Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices

A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electro-chemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37 degrees C. Increasing the concentration of tyrosine in medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM alpha-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K+ to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K+-induced increase in DOPA synthesis was blocked by omission of Ca2+ and addition of 1 mM EGTA into the medium, suggesting Ca2+ dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.     

Characterization of in vitro dopamine synthesis in the median eminence of rats with haloperidol-induced hyperprolactinemia and bromocriptine-induced hypoprolactinemia

In order to investigate the mechanism of PRL action on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons, in vitro dopamine synthesis in the median eminence of hypothalamic slices was compared between hyperprolactinemic and hypoprolactinemic rats, Hyper- and hypoprolactinemia were induced in ovariectomized rats by repetitive injections of the dopamine antagonist haloperidol (Halo) and the dopamine agonist bromocriptine (Bromo), respectively. In vitro dopamine synthesis in TIDA neurons was estimated by measuring 3,4-dihydroxyphenylalanine (DOPA) accumulated in the median eminence after incubation of hypothalamic slices with a DOPA decarboxylase inhibitor. Treatment with Halo or Bromo produced increases or decreases, respectively, in the concentration of PRL in serum and in in vivo DOPA accumulation in the median eminence, as compared with vehicle treatment. The basal rate of in vitro DOPA accumulation in the median eminence was increased in Halo-treated rats and was decreased in Bromo-treated rats. The increase in basal DOPA accumulation after Halo treatment was inhibited by Ca2+ removal from medium or tetrodotoxin addition. A CA2+ -dependent increase in DOPA accumulation in the median eminence by depolarization was greater in Halo-treated rats than in Bromo-treated rats. This difference in DOPA accumulation was due to the changes in PRL secretion after Halo and Bromo treatments, since hypophysectomy abolished it. Incubation of hypothalamic slices in Na+-free media to increase the intracellular concentration of Ca2+ through inhibition of Na+-Ca2+ exchange caused an increase in DOPA accumulation. The rate of DOPA accumulation in Na+-free media was increased in Halo-treated rats and was decreased in Bromo-treated rats. On the other hand, neither Halo nor Bromo treatment altered the increase in DOPA accumulation induced by (Bu)2cAMP or forskolin. These results support the view that PRL stimulates dopamine synthesis in TIDA neurons by mechanisms which include an increase in the firing rate of TIDA neurons and increased depolarization-induced synthesis due to an enhanced response of the component that regulates dopamine synthesis to intracellular Ca2+.     

Morphine and endorphins modulate dopamine turnover in rat median eminence.

The is evidence that some of the actions of both endogenous and exogenous opioids (e.g., stimulation of prolactin release) are mediated by interaction with catecholaminergic systems. Morphine (1.67, 5, and 15 mg/kg of body weight, intraperitoneally) altered dopamine turnover as measured by the alpha-methyl-p-tyrosine method in the median eminence, neostriatum, and frontal cortex of male Sprague-Dawley rats. The turnover rate of dopamine was reduced in the median eminence and frontal cortex but accelerated in the neostriatum. In the frontal cortex all doses were effective in decreasing dopamine turnover; however, in the median eminence the lowest dose of morphine did not significantly alter dopamine turnover. All three doses accelerated dopamine turnover in the neostriatum. Naloxone effectively reversed the effects of morphine at all doses in all brain areas, whereas it had no effect on turnover when given alone. In the median eminence, neostriatum, and frontal cortex, intraventricular injection of [D-Ala2,D-Leu5]-enkephalin (25 micrograms) or beta-endorphin (15 micrograms) produced the same effects on dopamine turnover as morphine. The actions of these peptides were blocked by naloxone. It is hypothesized that opiates and opioid peptides increase prolactin release by reducing the activity of the tuberoinfundibular dopaminergic system.     

N-tosyl-L-phenylalanine chloromethyl ketone inhibits LHRH-degrading activity and increases in vitro LHRH release from the immature rat median eminence

Median eminence (ME) luteinizing-hormone-releasing hormone (LHRH)-degrading activity (LHRH-DA) may play a role in regulating the availability of releasable LHRH. Incubation of LHRH with ME tissue supernatant yields LHRH(1-5) and LHRH(6-10) degradation fragments, as detected by high-performance liquid chromatography (HPLC) analysis, suggesting a 5-6 cleavage of the decapeptide. Since these fragments are also present after incubation of LHRH with alpha-chymotrypsin (alpha-CH), we examined the possibility that the irreversible inhibitor of alpha-CH, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), might inhibit LHRH-DA and affect LHRH release. Irreversible inhibitors of trypsin-like proteases [N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and phenylmethylsulfonylfluoride (PMSF)] were used as controls. LHRH-DA was determined by HPLC estimation of the loss of synthetic LHRH incurred when the peptide was incubated with aliquots of ME supernatant in the presence or absence of the inhibitors. LHRH release from ME fragments was assessed by radioimmunoassay after incubating the tissue with the inhibitors in Krebs-Ringer bicarbonate buffer. The LHRH-DA in both the incubation medium and the ME tissue was determined at the end of the incubation. TPCK (0.5-100 microM) added to ME tissue supernatant inhibited LHRH-DA in a dose-dependent manner. In contrast, when TPCK was added to medium in which intact ME were being incubated to assess LHRH release, the LHRH-DA of these ME was inhibited only at the 25-, 50- and 100-microM doses of TPCK, suggesting a relative inability of the inhibitor to reach endopeptidase pools in intact tissue. These same doses of TPCK increased LHRH release from the incubated ME.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative in vitro studies on corticotropin releasing activity of vasopressin and hypothalamic median eminence extract.

The in vitro corticotropic releasing effects of vasopressin (VP) and hypothalamic median eminence (HME) extract were compared as a function of their concentration and preincubation and incubation times. Whereas HME extract augmented the ACTH secretion from non-preincubated adenohypophyses, VP released corticotropin from the pituitaries only after a 2 h preincubation period. The disappearance of endogenous VP during the preincubation time rendered the gland responsive. The maximal stimulation of ACTH secretion by VP was markedly less than that induced by HME extract. The results suggest the presence of VP receptor sites in the anterior pituitary (AP) which are probably different from the receptor sites of the hypothalamic corticotropin releasing factor (CRF).     

Pro-opiomelanocortin-containing neurons in rat median eminence.

A significant number of pro-opiomelanocortin (POMC)-containing cells were detected in the rat median eminence (ME) by immunocytochemistry using an antibody raised against a synthetic peptide corresponding to the cleavage site between adrenocorticotropin and beta-lipotropin moieties. Distribution of POMC-positive cells was restricted to the internal zone of the anterior parts of the ME. Such cells were observed as early as the 14th day of gestation in the area of the primitive ME, long before glial fibrillary acidic protein-positive cells appeared postnatally in this structure. Dissociated cell cultures obtained from the ME of 1-day-old rats produced cells immunoreactive for neurofilaments and the POMC moiety. Such cells displayed a neuronal morphology: the cell body was oval (13-18 microns) with long and fine beaded fibers. These findings clearly demonstrate the early appearance of POMC neurons in the developmental ME, a target organ of the hypothalamic infundibular neurons.     

Involvement of vasopressin in corticotropin-releasing effect of hypothalamic median eminence extract

Incubation of anterior pituitary (AP) fragments of rats was used to determine the specific role played by vasopressin (VP) in the overall effect of crude hypothalamic median eminence (HME) extract on ACTH release. Using the property of an AVP antiserum (AS) to completely abolish the CRF-like effect of hypothalamic VP without apparently affecting the effect of CRF, we show that under specific incubation conditions, the effect of the two secretagogues are additive at the pituitary level. ACTH secretion of pituitaries was enhanced when incubation was carried out in the presence of VP together with a maximum effective dose of VP-free HME extract (from Brattleboro rats). These observations favor the hypothesis that VP and CRF have different receptor sites in the anterior pituitary.     

Effects of dopamine on immunoreactive growth hormone-releasing factor and somatostatin secretion from rat hypothalamic slices perifused in vitro

The effects of dopamine (DA) on the release of GRF and somatostatin (SRIF) from the hypothalami of adult male rats were examined in an in vitro perifusion system using horizontal hypothalamic slices, 400 micron thick, including the median eminence and arcuate nuclei. When hypothalamic slices from five animals were perfused in a chamber with artificial cerebrospinal fluid (ACSF) at a flow rate of 100 microliters/min under a gaseous phase of 95% O2 and 5% CO2 at 37 C, rat (r) GRF- and SRIF-like immunoreactivities (-LI) were constantly detected in 30-min perifusates at least until 240 min of perifusion, and during the perifusion with 60 mM K+, the concentrations of rGRF-LI and SRIF-LI were increased 2.1 and 3.2 times, respectively, over basal values. Under the perifusion with ACSF containing normal goat gamma-globulin, the addition of 10(-8) M DA resulted in a significant increase in SRIF-LI from 8.2 +/- 0.3 to 14.3 +/- 1.5 pg/hypothalamus.30 min, but conversely, it caused a slight but significant decrease in rGRF-LI from 4.5 +/- 0.9 to 2.0 +/- 0.3 pg/hypothalamus.30 min. On the other hand, 10(-8) and 10(-6) M DA significantly stimulated rGRF-LI release from hypothalamic slices perifused with ACSF containing anti-SRIF goat gamma-globulin. These findings suggest that DA is a secretagogue for both SRIF and rGRF in the hypothalamus, but the rGRF-stimulating effect of DA is masked unless the action of endogenous SRIF is attenuated.     

In vitro release of vasopressin and oxytocin from rat median eminence tissue

Release of vasopressin (AVP) and oxytocin (OT) from rat median eminence and posterior pituitary tissue was studied in vitro by incubation in Krebs-56 mM KCl buffer. Both total tissue content and releasable pool of each hormone was measured in control rats, adrenalectomized rats and dexamethasone-treated rats. Adrenalectomy resulted in significantly increased release of AVP, but not OT, from median eminence tissue, whereas dexamethasone treatment failed to affect release of either hormone. Neither treatment had any effect on AVP or OT release from posterior pituitary tissue. Similarly, neither treatment caused any significant changes in total median eminence or posterior pituitary AVP and OT contents relative to controls, although dexamethasone-treated rats had a significantly lower posterior pituitary OT content than adrenalectomized rats. KCl-stimulated hormone release from median eminence tissue most likely represents an estimate of AVP and OT in zona externa terminals rather than in zona interna axons, because release was blocked by CoCl2 indicating calcium-dependent exocytosis. Immunohistochemical staining of median eminence tissue correlated well with the results of in vitro hormone release, in that increased AVP staining in the zona externa of adrenalectomized rats was also the only significant change noted using this methodology. Since increased levels of releasable AVP in the median eminence probably reflects similarly increased AVP levels in the hypothalamo-hypophyseal portal vessels of adrenalectomized rats, these results support a potential physiologic role for median eminence AVP, but not OT, in the chronic stimulation of adrenocorticotropin hormone secretion following adrenalectomy.     

Novel hypothalamic arachidonate products stimulate somatostatin release from the median eminence

Arachidonic acid (AA) stimulates the in vitro release of somatostatin (SRIF) from the hypothalamic median eminence (ME). This effect is inhibited by 5, 8, 11, 14-eicosatetraynoic acid (ETYA) but not by indomethacin (ID). Microsomal fractions from the rat hypothalamus catalyze an NADPH-dependent metabolism of AA to form several oxygenated products of which the major metabolites are 5, 6-epoxyeicosatrienoic acid (5, 6-EET) and its hydration product, 5, 6-dihydroxyeicosatrienoic acid (5, 6-DHET). Both novel arachidonate metabolites, particularly 5, 6-EET, are potent in vitro stimuli for the release of SRIF. To a lesser extent, 5, 6-EET is also capable of evoking luteinizing hormone-releasing hormone (LHRH) release. The results suggest that these "epoxygenase" metabolites of AA may be physiologically involved in the control of SRIF release.     

Origin of insulin-receptive nerve terminals in rat median eminence

The origin of insulin-receptive axon terminals in the rat median eminence was determined by combining surgical and chemical ablation techniques and the in vivo radioautographic approach, in which labeling of the median eminence with blood-borne [125I]insulin served as a quantifiable marker for the presence of receptive axonal elements. Whereas unilateral deafferentation of the median eminence from the ipsilateral brain produced as much as a 50% ipsilateral loss of insulin-binding sites, transection of axonal projections to median eminence from neurons located lateral to the ventromedial hypothalamic nucleus produced no detectable loss in insulin-binding capacity. Unilateral electrocoagulation of various regions of the medial basal hypothalamus indicated that insulin-receptive axon terminals arise primarily from neurons in and about the hypothalamic arcuate nucleus and from the posterior ventrolateral subdivision of the hypothalamic ventromedial nucleus. A primary site of origin from the arcuate nucleus was confirmed in rats treated neonatally with monosodium L-glutamate, which, in addition to a selective destruction of arcuate neurons, produced a profound reduction in the insulin-specific binding capacity of the median eminence. The results of this study indicate that insulin-binding axon terminals arise from a unique class of tuberoinfundibular neuron with hormone-receptive capacity. These neurons may function to mediate direct interaction of circulating insulin with central autonomical, behavioral, and neuroendocrine systems.     

Neurons in the subependymal layer of the rat median eminence.

A considerable number of small and medium-size neurons are described in the subependymal layer of the caudal part of the middle portion of the rat median eminence. They are arranged mainly in rostro-caudally oriented rows. Fine structure of these nerve cells is similar to that of the arcuate neurons. Small axon terminals establish synaptic contacts with these cells. The possible functional significance of these neurons is discussed.     

Direct inhibitory effect of long term estradiol treatment on dopamine synthesis in tuberoinfundibular dopaminergic neurons: in vitro studies using hypothalamic slices

The mechanism of the inhibitory effect of long term treatment with estradiol on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons was studied by using hypothalamic slices from ovariectomized rats. Treatment with 2 mg estradiol valerate (EV) at a 3-week interval increased the weight of the anterior pituitary gland and the concentration of serum PRL. In vivo and in vitro dopamine synthesis in TIDA neurons were estimated in EV-treated animals by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after injections of 3-hydroxybenzylhydrazine (NSD 1015), a DOPA decarboxylase inhibitor, and after incubation of hypothalamic slices with NSD 1015, respectively. In vivo DOPA accumulation in the median eminence was less in EV-treated rats than in control rats. The basal rate of in vitro DOPA accumulation in the median eminence of hypothalamic slices from EV-treated rats was lower than that in control rats. Ca2+-dependent DOPA accumulation in the median eminence, determined by incubation in medium containing depolarization agents such as 50 mM K+ and veratridine, was decreased in EV-treated rats. Furthermore, cAMP-dependent DOPA accumulation, determined by incubation with Bu2cAMP or forskolin, was also suppressed in EV-treated rats. The decreased depolarization-induced DOPA accumulation in the median eminence recovered after cessation of EV treatment. Hyperprolactinemia lasting for 6 weeks, achieved by transplantation of anterior pituitaries under the kidney capsule, increased the rate of depolarization-induced DOPA accumulation in the median eminence. On the other hand, EV treatment was effective in inhibiting depolarization-induced DOPA accumulation in hypophysectomized rats regardless of the presence of anterior pituitary transplants. These results suggest that chronically administered estradiol inhibits dopamine synthesis in TIDA neurons via a direct action on the hypothalamus and overcomes the facilitatory action of PRL on dopamine synthesis; and estradiol inhibits all three distinct systems that regulate basal, Ca2+-dependent, and cAMP-dependent dopamine synthesis in TIDA neurons.     

Autoradiographic identification of prolactin binding sites in rat median eminence

Binding sites for prolactin were localized and quantified in the female rat brain by in vitro autoradiographic analysis with iodine-125-labeled ovine prolactin. Following incubation, labeled prolactin bound preferentially to the median eminence and choroid plexus of the lateral and third ventricles. The addition of excess unlabeled ovine prolactin blocked binding of the labeled hormone in the choroid plexus, and attenuated prolactin binding in the median eminence. These results provide evidence for median eminence. These results provide evidence for prolactin-specific recognition sites in the median eminence, a region intimately involved in the hypothalamic regulation of prolactin secretion.