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.     

Suppression of spontaneous LH surges in estrogen-treated ovariectomized rats by microimplants of antiestrogens into the preoptic brain

Studies by others have shown that parenteral administration of antiestrogens blocks the positive feedback effect of estrogen on the luteinizing hormone (LH) surge mechanism. Since all estrogen-accumulating cells could be affected by this treatment, it is difficult to identify the site(s) at which this steroid acts to affect LH surges. In the present study we attempted to deprive specific hypothalamic neurons of estrogen by stereotaxically implanting antiestrogen-containing microcannulae into the brains of ovariectomized (OVX) rats which, otherwise, were completely estrogenized. The animal model used in these studies was the 14-day OVX rat into which 2 estradiol-containing Silastic capsules were inserted s.c. on day 14 (day 0). Microcannulae were placed into either the medial or lateral preoptic nuclei (MPN, LPN) on day 0 and the effects on LH release were examined 2 days later (day 2). When empty cannulae were placed into the MPN or LPN, 6 of 7 and 8 of 8 rats, respectively, had normal spontaneous LH surges. In contrast, when cannulae containing either CI-628, LY 10074 or Keoxifene were implanted into MPN only 33.3, 0, and 14.3% of the rats, respectively, had LH surges by 16.00 h on day 2 (time of LH peak). When antiestrogen-containing cannulae were placed into the LPN, all rats displayed normal LH patterns of release and concentrations. The antiestrogens did not prevent estrogen from suppressing elevated high post-ovariectomy plasma LH concentrations (negative feedback). To evaluate whether Keoxifene affected releasable luteinizing hormone-releasing hormone (LH-RH), we examined the effects of MPN-Keoxifene implants on LH secretion evoked by electrochemical stimulation (ECS) of the MPN or the medial basal hypothalamus (MBH). In ketamine-anesthetized rats with empty cannulae, plasma LH increased significantly to reach peak concentrations 30-45 min after ECS. Similar LH concentrations and release patterns occurred in rats with the antiestrogen implant. Other studies examined the effects of MPN-Keoxifene implants on norepinephrine (NE) concentrations and rate constants following administration of alpha-methyl-p-tyrosine. NE concentrations and rate constants in the MPN and median eminence did not differ significantly in rats which had received empty versus Keoxifene-containing microcannulae. In the final series of studies we examined the response of LH-RH neurons to an intracerebroventricular (i.c.v.) infusion of norepinephrine (20 micrograms). Plasma LH peaked within 10 min after i.c.v. NE and, thereafter, declined towards baseline. Keoxifene did not affect LH-RH neuronal responsiveness to i.c.v. NE.(ABSTRACT TRUNCATED AT 400 WORDS)     

An analysis of serotonin secretion in hypothalamic regions based on 5-hydroxytryptophan accumulation or push-pull perfusion. Effects of mesencephalic raphe or locus coeruleus stimulation and correlated changes in plasma luteinizing hormone

This study evaluates the effect of electrical stimulation (ES) of the dorsal (DRN) and median raphe (MRN) nuclei serotoninergic systems on luteinizing hormone (LH) release in estrogen-treated, ovariectomized rats. To show that ES increased serotonin (5-hydroxytryptamine: 5-HT) secretion into hypothalamic regions known to contain luteinizing hormone-releasing hormone (LH-RH) cell bodies and terminals. 5-hydroxytryptophan (5-HTP) accumulation was measured in microdissected hypothalamic areas after blockade of aromatic-L-amino-acid decarboxylase with NSD-1015. DRN-ES produced a significant increase in 5-HTP accumulation in the medial preoptic (MPN) and paraventricular nuclei (PVN), but not in the suprachiasmatic nucleus (SCN), arcuate nucleus (ARC) or median eminence (ME). In contrast, MRN-ES produced a significant rise in 5-HTP accumulation only in the PVN and ARC, not in the SCN, MPN or ME. Because the DRN receives noradrenergic innervation from the locus coeruleus (LC) we also evaluated the effect of LC stimulation on 5-HT secretion into these hypothalamic regions. LC stimulation, like DRN-ES, resulted in increases in 5-HTP accumulation in MPN and PVN, but not in SCN or ME. In addition, using push-pull perfusion methodology, we observed that LC-ES results in a 240% increase in 5-HT and a decrease of approximately 40% in 5-hydroxyindoleacetic acid (5-HIAA) in MPN perfusates collected 10-20 min after LC-ES began. With evidence that DRN- and MRN-ES evoke 5-HT release, we next examined whether such stimulation affects basal LH release. Because we were unable to find any effect, we determined whether 5-HT release would augment or suppress electrochemically evoked LH release. MPN electrochemical stimulation (ECS) induced a significant increase in plasma LH with a peak seen at 45 min. When the MPN was ECS and 30 min later the DRN was ES for 30 min no augmenting or suppressive effect was seen during the first 60 min. However, while plasma LH declined towards baseline in the MPN-ECS group, it remained significantly elevated in MPN-ECS + DRN-ES rats. MPN-ECS + MRN-ES had no such effect: instead. LH levels were transiently decreased 45 min after beginning MPN-ECS. This study provides additional information on hypothalamic sites which receive axonal projections from the DRN and MRN and clearly indicate that an increase in 5-HT secretion occurs in the MPN following DRN- or LC-ES.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of progresterone-induced increase in hypothalamic luteinizing hormone-releasing hormone levels and serum gonadotropins by intrahypothalamic implantation of 6-hydroxydopamine

The present study was undertaken to identify the hypothalamic locus where norepinephrine (NE) nerve terminals communicate with leteinizing hormone-releasing hormone (LH-RH)-containing neurons involved in the stimulatory feedback action of gonadal steroids on LH and FSH release. Ovariectomized rats received estradiol benzoate (10 μg/rat s.c.) on day 0. Intracranial implants containing either 6-hydroxydopamine (6-OHDA), to destroy NE terminals, or cocoa butter (controls) were placed bilaterally in the suprachiasmatic nucleus (SCN), medial basal hypothalamus (MBH) or olfactory tubercle (OT) on day 1. Progesterone (P, 5 mg/rat s.c.) was administered at 10.00 h on day 2 to elicit increases in serum LH and FSH and the MBH LH-RH levels in the afternoon. Implantation of 6-OHDA in the SCN resulted in a marked depletion of NE in and around the region of the SCN in the preoptic-anterior hypothalamic area (POA-AH) without adversely affecting dopamine (DA) concentrations, and blocked the P-induced afternoon increase in the MBH LH-RH and serum gonadotropin levels. Similar reduction in the MBH NE concentrations occurred following placement of 6-OHDA in the MBH; however, these as well as implants in the OT were ineffective in suppressing the P-induced effects. These studies show that functional integrity of the SCN regions is critical in manifestation of the P-induced rise in the MBH LH-RH activity, and this region in the POA-AH, therefore, may be the primary locus of synaptic communication between NE terminals and LH-RH neurons.     

Blockade of progesterone-induced increase in hypothalamic luteinizing hormone-releasing hormone levels and serum gonadotropins by intrahypothalamic implantation of 6-hydroxydopamine.

The present study was undertaken to identify the hypothalamic locus where norepinephrine (NE) nerve terminals communicate with luteinizing hormone-releasing hormone (LH-RH)-containing neurons involved in the stimulatory feedback action of gonadal steroids on LH and FSH release. Ovariectomized rats received estradiol benzoate (10 microgram/rat s.c.) on day 0. Intracranial implants containing either 6-hydroxydopamine (6-OHDA), to destroy NE terminals, or cocoa butter (controls) were placed bilaterally in the suprachiasmatic nucleus (SCN), medial basal hypothalamus (MBH) or olfactory tubercle (OT) on day 1. Progesterone (P, 5 mg/rat s.c.) was administered at 10.00 h on day 2 to elicit increases in serum LH and FSH and the MBH LH-RH levels in the afternoon. Implantation of 6-OHDA in the SCN resulted in a marked depletion of NE in and around the region of the SCN in the preoptic-anterior hypothalamic area (POA-AH) without adversely affecting dopamine (DA) concentrations, and blocked the P-induced afternoon increase in the MBH LH-RH and serum gonadotropin levels. Similar reduction in the MBH NE concentrations occurred following placement of 6-OHDA in the MBH; however, these as well as implants in the OT were ineffective in suppressing the P-induced effects. These studies show that functional integrity of the SCN regions is critical in manifestation of the P-induced rise in the MBH LH-RH activity, and this region in the POA-AH, therefore, may be the primary locus of synaptic communication between NE terminals and LH-RH neurons.     

Cellular mechanisms of acute estrogen negative feedback on LH secretion: norepinephrine, dopamine and 5-hydroxytryptamine metabolism in discrete regions of the rat brain

Rats on diestrous day 1 were ovariectomized (OVX) and killed 10 days later. LH was measured by RIA and the metabolism of NE, DA and 5-HT were assayed concurrently in the suprachiasmatic (SCN), medial preoptic (MPO), dorsomedial (DMN), rostral (ANr) and caudal (ANc) arcuate nuclei as well as the median eminence (ME) utilizing HPLC with electrochemical detection. Serum LH increased 10-12 fold 10 days following OVX compared to diestrous controls. The injection of estradiol benzoate (Eb, 20 micrograms in corn oil/rat, SC) did not affect LH concentrations at 30 minutes but decreased serum LH both 60 and 180 min following its administration. OVX caused an increased NE metabolism (estimated by the concentration of the NE metabolite, 3-methoxy-4-hydroxyphenylethylene glycol) in the SCN, MPO, ME, and DMN and a decreased NE metabolism in the ANc compared to diestrous control values. All of these changes were reversed or attenuated 180 minutes following Eb treatment. Observed changes in the DA and 5-HT neuronal systems were more restricted and less dramatic with the largest effects on DA metabolism occurring in the DMN and ME and the clearest changes in 5-HT metabolism occurring in the MPO, ANr, and ANc. The results demonstrate that the inhibition of LH secretion following the injection of Eb to OVX rats is accompanied by changes in metabolism in NE neurons in preoptic (SCN and MPO) and medial (ME, DMN, and ANc) hypothalamic areas, as well as in DA neurons in the DMN and ME, and in 5-HT neurons in the MPO, ANr, and ANc.     

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.     

The role of hypothalamic serotonin (5HT) before ovulation in immature rats treated with pregnant mare serum (PMS).

(1) PCPA methyl ester (10 mg/rat i.p.) inhibits induced ovulation in immature rats treated with pregnant mare serum (PMS). It also suppresses the preovulatory surges of LH and FSH, but not those of oestradiol or progesterone. (2) There is an increase in hypothalamic 5HT levels in the aftermoon and hypothalamic 5HIAA levels in the evening of the two days studied (days 28 and 29 of life). This occurs whether or not PMS was given on day 27. (3) The antiovulatory effects of PCPA are only seen when it is given on the afternoon or evening of the day before the pre-ovulatory gonadotrophin surge, i.e. on day 28 over the period of raised hypothalamic 5HT metabolism. (4) PCPA reduces 5HT metabolism in the hypothalamus within 2 hr of administration and its anti-ovulatory effect can be overcome by 5-hydroxytryptophan. This indicates that hypothalamic 5HT activity is essential for the gonadotrophin surge. (5) The anti-ovulatory effect of PCPA can be overcome by progesterone, LH and FSH but not oestradiol.     

Diurnal rhythm of vasopressin mRNA species in the rat suprachiasmatic nucleus: independence of neuroendocrine modulation and maintenance in explant culture

Vasopressin mRNA in the suprachiasmatic nucleus (SCN) of the rat brain exhibits diurnal variation in poly(A) tail length; a single species of mRNA identical in size to that in other hypothalamic nuclei is expressed in the light phase of the daily cycle whereas a second, smaller species is expressed in the dark phase. We have investigated the neuroendocrine factors which may regulate this rhythm by comparing vasopressin mRNA size with Northern analysis of RNA extracted from SCN tissue taken at 09.00 h (light phase) and 21.00 h (dark phase). The consistent rhythmic variation observed in normal male rats was not modified in either adrenalectomized or castrated animals or in ovariectomized female rats. The rhythm was also not disrupted following treatment with the serotonin-depleting agent parachlorophenylalanine, or following treatment with either melatonin or the benzodiazepine, triazolam. We next investigated whether the daily pattern of expression was maintained in isolated SCN. Microdissected blocks containing the paired SCN were explanted into culture at various times of the day for a period of 4 h. Vasopressin mRNA extracted from light phase cultures (11.00-15.00 h) exhibited no size change from control SCN mRNA taken at either 11.00 h or 15.00 h. In contrast, mRNA from cultures maintained over the period 16.00-20.00 h (lights off: 18.00 h) exhibited a marked size difference from 16.00 h controls, being similar to the smaller species observed in 20.00 h controls. Similarly, vasopressin mRNA from cultures maintained over the period 05.00-09.00 h (lights on 06.00 h) was similar in size to 09.00 h controls and contrasted to the pattern of expression observed in 05.00 h controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated norepinephrine turnover in peripheral tissues after ventromedial hypothalamic stimulation in rats

To obtain evidence for a functional connection between the ventromedial hypothalamic nucleus (VMH) and the sympathetic nervous system, effects of electrical stimulation of the VMH, the lateral hypothalamic area (LH) and the paraventricular hypothalamic nucleus (PVN) on norepinephrine (NE) turnover in the heart, liver, pancreas, spleen, submandibular gland and the interscapular brown adipose tissue were examined in anesthetized rats. Stimulation of the VMH elicited a 3-8-fold increase in the rate of NE turnover in all organs examined, whereas stimulation of the LH or the PVN had no appreciable effects. The effect of VMH stimulation was abolished after sympathetic ganglionic blockade with hexamethonium. Epinephrine turnover in the adrenal gland was accelerated by stimulation of not only the VMH but also the LH. It was concluded that the VMH is intimately associated with sympathetic facilitation in peripheral tissues.     

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.     

Gonadotropin responses to naloxone may depend upon spontaneous activity in noradrenergic neurons at the time of treatment

The opiate system is thought to modulate gonadotropin secretion by its effect on catecholamine secretion. This action may be produced by opiates regulating the amount of catecholamine released from presynaptic terminals at a given frequency of depolarization and/or by increasing the rate of impulse traffic within catecholamine neurons. We examined the effects of naloxone, an opiate receptor antagonist, on luteinizing hormone (LH) and prolactin (Prl) secretion in 3 sex steroid-treated, gonadectomized rat models in which we have considerable information on the rates of turnover of norepinephrine (NE) and dopamine (DA) in the hypothalamus. In 7 day ovariectomized rats treated for 2 days with estradiol (E2), the injection of naloxone (10 mg/kg) at 09.15 h produced a small 3-fold rise in LH and a short-lived decline in Prl. In contrast, naloxone, given at 12.15 h, markedly amplified (10-fold) and advanced the time of the LH surge but did not affect afternoon Prl surges. Hypothalamic NE turnovers are low in the morning and high in the afternoon for such animals. Other ovariectomized (OVX) rats received E2 for 2 days and progesterone (P4) on day 2. Such treatment extinguishes the LH surges which normally occur the next day (day 3) but does not affect phasic Prl secretion. Naloxone, given at 09.15 h to E2P4-treated rats on day 3, did not affect basal LH levels but serum Prl declined for about 1 h. When given at 12.15 h, naloxone produced a small 3-fold rise in LH but did not affect phasic Prl release.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ovarian steroids influence the activity of neuroendocrine dopaminergic neurons

The secretion of prolactin (PRL) from the anterior lobe (AL) of the pituitary gland is tonically inhibited by dopamine (DA) of hypothalamic origin. While ovarian steroids play a role in the regulation of the secretion of PRL, their effect on all three populations of hypothalamic neuroendocrine dopaminergic neurons is not fully understood. In this study we describe the effects of ovarian steroids on regulation of the release of DA from tuberoinfundibular dopaminergic (TIDA), tuberohypophyseal dopaminergic (THDA) and periventricular-hypophyseal dopaminergic (PHDA) neurons. Adult female rats were bilaterally ovariectomized (OVX) and, 10 days following ovariectomy (day 0), injected with corn oil (vehicle), estrogen, or estrogen plus progesterone (day 1). Animals were sacrificed every 2 h from 09.00 to 21.00 h by rapid decapitation. Trunk blood was collected and the concentration of PRL in serum was determined by radioimmunoassay. The median eminence (ME) and the AL, intermediate (IL) and neural (NL) lobes of the pituitary gland were dissected and the concentration of DA and DOPAC in each was measured by HPLC-EC. OVX rats presented small but significant increases in the secretion of PRL at 15.00 and 17.00 h. Replacement of estrogen or estrogen plus progesterone increased the basal concentration of PRL. Moreover, injection of estrogen only, or estrogen plus progesterone increased the concentration of PRL in serum at 15.00 h through 19.00 h, respectively, followed by a decrease to baseline thereafter. The turnover of DA in the ME and NL of OVX rats increased at 13.00 and returned to low levels. Turnover of DA in the IL of OVX rats increased in the morning by 11.00 h and remained elevated before decreasing by 17.00 h. The turnover of DA in the ME, IL and NL of OVX rats increased by 19.00 h. Injection of estrogen advanced the increase of TIDA activity by 2 h in the ME compared to OVX rats. Moreover, administration of estrogen suppressed the activity of THDA and PHDA neurons in the afternoon compared to OVX rats. In estrogen plus progesterone-treated rats, the activity of hypothalamic neuroendocrine dopaminergic neurons terminating in the ME, IL, and NL was inhibited prior to the increase in the secretion of PRL. The concentration of DA in the AL diminished prior to the estrogen-induced increase of PRL. Administration of progesterone, in concert with estrogen, delayed the increase of PRL in serum and the decrease of DA in the AL, compared to estrogen-treated rats, by 4 h. These data suggest a major role for ovarian steroids in controlling increases in the secretion of PRL by not only stimulating PRL release from lactotrophs, but also by inhibiting the activity of all three populations of hypothalamic neuroendocrine DAergic neurons.     

Ontogeny of catecholamine turnover rates in limbic and hypothalamic structures in relation to serum prolactin and gonadotropin levels

Norepinephrine (NE) and dopamine (DA) concentrations and turnover rates have been studied in the n. accumbens, medial preoptic area (MPO) and anterior and posterior parts of the mediobasal hypothalamus of developing rats. Turnover rates are determined by injection of -methyl-p-tyrosine 30 and 90 min prior to decapitation. NE concentrations and turnover in the n. accumbens were low in all age groups with slightly increased values between days 20 and 35 after birth whereas DA concentrations and turnover rates were low at day 15 and 20 and at high adult values by day 25 after birth. Medial preoptic and anterior mediobasal hypothalamic catecholamines exhibited a rather unique pattern. Concentrations and turnover rates were low in 15-day-old animals and increased between days 20 and 30 to very high values. Such high values were never observed in adult diestrous animals. The same pattern was also observed in the posterior mediobasal hypothalamus for NE concentrations and turnover rates whereas the respective values for DA showed relatively large fluctuations. On the basis of catecholamine measurements 30 and 90 min after blockade of tyrosine hydroxylase an attempt was also made to differentiate turnover rates of the functional and of the storage pool.

Serum LH levels in the 15-day-old animals showed large fluctuations. FSH levels were high and prolactin levels were low. At the time of increased preoptic and hypothalamic NE and DA turnover rates, serum prolactin levels were also high whereas serum LH levels were lowest between days 20 and 30 and then slightly increased. Serum FSH levels were uniformly low. The possibility is discussed that high NE turnover may stimulate pituitary LH and prolactin release by hypothalamic mechanisms. High serum prolactin levels may stimulate DA turnover which is inhibitory to pituitary LH release, thus counteracting the stimulatory effect of NE on LH-RH release. The dopaminergic inhibition of LH may be relieved at the time of puberty partially because the DA receptors become desensitized to the action of DA.     

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.     

Diabetes-induced changes in monoamine concentrations of rat hypothalamic nuclei

Streptozotocin-induced diabetes produced marked alterations of monoamine concentrations in several hypothalamic nuclei of male and female rats. Norepinephrine (NE) concentrations were significantly elevated in the median eminence (ME), supraoptic nucleus (SON) and periventricular nucleus (PEVN) in both sexes of diabetic rats. NE concentrations in the suprachiasmatic nucleus (SCN) and ventromedial nucleus (VMN) of male and female diabetic animals remained unaltered. Serotonin (5-HT) concentrations were increased in PEVN of male and female diabetic rats. No significant changes in hypothalamic dopamine (DA) concentrations were observed. Insulin treatment reversed the diabetes-related changes in monoamine concentrations in most of the nuclei. The significance of these biochemical changes relative to the endocrine and behavioral abnormalities in diabetes is discussed.     

Alteration in hypothalamic neuropeptide Y (NPY) secretion may underlie female reproductive ageing: induction of steroid-induced luteinising hormone surge by NPY in ovariectomised aged rats

A large body of evidence suggests that a defect in the hypothalamic function may be the primary cause of reproductive ageing in female rats. We have previously shown that luteinising hormone (LH)-surge associated changes in hypothalamic neuropeptide Y (NPY) gene expression and median eminence (ME) NPY levels seen in young rats do not occur in middle-aged (MA) rats. The present study examined whether hypothalamic NPY release is altered during the steroid-induced LH surge in ovariectomised (OVX) MA rats, and whether exogenous NPY initiates steroid-induced LH surge in OVX old rats. In the first study, NPY release from the ME-arcuate nucleus, as assessed by the push-pull cannula technique, was significantly increased before and during the progesterone-induced LH surge in oestrogen (E(2))-primed ovariectomised young rats (2-3 months old). This antecedent increase in NPY release seen in young rats was not apparent in MA rats (11-13 months old) in association with a delayed and attenuated LH surge. In the second study, whereas progesterone failed to induce LH surges in E(2)-primed ovariectomised old rats (23-25 months old), intracerebroventricular NPY (0.1-0.5 microg) injections at 1100, 1200 and 13.00 h resulted in LH surge induction in E(2) + progesterone-primed ovariectomised old rats. Because increased hypothalamic NPY synthesis and release is obligatory for the preovulatory LH discharge in young rats, the present findings suggest that alteration in NPY release from the ME-arcuate nucleus contributes to the delayed and reduced LH surges in MA rats and may be involved in the subsequent loss of the LH surges in old rats.     

Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized long evans rats

The ability of several stimuli which augment central catecholamine (CA) neuronal activity to reinitiate estrous cycles in old constant estrous (CE) rats suggests CA neuronal function is impaired with advanced age. We examined the effects of age on dopamine (DA) and norepinephrine (NE) levels and turnover rates within microdissected brain regions of previously normally cycling young (3-4 months old) and middle-aged (10 months old) and CE old (20-22 months old) Long Evans 2 weeks after ovariectomy. Steady-state DA concentrations were significantly decreased in old compared to young rats in the nucleus accumbens (34%), anterior hypothalamic nucleus (54%, NHA ), neurointermediate pituitary lobe (51%, NIL) and median eminence (74%, ME). The rate constant of DA loss, an estimate of neuronal activity, decreased in old versus young rats only in the preoptic area suprachiasmatica (60%, POAs ) and NHA (60%) and was unchanged or augmented in the 7 other regions. In contrast, a decline in DA turnover rate of 29-67% was observed in 6 of 9 regions in middle-aged rats and 45-81% in 5 of 9 regions in old rats. Steady-state NE concentrations similarly were significantly decreased in old versus young rats in the POAs (54%), medial forebrain bundle (44%), nucleus suprachiasmatica (49%) and ME (59%). The rate constant of NE loss progressively decreased with increasing age only in the POAs and was unchanged or augmented in other regions. Turnover rate of NE was decreased from 21 to 98% in 4 of 8 regions from old animals. A strong positive correlation was noted between the rate constant of NE (but not DA) loss measured in young rats and the magnitude of the age-related depletion in NE concentrations within specific brain regions. Collectively these data indicate that with increasing age: CA neuronal function is differentially altered in nuclei located along the preoptico-tuberal pathway; substantial declines in both DA and NE concentrations are the primary contributor to the reduced amine turnover noted in several of these regions; and the observed age-related alterations in CA turnover may contribute to impaired LH response and the persistent hyperprolactinemia in old CE rats.     

Decreased dopamine turnover in the median eminence in response to suckling in the lactating rat

The effects of suckling on the turnover of dopamine (DA) and norepinephrine (NE) were studied in terminal projection fields of the tuberoinfundibular (median eminence, ME), nigrostriatal (caudate nucleus, CN), incertohypothalamic (medial preoptic nucleus, MPN) and mesolimbic (nucleus accumbens, NA) dopaminergic neurons. Decreased dopamine turnover in the median eminence was found in suckled compared with nonsuckled rats at 10 days postpartum. This effect was specific as dopamine turnover in the CN, NA and MPN and norepinephrine turnover in the ME, NA and MPN were not affected by suckling. The suckling-induced prolactin response is markedly blunted in rats 20 days postpartum. In these rats, median eminence dopamine turnover did not decrease significantly in response to suckling. These results are consistent with the hypothesis that median eminence dopamine is a physiological prolactin inhibitory factor mediating suckling-induced prolactin release.