[Quantitative study by radioautography and electron microscopy of the utilization of DL-leucine-H3 by duck hypophyseal cells in organ culture]. / Etude quantitative par radioautographie au microscope electronique de l'utilisation de la DL-leucine-3H par les cellules de l'hypophyse du canard en culture organotypiqe.

The synthesis, intracellular transport, storing, and excretion of proteins by duck hypophyseal cells in organ culture were studied with tritiated DL-leucine and high resolution radioautography (pulse-labeling experiments). Quantitative study of the radioautographs allowed a determination of the relative proportions of cytoplasmic radioactivity located in each cellular compartment (ergastoplasm, Golgi apparatus, and protein granules) as well as the variations in these proportions as a function of time. The number of labeled protein granules as opposed to the total number of granules in the cell was also determined (RSg). These data were separately analyzed for the two types of cells present in the explants: prolactin cells and "MSH" cells. The synthetic process follows a course common to both cell types, each of which is distinguished by its particular modalities. The labeled proteins, synthesized within several minutes in the ergastoplasm, are concentrated in the Golgi zone within 30 min. They then migrate out of this area, the emptying of which is accomplished in about 4 hr. These proteins become equally distributed between the protein granules, on the one hand, and the cytoplasm ("sedentary" proteins), on the other. The RSg reaches its maximum when the Golgi zone is emptied, but this figure remains very low (3%). The RSg then decreases slowly (1% in 40 hr). It is concluded that hypophyseal cells are able to store protein in their granules and that their processes of synthesis and excretion are not continuous. The prolactin cells differ from the "MSH" cells in that they have a slower migration of newly synthesized proteins, and these proteins pass via the dilated ergastoplasmic cisterns in which they may possibly be stored.

Synthesis and renewal of proteins in duck anterior hypophysis in organ culture.

In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material.

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique.

The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHalpha and oLHbeta) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHalpha and for A-oLHbeta and, from castrated males, they displayed an increased affinity for A-oLHbeta. Another cell type which was blue after the AB-PAS method reacted with the A-oLHalpha only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHalpha, acquired an affinity for A-oLHbeta. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHbeta, and A-oLHalpha and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHbeta. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHbeta.

Antibodies against a lysosomal membrane antigen recognize a prelysosomal compartment involved in the endocytic pathway in cultured prolactin cells.

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526). They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 min) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).

Membrane effects of thyrotropin-releasing hormone and estrogen shown by intracellular recording from pituitary cells.

The effects of thyrotropin-releasing hormone and 17 beta-estradiol on the electrical membrane properties of a prolactin-secretin pituitary cell line (GH3/B6) were studied with intracellular microelectrode recordings. Of the cells tested, 50 percent were excitable and displayed calcium-dependent action potentials when depolarized. When injected directly on the membrane of an excitable cell, thyrotropin-releasing hormone and 17 beta-estradiol induced action potentials within 1 minute. The spiking activity was preceded by a progressive increase of the input resistance without any detectable change in the resting membrane polarization. The results reveal a rapid effect of both substances on the membrane of GH3/B6 cells. In the case of thyrotropin-releasing hormone, which has both a short-term effect on release of prolactin and a long-term effect on its synthesis, the induced electrical activity may be associated with the stimulation of prolactin production. The physiological implication of 17 beta-estradiol-induced, calcium-dependent spiking activity remains to be elucidated.

Ontogeny of thyrotropin-releasing hormone biosynthesis and release in hypothalamic neurons.

Thyrotropin-releasing hormone (TRH) is expressed at early postmitotic stages of hypothalamic neuron development, in the mouse and rat, as revealed by the presence of the mature peptide, of pro-TRH mRNAs, and of large precursor forms. This indicates a coordinate expression of several genes encoding, respectively, pro-TRH, its processing enzymes, and the cell machinery for intracellular transport, sorting, and release of TRH. During development, an acceleration of pro-TRH processing is revealed by an increased proportion of the mature peptide. This is correlated with changes in the respective distribution of pro-TRH and TRH along neurites and the ontogenesis of neurosecretory granules.

Properties of monoclonal antibodies to thyroliberin (TRH) induced by different immunogens: comparison with pituitary TRH receptor.

Thyroliberin E-H-P-NH2 (TRH) is a small neuropeptide pGlu-His-Pro-NH2 widely distributed in neural sites. The aim of this work was to obtain an antibody molecule with the nearest properties to that of TRH-receptor in GH3 cells. Different TRH-protein conjugates were prepared and utilized to induce monoclonal antibodies in mice. Several monoclonal antibodies were obtained using E-H-P-NH2 (TRH) coupled either to the histidyl residue (immunogen I) or to the prolyl residue (immunogen II). Antibodies generated using immunogen I and immunogen II were characterized in a radioimmunoassay system and an enzyme immunoassay system respectively. Their selectivities regarding a series of TRH related peptides were compared to those of rabbit polyclonal antibodies using three differently labelled TRH (tritiated-TRH, mono-iodinated-TRH and TRH-OH-acetyl-cholinesterase) as tracers and to prolactin secreting cells TRH receptors using 3H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.

The effects of two drugs known to affect microtubules (nocodazole, a depolymerizing agent, and taxol, a polymerizing and stabilizing agent) have been tested in GH3B6 prolactin (PRL) cells, a rat pituitary cell line. Under basal condition, GH3B6 cells displayed a dense and entangled microtubule (MT) network, and a tight perinuclear cage of cytokeratin fibers with branching bundles in the cytoplasm. Nocodazole induced a disappearance of MT in the cytoplasm accompanied by the formation of tubulin blebs at the cell periphery, and a slackening of the perinuclear cage of cytokeratin. Taxol induced the formation of straight MT bundles in the cytoplasm, and a tightening of the cytokeratin cage. In parallel, nocodazole induced a fragmentation of the Golgi apparatus which appeared, after staining with antibodies against PRL or against mannosidase II, a Golgi membrane antigen, as small subunits dispersed in the cytoplasm. Taxol induced a perturbation of the Golgi apparatus which, however, remained located near the nucleus. Surprisingly, despite their obvious effects on the subcellular organization, the two MT drugs did not perturb the basal and thyroliberin (TRH)-stimulated PRL release. Moreover, they do not seem to affect the intracellular transport and release of neosynthesized PRL as appreciated by "pulse-chase" experiments. These observations demonstrate that, although MT assume an important role in the spatial compartmentalization of GH3B6 cells, they are not directly involved in the different steps of the intracellular PRL transport from its synthesis site to its release site, as well as in the associated membrane traffic.

Effects of thyrotropin-releasing hormone on prolactin compartments in clonal rat pituitary tumor cells.

PRL compartments were studied in a clonal strain of rat pituitary tumor cells (GH3B6). The cells were pulse-labeled for 10 min with 35S-methionine and then chased for 20 h in the absence or presence of TRH (30 nM) or cycloheximide (3.6 X 10(-5) M), or both. The specific radioactivity (SA) of PRL was followed in the cells and chase medium as a function of chase time and treatments. The transit of labeled and unlabeled PRL has been investigated in cells treated with monensin (1 microM), a drug which is known to perturb the Golgi zone. Newly synthesized PRL was rapidly (15 min of chase) and preferentially released in basal conditions. The pattern of the decay of the SA of PRL released in the medium suggested the existence of at least two PRL pools with different half-lives: 15 min and 3 h, respectively. TRH induced the preferential release of a PRL pool synthesized before the labeling pulse. Monensin decreased the basal release of total radioimmunoassayable PRL without affecting that of the newly synthesized PRL. In contrast, it did not affect the stimulating effect of TRH on the release of unlabeled PRL. These results are in favor of the existence of different intracellular routes for the basal release of PRL (mostly newly synthesized) and the TRH-stimulated release of PRL (mostly stored). Moreover, after 20 h of chase a large fraction (approximately 80%) of the labeled immunoprecipitated material remained intracellularly located and not degraded. This material was not mobilizable by TRH even in the presence of cycloheximide. Polyacrylamide gel electrophoresis analysis revealed that it consisted of large immunoreactive proteins (mol wt, 45,000 and 50,000) instead of mol wt 23,000 PRL which was found in the medium.

Effects of thyrotropin-releasing hormone on prolactin compartments in normal rat pituitary cells in primary culture.

PRL compartments have been studied in normal rat pituitary cells cultured for 6 days. The cells were pulse-labeled for 15 min with 35S-methionine and then chased for 24 h in the absence or presence of cycloheximide (3.6 X 10(-5) M). TRH (30 nM) was introduced into the medium either at the beginning or after increasing durations of chase. The findings were compared with those obtained with GH3B6 cells in similar experimental conditions. Despite the fact that normal PRL cells differ from GH3B6 cells by a large intracellular PRL store, several similarities were found between the two systems: newly synthesized PRL was rapidly and preferentially released in basal conditions, the pattern of the decay of the specific radioactivity of PRL released into the medium suggested the existence of at least two PRL pools with different half-lives: 2.5 h and 22 h, respectively, TRH induced the preferential release of stored PRL synthesized before the pulse, only 20% of the pulse-labeled PRL was released into the medium after 24 h of chase. However, normal PRL cells differed in several respects from GH3B6 cells: the turnover time of the two PRL pools is 8 times greater in normal PRL cells, an asynchrony in the time of appearance of labeled PRL in the medium was observed, suggesting a functional heterogeneity of these cells, at the end of the chase, 40% of the pulse-labeled PRL was lost in the case of normal cells, but not of GH3B6 cells, and this was prevented by cycloheximide, polyacrylamide gel electrophoresis analysis of this labeled immunoprecipitated intracellular material revealed the existence, in addition to the mol wt of 23,000 PRL and the large PRL-like forms (mol wt, 45,000 and 50,000), as observed with GH3B6 cells, of smaller proteins (mol wts, 39,000, 36,000, 20,000, 18,000, 15,000), which might represent degradation products.

Preferential role of calcium in the regulation of prolactin gene transcription by thyrotropin-releasing hormone in GH3 pituitary cells.

TRH induces two separate events in pituitary PRL cells. It increases the release of stored PRL and enhances the rate of PRL gene transcription, which results in an increased steady state concentration of PRL messenger RNA (mRNA) and a concomitant augmentation of PRL production. The mechanisms underlying the release process involve the activation of phosphatidylinositol turnover which generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. In order to determine whether these intracellular messengers also mediate the stimulation of PRL gene expression by TRH, we have correlated the level of receptor occupancy with the rate of gene transcription and investigated the action of drugs which increase cytosolic calcium or activate protein kinase C. We have determined that sustained stimulation of transcription requires the persistent occupancy of a limited number of TRH receptor sites and that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores (A23187, ionomycin), and the calcium channel agonist BAY K 8644 enhance PRL gene transcription. However, TPA is less potent and ionomycin requires a low concentration of TPA to fully mimic TRH action, whereas BAY K 8644 alone displays the same potency as TRH. The effects of BAY K 8644 and TRH are not additive and thus suggest that the influx of calcium plays a predominant role in the regulation of PRL gene transcription by TRH.

Evidence for high molecular weight immunoreactive thyrotropin-releasing hormone (TRH) precursor forms in the developing mouse hypothalamus. Simultaneous immunolocalization with TRH in cultured neurons.

Two antisera (Anti-P7 and Anti-P10) were raised against (-Gln-His-Pro-Gly-) elongated peptides: P7 Gln-His-Pro-Gly-Lys-Arg-Phe) and P10 (Ser-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Phe). They recognized TRH extended peptides but not TRH. A RIA against P7 and a highly sensitive enzyme immunoassay against P10 were used to identify two major high mol wt forms of 25-35 K and 6-8 K in chromatography fractions of adult and fetal mouse as well as adult rat hypothalami. The existence of the largest form was confirmed by immunoblotting with Anti-P7. During mouse hypothalamus development in vivo and in vitro, the ratio of TRH content vs. P10-associated immunoreactivity increased several times. This suggests that these Pro-TRH peptides are precursors of TRH biosynthesis and indicate an acceleration of TRH processing during development. Double immunostaining with A-TRH and A-P7 of hypothalamic cells taken on the 16th fetal day and cultured for 6, 12, and 18 days in vitro (DIV) revealed three populations of neurons: 1) a very minor population (approximately 2%) of small round cells positive with A-TRH only; 2) a major population of neurons positive with both A-TRH and A-P7. 3) multipolar neurons positive with A-P7 only (up to approximately 45% after 18 DIV). The respective distribution of TRH and P7 along neurites also varied with time in culture. Whatever perikarya staining, TRH was restricted to short neurites and growth cones before synapse formation and, during synapse development, to varicosities and terminal boutons. However even at the latest stage examined some varicosities and terminal boutons were positive with A-P7 only. These results suggest a preferential processing of pro-TRH at a post-Golgi step during axonal transport to growth cones and synaptic boutons.

Differential implication of deoxyribonucleic acid methylation in rat prolactin and rat growth hormone gene expressions: a comparison between rat pituitary cell strains.

In order to assess the potential role of DNA methylation in the expression of rat PRL (rPRL) as compared to rat GH (rGH) gene, the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI were examined in DNA isolated from rat pituitary cell lines producing either high levels of rPRL (GH3B6) or of rGH (GC) and in a stable variant cell strain which produces minute amounts of both hormones (GH3CDL cells). The rPRL and the rGH genes were found hypomethylated in GH3B6 and GC cells, respectively, whereas in GH3CDL cells both genes were methylated, indicating a correlation between the extent of gene methylation and the level of expression. However the use of 5-azacytidine (5-azaC), which decreases DNA methylation, suggested a variable importance of gene methylation in the control of rPRL and rGH gene expression. 5-AzaC was unable to increase rPRL production to a detectable level in GC cells, whereas the cytidine analog markedly increased rPRL production and rGH production in GH3CDL cells. Further analysis using GH3CDL cells showed that the extent of the 5-azaC-induced rPRL and rGH gene demethylation was consistent with the 5-azaC-induced increase of gene expressions. However, in these cells, the stimulation of rPRL and rGH production unexpectedly increased as a function of time elapsed after drug withdrawal. The maximal stimulation, 30-fold and 7-fold, respectively, was observed 3 weeks after a 60-h exposure to 5-azaC. This pattern suggests that other events are required for the full expression of rPRL and rGH genes in addition to their own demethylation.

Evidence of thyrotropin-releasing hormone (TRH) and TRH-binding sites in human nonsecreting pituitary adenomas.

Immunoreactive TRH (IR-TRH) and TRH-binding sites were sought in nonsecreting pituitary adenomas. [3H]TRH bound specifically to cellular membranes from 11 of 12 such adenomas studied, with a dissociation constant (Kd) of 50 +/- 5 (+/- SEM) nmol/L and a maximum number of binding sites of 76 +/- 16 fmol/mg membrane protein (range, 32-229 fmol/mg protein). IR-TRH was detected in all 8 of the tumors in which it was sought. The identity of the IR-TRH was verified by high pressure liquid chromatography. The tumor IR-TRH concentration varied from 45-248 fmol/mg cell protein (mean, 109 +/- 28 fmol/mg cell protein), about half that in normal human pituitary (229 +/- 55 fmol/mg protein). There was no correlation between the number of binding sites and the IR-TRH content. The role of TRH in nonsecreting pituitary adenomas is unknown at this time.

Effects of polyunsaturated fatty acids and hormones on synaptogenesis in serum-free medium cultures of mouse fetal hypothalamic cells.

The effects of soluble factors on synaptogenesis by mouse fetal hypothalamic cells cultured in chemically defined conditions have been examined using transmission electron microscopy. Hypothalami taken on the 16th day of gestation were mechanically dissociated and cells were seeded in a minimum serum-free medium supplemented or not with the following components: triiodothyronine, corticosterone and a mixture of polyunsaturated fatty acids (arachidonic acid plus docosahexaenoic acid bound to defatted bovine serum albumin). In the minimum serum free medium synapses were found after 10 days in culture. However, the development of synaptic vesicles was very limited, whereas that of the presynaptic and postsynaptic densities was apparently normal. Supplementation of the minimum serum-free medium with triiodothyronine, corticosterone and polyunsaturated fatty acids added simultaneously, permitted a full development of synapses as attested to by the increase in number and the regular shape and diameter of synaptic vesicles as well as by the complexity and diversity of synapse configurations. Among those three factors, polyunsaturated fatty acids clearly played a key role. The ability of synapses formed in culture to respond to potassium evoked depolarization was examined on cultures grown for 12 days in the simultaneous presence of the three above mentioned supplements. Exposure for 3 min to 60 mM potassium chloride induced in synaptic boutons vesicular depletion, apposition of vesicle clusters onto the presynaptic grid, appearance of a rich filamentous network and of some coated vesicles. Return to 3mM potassium chloride induced in 3 min a massive restoration of the population of vesicles which slightly differed from synaptic vesicles in control cultures. These results show that: (1) the formation of synaptic vesicles in this system is regulated by soluble factors among which polyunsaturated fatty acids play a major role, and (2) synapses formed de novo in chemically defined conditions of culture display the same ability to respond to and to recover from potassium evoked depolarization as adult axon terminals. Thus, they offer a suitable model for analysis of the mechanisms involved in membrane traffic in central neurons.

Immunoelectron microscopic localization of synaptophysin in a Golgi subcompartment of developing hypothalamic neurons.

Synaptophysin, previously identified as an integral membrane glycoprotein (mol. wt 38,000) characteristic of presynaptic vesicles of mature neurons, provides a molecular marker to study the origin, formation and traffic of synaptic vesicles. Using the monoclonal antibody SY38 against this polypeptide we have localized synaptophysin by immunofluorescence and electron microscope immunoperoxidase methods in cultured mouse hypothalamic neurons taken from 16-day-old fetuses which achieve synaptogenesis after 10-12 days in vitro. We have compared the localization of synaptophysin in perikarya and nerve endings as a function of age (2-19 days in vitro) and of treatment of mature neurons with nocodazole. Using immunofluorescence microscopy, synaptophysin was already detected in neuronal soma at 2 days in vitro, where the initiation of neurite development is observed. At the electron microscope level, virtually all mature synaptic boutons and varicosities showed an extensive synaptophysin labeling of synaptic vesicles at 12-13 days in culture whereas neurites showed only very few labeled vesicles. In neuronal soma taken before synapse formation (6 days in vitro), synaptophysin was selectively localized in membranes of the innermost cisternae of the Golgi zone and in vesicles of variable size and shape in the core of the Golgi zone. In contrast, after synapse formation, synaptophysin labeling was barely detected in the Golgi zone of neurons but a very strong labeling of synaptic vesicles in synaptic boutons was observed. Treatment of mature neurons (12 days in vitro) with nocodazole (10(-5) M) resulted in a conspicuous synaptophysin staining of the innermost trans-Golgi cisternae and numerous vesicles in the cytoplasm. Furthermore, an accumulation of labeled synaptic vesicles on the presynaptic membrane of nerve terminals was found. The data suggest that synaptophysin is released from the Golgi apparatus in a vesicular form, after glycosylation, and is then transported to nerve endings by a mechanism which requires integrity of microtubules.

Differential expression and subcellular localization of secretogranin II and synaptophysin during early development of mouse hypothalamic neurons in culture.

Mature neurons contain two distinct regulated secretory pathways, characterized electron microscopically by so-called large dense core vesicles and small synaptic vesicles, respectively. Each vesicle type is characterized by vesicle-specific proteins, such as the granins (chromogranins/secretogranins) for the matrix of large dense core vesicles and synaptophysin for the membrane of small synaptic vesicles. So far, no data exist on the biogenesis of these two distinct vesicle types during neuronal development. We have used secretogranin II and synaptophysin as markers for the biogenesis of these two vesicle types during the development of mouse hypothalamic neurons in culture, using immunocytochemistry and biochemical analyses. By immunofluorescence, we found that secretogranin II appears as early as synaptophysin, but in a subset of neurons only, and with different subcellular localizations. It was observed in cytoplasmic areas where little or no synaptophysin immunofluorescence was detected, such as lamellipodia, emerging neurites and growth cones. At later stages, the proportion of secretogranin II-containing varicosities remained steady whereas that of synaptophysin-containing varicosities increased dramatically. By quantitative analysis we found that the level of expression of synaptophysin increased several-fold during synaptogenesis whereas that of secretogranin II decreased. These data suggest that large dense core vesicles and small synaptic vesicles can be formed separately and expressed at different levels. They provide evidence for a differential biogenesis of these two distinct vesicle types.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies.

The development of the catecholaminergic system in the hypothalamus and in the septal region was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic structures were visualized with pre-embedding immunocytochemistry using antiserum to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to make the immunocytochemical technique more sensitive. In this paper only the data on the appearance and distribution of the tyrosine hydroxylase-immunopositive neurons (cell bodies) are presented, whereas the catecholaminergic innervation of the hypothalamus with the tyrosine hydroxylase-immunopositive fibers is the topic of an accompanying paper. Sparse tyrosine hydroxylase-immunopositive neurons were first observed in the anlage of the hypothalamus and septal region on the 13th fetal day. Their number increased progressively with age and by the 15th fetal day they already gave rise to a large dorsal accumulation. From the 18th fetal day on, tyrosine hydroxylase immunopositive neurons began to occupy their definitive positions, mainly concentrating within the hypothalamus: in the zona incerta, periventricular and arcuate nuclei. To a lesser extent, they were concentrated in the medial preoptic area, suprachiasmatic, supraoptic, paraventricular, dorsomedial, and anterior hypothalamic nuclei. The data on the distribution of the tyrosine hydroxylase-immunopositive neurons both in the hypothalamus and in the septal region during ontogenesis are summarized in the precise atlas. Primarily small bi- and unipolar catecholaminergic neurons first observed in the youngest fetuses undergo cytodifferentiation during ontogenesis, giving rise to at least two different populations localized ventrally, mainly in the arcuate nucleus, and dorsally, in the zona incerta. The neurons of the former population remain similar to those of the youngest fetuses, whereas the neurons of the latter increase significantly in size, forming several long, highly ramified processes.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.