed to the functional improvements observed.     

Oestrogen receptor-alpha and -beta immunoreactivity in gonadotropin-releasing hormone neurones after ovariectomy and chronic exposure to oestradiol

Oestradiol exerts negative- and positive-feedback actions on luteinizing hormone (LH) secretion by modulating gonadotropin-releasing hormone (GnRH) release. Furthermore, a chronic increase in circulating oestradiol in either young ovariectomized (OVX) rats, or in middle-aged persistent oestrous (PE) rats, causes a gradual attenuation of LH surges until the positive-feedback action of oestradiol disappears. Based on these findings, and on the equivocal evidence regarding a direct action of oestradiol on GnRH neurones, we tested the hypothesis that chronic oestradiol abolishes LH surges by decreasing the proportion of GnRH neurones containing oestrogen receptor (ER)alpha or beta. Regularly cycling rats were ovariectomized, and half immediately received oestradiol. Three days, or 2 or 4 weeks later, rats were perfused at 18.00 h, and GnRH was colocalized with ERalpha or ERbeta by immunocytochemistry. ERbeta was expressed in 76% of GnRH neurones, whereas virtually no GnRH cells were immunopositive for ERalpha. The proportion of GnRH cells expressing ERalpha or beta in OVX rats was not altered by oestradiol or time after OVX, and this was the case regardless of their medial to lateral, or rostral to caudal location. The results indicate that the mechanisms for the positive-feedback action of oestradiol, and the loss of LH surges in OVX rats after chronic oestradiol, are not mediated by changes in the proportion of oestrogen-receptor containing GnRH neurones.     

Cyclic changes in the release of norepinephrine and dopamine in the medial basal hypothalamus: effects of aging

Push-pull perfusion and HPLC were used to measure the release of norepinephrine (NE) and dopamine (DA) in the medial basal hypothalamus of young (4–5 months old), middle-aged (8–10 months old), and old (22–24 months old) rats. In the young animals, the afternoon of proestrus was characterized by a gradual increase in NE release and a simultaneous gradual decrease in DA release. The peak in NE release and the nadir in DA release occurred at about the time when the proestrous surges in serum LH and PRL are known to occur. No changes in NE and DA releases occurred in the afternoon of diestrus when serum LH and PRL are known to remain stable. In the middle-aged proestrous animals, the patterns of NE and DA releases were similar to those in the young proestrous animals, but the peak in NE release was attenuated and did not reach statistical significance. This corresponded with the reported attenuation in the LH surge in middle age. In the old persistently diestrous animals, NE and DA were released at constant rates, which correlated with the well-documented constant levels of serum LH and PRL in old age. These data provide an explanation for the simultaneous proestrous surges of LH and PRL and lead us to conclude that NE plays a facilitatory role in the LH surge, while DA, through its inhibitory action, regulates the PRL surge. These studies, by monitoring NE and DA releases from adulthood through middle-age to old age, indicated that cyclicity in catecholamine (CA) activities begins to be dampened in middle-age and eventually completely disappears in the acyclic period of old age which is also characterized by a marked deficiency in CA activities.