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The aim of the present work was to study the cellular localization of 5-HTIA receptor protein and mRNA in rat embryonic brain cultures. Primary cultures of the whole brain from rat fetuses at embryonic day (ED) 12 and of the brain stem at ED 14-ED 16 were stained with specific anti-5-HTIA receptor antibodies or a 40-mer biotin-labelled deoxyoligonucleotide complementary to the 5-HTIA receptor mRNA. The use of a biotinylated probe allowed the morphology of the cells to be preserved. 5-HTIA receptor mRNA was already detected in primary cultures from the brain of ED 12 embryos whereas the receptor protein first appeared two days later, at ED 14. Both 5-HTIA receptor mRNA and protein were found within neuron-like cells (labelled with antibodies against neuron specific enolase, microtubule-associated protein 2 or aromaticl-aminoacid decar☐ylase) but not in glial cells (specifically labelled with antibodies against glial fibrillary acidic protein, myelin basic protein or carbonic anhydrase II). Double staining with the 5-HTIA receptor mRNA probe and anti-5-HT antibodies suggests that 5-HTIA (auto)receptors are expressed by serotoninergic neurons during early ontogenesis.

Previous studies, aimed at identifying which diffusible signals may influence the differentiation of embryonic neurons towards the monoaminergic phenotypes during brain development, have shown that serotonin itself could promote the 'serotoninergic-like properties' of hypothalamic cells from mouse embryos. We presently reinvestigated such 'autocrine/paracrine' regulatory mechanisms by exposing dissociated cell cultures from embryonic rat hypothalamus and brain stem to dopamine--or related agonists--in an attempt to influence their differentiation towards the catecholaminergic phenotype. Chronic treatment of cells by dopamine or apomorphine (a mixed D1/D2 agonist), but not selective D1 and D2 agonists, significantly increased the number of cells that expressed tyrosine hydroxylase (TH: as assessed with a specific anti-TH antiserum) and the activity of aromatic L-amino acid decarboxylase (AADC) in the cultures. Furthermore, apomorphine treatment also decreased the levels of cholecystokinin-like material in primary cultures from the brainstem (but not the hypothalamus) where both dopamine and cholecystokinin are--partly--colocalized in mesencephalic dopaminergic neurons. The maximal effects of both dopamine and apomorphine on TH expression and AADC activity occurred earlier in the brainstem (on cells from 14- to 15-day-old embryos) than in the hypothalamus (on cells from 15- to 16-day-old embryos), in line with the well established caudo-rostral maturation of the rat brain. Furthermore both the expression and the dopamine-induced modulation of AADC activity and TH immunoreactivity appeared to occur independently of each other. Present and previous data are in agreement with a possible autocrine/paracrine action of dopamine and serotonin during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

The principal finding of this study is the establishment of culture conditions that permit the survival of a very limited population of muscle precursor cells from the embryonic and neonatal rat brain stem, with these cells displaying the capacity to differentiate into contractile striated fibers under these in vitro conditions. Examination of desmin and troponin T expression by immunoperoxidase analysis revealed the presence of the two muscle proteins in the mononucleated precursors and in differentiated multinucleated myotubes. In vivo, examination of sections at corresponding stages revealed the simultaneous presence of troponin T, desmin, and lectin binding sites--a property of endothelial cells--association which appears to be transitory, since it is no longer detectable in the adult brain. The presence of the choroid plexus neuroepithelium, in close vicinity with TnT+, desmin+cells located in a limited zone of the brain stem, as well as the stage of expression for these markers suggest an interaction between brain myogenic cells and the onset of the embryonic circulation. The question of the embryological origin of the brain myogenic cells is discussed.

Immunocytochemical observations with purified antibodies revealed the presence of met-enkephalin-like material in the primitive nerve cell line F7 derived from the fetal mouse hypothalamus and previously shown to synthesize somatostatin (1). Radioimmunoassays associated with Bio-gel P 2 chromatography confirmed that met-enkephalin itself accounted (at least partly) for the positive immunocytochemical reaction. Trypsinization (± carboxypeptidase B treatment) of cell extracts significantly increased their met-enkephalin-like immunoreactivity therefore suggesting that met-enkephalin precursors were also present in the F7 clone. Parallel studies on the hypothalamus of fetal mice indicated that met-enkephalin but apparently no precursor was already present at embryonic day 15. The clonal cells F7 may be an appropriate model for investigating the functional correlate of the co-occurrence of metenkephalin and somatostatin in the same cells.

Dissociated fetal hypothalamic cells mainly taken from 14 day-old mouse fetuses were grown in vitro for increasing time (9 to 60 days). Soon after inoculation the cells partly reaggregated and attached. The small reaggregates were then interconnected by fibers bundles. After the first week the cultures were composed of a continuous basal monolayer of flat and transparent cells, over which various types of refractile cells were lying in discontinuous areas. The ultrastructural study enabled us to identify these cell types, to describe their spatial relationships, and to follow their evolution with time in culture. The basal cell formed several superimposed layers. With increasing age, they displayed typical features of astrocytes and of ependymal cells. The latter exhibited rhythmic ciliary movements in culture. The overlying cells corresponded to three types which were associated in small clumps: primitive neuro-epithelial cells, maturing as well as mature neurons and typical neurosecretory cells. The latter cells were found as early as 9 days of culture of 14 day-old fetal hypothalamic cells and retained their typical features up to two months. Neuronal processes formed very dense networks at the surface of the cultures and terminated within the basal layers. Axon and dendrites were precociously found and were still present after two months. Within axon terminals dense-core vesicles appeared at the same time as neurosecretory cells. Synaptic vesicles and synaptic junctions were found later on.

A serum-free medium has been devised which permits proliferation of the mouse primitive nervous cell line F7. When cholesterol, eye-derived growth factor and brain extract are added in this medium for 48 h, 80-90% of oligodendrocyte-like cells are generated. These cells have diminished substrate adhesion. They acquire the capacity to synthesize carbonic anhydrase II and myelin basic protein, two specific proteins of oligodendrocytes. These observations suggest that F7 clonal cell line, which has been previously shown to be a neurophysin cell precursor, is also a precursor for oligodendrocytes, and represents a bipotent stem cell line for both neuronal and glial cell lineages.

A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of beta-glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH. The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.

The cellular localization of a neuronal and a glial cell specific protein (14-3-2 and S-100, respectively) has been explored in mouse hypothalamus in order to trace cell lineages. This study was performed on fixed slices, at the light microscope level, by using either the indirect peroxidase-labeled immunoglobulin technique or immunofluorescence. In the adult, only S-100 immunoreactivity was found in the ependymal layer. In contrast, the magnocellular neurons of the preoptic area displayed strong 14-3-2 immunoreactivity. At neonatal stages (fetal day 17-postnatal day 3), both 14-3-2 and S-100 immunoreactivities developed simultaneously in the same cells lining the ventral part of the third ventricle. Transient detachment of some of these ventricular cells could be visualized before migration in the hypothalamus where they remained as bipotential cells up to postnatal day 10. Later in the development, they differentiated into separate cells, one type containing 14-3-2 and the other S-100, like neurons and glial cells. These results argue for a developmental stage during which cells lining the ventricle are bipotential and may thus be candidates for the role of stem cells for both neuronal and glial lineages.

International audience; Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization-selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3' untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross-reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.

A mouse carbonic anhydrase (CA II) complementary(c) DNA probe was used for in situ hybridization on mouse brain cultured cells in order to follow CA II gene expression during brain development. An improved method was established using biotinated probes that resulted in a high sensitivity and an absence of background; this method could be combined with immunohistochemistry. Hypothalamic cells of embryonic day (ED) 12-14 mice were cultured for various periods. Chronologic appearance of CA II messenger(m)RNA and protein was studied. The CA II gene transcripts are detectable as early as ED 12-13, although the protein they encode is not detectable until ED 17-18. Gene expression is restricted to 0.1% of the total population. Northern blot analysis confirmed the presence of CA II transcripts in embryonic hypothalamus. At postnatal stage, the majority of glial cells express both the CA II mRNA and the protein. Our results favour the early appearance of a glial lineage in a precise area of the developing CNS. The precocity of CA II gene transcription makes in situ hybridization an invaluable approach in defining the onset of nerve cell lineages during embryonic development.

Some cells from cultured embryonic mouse hypothalamus were found to express aromatic-L-amino acid decarboxylase (EC 4.1.1.28) activity and serotonin uptake and storage. These neuron-like cells differed from serotoninergic neurons in cultured embryonic mouse brain stem since they did not contain tryptophan hydroxylase. We studied the effect of the serotonin agonist 8-hydroxy-2-[di-(n-propyl)amino]tetralin on neuronal differentiation of hypothalamic cells from 12- to 15-day embryos. Repeated treatment of cultures with the serotonin agonist for 10 days resulted in an increased number of serotonin cells containing high levels of decarboxylase activity. Both the increase in cell numbers and the elevated decarboxylase activity could be suppressed by the addition of the serotonin antagonist metergoline to the culture medium. These data show that serotonin (or an agonist), acting on specific receptors, can initiate and amplify its own synthesis in embryonic hypothalamic neurons, as observed in the primitive hypothalamic nerve cell line F7 [De Vitry, F., Catelon, J., Dubois, M., Thibault, J., Barritault, D., Courty, J., Bourgoin, S. & Hamon, M. (1986) Neurochem. Int. 9, 43-53]. Such an autocrine-like mechanism may be active during nervous system development and may represent an example of learning at the cellular level.

In vitro study of nerve cell differentiation has been primarily concentrated on the examination of cell lines that, at least partially, express terminal steps of differentiation. The isolation and characterisation of precursor populations would be an aid to further understanding of nerve cell differentiation. I present here evidence for a clonal line of such a nerve cell precursor type which, after in vivo passage, is able to give distinct neuronal phenotypes. These phenotypes are potentially lineage related, and show qualitative differences in the expression of their hypothalamic neuronal function.

Electrophysiological and pharmacological studies have been performed on a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin (HT9-C7). These neurons possessed low resting membrane potential (RMP) and weak membrane resistance (MR). They did not exhibit spontaneous activity. Electrical stimulation or microiontophoretic application of putative neurotransmitters did not induce action potentials. Nevertheless, dopamine and gamma-aminobutyric acid appeared to exert a slight hyperpolarizing effect on RMP. Radioimmunoassays, carried out on the culture medium after electrical stimulation, did not reveal any measurable quantities of vasopressin. However, an electron microscopic analysis of the cytoplasmic processes of these cells did not reveal axonal outgrowth. It can be assumed that the weak electrophysiological and pharmacological properties of these neurons have to be related to their weak morphological differentiation. Two hypotheses might account for the absence of most characteristics of in situ magnocellular hypothalamic neurons in HT9-C7 cells: the lack of pituicytes, the target cells for the axon terminal of magnocellular neurons, and the SV40 transformation itself which may impede neuronal maturation.

A serum-free medium supplemented with a glial conditioned medium, a brain extract from 8-to 10-day-old mice, hormones, and eye-derived growth factor has been devised which permitted the mouse primitive hypothalamic nerve cell line F7 to express some biochemical properties typical of monoaminergic neurons. Maximal expression was obtained when the culture conditions were applied for 2 days. Most (90-95%) cells then synthesized [(3)H]serotonin from [(4)H]5-hydroxytryptophan (but not from [(3)H]tryptophan). No synthesis was detected in the presence of carbidopa (20 ?M), therefore suggesting the involvement of l-aromatic-amino-acid decarboxylase in this process. In addition, F7 cells cultured in such serum-free medium exhibited the capacity of accumulating exogenous serotonin by an ouabain-sensitive mechanism. These data further supported that active molecules in the cell environment can induce, in a primitive cell line, some of the enzymatic activities associated with monoaminergic neurons. Since other well-defined culture conditions can promote the differentiation of the same clone into oligodendrocytes (De Vitry et al., 1983), it can be concluded that the F7 cell has the properties of an embryonic stem cell of the CNS which, depending on external signals, may switch into different alternative developmental neural pathways. We postulate that the stabilization of neuron-like properties due to repetitive cell stimulation by active signals in the environment may represent an example of learning at the cellular level.

A primitive nerve cell line (F7 clone), obtained by SV 40 transformation of mouse fetal hypothalamic cells, has been previously shown to share some properties with a progenitor cell for neurophysin synthesizing neuron. This paper describes the immunological detection, at the light microscope level, of somatostatin in this cell line. A SV 40 transformed neurosecretory clone, synthesizing neurophysin and vasopressin, was used as control.

The differentiation of tanycytes during development of the mouse hypothalamus has been followed by immunohistochemistry with a GFA protein antiserum. Radially oriented processes of the tanycytes were stained with GFA antiserum in the ventral and the dorsal lining of the third ventricle in the mouse neonate and at later ages. Typical astrocytes in the hypothalamus could not be visualized before 16-20 days postnatally. These results indicate that tanycytes, which acquire their final position early in embryonic development, exhibit a structural feature of fully differentiated astrocytes early in postnatal development.

Astrocytic cells of unusual aspect can be detected in the cerebellum of normal mice during the first 4 weeks of life. They are visualized with anti-GFAP (glial fibrillary acidic protein), anti-S100 and anti-vimentin immune sera. Their perikaryons, located in the white matter or in the granular layer, extend long processes which are inserted onto the pial surface. These cells may be transitional forms between the radial glial cells and some of the differentiated astroglial elements. These unusual astrocytes are more numerous and heavily stained in the reeler mutant than in the normal mouse and it is suggested that our observations signify some degree of glial immaturity in the cerebellum of the mutant.

Using immunocytochemical methods carbonic anhydrase II (CAII) has been detected in the embryonic mouse central nervous system as early as in stage E16. In the spinal cord and the brain stem, the enzyme first appeared in transitory cells probably derived from the radial glia. In the cerebrum such transitory cells were never stained with anti-CAII sera. The bodies of CAII-positive cells were never visualized in the ventricular layer. The controversial cellular specificity of CAII made it impossible to specify the glial lineage to which these transitory CAII-positive cells are committed.

Publisher Summary This chapter discusses the hypothalamic neurons in cell culture. A cell-culture approach to the study of hypothalamic neurons is fully justified by the complexity of the anatomy, cell architecture, and function of the hypothalamus as established over the past 50 years. The hypothalamus forms the ventral and lateral walls of the third ventricle and has an uncommon neurovascular relationship with an endocrine gland, the pituitary. From a functional point of view, the hypothalamus secretes many chemical messengers in response to external as well as internal stimuli. Such a neuronal function implies the synthesis, migration, storage, and release of peptide hormones, as well as the existence of specific receptors. The culture of dissociated hypothalamic cells offers a unique means of obtaining simplified models. It differs fundamentally from organ culture in which the predetermined in vivo pattern of tissue organization is maintained. In contrast, the culture of dispersed hypothalamic cells permits work at a single-cell level, provided that the cells completely retain their features of differentiation under such conditions.