Fetal Rat Hypothalamus Research Articles

Microglia Play a Role in Ethanol‐Induced Oxidative Stress and Apoptosis in Developing Hypothalamic Neurons

Animals exposed to alcohol during the developmental period develop many physiological and behavioral problems because of neuronal loss in various brain areas including the hypothalamus. Because alcohol exposure is known to induce oxidative stress in developing neurons, we tested whether hypothalamic cells from the fetal brain exposed to ethanol (EtOH) may alter the cell-cell communication between neurons and microglia, thereby leading to increased oxidative stress and the activation of apoptotic processes in the neuronal population in the hypothalamus. Using enriched neuronal and microglial cells from fetal rat hypothalami, we measured cellular levels of various oxidants (O2 -, reactive oxygen species, nitrite), antioxidants (glutathione [GSH]), antioxidative enzymes (glutathione peroxidase [GSH-Px], catalase, superoxide dismutase) and apoptotic death in neurons in the presence and absence of EtOH or EtOH-treated microglial culture medium. Additionally, we tested the effectiveness of antioxidative agents in preventing EtOH or EtOH-treated microglial conditioned medium actions on oxidative stress and apoptosis in neuronal cell cultures. Neuronal cell cultures showed increased oxidative stress, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidant and antioxidative enzymes, as well as, increased apoptotic death following treatment with EtOH. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Antioxidative agents protected developing hypothalamic neurons from oxidative stress and cellular apoptosis which is caused by EtOH or EtOH-treated microglial culture medium. These data suggest that exposure of developing hypothalamic neurons to EtOH increases cellular apoptosis via the effects on oxidative stress of neurons directly and via increasing production of microglial-derived factor(s).

T3 differentially regulates TRH expression in developing hypothalamic neurons in vitro

Triiodothyronine (T3) plays an important role during development of the central nervous system. T3 effects on gene expression are determined in part by the type of thyroid hormone receptors (TRs) expressed in a given cell type. Previous studies have demonstrated that thyrotropin releasing hormone (TRH) transcription in the adult hypothalamus is subjected to negative regulation by thyroid hormones. However, the role of T3 on the development of TRH expression is unknown. In this study we used primary cultures derived from 17-day-old fetal rat hypothalamus to analyze the effects of T3 on TRH gene expression during development. T3 increased TRH mRNA expression in immature cultures, but decreased it in mature cultures. In addition, T3 up-regulated TRα1 and TRβ2 mRNA expression. TRα1 expression coincided chronologically with that of TRH in the rat hypothalamus in vivo. Maturation of TRH expression in the hypothalamus may involve T3 acting through TRα1.

Factors accounting for perinatal occurrence of pulsatile gonadotropin-releasing hormone secretion in vitro in rats.

Our aim was to study the inhibitory and facilitatory factors possibly accounting for the undetectable activity of the GnRH pulse generator in late fetal life in vitro and its awakening in early postnatal life. Gamma aminobutyric acid (GABA(A)) receptor antagonism using SR 95 531 did not cause any secretory pulse in fetal explants, whereas a significant stimulation of GnRH pulse frequency was obtained at 5 and 15 days. GnRH secretory response to repeated N-methyl-D-aspartate (NMDA) stimulation showed progressive disappearance, indicating that the inhibitory autofeedback was operating. GnRH release caused by glutamine was respectively 9% and 20% of that evoked by glutamate in fetal and 5-day-old rats whereas both amino acids were equally active at 15 days. Explants obtained after cesarean section performed at onset of labor did not show any secretory pulse, while pulses could be observed with explants obtained 2 h after vaginal delivery. Incubation of fetal explants with oxytocin (10(-8) M) or prostaglandin E2 (PGE2) (10(-6) M) resulted in occurrence of GnRH secretory pulses. A facilitatory effect of the oxytocin was shown to persist on Days 1, 5, and 15 and inhibitory effects of an oxytocin receptor antagonist provided some evidence of endogenous oxytocin involvement. We conclude that, in the fetal rat hypothalamus, GnRH inhibitory autofeedback and GABAergic inputs do not account for the absence of pulsatile GnRH secretion in vitro. A low rate of glutamate biosynthesis from glutamine is a possibly limiting factor. Oxytocin and PGE2 can play a facilitatory role in the postpartal occurrence of pulsatile GnRH secretion.

Subcellular distribution of histamine, GABA and galanin in tuberomamillary neurons in vitro

Histamine acts as a neurotransmitter in the brain and regulates e.g. sleep, hibernation, vigilance, and release of several other transmitters. All histaminergic neurons are found in the tuberomamillary nucleus (TM), and send axons to almost all parts of the CNS. Despite the obvious importance of these neurons, their development, transmitter storage, and compartmentalization of cotransmitters are poorly known. Histaminergic neurons from fetal rat hypothalamus were studied in primary explant cultures and analyzed by confocal microscopy. Most histaminergic neurons were oval in shape, but round and triangular ones were also found. The average size of the 212 analyzed neurons was 19.2 μm (length), 12.5 μm (width) and 11.7 μm (thickness). The cells possessed two to five microtubule-associated protein (MAP2) positive processes, putative dendrites, and in general one MAP2-negative thin process, a putative axon. Granular histamine-immunoreactivity was found in the cell bodies, axons, and dendrites. In tuberomamillary neurons, most histamine-containing structures displayed immunoreactivity for vesicular monoamine transporter 2 (VMAT2), indicating that the two markers may coexist in the same structures. Lack of VMAT2 in some histamine-immunoreactive structures indicates that another transporter for histamine may exist. In the same neurons, γ-aminobutyric acid (GABA)-immunoreactivity was found in structures, distinct from those containing histamine, indicating that the two transmitters may be differentially localized, regulated and released. Galanin-immunoreactivity in the cultured tuberomamillary neurons was partially located in the same structures as VMAT2. The results suggest that histamine and GABA, the two principal transmitters of tuberomamillary neurons, are not costored in the same structures in tuberomamillary neurons.

Immunohistochemical analyses of thyroid-specific enhancer-binding protein in the fetal and adult rat hypothalami and pituitary glands

Thyroid-specific enhancer-binding protein (T/EBP), also known as NKX2.1 or TTF-1, regulates the expression of thyroid- and lung-specific genes. The t/ebp/Nkx2.1-null mutant mouse was stillborn but lacked the thyroid gland, pituitary gland, ventral region of the forebrain and normal lungs. These data demonstrated that T/EBP/NKX2.1 plays an important role not only in tissue-specific gene expressions in adults but also in genesis of these organs during development. Although the expression of t/ebp/Nkx2.1 in the brain has been reported, its function in the brain remains unknown. The present study was designed to determine the localization of T/EBP/NKX2.1 in the hypothalamus and pituitary gland of fetal and adult rats by immunohistochemistry as the first step toward understanding the function of T/EBP/NKX2.1 in the rat brain. In the fetal rat hypothalamus, T/EBP/NKX2.1 was localized widely in the ventral hypothalamic areas. In the adult rat brain, T/EBP/NKX2.1 was localized in the ventromedial hypothalamic nucleus, medial tuberal nucleus, arcuate nucleus and mammillary body. No T/EBP/NKX2.1 immunoreactivity was observed in the anterior or intermediate lobe of the pituitary gland in either fetal or adult rats. On the other hand, immunoreactive T/EBP/NKX2.1 was found in the posterior lobe of the pituitary gland. This paper presents results of detailed analyses of the distributions of T/EBP/NKX2.1 protein in the fetal and adult rat hypothalami and pituitary glands, and these results should provide important information for understanding the function of T/EBP/NKX2.1 in the brain.

BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.

Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.

Estrogen inhibits hypothalamic pro-opiomelanocortin gene expression in hypothalamic neuronal cultures.

Many in vitro studies show estrogen regulation of the hypothalamic pro-opiomelanocortin (POMC) system, including a decrease in hypothalamic POMC mRNA after estradiol treatment. Because such in vivo experiments do not allow one to determine whether peripheral, interacting systems or extra-hypothalamic brain regions are involved in this regulation, we sought to establish whether estrogen acts directly in hypothalamus to decrease POMC mRNA. Using an in vitro approach, we studied effects of estradiol (E2) on POMC/cyclophilin mRNA concentrations (RNAse protection assays) in neuronal cultures derived from day 17 fetal rat hypothalamus. Chemically defined medium was deprived of progesterone for 2 days prior to E2 treatment and for the duration of the study. E2 (10(-13)-10(-9) M) dose-dependently decreased POMC mRNA concentrations during a 2-day treatment. Whereas the lowest dose (10(-13) M) of E2 resulted in a statistically significant 44% decrease in POMC mRNA concentrations relative to control cultures, this inhibitory effect was lost because higher doses (10(-11) and 10(-9) M) did not produce statistically significant decrements (22 and 16%, respectively) in POMC mRNA concentrations. Additional time course studies revealed that this decrease in POMC mRNA can be seen as early as 4 h after E2 (10(-13) M) treatment. We conclude that E2 inhibition of POMC mRNA concentrations in hypothalamic neuronal cultures indicates that this inhibition can occur directly in hypothalamus.

Interaction of insulin-like growth factor-I and estradiol signaling pathways on hypothalamic neuronal differentiation

Neurotrophic effects of estradiol and insulin-like growth factor-I were assessed in primary cultures from fetal rat hypothalamus. Cultured neurons were immunostained with an antibody for the microtubule-associated protein-2. While both estradiol and insulin-like growth factor-I increased the number of microtubule-associated protein-2- immunoreactive neurons and the extension of immunoreactive processes, the effect of these two factors was not additive. The estradiol-induced increases in neuronal numbers and extension of neuronal processes were blocked by either the estrogen receptor antagonist ICI 182,780 or by an anti-sense oligonucleotide to the estrogen receptor. Furthermore, incubation of the cultures with an anti-sense oligonucleotide directed against the insulin-like growth factor-I messenger RNA also blocked the effect of estradiol. In turn, the effects of insulin-like growth factor-I were blocked by the estrogen receptor antagonist ICI 182,780 and by the anti-sense oligonucleotide to the estrogen receptor. These findings suggest that estradiol-induced activation of the estrogen receptor in developing hypothalamic cells requires the presence of insulin-like growth factor-I, and that both estradiol and insulin-like growth factor-I use the estrogen receptor as a mediator of their trophic effects on hypothalamic neurons.

A new organotypic culture method to study the actions of steroid hormones on the nervous system.

A new organotypic culture method for growing slices of nervous system tissue, based on the use of hyaluronic acid as a growth supporting milieu, is described. This method allows cultures derived from either fetuses or newborns to grow and develop with markedly reduced amounts of added serum. Organotypic cultures from fetal rat hypothalamus were exposed to 17 beta estradiol and compared to control cultures exposed to the ethanol vehicle. When exposed to estradiol, cultures showed an outgrowth of thick nerve fibers that was accompanied by an elevation in the number of microtubules present in the neuronal processes, an increment in the number of synapses, and an increased morphological differentiation of synaptic terminals. Freeze-fracture analysis of neuronal membranes from estradiol-treated cultures revealed a significant increase in the number of exoendocytotic images and a decrease in the number of intramembranous particles. Estradiol's effects parallel those found in in vivo studies, indicating that hyaluronic acid-based organotypic cultures represent an appropriate model to study hormonal influences on the developing nervous system.

Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus.

Recent evidence indicates that, in addition to their well known effects on neurons, gonadal steroids may exert part of their neural effects through astroglia. In adult female rats astroglia participate in the phasic remodelling of synapses that takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the influence of estradiol. Astroglia also appear to be involved in the genesis of sex differences in synaptic connectivity. Gonadal steroids influence hypothalamic astroglia differentiation in vitro and in vivo. In monolayer mixed neuronal-glial cultures from fetal rat hypothalami, estradiol induces a progressive differentiation of astrocytes from a flattened epithelioid morphology to bipolar, radial and stellate shapes. This effect of estradiol on astroglia is dependent on the expression of specific molecules on the neuronal surface, such as the polysialic acid-rich form of the neural cell adhesion molecule. In the rat arcuate nucleus in situ, perinatal androgen influences astroglia gene expression and differentiation, resulting in a sex difference in astroglia organization by postnatal day 20. By this day, the amount of neuronal surface covered by astroglial processes is higher in males than in females. This difference in the coverage of neuronal surface by astroglia may be directly related to the reduced number of synaptic contacts that is established on the soma of male neurons compared to females.

Role of glutamate in regulating hypothalamic proglucagon-derived peptide secretion in vitro

We have previously demonstrated expression of the proglucagon gene and synthesis and secretion of the proglucagon-derived peptides (PGDP s) in the fetal rat hypothalamus. The excitatory amino acid glutamate has been found to be a key regulator of hypothalamic neuroendocrine hormone secretion. Therefore, the effects of glutamate on hypothalamic PGDP secretion and synthesis were examined in the present study, using the hypothalamic culture model. Glutamate (10 μM) significantly stimulated PGDP secretion (P < 0.01), but had no effect on the total PDGP content of the cultures over 24 hr of incubation. Similarly, the metabotropic/ionotropic glutamate receptor agonist, quisqualic acid (10 μM) stimulated PGDP secretion only (P < 0.05). In contrast, the ionotropic glutamate receptor agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 μM) and antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; 10 μM), had no effects on either secretion or content of the hypothalamic PGDPs. These findings suggest that excitatory amino acids, and glutamate in particular, regulate secretion but not synthesis of the PGDPs in the hypothalamus.

Estradiol promotion of changes in the morphology of astroglia growing in culture depends on the expression of polysialic acid of neural membranes.

Gonadal steroids are known to affect astroglial morphology in developing and adult animals. Earlier studies of mixed neuronal-glial cultures from fetal rat hypothalamus showed that glial fibrillary acidic protein (GFAP)-immunoreactive cells with a polygonal shape were transformed into process-bearing cells upon exposure to the ovarian hormone estradiol. This effect was dependent on a direct contact of astroglia with living hypothalamic neurons. The present study shows that somata and processes of neurons in such cultures were immunoreactive for polysialic acid (PSA); astroglia were immunonegative. PSA appears to participate in the estradiol-induced shape changes since treatment with endoneuraminidase, an enzyme that specifically removes PSA from the cell surface, abolished PSA immunostaining and prevented the 17 beta-estradiol-induced morphological changes of astroglia. In contrast, treatment with endoneuraminidase did not affect astroglial shape changes induced by basic fibroblast growth factor (bFGF), nor those induced by the addition of neurons to glial cultures. These results suggest that PSA on neuronal membranes, probably linked to the highly sialylated isoform of the neural cell adhesion molecule, is necessary for the expression of certain hormonally-regulated neuro-glial interactions.

Estradiol Modulates Insulin‐Like Growth Factor I Receptors and Binding Proteins in Neurons from the Hypothalamus

Trophic effects of 17 beta-estradiol (beta E2) on in vitro developing hypothalamic cells have been reported. Insulin-like growth factor I (IGF-I) is also a potent trophic factor for cultured hypothalamic cells. An interaction between sexual steroids and insulin-like growth factors (IGFs) in modulating growth of hypothalamic cells has been suggested. Thus, we tested whether beta E2 modulates the levels of IGF-I, its membrane receptor and its binding proteins in rat hypothalamic cultures. Using both neuron- and glial-enriched cultures obtained from fetal rat hypothalami we found that addition of beta E2 elicited a significant increase in IGF-I receptor levels in neurons, without affecting its affinity. On the other hand, the three different IGF-binding proteins (IGFBPs) found in the conditioned medium of the cultures were differentially modulated by beta E2 in the two types of cells studied. Overall, neuronal cultures produced greater amounts of IGFBPs after treatment with beta E2, with IGFBP2 reaching significantly higher levels. On the contrary, treatment with beta E2 did not significantly alter the amounts of IGFBPs produced by glial cells. Finally, the levels of immunoreactive IGF-I found either in the medium or in cellular extracts in both neuronal and glial cultures were not modified by treatment with beta E2. These results strongly support previous observations of a trophic synergistic interaction between IGFs and beta E2 on hypothalamic cells. Thus, an increase in IGF-I receptors and/or IGFBPs after exposure to beta E2 may result in an enhanced response of hypothalamic neurons to IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Morphological and functional development of the suprachiasmatic nucleus in transplanted fetal hypothalamus

The development of the suprachiasmatic nucleus (SCN) in fetal rat hypothalamus transplanted to the adult brain was studied using morphological and functional methods. Anterior hypothalamic tissue was transplanted into the third ventricle, lateral ventricle or subarachnoid space of intact, adult hosts from E17 fetuses. These transplants developed the cytoarchitectonic and immunohistochemical staining characteristics of SCN, clusters of parvocellular neurons expressing vasopressin- and vasoactive intestinal polypeptide-like immunoreactivity in adjacent cellular populations, irrespective of the exact location of the transplanted tissue in the host brain. The functional status of the transplants placed in the rostral third ventricle and the foramen of Monroe was analyzed and compared to host SCN using in vitro recording of neuronal firing rate and measurement of metabolism using the 2-deoxyglucose (2-DG) technique. During subjective day, neuronal firing rates and 2-DG uptake were high in discrete cell groups within the transplants which were subsequently demonstrated to exhibit the cytoarchitectonic and immunohistochemical characteristics of SCN. The firing rates and 2-DG uptake in these areas were lower during the subjective night. This pattern of activity closely resembles that of the intact SCN. In contrast, neither transplanted anterior hypothalamic area, lacking an identifiable SCN-like structure, nor posterior hypothalamic area showed day-night differences in firing rate or 2-DG uptake. These observations indicate that SCN transplanted into intact adult hosts exhibits morphological and functional differentiation nearly identical to the host and that the transplanted SCN maintains circadian function which is probably entrained to the host SCN.

Control of proglucagon-derived peptide synthesis and secretion in fetal rat hypothalamus.

The hypothalamus has the highest concentration of proglucagon-derived peptides (Pgdp's) in the brain, however, the control of the synthesis and secretion of these peptides is not understood. The goal of our studies was to examine in detail the regulation of synthesis and secretion of Pgdp's in the hypothalamus. Hypothalamic cultures were prepared from fetal rats on day 19-21 of gestation and Pgdp's in media and cells were determined by radioimmunoassay after treatment with test agents. Dibutyryl cyclic AMP or forskolin, activators of protein kinase A, markedly stimulated both Pgdp synthesis (by 5-fold) and secretion (by 10-fold) after 24 h of treatment (p < 0.05). The effects of protein kinase A stimulation on Pgdp's in the hypothalamus were greater than seen in our previous studies with the Pgdp-producing pancreatic A and intestinal L cells. Therefore there are tissue-specific differences with regard to the magnitude of the response of Pgdp's to protein kinase A stimulation. Consistent with an involvement of protein kinase A in hypothalamic Pgdp synthesis and secretion, somatostatin-14, an inhibitor of protein kinase A, was found to inhibit Pgdp synthesis and secretion in a dose-dependent fashion (p < 0.05). Phorbol myristate acetate (PMA), a stimulator of protein kinase C, did not significantly affect the synthesis or secretion of Pgdp's at 6 h, but significantly stimulated Pgdp secretion after 24 h (p < 0.05). The inactive phorbol ester, phorbol triacetate was without effect on Pgdp synthesis or secretion after 24 h of incubation (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of Ca 2+ channel antagonists (cadmium, ω-conotoxin GIVA, and nitrendipine) on the release of angiotensin II from fetal rat brain in vitro

We have shown previously that K + stimulation of dissociated cell cultures of fetal rat brain results in a graded release of angiotensin II (ANG II) that is dependent on the availability of extracellular Ca 2+. In this study, using dissociated cell cultures of fetal rat hypothalamus, thalamus, septum, and midbrain (HTSM), we further examined the role of calcium channels on ANG II release using specific channel blockers (cadmium, ω-conotoxin, and nitrendipine) and a calcium ionophore (A23187). Levels of ANG II release were quantitated by radioimmunoassay and HPLC. For control levels of ANG II release, cells were incubated in a stock buffer containing 89 mM choline chloride/58 mM KCl/2 mM CaCl 2. Pretreatment of the cells with either 100 μM Cd 2+ (to block N-, L-, and T-type calcium channels), 100 nM ω-conotoxin (to block N- and L-type calcium channels), or 500 nM nitrendipine (to block L-type calcium channels) decreased ANG II release by approximately 71%, 71%, and 22%, respectively, when compared to control levels. In contrast, pretreatment of the cells with 1.6 μM A23187 (a calcium ionophore) increased ANG II release by approximately 90% over control levels. These findings suggest that angiotensin release is dependent on the intracellular entry of Ca 2+ ions through primarily N-type channels, and to a lesser extent, L-type channels.

Gap junctional intercellular communication between cultured ependymal cells, revealed by lucifer yellow CH transfer and freeze‐fracture

In order to analyze intercellular communication between ependymal cells in mammalian brain, we have studied gap junctional communication of ependymal and glial cells in long term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus obtained in serum supplemented media with two complementary methods: 1) dye transfer of Lucifer Yellow CH after intracellular microinjection of the different cellular types, and 2) freeze-fracture of the same cultured ependymal cells. In our culture conditions, we have shown that the GJIC capacity to transfer dye was very different according to cellular types microinjected with Lucifer Yellow CH in the following respects: 1) in ependymal cells, GJIC was always important: ciliated ependymal cells, which are numerous in hypothalamic ependymal cultures (10-120 coupled cells), choroidal ependymocytes in plexus cultures (15-250 coupled cells), and non-choroidal ependymocytes in diencephalic roof cultures (10-30 coupled cells), and 2) in astroglial cells found in these primary cultures, no GJIC was observed in spite of the presence of well-differentiated gap junctions revealed by freeze-fracture replicas. All these results show a strong GJIC in ependymal cells and indicate the very good functional state of these cells in vitro.

Cultured rat neurons and astrocytes express immunologically related epitopes of the GABAA/benzodiazepine receptor

gamma-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mammalian central nervous system (CNS) and an understanding of the development of receptors for this compound is of considerable interest. In this study, 3 monoclonal antibodies directed against the GABAA/benzodiazepine (GABA/BDZ) receptor were utilized in immunocytochemical experiments to localize receptor sites on neurons in dissociated cultures from fetal rat hypothalamus. Immunoreactivity was restricted to distinct patches on the plasma membrane of neurons. Analogous patches were observed on the surface of type 1 astrocytes, but not on type 2 astrocytes or oligodendrocytes. In membrane extracts from whole brain these antibodies reacted with two proteins (50 and 55 kDa) whereas extracts of cultured astrocytes yielded a single protein of approximately 63 kDa. Thus the identity of the glial protein remains unclear, but this study provides evidence for the presence of shared epitopes of the GABA/BDZ-receptor on both neurons and glia, and suggests that the astrocytic receptors may be important to nervous system function. Moreover it indicates that in vitro GABA/BDZ-receptors may form aggregates which would account for the restricted patterns of GABA sensitivity reported in some electrophysiological studies.

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.