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The relationship between the cell theory formulated by Schwann (1839) and by Virchow (1855) on the one hand, and, on the other hand, the neuron theory, as formulated by Waldeyer (1891) and by Cajal (1906), are discussed from a historical point of view. Both of them are the result of technical and conceptuel progress. Both of them had to fight against the dominant dogma before being accepted. The cell theory opposed the school of Bichat, the vitalist philosophy and the positivist philosophy of Auguste Comte. The neuron theory, which is clearly based on the cell theory, was mostly concerned with the mode of interneuronal communication; it opposed the concept of contiguity to Golgi's concept of continuity. At present, the cell theory remains central in every field of Biology. By contrast, the neuron theory, which until the middle of the XXth century opened the study of the nervous system to a necessary reductionnist approach, is no longer central to recent developments of neurosciences.

This talk, given as an introduction to a symposium organised to honor Andre Calas, calls forth his personality, recalls the major events in his career and summarizes the evolution of his research.

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.

Compte tenu du role primordial du recepteur de la GnRH dans le fonctionnement de l’axe de reproduction, la connaissance des assemblages moleculaires qui president a l’expression histospecifique et a la regulation de ce gene doit permettre, a terme, de mieux comprendre la physiologie et la physiopathologie de la fonction gonadotrope. Pour elucider ces mecanismes, nous avons mis en œuvre deux approches complementaires in vivo et in vitro. Nous avons tout d’abord isole le promoteur hypophysaire de ce gene et analyse son activite par transfection transitoire dans deux lignees de type gonadotrope, les cellules αCT3-1 et LβT2. Nous avons ainsi defini une premiere combinatoire de facteurs de transcription impliques dans l’expression histospecifique maximale du gene du recepteur de la GnRH, ou le facteur steroidogenique SF-1 joue un role decisif en interagissant avec des facteurs de transcription apparentes aux familles GATA et LIM a homeodomaine. De meme, en demontrant la fonction stimulatrice du PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) sur l’activite du promoteur, nous avons mis en evidence l’implication critique de SF-1 et son interaction avec un facteur apparente a CREB. Ces avancees nous ont alors conduits a analyser l’activite de ce promoteur par transgenese chez la Souris en utilisant le gene rapporteur de la phosphatase alcaline placentaire humaine. En accord avec les donnees obtenues in vitro, ce promoteur hypophysaire s’est avere capable de conferer au transgene une expression specifique des cellules gonadotropes. Il est egalement capable de cibler l’expression de la phosphatase alcaline dans plusieurs zones du systeme nerveux central, notamment le complexe hippocampo-septal. Certains de ces tissus n’expriment pas SF-1, suggerant qu’in vivo son role ne serait pas aussi determinant que le predisaient les experiences realisees in vitro. De maniere surprenante, au cours de l’ontogenese hypophysaire, le transgene s’exprime des 13,5 jours postconception alors que le facteur SF-1 n’est pas encore present. In vivo, SF-1 ne serait donc pas necessaire a l’activation du gene du recepteur de la GnRH pendant les etapes precoces du developpement. Ces resultats sont coherents avec les donnees obtenues apres invalidation generale ou ciblee dans l’hypophyse du gene codant SF-1, suggerant que le recepteur de la GnRH s’exprime malgre l’absence de ce facteur. Connaitre le ou les facteurs responsables de l’activation du gene du recepteur de la GnRH lors des etapes precoces du developpement devrait permettre de mieux cerner le role de SF-1, non seulement dans l’expression du gene du recepteur mais, plus generalement, dans la physiologie et la physiopathologie de la fonction gonadotrope.

Les donnees issues du decryptage du genome humain puis de la proteomique naissante doivent etre integrees dans les structures de la cellule vivante si l’on veut parvenir a une meilleure comprehension de son fonctionnement normal et pathologique. L’organisation compartimentee de la cellule eucaryote, pressentie des la fin du XIXe siecle, est maintenant definitivement etablie dans l’ensemble du monde vivant. Cet article situe dans une perspective historique les donnees de base indispensables a la comprehension des recherches modernes sur les mecanismes du transport intracellulaire.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteinsor = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

The mechanisms of membrane recycling and endocytic activity have been investigated by morphological methods, following chemical depolarization, in cultured mouse hypothalamic neurons that have developed in vitro fully differentiated synapses. Previous studies with this cell system have shown that short-term (3 min) exposure to a depolarizing concentration of KCl induces a depletion of synaptic vesicles in synaptic boutons. Subsequent return to control medium is followed by a restoration of synaptic vesicle numbers. We have now used horseradish peroxidase (HRP) as a tracer for endocytosis and wheat-germ agglutinin coupled to HRP-gold as a marker for adsorptive endocytosis. In parallel, synaptophysin, a marker for small synaptic vesicles, was simultaneously localized by electron microscopic immunocytochemistry in some experiments. The morphometric analyses and the time course of HRP uptake combined with the distribution of synaptophysin immunostaining indicate that short-term (3 min) depolarization leads to a depletion of synaptic vesicles together with an enhanced endocytic activity via vesicles that possess both the diameter and the major membrane protein, synaptophysin, characteristic of synaptic vesicles. These findings suggest a local, direct recycling mechanism of synaptic vesicles. In contrast, long-term depolarizations (15–30 min) appear to involve additional membrane compartments.

The distribution of three components of basement membranes (BM): heparan-sulfate proteoglycan (HSPG), entactin (ENT), and type IV collagen (Coll. IV), was studied in the adult rat anterior pituitary and compared to the distribution of laminin (LAM) described in an earlier report (Vila-Porcile et al., 1987, J. Histochem. Cytochem., 35, 287). Several immunocytochemical methods were applied at both light and electron microscope levels. The three components were detected in all the pituitary BM and in endothelial and perivascular connective cells, as previously observed for LAM. In contrast to the prominent labeling previously observed for LAM within epithelial cells, however, only a faint immunoreaction could be detected for the other components, with nonetheless a discrete signal for Coll. IV. These findings indicate that 1) the four studied components are present in all the BM of the rat anterior pituitary; 2) pituitary endocrine cells contain LAM, and to a lesser extent Coll. IV, and thus could participate to the elaboration of the BM; and 3) the presence of the four components in non-epithelial cells also suggests their cooperation in BM building. The questions of the turnover and intracellular pathways of each of these components were addressed but remain unanswered.

Thyroliberin E-H-P-NH4 (TRH) is a small neuropeptide p Glu-His-Pro-NH 2 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-NH 2 (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 3 H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

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.

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17β-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC50 (18–59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.

The subcellular distribution of clathrin has been examined in developing hypothalamic neurons cultured in a chemically defined medium up to synapse formation (12-13 days in vitro) and exposed, or not, to a depolarizing concentration of KCl (60 mM for 3 min) followed, or not, by a return to control KCl concentration (3 mM KCl for 3 min). Previous studies have shown that such treatments induce in synaptic boutons a rapid vesicle depletion followed by massive restoration. Using an enzyme immunoassay, we have compared the relative proportion of assembled and unassembled pools of clathrin as a function of exposure to depolarizing or repolarizing concentrations of KCl. In parallel we have localized clathrin at the electron microscopic level using immunoperoxidase. Clathrin concentration in culture is lower (0.36 vs 0.75%) and the proportion of unassembled clathrin is much higher than in the adult brain (82 vs 14%). These proportions were not affected by depolarizing or repolarizing treatments. Morphologically clathrin was exclusively detected in two neuron compartments: perikarya and synaptic boutons. In perikarya clathrin was localized as a thick coat on plasma membrane coated pits and in the Golgi zone on coated buds and vesicles, presumably located in a trans compartment. In synaptic boutons clathrin immunoreaction was found as an irregular thin rim around synaptic vesicles, whatever the polarization state of the cells, but coated vesicles were extremely rare. Taken together these findings raise the problem of the functional meaning and localization of the large unassembled pool of clathrin in such neurons and question its role in vesicular traffic in synaptic boutons.

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.

Three DNA constructs, the natural human growth hormone gene (hGH-hGH) its 500 bp promoter linked to the chloramphenicol acetyl transferase reporter gene (hGH-CAT), and its structural part linked to the herpes virus thymidine kinase promoter (TK-hGH) were introduced into rat pituitary GC cells by DEAE-dextran transfection. Transient expression was followed as a function of triiodothyronine (T3) concentration. The hGH-CAT expression was specifically inhibited by T3 following a typical dose-response curve while hGH-GH gene expression was not significantly modified. The transient expression of TK-hGH increased as a function of T3 concentration. These results indicate that T3 exerts two opposite effects on hGH gene expression. First, it down-regulates expression by acting on the promoter; second, it up-regulates expression by acting on the structural part of the gene. These action could be due to regulation of transcription and mRNA stabilization, respectively.