Median Eminence Area Research Articles

Ketogenic diet promotes the proliferation of oligodendrocyte precursor cells in ME region by activating fatty acid oxidation.

Hypothalamic median eminence (ME) is a potential niche for neurons and oligodendrocytes, and trophic factors may regulate hypothalamic function by inducing cellular changes in the ME region. To determine whether diet-induced plasticity exists in hypothalamic stem cells dormant under physiological conditions, we used a combination of a normal diet, a high-fat diet, and a ketogenic diet (a low-carb, high-fat diet) to compare the proliferation of tanycytes (TCs) and oligodendrocyte precursor cells (OPCs) in the ME area of mice under the different diets. The results showed that the ketogenic diet could induce and promote the proliferation of OPCs in the ME area, and blocking the fatty acid oxidation program could inhibit the proliferation of OPCs induced by a ketogenic diet. This study preliminarily revealed the diet-induced effect on OPCs in the ME region and provided enlightenment for further study on the function of OPCs in the ME region.

P.1.012 Changes in hippocampal plasticity during pregnancy and post partum are reversed by offspring separation

During the last two decades attention has been focussed on the role of different neuropeptides in hypothalamic control of feeding behavior. Several hypothalamic peptides that participate in the control of ingestive behavior are produced in neuronal cell bodies of the arcuate nucleus and/or the lateral hypothalamic area. Apart from producing orexigenic or anorexigenic compounds of peptidergic nature, these neurons also produce excitatory and inhibitory amino acid neurotransmitters. The role of GABA and glutamate in regulating energy balance has received less attention in comparison to neuropeptides. The arcuate nucleus–median eminence area, a region with a weak blood–brain barrier, contains at least two neuronal cell populations that exert opposing actions on energy balance. The majority of the neurons located in the ventromedial aspect of the arcuate nucleus, which produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP), contain in addition the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter (VGAT), thereby supporting their GABAergic nature. Some neurons producing pro-opiomelanocortin (POMC)- and cocaine- and amphetamine-regulated transcript (CART), located in the ventrolateral division of the arcuate nucleus have recently been reported to contain the vesicular glutamate transporter 2 (VGLUT2), a marker for glutamatergic neurons, and the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) as well as the vesicular ACh transporter (VAChT), supporting also a cholinergic phenotype. In the lateral hypothalamic area, hypocretin/orexin neurons express VGLUT1 or VGLUT2, but not GAD, whereas some melanin-concentrating hormone (MCH) cells contain GAD. These observations support the view that several classical transmitters, relatively neglected feeding transmitters candidates, are present in key neurons that regulate body weight and consequently may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in regulation of energy balance.

Calcitonin receptor bindings in the hen hypothalamus before and after oviposition

To demonstrate the presence of a receptor for calcitonin (CT) in the hen hypothalamus and to determine when CT acts on this tissue during the oviposition cycle, bindings of (125)I labeled CT in the plasma membrane fraction of the hen hypothalamus were measured by radioligand binding assay. The specific CT binding component in the plasma membrane fraction of the hypothalamus containing the preoptic area (HPOA) possessed properties of a receptor: binding specificity to CT, saturable binding, high affinity, and limited capacity. As for the median eminence area, no specific binding component was found in the present study. Therefore, the binding component for CT in the plasma membrane fraction of HPOA is likely to be a receptor for CT. In laying hens, the binding affinity of CT receptor increased at 30 min before oviposition and the binding capacity was decreased at 30 min before oviposition but not changed in nonlaying hens during a 24-h period. These results suggest that the action of CT on the hen HPOA may increase 30 min before oviposition.

Neurotransmitters in key neurons of the hypothalamus that regulate feeding behavior and body weight

During the last two decades attention has been focussed on the role of different neuropeptides in hypothalamic control of feeding behavior. Several hypothalamic peptides that participate in the control of ingestive behavior are produced in neuronal cell bodies of the arcuate nucleus and/or the lateral hypothalamic area. Apart from producing orexigenic or anorexigenic compounds of peptidergic nature, these neurons also produce excitatory and inhibitory amino acid neurotransmitters. The role of GABA and glutamate in regulating energy balance has received less attention in comparison to neuropeptides. The arcuate nucleus–median eminence area, a region with a weak blood–brain barrier, contains at least two neuronal cell populations that exert opposing actions on energy balance. The majority of the neurons located in the ventromedial aspect of the arcuate nucleus, which produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP), contain in addition the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter (VGAT), thereby supporting their GABAergic nature. Some neurons producing pro-opiomelanocortin (POMC)- and cocaine- and amphetamine-regulated transcript (CART), located in the ventrolateral division of the arcuate nucleus have recently been reported to contain the vesicular glutamate transporter 2 (VGLUT2), a marker for glutamatergic neurons, and the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) as well as the vesicular ACh transporter (VAChT), supporting also a cholinergic phenotype. In the lateral hypothalamic area, hypocretin/orexin neurons express VGLUT1 or VGLUT2, but not GAD, whereas some melanin-concentrating hormone (MCH) cells contain GAD. These observations support the view that several classical transmitters, relatively neglected feeding transmitters candidates, are present in key neurons that regulate body weight and consequently may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in regulation of energy balance.

EFFECT OF CHRONIC HYPERPROLACTINEMIA ON DAILY CHANGES OF GLUTAMATE AND ASPARTATE CONCENTRATIONS IN THE MEDIAN EMINENCE AND DIFFERENT HYPOTHALAMIC AREAS OF MALE RATS

The 24h changes of glutamate (GLU) and aspartate (ASP) werestudied in the median eminence (ME) and hypothalamic areas. It was analyzedwhether prolactin may change their daily patterns. The hypothalamic concentrationof these amino acids was measured by high-performance liquid chromatography(HPLC) with fluorometric detection. Plasma prolactin levels increased overthe 24h light-dark cycle after pituitary grafting compared to controls, andits circadian rhythm was disrupted. In controls, aspartate and glutamate inthe hypothalamic areas studied followed a specific daily variation or showedno rhythmicity. In the median eminence, hyperprolactinemia seem to phase advancethe aspartate or glutamate peaks from 16:00 to 12:00. In the mediobasal hypothalamus,hyperprolactinemia altered daily changes of aspartate and significantly decreasedits concentration. Also, it seems to delay the nocturnal glutamate peak comparedto controls. In the posterior hypothalamus, hyperprolactinemia did not changeaspartate and glutamate concentrations and their daily changes, although itincreased the glutamine concentration. These data show the existence of 24hchanges of amino acid concentration in three of the hypothalamic regions studied.Increased plasma prolactin levels differentially affected these patterns dependingon the hypothalamic area analyzed. (ChronobiologyInternational, 17(5), 631–643, 2000)

Induction of c-fos gene expression by the selective sigma receptor ligand EMD 57445 in rat brain

Based on animal studies it has been reasoned that ligands to sigma binding sites might be effective in the treatment of schizophrenic disorders and may also be used to investigate this largely elusive disorder on a molecular level. Expression patterns of c-fos in rat brain were studied following treatment with single doses of the sigma ligand EMD 57445 (0.3, 1, 3, 30 mg/kg s.c.). Specific c-fos gene expression was detected at all concentrations tested in various cortical areas. The signals observed were dose-dependent with the highest intensities in the piriform cortex. Strong signals were also detected in hippocampal areas CA 1,2,3 and the gyrus dentatus, as well as in the medial habenula nuclei. In the caudate putamen, nucleus accumbens and lateral septal nucleus signals were detectable after administration of doses ≥1 mg/kg. Furthermore, c-fos hybridization was visible in the amygdala, in the mammillary bodies, the islands of Calleja and in the olfactory tubercle. In the hypothalamus, c-fos expression was seen in the median eminence area after 30 mg/kg EMD 57445. No hybridization signals were obtained in brainstem or cerebellum. Since c-fos expression induced by EMD 57445 resembled the pattern obtained with atypical neuroleptics and studies on animal behavior point to antipsychotic activity, it is concluded that the drug might be suitable in the treatment of schizophrenia.

Dopamine transporter immunohistochemistry in median eminence, amygdala, and other areas of the rat brain

Dopamine transporter immunohistochemistry in median eminence, amygdala, and other areas of the rat brain

Presence of vasoactive intestinal peptide receptor in the hen hypothalamus.

Radioligand assays of the membrane fraction of hen hypothalamic tissues involving the preoptic (HPOA) or median eminence (HMEA) areas revealed the presence of a specific binding component to chicken vasoactive intestinal peptide (cVIP) having properties of a receptor. The equilibrium dissociation constant (Kd) was 0.70 +/- 0.07 nM (Mean +/- SEM; N = 5) in HPOA and 1.02 +/- 0.15 nM (N = 5) in HMEA as estimated by Scatchard analysis of saturation studies, and was 0.91 +/- 0.11 nM (N = 3) (HPOA) and 1.25 +/- 0.09 nM (N = 3) (HMEA) as determined by a kinetic analysis. The maximum binding capacity (Bmax) obtained by Scatchard analysis was 167 +/- 19 fmol/mg protein (N = 5) (HPOA) and 133 +/- 17 fmol/mg protein (N = 5) (HMEA). The Kd and Bmax values obtained by Scatchard analysis were similar in the two areas of the hypothalamus and in both laying and nonlaying hens. Administration of cVIP in vivo caused a decrease in specific cVIP binding. These results suggest the presence of a VIP receptor in the hen hypothalamus.

Estrogen Receptor Binding in the Hen Hypothalamus and Pituitary During the Ovulatory Cycle

The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) of estrogen receptor in soluble (cytosolic) and insoluble (nuclear) fractions in a hypotonic buffer solution of hypothalamus containing preoptic (HPOA) and median eminence (HMEA) areas and of anterior pituitary (AP) of laying and nonlaying hens were examined by Scatchard analysis of specific [3 H]estradiol-17β ([3 H]-E2) binding. The Kd of receptor in all of the tissues was different neither between soluble and insoluble fractions, nor between laying and nonlaying hens. The Bmax in laying hens was greater in the insoluble fraction and lower in the soluble fraction than that in nonlaying hens, but the total binding capacity (sum of Bmax in the soluble and insoluble fractions) was not different between laying and nonlaying hens. In laying hens, the specific [3 H]-E2 binding in the insoluble fraction of HPOA was found to increase at 21 h before ovulation and again at 8 to 6 h before ovulation, and of HMEA and AP at 18 to 11 h before ovulation. No change in the specific [3 H]-E2 binding in the insoluble and soluble fractions was found in any of the tissues of nonlaying hens during a 24-h period. The results suggest that in laying hens, estrogen may act on the hypothalamus and pituitary at restricted hours during the ovulatory cycle.

A putative role for extracellular ATP: Facilitation of 67copper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for 67Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates 67Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 μM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, α,β-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.

Postovariectomy Decline in the in vitro Release of LH-Releasing Hormone Is Not Manifested in Aging Female Rats

Aging of the female reproductive system is associated with a transition from a regular cyclic to an irregular cyclic and finally to an acyclic mode of function. We addressed the question: is the transition associated with a decline in the secretory response of the LHRH neuron to an external stimulus and with a change in steroid regulation of the secretory function of the LHRH neuron? Three groups of virgin female rats ranging from 2 to 7 months of age were used: intact, ovariectomized (8 days) and ovariectomized treated with estradiol-17 beta for 49-50 h; explants of the median eminence area (MEA) were incubated in vitro and LHRH release in response to the combined actions of copper and prostaglandin E2 was used as a criterion for secretory function. Based on vaginal cytology, young (2-3 months) females exhibited regular estrous cycles, the transition to irregular cycles manifested at 5 months of age (2/3 of animals) and to acyclicity at 7 months (all animals). In intact animals, there was a doubling in the magnitude of basal as well as stimulated LHRH release which paralleled the increase in MEA content of the peptide. By comparison to intact, stimulated LHRH release was markedly (greater than or equal to 50%; p less than 0.001) attenuated in 2- and 3-month-old ovariectomized rats but not in 5- and 7-month-old rats; thus, stimulated LHRH release at 5-7 months was approximately 2.5-fold greater than at 2-3 months of age. At each age group, estradiol treatment did not alter the magnitude of stimulated LHRH release. Ovariectomy with or without estradiol did not alter basal release nor the MEA content of LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid, Ca 2+-independent, onset of prostaglandin E 2 stimulation of the release of luteinizing hormone releasing hormone from copper-treated median eminence explants

Prostaglandin E 2 (PGE 2) is known to stimulate the release of luteinizing hormone releasing hormone (LHRH) from explants of the median eminence area (MEA). We have previously shown that a short preincubation of the MEA with copper markedly amplifies PGE 2 stimulation of LHRH release and that the Ca 2+-cAMP pathway is involved in this release process. In this study, we defined the kinetics of the onset of PGE 2 action and examined the Ca 2+ requirement for the onset and manifestation of PGE 2 action. MEA explants from immature female rats were incubated with copper (150 μM copper-histidine complex); thereafter, expiants were exposed to 10 μM PGE 2 for periods of 2−7 min with or without Ca 2+ (Ca 2+-free buffer containing 1 mM EGTA). A 2 min PGE 2 exposure was as effective as a 7 min PGE 2 exposure in stimulating LHRH release; the latter was manifested during the period between 2–7 min. When MEA were exposed to PGE 2 for 5 min, maximal rate of release was attained within this 5 min period. When MEA were exposed to PGE 2 for 2 min and then to forskolin (100 μM) for 5 min, there was no further increase in the rate of LHRH release (although exposure to forskolin alone maximally stimulated LHRH release). In the presence of EGTA (no Ca 2+), PGE 2 stimulation of LHRH release was abolished and this effect of EGTA was totally reversed by the addition of Ca 2+ 2 min after PGE 2 exposure and partially reversed (40%) by the addition of Ca 2+ 5 min after PGE 2 exposure in the presence of EGTA. In summary, (i) a 2 min exposure of copper-treated MEA expiants to PGE 2 suffices to maximally stimulate LHRH release presumably by maximally activating adenylate cyclase; (ii) maximal rates of LHRH release are attained within 3–5 min after PGE 2 exposure; (iii) there is a differential Ca 2+ requirement for the onset and for the manifestation of PGE 2 action: the initial 2 min interaction does not require extracellular Ca 2+; the subsequent events leading to LHRH release require Ca 2+ and the Ca 2+ requiring element(s) is short-lived (3–5 min).

Presence of androgen receptors in the hen hypothalamus and pituitary.

Soluble and insoluble fractions in a hypotonic buffer solution of hypothalamic-preoptic and median eminence areas, and of the anterior pituitary of the hen were found to contain a specific androgen binding component having the properties of a receptor. Administration of testosterone in vivo caused a marked decrease in specific [3H]R1881 (a synthetic androgen) bindings in the soluble fraction with a concomitant increase in the bindings in the insoluble fraction, whereas the sum of the bindings did not change. A similar relationship between the bindings of the two fractions was also observed during an ovulatory cycle. The results may provide an evidence for a direct action of androgens on the hen hypothalamus and pituitary.

Progesterone augments copper-prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from explants of the median eminence of immature female rats: an estrogen-dependent process.

We have previously shown that extracellular copper (Cu) amplifies prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA) of adult male rats, and that amplification is a post-PGE2 receptor event involving the adenylate cyclase system. We addressed the question: Is the process of Cu-amplified PGE2 stimulation of LHRH release regulated by ovarian steroids and, if so, is the regulatory steroid estrogen and/or progesterone? Immature female rats (30-32 days old) were ovariectomized and 6 days later treated with a combination of these steroids: E, sc implant of 17 beta-estradiol in a Silastic capsule for 3 days; EEi, E plus im injection of 17 beta-estradiol (2 micrograms/rat) 24 h before killing; and P, either im injection of 0.08 mg/kg progesterone 1 h before killing or 10(-9) M P included in the incubation buffer starting 1 h before Cu/PGE2 exposure. Controls were treated with the vehicle. MEAs were incubated for 5 min with 150 microM Cu, for 15 min with 10 microM PGE2, and then for 45 min with buffer (Cu/PGE2); LHRH release into the medium was measured by RIA. Cu/PGE2-stimulated LHRH release from MEA of intact rats was about 10 times greater than basal release. Ovariectomy led to a 50% reduction in Cu/PGE2-stimulated release [sham, 20.6 +/- 2.0 (mean +/- SE); ovariectomized, 9.4 +/- 1.8], pg/30 mm/MEA; and neither E, EEi, nor P significantly altered this response. In contrast, administration of P to either E- or EEi-primed rats augmented Cu/PGE2 stimulation of LHRH release 3.5-fold (E vs. EP or EEi vs. EEiP); however, P did not augment stimulation of LHRH release by Cu alone or PGE2 alone. Also, inclusion of P in the incubation buffer was as effective as in vivo P in augmenting Cu/PGE2 stimulation of LHRH release from the MEA of EEi-primed rats. On the other hand, in vitro P by itself did not alter LHRH release. These effects of P on the response of the MEA to Cu/PGE2 were not accompanied by a significant increase in the MEA content of LHRH. The process of Cu/PGE2 stimulation of LHRH release is regulated by ovarian steroids, so that ovariectomy leads to a marked reduction of the response of the MEA to Cu/PGE2, and P augments this response in an estrogen-dependent manner. Moreover, it is the secretory process elicited by the combined effects of Cu and PGE2 that is augmented by P.(ABSTRACT TRUNCATED AT 400 WORDS)

A correlation between the ligand specificity for 67copper uptake and for copper-prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants.

We have previously shown that copper (Cu) leads to a 3- to 4-fold amplification of prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA), that this amplification is a saturable function of [Cu], and that complexed Cu but not ionic Cu is the active form of the metal. This implicates a ligand-specific site in Cu action. In this study we address the following questions. Is there a ligand specificity for Cu amplification of PGE2 stimulation of LHRH release, and if so, does it correlate with the ligand specificity for Cu uptake? MEAs of 1-month-old female rats were incubated for 5 min with 150 microM Cu solution and then for 15 min with 10 microM PGE2 (Cu/PGE2); LHRH released into the medium was evaluated by RIA. To assess Cu uptake, MEAs were incubated with 100 microM67 Cu solution for 15 min, and 67Cu accumulation by the MEA was evaluated. The Cu was complexed to one of the following ligands: histamine, His, Cys, Thr, Gly, glutathione, Gly-His-Lys, or albumin. There was a high degree of correlation (r = 0.943) between the ligand specificity for Cu/PGE2 stimulation of LHRH release (Cu action) and 67Cu uptake. Complexation of ionic Cu with His facilitated Cu action and 67Cu uptake 3-fold each, and this was completely prevented by the inclusion of His in a 100-fold excess over the concentration of the Cu/His complex. Histamine, the amino acids, and the peptides facilitated Cu action and 67Cu uptake, whereas albumin did not do so. Of these facilitatory ligands, histamine and His were the most effective and Gly-His-Lys was the least effective. In summary, both 67Cu uptake and Cu action are ligand-dependent and ligand-specific; the Cu interactive sites have a common recognition for the Cu-ligand complex and for the ligand itself; and the ligand specificity for 67Cu uptake and for Cu action are highly correlated. These results are consistent with the ligand specificity for Cu uptake being the primary determinant of the ligand specificity for Cu/PGE2 stimulation of LHRH release.

Evidence for synergism between copper and prostaglandin E 2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na +/Cl − requirements

Copper (Cu) and PGE 2 are known to stimulate LHRH release from expiants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca 2+ dependence. Moreover, exposure to Cu and PGE 2 results in an amplified release of LHRH which is partially Ca 2+ dependent, thus, resembling the release process stimulated by PGE 2 alone. We have shown that LHRH release stimulated by Cu alone is Na +/Cl − dependent. By defining the Na +/Cl − dependence of PGE 2- and Cu/PGE 2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE 2 actions. MEA of adult male rats were incubated for 5 min with 150 μM Cu and then for 15 min with 10 μM PGE 2 (Cu/PGE 21). Controls were incubated with Cu or PGE 2. LHRH release into the medium was evaluated by RIA. Substituting Cl − in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE 2 stimulation of LHRH release, but it drastically inhibited Cu/PGE 2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE 2 and Cu/PGE 2 stimulation of LHRH release were both inhibited when Na + was substituted with Li +, or when 0.5 mM ouabain was included in the Na +-containing buffer; neither 10 μM tetrodotoxin (TTX) nor 100 μM amiloride were inhibitory. To ascertain if Na + is required for Cu uptake, we evaluated the uptake of 67Cu by MEA expiants and found that neither ouabain nor Li + inhibited uptake, indicating that the extracellular Na + and the activity of Na +/K + ATPase are required for the process of LHRH release. In summary, (i) Cu and PGE 2 stimulation of LHRH release are distinct processes, based on their Ca 2+ and Cl − requirements; and (ii) the process of Cu/PGE 2 stimulation of LHRH release exhibits the ionic requirements of both secretagogues. This is consistent with Cu and PGE 2 synergistically acting on the LHRH neuron, which results in amplification of the release processes initiated by each of the secretagogues.

Copper amplification of prostaglandin E2 stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event.

We have shown that copper amplifies prostaglandin E2 (PGE2) stimulation of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA) and that this process is calcium-dependent. Since a Ca-cAMP pathway has been implicated in PGE2 action on the LH-RH neuron, in this study we wished to ascertain if copper exerts its effect on the PGE2 receptor or on a postreceptor component involved in PGE2 action. MEA of adult male rats were incubated for 5 min with 200 microM Cu/histidine (CuCl2 mixed with L-histidine at an equimolar ratio) and then incubated for 15 min either with 10 microM PGE2 (Cu/PGE2), 100 microM forskolin (Cu/forskolin), or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Cu/cAMP). Controls were incubated without Cu/histidine or with Cu/histidine alone. Basal release of LH-RH was 4.6 +/- 0.45 pg/15 min per MEA (mean +/- SEM). Net stimulated release during the 15-min exposure to PGE2, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate was 3.6 +/- 0.52, 3.1 +/- 0.39, and 1.6 +/- 0.42 pg/15 min per MEA, respectively. Net stimulated release after exposure to Cu/PGE2, Cu/forskolin, or Cu/cAMP was 12.7 +/- 2.2, 9.9 +/- 1.46, and 1.4 +/- 1.9 pg/15 min per MEA, respectively, indicating that copper amplifies the action of PGE2 and forskolin but not cAMP action. When MEA were exposed to a mixture of PGE2 and forskolin for 15 min, the effects of these two secretagogues on LH-RH release were not additive, regardless of whether the MEA were pretreated with Cu/histidine. In contrast to PGE2 and forskolin, copper did not amplify K+ stimulation of LH-RH release and, moreover, when Cu/histidine-treated MEA were exposed to a mixture of PGE2 and 30 mM K+, the effects of these two secretagogues were additive. These results are supportive of the proposition that PGE2 stimulation of LH-RH release is mediated by the Ca-cAMP pathway and that copper amplification of PGE2 action is a postreceptor event.

Dual effects of (D-Ala2,Met5)-enkephalinamide on CRF and ACTH secretion.

An intra-third ventricular administration of (D-Ala2,Met5)-enkephalinamide (DALA) did not elevate plasma ACTH and corticosterone levels in unanesthetized freely moving rats, but intra-third ventricular administration of DALA and methionine (Met)-enkephalin potentiated a mild stress (hanging for 10 or 30 sec)-induced plasma ACTH and corticosterone elevations in unanesthetized freely moving rats. DALA and Met-enkephalin seemed to stimulate CRF release from the median eminence to increase plasma ACTH, as the CRF concentration in the median eminence area was reduced after injection in these stressed rats. When hypothalamic tissues were perifused in vitro, DALA (1-100 ng/ml) reduced the release of CRF. These results suggest that the opiates seem to have a dual effect on the CRF-ACTH system depending on which action overrides the other.

Chronic push-pull brain perfusion in unrestrained rhesus macaques.

A system was developed to permit perfusion of local brain regions and simultaneous peripheral blood sampling in free-moving caged monkeys. The system comprises a calvarial headpiece that contains multiple push-pull cannulas (PPC), a flexible stainless steel tether, a four-channel fluid swivel, and a surgical procedure for simultaneous multisite brain cannulation. Rhesus macaques were fitted surgically with an indwelling jugular catheter and PPC directed into the third ventricle, median eminence, and preoptic area. These animals were tethered for periods of 14-70 h during which brain perfusates and peripheral blood samples were collected at 10- to 30-min intervals through the tether-swivel assembly. Levels and pulsatile patterns of gonadotropin-releasing hormone in 10-min perfusate samples and luteinizing hormone and cortisol in sequential plasma samples were quantified by specific radioimmunoassays. The normal endocrine profiles in these animals suggest that this system provides a valuable method to study patterns of neurosecretions in an unrestrained simian.

Amplification of prostaglandin E 2 stimulation of luteinizing hormone-releasing hormone release from median eminence explants: a metal(II)-specific effect of chelated copper

We have previously demonstrated that extracellular copper amplifies prostaglandin (PG) E 2 stimulation of the release of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area(MEA) 1. Two questions were addressed: what is the active form of copper and the metal(II) specificity for copper action? MEA explants were incubated for 5 min in the presence of CuCl 2 (ionic) or copper complexed to histidine (CuHis) at a concentration of 200 μM each and then for 15 min in the presence of 10 μM PGE 2. It was found that chelated copper but not ionic amplified PGE 2 action, and that the magnitude of PGE 2 stimulation of LH-RH release was 3–4 fold in copper-treated than untreated tissue. Moreover, PGE 2-stimulated release was directly related to the dose of CuHis. To test the metal specificity, MEA explants were incubated for 5 min with one of the following metal(II) complexes: CuHis, NiHis, FeHis, ZnHis, CdHis, MnHis, or BaHis (200 μM each) and then for 15 min with 10 μM PGE 2. Controls were incubated with metal(II) complex or PGE 2. Of these complexes, only CuHis and to a lesser extent NiHis stimulated LH-RH release. However, CuHis was the only complex that amplified PGE 2 stimulation of LH-RH release. Thus, amplification is specific for copper. The finding that chelated copper but not ionic copper amplifies PGE 2 is suggestive that the copper-interactive sites on the LH-RH neurons are not exposed to the extracellular space but that they are either embedded in the plasma membrane, facing the intracellular space, or in the cytoplasm. Such an orientation is consistent with copper modulation of the functional state of a postreceptor component involved in the cascade of events initiated by PGE 2 and leading to LH-RH release.