Enzyme immunoassays for thyroliberin (TRH) and TRH-elongated peptides in mouse and rat hypothalamus

Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.     

Immunological evidence for thyroliberin (TRH) neurons in primary cultures of fetal mouse brain cells. Ontogenic aspects

Primary cultures of dissociated cells were initiated from fetal mouse hypothalami and brain hemispheres, on the 13th and the 16th day of gestation (respectively 12-day and 15-day-old fetuses). After 10 days in vitro, the cultured cells were collected, pooled in an appropriate medium and thyroliberin (TRH) was assayed in the cell extracts using a specific radioimmunoassay. TRH was found in every type of culture. For hypothalami, higher levels of TRH were found when starting from older embryos, while in brain hemisphere cultures the TRH content increased in culture of 12-day-old fetal cells only, whereas it decreased in cultures of 16-day-old fetal cells. Immunocytochemical staining allows visualization of TRH positive cells in all cultures except in hypothalamic cultures from 12-day-old embryos. This is consistent with the radioimmunoassay data. TRH was localized exclusively in some of the overlying cells, whereas the basal cells were always negative. Specificity of the staining was assessed by immunochemistry and radioimmunoassay. At the electron microscope level, the positive cells display neuronal features. The immunoprecipitate was found in both perikaryon and axons as well as in axonal dilatations.     

Release of immunoreactive TRH in serum-free cultures of mouse hypothalamic cells

Serum-free cultures of mouse hypothalamic cells were used as a model for studying TRH (thyroliberin) secretion in vitro. Supplementation of the culture medium with triiodothyronine, corticosterone and polyunsaturated fatty acids is necessary to obtain a substantial release capacity of TRH neurons. Under these conditions depolarization of the cells with 60 mM K+ results in a calcium-dependent release of immunoreactive TRH.     

Biochemical neuroendocrinology. I. Biosynthesis of thyrotropin releasing hormone (TRH) by organ cultures of mammalian hypothalamus

Organotypic cultures of the hypophysiotropic area of guinea pig hypothalamu were maintainedin vitro for periods up to 1 week. These cultures showed a high degree of metabolic integrity, and were able to incorporate [³H]proline into TRH. The identification of labeled TRH was achieved by subjecting aqueous methanol extracts of the cultured tissue to an extensive purification procedure.     

Involvement of DHP voltage-sensitive calcium channels and protein kinase C in thyroliberin (TRH) release by developing hypothalamic neurons in culture

The intracellular mechanisms regulating the process of thyroliberin (TRH) release were studied using fetal hypothalamic neurons grown in serum-free medium. In particular, we compared the effects of dihydropyridine (DHP) derivatives, omega-conotoxin and phorbol esters on basal and K+-evoked TRH release from 12 days in vitro (DIV) neurons. BAY K 8644, a DHP calcium channel agonist increased in a dose-related manner basal and K+-evoked TRH release. PN 200-110, an antagonist of DHP-sensitive calcium channels, completely suppressed the effect of BAY K 8644, whatever the extracellular K+ concentration, but did not modify basal or K+-evoked TRH release. In contrast, omega-conotoxin partially inhibited the two latter processes. The active phorbol ester 12-O-tetradecanoyl-phorbol-beta-acetate (TPA), and to a lesser extent Sn-1,2-dioctanoylglycerol (DAG), triggered TRH release. This effect was specific, time and dose dependent and only partly dependent on extracellular calcium. Simultaneous addition of BAY K 8644 and TPA to the cells displayed a synergistic effect. The same compounds were studied on younger neurons (6-DIV cultures): BAY K 8644 stimulated TRH release whereas neither 60 mM K+ nor TPA did. These results suggest that TRH release can be mediated at least by two intracellular routes: (i) increase of intracellular calcium mediated by the opening of different types of voltage sensitive calcium channels, and (ii) activation of protein kinase C (PKC). The asynchrony in the maturation of the intracellular mechanisms underlying TRH release may be explained by different subcellular localizations of these mechanisms in neurons and is discussed in relation to synapse differentiation.     

Cell types and cell-substrate interactions in serum-free dissociated cultures of rat hypothalamus

We have developed a system for the long-term cultivation of hypothalamic neurons at low density in defined serum-free medium, and have identified the major classes of cells in these cultures. Cells prepared from the dissociation of embryonic rat hypothalami are plated, in serum-free medium, onto monolayers of cortical astrocytes. Neurons adhere and begin to extend neurites soon after plating, and survive for up to several weeks in culture. Cell-type-specific immunological markers were utilized to identify neurons and the major classes of glial cells in these cultures. The culture of hypothalamic neurons in serum-free conditions provides a valuable system for the study of the cellular basis of hypothalamic neuronal heterogeneity and functional diversity.     

Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium

Grown in serum-free medium, dissociated cells from fetal mouse hypothalami release insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) into the culture medium. Addition of triiodothyronine (10-12-10-8 M), which enhances neuron maturation, resulted in a significant increase in IGF concentration. By contrast, there was no significant effect on IGF BP. These results suggest a role for thyroid hormone in the control of IGF biosynthesis in nerve cells.     

Subcellular distribution of thyrotropin-releasing hormone (TRH) in rat brain and hypothalamus

TRH localization in rat brain and hypothalamic tissue was studied by determining of immunoassayable TRH in subcellular fractions prepared from homogenates of these tissues. In both hypothalamus and brain, most of the TRH was found in a crude mitochondrial fraction and its concentration was higher in the synaptosomal fraction isolated by density gradient centrifugation. Electron microscopy was used to verify the composition of the fractions. Exposure of the brain crude mitochondrial fraction to osmotic shock resulted in solubilization of TRH which then was sedimented by centrifugation at 96,000 × g. TRH added to brain homogenates in vitro was rapidly destroyed by incubation at 37°C, but no endogenous TRH was lost by such incubation. The localization of TRH of hypothalamic and brain tissue to subcellular fractions consisting largely of synaptosomes and synaptic vesicles is compatible with a neurotransmitter function for this substance.     

Distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus

In the present study, we describe for the first time the distribution of thyrotropin-releasing hormone (TRH)-containing cells and fibers in the human hypothalamus using brain material obtained with a short postmortem delay. Following fixation in paraformaldehyde, glutaraldehyde and picric acid, excellent staining was obtained with two different TRH antisera. Many TRH-containing neurons were present in the paraventricular nucleus (PVN), especially in the dorsocaudal part of this nucleus. They were mostly parvicellular, but a few magnocellular TRH-positive neurons were observed as well. The PVN also contained a dense network of TRH fibers. The supraoptic nucleus (SON) did not show any TRH immunoreactivity, excluding the possibility of cross-reactivity of the antiserum with neurohypophysial hormones or their precursors. In addition, TRH cells were found in the suprachiasmatic nucleus (SCN), which is the circadian clock of the brain, in the sexually dimorphic nucleus (SDN) and dorsomedially of the SON. We observed small numbers of TRH cells throughout the hypothalamic gray in all subjects studied. A high density of TRH-containing fibers was seen not only in the median eminence but also in other hypothalamic areas, e.g., in the ventromedial nucleus (VM) and in the perifornical area. The results generally agree with earlier data in the rat, with the exception of the absence of TRH cells in the SON. The large number of sites of TRH-containing fiber terminations on neurons suggests important physiological functions of this neuropeptide as a neurotransmitter or neuromodulator in the human brain in addition to its role as a neurohormone in pituitary secretion of thyroid-stimulating hormone (TSH).     

Functional maturation of somatostatin neurons and somatostatin receptors during development of mouse hypothalamus in vivo and in vitro

Ontogenesis of somatostatin (SRIF) neurons and receptors was studied in fetal hypothalamic cell cultures kept in serum-free medium, and compared to the in vivo developmental pattern. Initial rise in neuronal content of SRIF occurred later in vitro than in vivo. In vitro, K(+)-induced SRIF release was only present after synaptogenesis. SRIF binding sites were measurable as early as 1 day after birth and at an equivalent time in culture, after 6 days in vitro (DIV); their affinity was in the nanomolar range. In cultured cells, binding reached a maximum at two weeks in vitro and decreased sharply thereafter as a consequence of binding site occupancy by the endogenous ligand. Indeed, pretreatment with cysteamine decreased SRIF concentration in the neuronal cultures and twice as many binding sites as in control cultures of 21 DIV were measured. Competition kinetics using unlabelled SMS 201-995 to displace [125I]SRIF revealed two distinct binding sites in the neuronal preparations (IC50 = 11 +/- 3 pM and 4.5 +/- 0.8 nM). In contrast, only the lower affinity site was present on glial cell preparations (1.7 +/- 0.4 nM). SRIF inhibited adenylate cyclase activity in glia and neurons, and the onset of SRIF coupling to the second messenger occurred earlier in vitro than in vivo. Pertussis toxin pretreatment was equally effective in neuronal and glial cell preparations to decrease SRIF binding and to inhibit adenylate cyclase activity.     

Medullary synaptic inputs to Thyrotropin-Releasing Hormone (TRH)-containing neurons in the hypothalamus: an ultrastructural study combining WGA-HRP anterograde tracing with TRH immunocytochemistry

Ascending projections from the A1/C1 cell group and from the A2 cell group in the medulla oblongata was studied in the light microscope by anterograde tracing ofPhaseolus vulgaris leucoagglutinin and in the electron microscope by anterograde tracing of wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) combined with thyrotropin-releasing hormone (TRH) immunocytochemistry in the hypothalamic paraventricular nucleus (PVN). WGA-HRP-labeled axon terminals originating from neurons in the A1/C1 or the A2 cell group were found to make synaptic contacts with TRH-containing cell bodies and dendrites in the medial parvocellular part of the PVN, usually forming axo-dendritic synapses. Of all the afferent synapses on TRH neurons in the PVN, 9.8–20.9% of the presynaptic axon terminals were WGA-HRP-positive. This indicates that each brain stem catecholaminergic cell group that contribute to innervation of the PVN is in a position to modulate the activity of TRH neurons.     

Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.     

Circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) contents in hypothalamic and extra hypothalamic brain nuclei of adult male rats

Using both the ‘punch’ microdissection and radioimmunological techniques, circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) concentrations can be statistically validated in some discrete brain regions of the (Wistar CFY) male rat investigated in May. Animals were synchronized with light from 06.00 to 18.00 h and darkness. Water and food were available ad libitum. Very well marked circadian rhythms were in evidence in brain areas where the neuronal peptides investigated are mostly present in nerve terminals in high concentrations: medial basal hypothalamus for luliberin and thyroliberin and substantia nigra for substance P. On the contrary, no significant rhythms were detected in a number of areas where luliberin (preoptic area) or substance P (preoptic area, central gray matter, amygdala, globus pallidus) synthetizing perikarya are present. This suggests that at least luliberin and substance P are not secreted according to a circadian rhythmicity.The most striking finding was that crest time locations were situated at only two different times of the day: 14.00 h for thyroliberin and 20.00 h for substance P and luliberin. This suggests that circadian variations are not solely dependent on the activity of the endocrine system.     

Effects of sex steroids on beta-endorphin release from rat hypothalamus in vitro.

The effects of sex steroids on immunoreactive beta-endorphin (EP) release from the rat hypothalamus in vitro were examined using a rat hypothalamic perifusion system and an EP RIA. Testosterone (1-100 ng/ml) and estradiol (10-100 pg/ml) stimulated EP release in a dose-dependent manner. Dehydroepiandrosterone (DHEA, 1-100 ng/ml) dose-dependently inhibited EP release. These results indicate an inverse relationship between the acute effect of gonadal sex steroids and that of adrenal androgen on hypothalamic EP release in vitro.     

In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase

Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimer's disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases.