71
Cell Differentiation and Development, 32 (1990) 71-82 Elsevier Scientific Publishers Ireland. Ltd.
CELDIF
00695
Are neuronal precursor cells committed to coexpress different neuroactive substances in early amphibian neurulae? F. Pituello, S. Boudannaoui, Centre de Biologie du Dkveloppement,
F. Foulquier and A.M. Duprat
CNRS- URA 675 afjilihe ci I’INSERM,
Vniversitk Paul-Sabatier,
Toulouse, France
(Accepted 16 July 1990)
Considering the initial expression of neurotransmitters and neuropeptides immediately after neural induction in amphibian embryos, we previously pointed out that a neuronal cell population emerges from neural plate (NP) and neural fold (NF) expressing very early specific cholinergic, catecholaminergic, GABAergic and peptidergic traits. The purpose of the present work was to investigate the extent to which the neuroblasts that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive transmitters rather than to different subpopulations of neurochemically homogeneous neurons. By means of double immunocytochemical localization with a monoclonal TOH-antibody and polyclonal antibodies against GABA or somatostatin, no coexistence of neurotransmitters and neuropeptide was ever found in neuronal subpopulations arising in vitro from NP or NF. The early emergence, under the same conditions, of distinct neuronal subpopulations as a consequence of neural induction strongly suggests that, at the gastrula stage, the neural precursor population most probably does not constitute a homogeneous set of cells. Neurogenesis;
Neurotransmitter;
Neuropeptide; Neural induction; Amphibian
Introduction Very early in the ontogeny of the amphibian nervous system, immediately after neural induction, there emerges in vivo and in vitro a neuronal cell population expressing different neurotransmitter-related phenotypes (Duprat et al., 1987, 1990a). Subsequent development of these neuronal characteristics does not require the further presence of chordamesodermal derivatives, such as
Correspondence address: A.M. Duprat, Centre de Biologic du DCveIoppement, CNRS-URA 675 affilike g Hnserm, Universitk Paul-Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France. 0922-3371/90/$03.50
somitic mesenchyme or notochordal cells. Thus, at the early neurula stage, embryonic cells isolated from neural plate (NP) or neural fold (NF) differentiate in vitro along choline&, adrenergic, GABAergic and peptidergic pathways without any further cues from the tissue responsible for the initial inductive stimulus (Duprat et al., 1985a, b, 1990b; Pituello et al., 1989a, b). The neurons that develop acquire the capacity to biosynthesize, store, take up and release various neurotransmitter substances. Over the past few years, in avian and mammalian models as well as in invertebrates, evidence has accumulated demonstrating the coexistence, later in nervous system development, in vivo and
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
72
in vitro, of different neurotransmitters and/or neuropeptides in the same neuron (Furshpan et al., 1976; Schultzberg et al., 1982; Belin et al., 1983; Bohn et al., 1984; Cournil et al., 1984; Maxwell et al., 1984, 1985; Kosaka et al., 1985; Garcia-Arraras et al., 1986; Melander et al., 1986; Potter et al., 1986; Gall et al., 1987; Leblanc et al., 1987; Moriss and Gibbins, 1987; Xue et al., 1987; Fisher et al., 1988; Kosaka et al., 1988; Ottersen et al., 1988; Pearson et al., 1988; Saland et al., 1988; Yazulla et al., 1988; Fauquet et al., 1989; Mize, 1989. The aim of the present study was to investigate to which extent the precursor cells that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive molecules rather than to different subpopulations of neurochemically homogeneous nerve cells. We first focused our interest on the possible coexistence within the same neuron of both GABAergic and adrenergic properties, as this has been previously observed in neurons of rat olfactory bulb (Kosaka et al., 1985; Gall et al., 1987). Subsequently, we also investigated the eventuality of coexpression of somatostatin and adrenergic properties in our in vitro model systems. Such a coexistence has been largely documented in other vertebrates (for review see Schultzberg et al., 1982), even at early steps of neuronal development (Maxwell and Sietz, 1985; Garcia-Arraras et al., 1986). By means of double-labeling with specific immunocytochemical markers (two polyclonal antibodies directed respectively against GABA and somatostatin and a monoclonal antibody directed against tyrosine hydroxylase), we have obtained clear evidence that there is no colocalization of any two of the neuroactive substances thus defined within the same neuron, whether it originates from NP or from NF. In the same way, using a specific marker for the whole neuronal catecholaminergic population (monoclonal antibody directed against TOH) and a specific marker for the noradrenergic neuronal population (polyclonal anti-dopamine P-hydroxylase antibody), we demonstrated the existence, both
in NP and NF cultures, of two separate laminergic lineages: a noradrenergic dopaminergic neuronal population.
catechoand a
Materials and methods
Animals Pleurodeles waltlii neurulae (stage 13 according to the table of Gallien and Durocher, 1957) were the source of cultured embryonic cells. Cell cultures Jelly was removed with fine forceps. The entire neurectoderm (NP and NF) or each component (NP or NF) separately, was excised with or without the underlying chordamesoderm as previously described (Duprat et al., 1984) (Fig. 1). Explants were then dissociated in Ca*+/Mg*+-free Barth’s medium (88 mM NaCl, 1 mM KCI, 2.4 mM NaHCO,, 2 mM Na,HPO,, 0.1 mM KH,PO, 0.5 mM EDTA, pH 8.7), and isolated cells were cultured at 20°C in Barth’s defined saline medium (Barth and Barth, 1959) supplemented with 1 mg/ml BSA (Sigma) and antibiotics. Cells were on plastic or plated (1.2 x lo5 cells/well) collagen-coated glass coverslips in four multiwell dishes or 35-mm diameter Petri dishes (Nunc). After the cells had spread (48 h), the medium was replaced by fresh Barth’s medium without BSA, and the cultures were maintained until complete cell differentiation, for 2-3 weeks (Duprat et al., 1984). immunocytodetection Antibodies. Monoclonal antibody RT 97 directed against the phosphorylated 200 kDa neurofilament subunit (Anderton et al., 1982) was provided by Dr. J. Wood (Sandoz Institute for Medical Research, London). The visualization of tetanus toxin binding sites was performed as previously described (Duprat et al., 1986) using the tetanus toxin and the rabbit anti-tetanus toxin antibody kindly provided by Dr. B. Bizzini (Institute Pasteur, Paris). Tyrosine hydroxylase (TOH) was evidenced using a monoclonal antibody raised against the quail enzyme, which also recognizes TOH from
73
I Fig. 1. Procedure
for cell cultures. chordamesodermal
II
III
(I) Cocultures (NP+NF+CM): neural plate plus neural fold cells were cocultured cells. (II) Isolated neural plate cells (NP). (III) Isolated neural fold cells (NF).
chick and pleurodele (Fauquet and Ziller, 1989) provided by Dr. M. Fauquet (Institut d’ Embryologie, Nogent/ Marne, France). Polyclonal antibody to dopamine /3-hydroxylase was obtained from Dr. D. Aunis (INSERM-U 44, Centre Neurochimie, Strasbourg, France). Somatostatin was visualized using a polyclonal antiserum provided by Dr. P. Brazeau (University Montreal, Canada) that reacts with the cyclic form of the peptide. GABA was detected using a specific polyclonal antibody raised against conjugated GABA (Pituello et al., 1989b) from Dr. M. Geffard (Institut de Neurochimie CNRS, Bordeaux, France). The corresponding secondary antibodies used were goat anti-rabbit-Ig, rabbit anti-sheep Ig, goat anti-mouse Ig conjugated with fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate (Nordic, Tilburg, The Netherlands; Immunotech, Marseille, France).
Immunocytochemistry Double labeling.
with
the
As previously described (Pituello et al., 1989a, b), in normal conditions, TOH-, somatostatinand GABA-like immunoreactivities were clearly but faintly discernible in neurons differentiated in vitro. To investigate a possible coexpression of two neuroactive substances using double labeling, it was necessary to improve the best conditions available for immunovisualization. Thus, in all cases the cellular contents of neurotransmitter-related molecules were increased using specific procedures. For TOH detection, cultures were first pretreated for 5 h with 10e5 M reserpine (Joh et al., 1973‘). Likewise, to enhance visualization of somatostatin, cultures were pretreated with colchicine (5 . JOT3 M) overnight. GABA uptake, performed by incubating the cultures for 5 h with 10e5 M GABA (Pituello et al., 1989b), was used to enhance visualization of GABAergic neurons. In our cultures, after such
74
75
treatment, the GABA immunoreactive neuronal population was not quantitatively different from that evidenced after immunostaining of endogenous GABA using intensification procedures such as the biotin-streptavidin technique (data not shown). After careful washing, cultures were fixed in 3.5% formaldehyde in Barth’s medium for 30 min at 20°C and then washed in Barth’s medium. Cells were permeabilized by treating with methanol at - 10°C for 6 min and then with 0.25 Triton X-100 in Barth’s solution containing 1% dry skim-milk for 2 min. After three thorough washings, cells were incubated overnight with TOH monoclonal antibody at 4°C and revealed with appropriate GAM-FITC or TRITC antibodies for 30 min at 2O’C. The cultures were then washed and incubated with polyclonal antibodies directed against GABA, somatostatin or dopamine p-hydroxylase for 60 min at 20°C before being incubated in appropriate GAR-TRITC or FITC antibodies for 30 min at 20°C. After rinsing and mounting in Mowiol 4-88, the cultures were observed with a Leitz Dialux microscope equipped with appropriate I, and N, filters. At least ten cultures were examined for each set of experiments. Controls. In all sets of experiments, immunoreactivity was abolished when the primary antibodies were either omitted or replaced (for polyclonal antibodies) by normal rabbit serum. In all immunolabeling experiments, non-neuronal cells originating from neurectoderm were negative.
Fig. 2. Non-coexistence of TOH-LI and GABA-LI in neuroblasts differentiated 14 days in vitro. (a) TOH-LI visualized in lCday-old cocultures. In a large neuronal aggregate only two cells expressed TOH-LI (arrow head). (b) GABA-LI neurons dispersed in an immunonegative neuronal cluster in cocultures. Cell bodies (arrow head) and neuritic processes (thin arrow) are fluorescent. (c and d) Double immunocytochemical labeling with the monoclonal TOH antibody (c) and the polyclonal antibody against GABA performed in cocultures (d). The neuronal subpopulation expressing GABA-LI is TOH immunonegative. Bar: 10 pm.
Results Colocalization of TOH and GABA TOH, the first enzyme in the pathway leading to catecholamine biosynthesis is considered to be a specific marker for catecholaminergic differentiation (Berod et al., 1982). TOH immunoreactivity is not detectable in neurectodermal cells, in the early neurula although TOH activity can just be detected using biochemical assays followed by HPLC and electrochemical detection (Boudannaoui et al., unpublished data). TOH-immunoreactive neurons are first faintly visible in 3-day-old differentiated cultures; fluorescence intensity increases over time (Fig. 2a). However, only a subset (2%) of the differentiated neuronal population is TOH-positive. Faint GABA immunoreactivity is also detected in a neuronal subpopulation (6% of the neuronal population) after 3 days in culture. A bright staining is observed in older cultures, both in the cell body and the neurites (Fig. 2b). In order to determine whether TOH and GABA coexist in certain neurons, double immunocytochemical labeling was performed as described in Materials and Methods. In all the culture types analyzed, i.e., cocultures, NP or NF cell cultures, the neuronal subpopulations expressing TOH and GABA were found to be totally distinct and nonoverlapping (Fig. 2c and d). These results were fully confirmed in cultures in which cells were stained first with the polyclonal antibody against GABA and then with the monoclonal TOH antibody. Colocalization of TOH and somatostatin We previously reported (Pituello et al., 1989a) that somatostatin immunoreactivity is undetectable at the early neurula stage by radioimmunoassay as well as by immunocytochemistry. However, somatostatin is present in 3-day-old cultures and strongly immunoreactive neurons can be observed later, either in small neuronal aggregates (Fig. 3a) or scattered through the culture. The somatostatinergic phenotype is expressed in a small subpopulation of cells (less than 1% of the neuronal population).
76
The possibility that TOH and somatostatin are coexpressed in same neurons was investigated. No doubly labeled cells were ever found in any of the experiments (Fig. 3c, d and e).
Coexpression of TOH and D/3H Dopamine P-hydroxylase (DPH) is the enzyme catalysing the synthesis of noradrenaline and is consequently a specific marker of noradrenergic metabolism (Lundberg et al., 1977). The simultaneous presence of TOH and Dj3H in the same neuron thus defines its phenotype as noradrenergic (Berod et al., 1982) whereas the presence of TOH alone, without D/3H, is indicative of the dopaminergic phenotype of the neuron. Double-labeling experiments were performed to study the characteristics of the catecholaminergic cell population that differentiated in vitro from cells taken at the early neurula stage (Boudannaoui et al., unpublished data) and to look for the possible presence of two different subpopulations. As shown in Fig. 4, certain neurons express TOH and not DBH (Fig. 4a, b and c) whereas others exhibit both TOH and DPH immunoreactivity (Fig. 4d, e and f). These data indicate here again the emergence of two other defined neuronal subpopulations, dopaminergic and noradrenergic. In all cases, the neuronal nature of TOH-, GABA-, DflH-, somatostatin-positive cells was confirmed using specific neuronal markers such as neurofilament polypeptides or tetanus toxin binding sites in agreement with our previous results (Duprat et al., 1986, 1987; Saint-Jeannet et al., 1989a; Huang et al., 1990).
Fig. 3. Absence of colocalization for Somatostatin-LI (SLI) and TOH-LI in 14-day-old cocultures. (a and b) SLI visualized in a neuronal aggregate. Nerve cell bodies (thick arrow) and their neurites (arrow head) are immunopositive (a). Phase contrast (b). (c, d and e) Double immunostaining with the monoclonal anti-TOH followed by the polyclonal antibody against somatostatin. A TOH-positive neuron in a small cluster (c). The same cell does not express SLI (d). Phase contrast (e). Bar: 10 pm.
Discussion During the past 10 years, it has become well established that many neuronal cells contain neurotransmitter (catecholaa ‘small-molecule’ mines, acetylcholine, GABA, serotonin) in addition to a neuropeptide (somatostatin, substance P, VIP,. . .) in both central and peripheral nervous systems (Schultzberg et al., 1982; Belin et al., 1983; Bohn et al., 1984; Cournil et al., 1984; Kosaka et al., 1985; Potter et al., 1986; Leblanc et al., 1987; Ottersen et al., 1988; Saland et al., 1988; Mize, 1989). Evidence has also been obtained demonstrating a similar coexpression in vitro in certain neurons derived from neural crest that differentiate in culture (Maxwell et al., 1984, 1985; Garcia-Arraras et al., 1986; Xue et al., 1987; Fauquet and Ziller, 1989). On the other hand, the absence of such colocalization was also documented in embryonic cells from mouse and Drosophila. Buse (1987) reported that ventricular cells from the neural plate of mouse (stage 12-Theiler) differentiated in vitro into distinct neuronal cell classes, without any colocalization of transmitters. An analogous early emergence of serotoninergic and dopaminergic neuronal subpopulations was recently reported in Drosophila (Huff et al., 1989). Considering the initial expression of neurotransmitters and neuropeptides in early neurogenesis in amphibian embryos, we recently pointed out that as early as at the late gastrula-early neurula stage, some embryonic neuroblasts from NP or NF have already acquired potentialities to differentiate in vitro into neurons (Duprat et al., 1986; Saint-Jeannet et al., 1989a) with specific choline& (Duprat et al., 1985a, b, 1990b), catecholaminergic (Boudannaoui et al., unpublished data), GABAergic (Pituello, 1989b) or peptidergic (Pituello et al., 1989a) traits. Mechanisms for functional biosynthesis, accumulation, release, uptake and degradation develop without any further influence of chordamesoderm. With regard to each phenotype considered, we showed that only a subset of the neuronal population was concerned. In this context it was of particular interest to determine whether as a con-
78
19
sequence of neural induction, these embryonic neuroblasts are committed to the neuronal pathway with a status allowing the coexpression of different neurotransmitter and/or neuropeptides, or with a status allowing the expression of one transmitter. The data presented here, obtained by means of double immunocytochemical detection with a monoclonal TOH-antibody and polyclonal antibodies, respectively, against GABA, somatostatin or D/3H, show that neurectodermal cells isolated in vitro at the late gastrula-early neurula stage, develop neurochemically distinct neuronal traits. These experiments demonstrated that GABA-like immunoreactivity develops in a subpopulation of neurons. This development occurs under the same conditions that are permissive for the development of somatostatin-containing neurons and dopamine-containing neurons or noradrenaline-containing neurons. No coexistence of neurotransmitters and neuropeptides was ever found in neuronal subpopulations arising in vitro from NP or NF of early amphibian neurulae. The same result was obtained when the neurons were cocultured with chordamesodermal cells indicating that the CM has no qualitative influence on these phenotypes expressed in culture. In conclusion, the early emergence of distinct neuronal subpopulations as a consequence of neural induction, together with recent data on neural potentialities of presumptive ectoderm (SaintJeannet et al., 1989b, 1990; Duprat et al., 1990a) underline once again that the neural precursor population at the gastrula stage most probably does not constitute a homogeneous set of cells.
Fig. 4. Evidence for two distinct catecholaminergic subpopulations in 14-day-old cocultures. (a, b and c) Dopaminergic neurons: in the large cellular cluster visualized in phase contrast (c), some neurons display a brillant immunostaining with the monoclonal antibody anti-TOH (a) but are negative with the polyclonal immunoserum against DBH. (d, e and f) Three neurons (thin arrow) defined as noradrenergic neurons by the double labeling with anti-TOH (a) and anti-DfiH (e) antibodies. Phase contrast (f). Bar: 10 pm.
The authors would like to thank Dr. P. Kan for her expert assistance in cell culture. Mrs C. Daguzan and C. Mont are gratefully acknowledged for photographic and secretarial help and Dr. J. Smith (Nogent/Marne) for reviewing the English manuscript. This work was supported by CNRS, MRT, EEC and CNES grants.
References Anderton, BH., Breinburg, D., Downes, M.J., Green, P.J., Tomlinson, B.E., Uhich, J., Wood, J.N. and Kahn, J.: Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants. Nature 298, 84-86 (1982). Barth, L.G. and Barth, L.J.: Differentiation of cells of Ram pipiens gastrula in unconditioned medium. J. Embryol. Exp. Morphol. 7, 210-222 (1959). Belin, M.F., Nanopoulos, D., Didier, M., Aguera, M., Steinbusch, H., Verhofstad, A., Maitre, M. and Pujol, J.F.: Immunohistochemical evidence for the presence of yaminobutyric acid and serotonin in one nerve cell. A study on the Raphe Nuclei of the rat using antibodies to glutamate decarboxylase and serotonin. Brain Res. 215, 329-339 (1983). Berod, T., Hartman, B.K., Keller, A., Joh, T.H. and Pujol, J.F.: A new double-labeling technique using tyrosine hydroxylase and dopamine /%hydroxylase immunohistochemistry: evidence for dopaminergic cells lying in the pons of the beef brain. Brain Res. 240, 235-243 (1982). Buse, E.: Ventricular cells from the mouse neural plate, stage Theiler 12, transform into different neuronal cell classes in vitro. Anat. Embryol. 176, 295-302 (1987). Bohn, M.C., Kessler, J.A., Adler, J.A. Markey, K., Goldstein, M. and Black, I.B.: Simultaneous expression of the SPpeptidergic and noradrenergic phenotypes in rat sympathetic neurons. Brain Res. 298, 378-381 (1984). Coumil, I., Geffard, M., Moulins, M. and Le Moal, M.: Coexistence of dopamine and serotonin in an identified neuron of the lobster nervous system. Brain Res. 310, 397-400 (1984). Duprat, A.M., Gualandris, L., Kan, P. and Foulquier, F.: Early events in the neurogenesis of amphibians. In: The role of cell interactions in early neurogenesis, Eds. A.M. Duprat, A. Kato, M. Weber, NATO AS1 Series, Life Sci. 77, 3-20 (1984). Duprat, A.M., Kan, P., Foulquier, F. and Weber, M.: In vitro differentiation of neuronal precursor cells from amphibian late gastrulae: morphological, immunocytochemical studies, biosynthesis, accumulation and uptake of neurotransmitters. J. Embryol. Exp. Morphol. 86, 71-87 (1985a).
80 Duprat, A.M., Kan, P., Gualandris, L., Foulquier, F., Marty, J., Weber, M.: Neural induction embryonic determination elicits full expression of specific neuronal traits. J. Embryo]. Exp. Morphol. 89, 167-183 (1985b). Duprat, A.M., Gualandris, L., Foulquier, F., Paulin, D., Bizzini, B.: Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians. Cell Differ. 18, 57-64 (1986). Duprat, A.M., Gualandris, L. Pituello, F., Saint-Jeannet, J.P., Boudannaoui, S.: S. Neural induction. Biol. Struct. Morhogen. (ex. Arch. Anat. Microsc. Morpbol., Exp.), 75, 211-227 (1987). Duprat, A.M., Saint-Jeannet, J.P., Pituello, F., Huang, S., Boudannaoui, S., Kan, P., Gualandris, L.: From presumptive ectoderm to neural cells in an amphibian. Int. J. Dev. Biol., 34, 149-156 (1990a). Duprat, A.M., Kan, P., Weber, M.: Initial expression of choline& traits in embryonic amphibian neuronal cells choline acetyltransferase and acetylcholinesterase activities. Ontogenez (1990b, in press). Fauquet, M. and Ziller, C.: A monoclonal antibody directed against quail tyrosine hydroxylase description and use in immunocytochemical studies on differentiating neural crest cells. J. Histochem. Cytochem., 37, 1197-1205 (1989). Fisher, R.S., Buchwald, N.A., Hull, C.D., Levine, M.S.: GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons. J. Comp. Neurol. 272, 489-502 (1988). Furshpan, E.J., McLeish, P.R., O’Lague, P.H., Potter, D.D.: Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for choline@, adrenergic and dual function neurons. Proc. Natl. Acad. Sci. USA 73, 4225-4229 (1976). Gall, CM., Hendry, S.H.C., Seroogy, K.B., Jones, E.G. and Haycock, J.W.: Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb. J. Comp. Neurol. 266, 307-318 (1987). Gallien, L. and Durocher, M.: Table chronologique du developpement chez Pleurodeless waltlii Michah. Bull. Biol. Fr. Belg. 91, 97-114 (1957). Garcia-Arraras, J.E., Fauquet, M., Chanconie, M. and Smith, J.: Coexpression of somatostatin-like immunoreactivity and catecholaminergic properties in neural crest derivatives: comodulation of peptidergic and adrenergic differentiation in cultured neural crest. Dev. Biol. 114, 247-257 (1986). Huang, S., Saint-Jeannet, J.P., Kan, P. and Duprat, A.M.: Extracellular matrix: an immunological and biochemical (CAT and TOH activity) survey of in vitro differentiation of isolated amphibian neuroblasts. Cell Differ. Dev. 30, 219-233 (1990). Huff, R., Furst, A. and Mahowald, A.P.: Drosophila embryonic neuroblasts in culture: autonomous differentiation of specific neurotransmitters. Dev. Biol. 134, 146-157 (1989). Joh, T.H., Geghman, C. and Reis, D.S.: Immunochemical demonstration of increased accumulation of tyrosine hy-
droxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine. Proc. Natl. Acad. Sci. USA 70, 2767-2771 (1973). Kosaka, T., Hataguchi, Y., Hama, K., Nagatsu, I. and Wu, J.Y.: Coexistence of immunoreactivities for glutamate decarboxylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: possible coexistence of y-aminobutyric acid (GABA) and dopamine. Brain Res. 343, 166-171 (1985). Kosaka, T., Tauchi, M. and Dahl, J.L.: Choline& neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivites in various brain regions of the rat. Exp. Brain Res. 70, 605-617 (1988). Leblanc, G.G., Trimmer, B.A. and Landis, S.C.: Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with radioactive intestinal peptide and choline acetyltransferase. Proc. Nat]. Acad. Sci. USA 84, 3511-3515 (1987). Lundberg, J., Bylock, A., Goldstein, M., Hansson, H.A. and Dahlstrom, A.: Ultrastructural localization of dopaminefi-hydroxylase in nerve terminals of the rat brain. Brain Res. 120: 549-552 (1977). Maxwell, G.D. and Sietz, P.D.: Development of cells containing catecholamines and somatostatin-like immunoreactivity in neural crest cultures: relationships of DNA synthesis to phenotypic expression. Dev. Biol. 108, 203-209 (1985). Maxwell, G.D., Sietz, P.D. and Jean, S.: Somatostatin-like immunoreactivity is expressed in neural crest cultures. Dev. Biol. 101, 357-366 (1984). Melander, T., Hokfelt, T., Rokaeus, A. and Cuello, A.C.: Coexistence of galanin-like immunoreactivity with catecholamines, S-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J. NeuroSci. 6, 3640-3653 (1986). Mize, R.R.: Enkephalin-like immunoreactivity in the cat Superior coiiiculw distribution, ultrastructure and colocalization with GABA. J. Comp. Neurol. 285, 133-155 (1989). Moriss, J.Y. and Gibbins, H.: Neuronal colocalization of peptides, catecholamines, and catecholamine-synthesizing enzymes in guinea pig paracervical ganglia. J. NeuroSci. 7, 3117-3130 (1987). Ottersen, O.P., Storm-Mathisen, J. and Somogyll, P.: Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res. 450, 342353 (1988). Pearson, J.C. and Jennes, L.: Localization of serotoninand substance P-like immunofluorescence in the caudal spinal trigemical nucleus of the rat. NeuroSci. Lett. 88, 151-156 (1988). Pituello, F., Deruntz, P., Pradayrol, L. and Duprat, A.M.: Peptidergic properties expressed in vitro by embryonic neuroblasts after neural induction. Development 105, 529-540 (1989a). Pituello, F., Kan, P., Geffard, M. and Duprat, A.M: Initial GABAergic expression in embryonic amphibian neuroblasts after neural induction. hit. J. Dev. Biol. 33, 445-453 (1989b).
81 Potter, D.D., Landis, S.C., Matsumoto, S.G. and Furshpan, E.J.: Synaptic functions in rat sympathetic neurons in microcultures. II. Adrenergic/cholinergic dual status and plasticity. J. NeuroSci. 6, 1080-1098 (1986). Saint-Jeannet, J.P., Foulquier, F., Goridis, C. and Duprat, A.M.: Expression of N-CAM precedes neural induction in Pleurodeles walfl (Urodele, Amphibian). Development 106, 675-683 (1989a). Saint-Jeannet, J.P., Huang, S. and Duprat, A.M.: Target cell contacts and neural commitment in Pleurodeles waltlii. Cell Differ. Dev. 27 (suppl.), 52 (1989b). Saint-Jeannet, J.P., Huang, S. and Duprat, A.M.: Modulation of neural commitment by changes in target cell contacts in Pleurodeles w&iii. Dev. Biol. 141, 93-103 (1990).
Saland, L.C., Wallace, J.A., Samora, A. and Gutierrez, L.: Colocalization of tyrosine hydroxylase (TH)- and serotonin (5-HT)- immunoreactive innervation in the rat pituitary gland. NeuroSci. Lett. 94, 39-45 (1988). Schultzberg, M., Hokfelt, T. and Lundberg, J.M.: Coexistence of classical transmitters and peptides in the central and peripheral nervous system. Br. Med. Bull. 38, 309 (1982). Xue, Z.G., Smith, J. and Le Douarin, N.: Developmental capacities of avian embryonic dorsal root ganglion cells: neuropeptides and tyrosine hydroxylase in dissociated cell cultures. Dev. Brain Res. 34, 99-109 (1987). Yazulla, S. and Yang, C.Y.: Colocalization of GABA and glycine immunoreactivities in a subset of retinal neurons in tiger salamander. NeuroSci. Lett. 95, 37-41 (1988).
Cell Differentiation and Development, 32 (1990) 71-82 Elsevier Scientific Publishers Ireland. Ltd.
CELDIF
00695
Are neuronal precursor cells committed to coexpress different neuroactive substances in early amphibian neurulae? F. Pituello, S. Boudannaoui, Centre de Biologie du Dkveloppement,
F. Foulquier and A.M. Duprat
CNRS- URA 675 afjilihe ci I’INSERM,
Vniversitk Paul-Sabatier,
Toulouse, France
(Accepted 16 July 1990)
Considering the initial expression of neurotransmitters and neuropeptides immediately after neural induction in amphibian embryos, we previously pointed out that a neuronal cell population emerges from neural plate (NP) and neural fold (NF) expressing very early specific cholinergic, catecholaminergic, GABAergic and peptidergic traits. The purpose of the present work was to investigate the extent to which the neuroblasts that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive transmitters rather than to different subpopulations of neurochemically homogeneous neurons. By means of double immunocytochemical localization with a monoclonal TOH-antibody and polyclonal antibodies against GABA or somatostatin, no coexistence of neurotransmitters and neuropeptide was ever found in neuronal subpopulations arising in vitro from NP or NF. The early emergence, under the same conditions, of distinct neuronal subpopulations as a consequence of neural induction strongly suggests that, at the gastrula stage, the neural precursor population most probably does not constitute a homogeneous set of cells. Neurogenesis;
Neurotransmitter;
Neuropeptide; Neural induction; Amphibian
Introduction Very early in the ontogeny of the amphibian nervous system, immediately after neural induction, there emerges in vivo and in vitro a neuronal cell population expressing different neurotransmitter-related phenotypes (Duprat et al., 1987, 1990a). Subsequent development of these neuronal characteristics does not require the further presence of chordamesodermal derivatives, such as
Correspondence address: A.M. Duprat, Centre de Biologic du DCveIoppement, CNRS-URA 675 affilike g Hnserm, Universitk Paul-Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France. 0922-3371/90/$03.50
somitic mesenchyme or notochordal cells. Thus, at the early neurula stage, embryonic cells isolated from neural plate (NP) or neural fold (NF) differentiate in vitro along choline&, adrenergic, GABAergic and peptidergic pathways without any further cues from the tissue responsible for the initial inductive stimulus (Duprat et al., 1985a, b, 1990b; Pituello et al., 1989a, b). The neurons that develop acquire the capacity to biosynthesize, store, take up and release various neurotransmitter substances. Over the past few years, in avian and mammalian models as well as in invertebrates, evidence has accumulated demonstrating the coexistence, later in nervous system development, in vivo and
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
72
in vitro, of different neurotransmitters and/or neuropeptides in the same neuron (Furshpan et al., 1976; Schultzberg et al., 1982; Belin et al., 1983; Bohn et al., 1984; Cournil et al., 1984; Maxwell et al., 1984, 1985; Kosaka et al., 1985; Garcia-Arraras et al., 1986; Melander et al., 1986; Potter et al., 1986; Gall et al., 1987; Leblanc et al., 1987; Moriss and Gibbins, 1987; Xue et al., 1987; Fisher et al., 1988; Kosaka et al., 1988; Ottersen et al., 1988; Pearson et al., 1988; Saland et al., 1988; Yazulla et al., 1988; Fauquet et al., 1989; Mize, 1989. The aim of the present study was to investigate to which extent the precursor cells that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive molecules rather than to different subpopulations of neurochemically homogeneous nerve cells. We first focused our interest on the possible coexistence within the same neuron of both GABAergic and adrenergic properties, as this has been previously observed in neurons of rat olfactory bulb (Kosaka et al., 1985; Gall et al., 1987). Subsequently, we also investigated the eventuality of coexpression of somatostatin and adrenergic properties in our in vitro model systems. Such a coexistence has been largely documented in other vertebrates (for review see Schultzberg et al., 1982), even at early steps of neuronal development (Maxwell and Sietz, 1985; Garcia-Arraras et al., 1986). By means of double-labeling with specific immunocytochemical markers (two polyclonal antibodies directed respectively against GABA and somatostatin and a monoclonal antibody directed against tyrosine hydroxylase), we have obtained clear evidence that there is no colocalization of any two of the neuroactive substances thus defined within the same neuron, whether it originates from NP or from NF. In the same way, using a specific marker for the whole neuronal catecholaminergic population (monoclonal antibody directed against TOH) and a specific marker for the noradrenergic neuronal population (polyclonal anti-dopamine P-hydroxylase antibody), we demonstrated the existence, both
in NP and NF cultures, of two separate laminergic lineages: a noradrenergic dopaminergic neuronal population.
catechoand a
Materials and methods
Animals Pleurodeles waltlii neurulae (stage 13 according to the table of Gallien and Durocher, 1957) were the source of cultured embryonic cells. Cell cultures Jelly was removed with fine forceps. The entire neurectoderm (NP and NF) or each component (NP or NF) separately, was excised with or without the underlying chordamesoderm as previously described (Duprat et al., 1984) (Fig. 1). Explants were then dissociated in Ca*+/Mg*+-free Barth’s medium (88 mM NaCl, 1 mM KCI, 2.4 mM NaHCO,, 2 mM Na,HPO,, 0.1 mM KH,PO, 0.5 mM EDTA, pH 8.7), and isolated cells were cultured at 20°C in Barth’s defined saline medium (Barth and Barth, 1959) supplemented with 1 mg/ml BSA (Sigma) and antibiotics. Cells were on plastic or plated (1.2 x lo5 cells/well) collagen-coated glass coverslips in four multiwell dishes or 35-mm diameter Petri dishes (Nunc). After the cells had spread (48 h), the medium was replaced by fresh Barth’s medium without BSA, and the cultures were maintained until complete cell differentiation, for 2-3 weeks (Duprat et al., 1984). immunocytodetection Antibodies. Monoclonal antibody RT 97 directed against the phosphorylated 200 kDa neurofilament subunit (Anderton et al., 1982) was provided by Dr. J. Wood (Sandoz Institute for Medical Research, London). The visualization of tetanus toxin binding sites was performed as previously described (Duprat et al., 1986) using the tetanus toxin and the rabbit anti-tetanus toxin antibody kindly provided by Dr. B. Bizzini (Institute Pasteur, Paris). Tyrosine hydroxylase (TOH) was evidenced using a monoclonal antibody raised against the quail enzyme, which also recognizes TOH from
73
I Fig. 1. Procedure
for cell cultures. chordamesodermal
II
III
(I) Cocultures (NP+NF+CM): neural plate plus neural fold cells were cocultured cells. (II) Isolated neural plate cells (NP). (III) Isolated neural fold cells (NF).
chick and pleurodele (Fauquet and Ziller, 1989) provided by Dr. M. Fauquet (Institut d’ Embryologie, Nogent/ Marne, France). Polyclonal antibody to dopamine /3-hydroxylase was obtained from Dr. D. Aunis (INSERM-U 44, Centre Neurochimie, Strasbourg, France). Somatostatin was visualized using a polyclonal antiserum provided by Dr. P. Brazeau (University Montreal, Canada) that reacts with the cyclic form of the peptide. GABA was detected using a specific polyclonal antibody raised against conjugated GABA (Pituello et al., 1989b) from Dr. M. Geffard (Institut de Neurochimie CNRS, Bordeaux, France). The corresponding secondary antibodies used were goat anti-rabbit-Ig, rabbit anti-sheep Ig, goat anti-mouse Ig conjugated with fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate (Nordic, Tilburg, The Netherlands; Immunotech, Marseille, France).
Immunocytochemistry Double labeling.
with
the
As previously described (Pituello et al., 1989a, b), in normal conditions, TOH-, somatostatinand GABA-like immunoreactivities were clearly but faintly discernible in neurons differentiated in vitro. To investigate a possible coexpression of two neuroactive substances using double labeling, it was necessary to improve the best conditions available for immunovisualization. Thus, in all cases the cellular contents of neurotransmitter-related molecules were increased using specific procedures. For TOH detection, cultures were first pretreated for 5 h with 10e5 M reserpine (Joh et al., 1973‘). Likewise, to enhance visualization of somatostatin, cultures were pretreated with colchicine (5 . JOT3 M) overnight. GABA uptake, performed by incubating the cultures for 5 h with 10e5 M GABA (Pituello et al., 1989b), was used to enhance visualization of GABAergic neurons. In our cultures, after such
74
75
treatment, the GABA immunoreactive neuronal population was not quantitatively different from that evidenced after immunostaining of endogenous GABA using intensification procedures such as the biotin-streptavidin technique (data not shown). After careful washing, cultures were fixed in 3.5% formaldehyde in Barth’s medium for 30 min at 20°C and then washed in Barth’s medium. Cells were permeabilized by treating with methanol at - 10°C for 6 min and then with 0.25 Triton X-100 in Barth’s solution containing 1% dry skim-milk for 2 min. After three thorough washings, cells were incubated overnight with TOH monoclonal antibody at 4°C and revealed with appropriate GAM-FITC or TRITC antibodies for 30 min at 2O’C. The cultures were then washed and incubated with polyclonal antibodies directed against GABA, somatostatin or dopamine p-hydroxylase for 60 min at 20°C before being incubated in appropriate GAR-TRITC or FITC antibodies for 30 min at 20°C. After rinsing and mounting in Mowiol 4-88, the cultures were observed with a Leitz Dialux microscope equipped with appropriate I, and N, filters. At least ten cultures were examined for each set of experiments. Controls. In all sets of experiments, immunoreactivity was abolished when the primary antibodies were either omitted or replaced (for polyclonal antibodies) by normal rabbit serum. In all immunolabeling experiments, non-neuronal cells originating from neurectoderm were negative.
Fig. 2. Non-coexistence of TOH-LI and GABA-LI in neuroblasts differentiated 14 days in vitro. (a) TOH-LI visualized in lCday-old cocultures. In a large neuronal aggregate only two cells expressed TOH-LI (arrow head). (b) GABA-LI neurons dispersed in an immunonegative neuronal cluster in cocultures. Cell bodies (arrow head) and neuritic processes (thin arrow) are fluorescent. (c and d) Double immunocytochemical labeling with the monoclonal TOH antibody (c) and the polyclonal antibody against GABA performed in cocultures (d). The neuronal subpopulation expressing GABA-LI is TOH immunonegative. Bar: 10 pm.
Results Colocalization of TOH and GABA TOH, the first enzyme in the pathway leading to catecholamine biosynthesis is considered to be a specific marker for catecholaminergic differentiation (Berod et al., 1982). TOH immunoreactivity is not detectable in neurectodermal cells, in the early neurula although TOH activity can just be detected using biochemical assays followed by HPLC and electrochemical detection (Boudannaoui et al., unpublished data). TOH-immunoreactive neurons are first faintly visible in 3-day-old differentiated cultures; fluorescence intensity increases over time (Fig. 2a). However, only a subset (2%) of the differentiated neuronal population is TOH-positive. Faint GABA immunoreactivity is also detected in a neuronal subpopulation (6% of the neuronal population) after 3 days in culture. A bright staining is observed in older cultures, both in the cell body and the neurites (Fig. 2b). In order to determine whether TOH and GABA coexist in certain neurons, double immunocytochemical labeling was performed as described in Materials and Methods. In all the culture types analyzed, i.e., cocultures, NP or NF cell cultures, the neuronal subpopulations expressing TOH and GABA were found to be totally distinct and nonoverlapping (Fig. 2c and d). These results were fully confirmed in cultures in which cells were stained first with the polyclonal antibody against GABA and then with the monoclonal TOH antibody. Colocalization of TOH and somatostatin We previously reported (Pituello et al., 1989a) that somatostatin immunoreactivity is undetectable at the early neurula stage by radioimmunoassay as well as by immunocytochemistry. However, somatostatin is present in 3-day-old cultures and strongly immunoreactive neurons can be observed later, either in small neuronal aggregates (Fig. 3a) or scattered through the culture. The somatostatinergic phenotype is expressed in a small subpopulation of cells (less than 1% of the neuronal population).
76
The possibility that TOH and somatostatin are coexpressed in same neurons was investigated. No doubly labeled cells were ever found in any of the experiments (Fig. 3c, d and e).
Coexpression of TOH and D/3H Dopamine P-hydroxylase (DPH) is the enzyme catalysing the synthesis of noradrenaline and is consequently a specific marker of noradrenergic metabolism (Lundberg et al., 1977). The simultaneous presence of TOH and Dj3H in the same neuron thus defines its phenotype as noradrenergic (Berod et al., 1982) whereas the presence of TOH alone, without D/3H, is indicative of the dopaminergic phenotype of the neuron. Double-labeling experiments were performed to study the characteristics of the catecholaminergic cell population that differentiated in vitro from cells taken at the early neurula stage (Boudannaoui et al., unpublished data) and to look for the possible presence of two different subpopulations. As shown in Fig. 4, certain neurons express TOH and not DBH (Fig. 4a, b and c) whereas others exhibit both TOH and DPH immunoreactivity (Fig. 4d, e and f). These data indicate here again the emergence of two other defined neuronal subpopulations, dopaminergic and noradrenergic. In all cases, the neuronal nature of TOH-, GABA-, DflH-, somatostatin-positive cells was confirmed using specific neuronal markers such as neurofilament polypeptides or tetanus toxin binding sites in agreement with our previous results (Duprat et al., 1986, 1987; Saint-Jeannet et al., 1989a; Huang et al., 1990).
Fig. 3. Absence of colocalization for Somatostatin-LI (SLI) and TOH-LI in 14-day-old cocultures. (a and b) SLI visualized in a neuronal aggregate. Nerve cell bodies (thick arrow) and their neurites (arrow head) are immunopositive (a). Phase contrast (b). (c, d and e) Double immunostaining with the monoclonal anti-TOH followed by the polyclonal antibody against somatostatin. A TOH-positive neuron in a small cluster (c). The same cell does not express SLI (d). Phase contrast (e). Bar: 10 pm.
Discussion During the past 10 years, it has become well established that many neuronal cells contain neurotransmitter (catecholaa ‘small-molecule’ mines, acetylcholine, GABA, serotonin) in addition to a neuropeptide (somatostatin, substance P, VIP,. . .) in both central and peripheral nervous systems (Schultzberg et al., 1982; Belin et al., 1983; Bohn et al., 1984; Cournil et al., 1984; Kosaka et al., 1985; Potter et al., 1986; Leblanc et al., 1987; Ottersen et al., 1988; Saland et al., 1988; Mize, 1989). Evidence has also been obtained demonstrating a similar coexpression in vitro in certain neurons derived from neural crest that differentiate in culture (Maxwell et al., 1984, 1985; Garcia-Arraras et al., 1986; Xue et al., 1987; Fauquet and Ziller, 1989). On the other hand, the absence of such colocalization was also documented in embryonic cells from mouse and Drosophila. Buse (1987) reported that ventricular cells from the neural plate of mouse (stage 12-Theiler) differentiated in vitro into distinct neuronal cell classes, without any colocalization of transmitters. An analogous early emergence of serotoninergic and dopaminergic neuronal subpopulations was recently reported in Drosophila (Huff et al., 1989). Considering the initial expression of neurotransmitters and neuropeptides in early neurogenesis in amphibian embryos, we recently pointed out that as early as at the late gastrula-early neurula stage, some embryonic neuroblasts from NP or NF have already acquired potentialities to differentiate in vitro into neurons (Duprat et al., 1986; Saint-Jeannet et al., 1989a) with specific choline& (Duprat et al., 1985a, b, 1990b), catecholaminergic (Boudannaoui et al., unpublished data), GABAergic (Pituello, 1989b) or peptidergic (Pituello et al., 1989a) traits. Mechanisms for functional biosynthesis, accumulation, release, uptake and degradation develop without any further influence of chordamesoderm. With regard to each phenotype considered, we showed that only a subset of the neuronal population was concerned. In this context it was of particular interest to determine whether as a con-
78
19
sequence of neural induction, these embryonic neuroblasts are committed to the neuronal pathway with a status allowing the coexpression of different neurotransmitter and/or neuropeptides, or with a status allowing the expression of one transmitter. The data presented here, obtained by means of double immunocytochemical detection with a monoclonal TOH-antibody and polyclonal antibodies, respectively, against GABA, somatostatin or D/3H, show that neurectodermal cells isolated in vitro at the late gastrula-early neurula stage, develop neurochemically distinct neuronal traits. These experiments demonstrated that GABA-like immunoreactivity develops in a subpopulation of neurons. This development occurs under the same conditions that are permissive for the development of somatostatin-containing neurons and dopamine-containing neurons or noradrenaline-containing neurons. No coexistence of neurotransmitters and neuropeptides was ever found in neuronal subpopulations arising in vitro from NP or NF of early amphibian neurulae. The same result was obtained when the neurons were cocultured with chordamesodermal cells indicating that the CM has no qualitative influence on these phenotypes expressed in culture. In conclusion, the early emergence of distinct neuronal subpopulations as a consequence of neural induction, together with recent data on neural potentialities of presumptive ectoderm (SaintJeannet et al., 1989b, 1990; Duprat et al., 1990a) underline once again that the neural precursor population at the gastrula stage most probably does not constitute a homogeneous set of cells.
Fig. 4. Evidence for two distinct catecholaminergic subpopulations in 14-day-old cocultures. (a, b and c) Dopaminergic neurons: in the large cellular cluster visualized in phase contrast (c), some neurons display a brillant immunostaining with the monoclonal antibody anti-TOH (a) but are negative with the polyclonal immunoserum against DBH. (d, e and f) Three neurons (thin arrow) defined as noradrenergic neurons by the double labeling with anti-TOH (a) and anti-DfiH (e) antibodies. Phase contrast (f). Bar: 10 pm.
The authors would like to thank Dr. P. Kan for her expert assistance in cell culture. Mrs C. Daguzan and C. Mont are gratefully acknowledged for photographic and secretarial help and Dr. J. Smith (Nogent/Marne) for reviewing the English manuscript. This work was supported by CNRS, MRT, EEC and CNES grants.
References Anderton, BH., Breinburg, D., Downes, M.J., Green, P.J., Tomlinson, B.E., Uhich, J., Wood, J.N. and Kahn, J.: Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants. Nature 298, 84-86 (1982). Barth, L.G. and Barth, L.J.: Differentiation of cells of Ram pipiens gastrula in unconditioned medium. J. Embryol. Exp. Morphol. 7, 210-222 (1959). Belin, M.F., Nanopoulos, D., Didier, M., Aguera, M., Steinbusch, H., Verhofstad, A., Maitre, M. and Pujol, J.F.: Immunohistochemical evidence for the presence of yaminobutyric acid and serotonin in one nerve cell. A study on the Raphe Nuclei of the rat using antibodies to glutamate decarboxylase and serotonin. Brain Res. 215, 329-339 (1983). Berod, T., Hartman, B.K., Keller, A., Joh, T.H. and Pujol, J.F.: A new double-labeling technique using tyrosine hydroxylase and dopamine /%hydroxylase immunohistochemistry: evidence for dopaminergic cells lying in the pons of the beef brain. Brain Res. 240, 235-243 (1982). Buse, E.: Ventricular cells from the mouse neural plate, stage Theiler 12, transform into different neuronal cell classes in vitro. Anat. Embryol. 176, 295-302 (1987). Bohn, M.C., Kessler, J.A., Adler, J.A. Markey, K., Goldstein, M. and Black, I.B.: Simultaneous expression of the SPpeptidergic and noradrenergic phenotypes in rat sympathetic neurons. Brain Res. 298, 378-381 (1984). Coumil, I., Geffard, M., Moulins, M. and Le Moal, M.: Coexistence of dopamine and serotonin in an identified neuron of the lobster nervous system. Brain Res. 310, 397-400 (1984). Duprat, A.M., Gualandris, L., Kan, P. and Foulquier, F.: Early events in the neurogenesis of amphibians. In: The role of cell interactions in early neurogenesis, Eds. A.M. Duprat, A. Kato, M. Weber, NATO AS1 Series, Life Sci. 77, 3-20 (1984). Duprat, A.M., Kan, P., Foulquier, F. and Weber, M.: In vitro differentiation of neuronal precursor cells from amphibian late gastrulae: morphological, immunocytochemical studies, biosynthesis, accumulation and uptake of neurotransmitters. J. Embryol. Exp. Morphol. 86, 71-87 (1985a).
80 Duprat, A.M., Kan, P., Gualandris, L., Foulquier, F., Marty, J., Weber, M.: Neural induction embryonic determination elicits full expression of specific neuronal traits. J. Embryo]. Exp. Morphol. 89, 167-183 (1985b). Duprat, A.M., Gualandris, L., Foulquier, F., Paulin, D., Bizzini, B.: Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians. Cell Differ. 18, 57-64 (1986). Duprat, A.M., Gualandris, L. Pituello, F., Saint-Jeannet, J.P., Boudannaoui, S.: S. Neural induction. Biol. Struct. Morhogen. (ex. Arch. Anat. Microsc. Morpbol., Exp.), 75, 211-227 (1987). Duprat, A.M., Saint-Jeannet, J.P., Pituello, F., Huang, S., Boudannaoui, S., Kan, P., Gualandris, L.: From presumptive ectoderm to neural cells in an amphibian. Int. J. Dev. Biol., 34, 149-156 (1990a). Duprat, A.M., Kan, P., Weber, M.: Initial expression of choline& traits in embryonic amphibian neuronal cells choline acetyltransferase and acetylcholinesterase activities. Ontogenez (1990b, in press). Fauquet, M. and Ziller, C.: A monoclonal antibody directed against quail tyrosine hydroxylase description and use in immunocytochemical studies on differentiating neural crest cells. J. Histochem. Cytochem., 37, 1197-1205 (1989). Fisher, R.S., Buchwald, N.A., Hull, C.D., Levine, M.S.: GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons. J. Comp. Neurol. 272, 489-502 (1988). Furshpan, E.J., McLeish, P.R., O’Lague, P.H., Potter, D.D.: Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for choline@, adrenergic and dual function neurons. Proc. Natl. Acad. Sci. USA 73, 4225-4229 (1976). Gall, CM., Hendry, S.H.C., Seroogy, K.B., Jones, E.G. and Haycock, J.W.: Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb. J. Comp. Neurol. 266, 307-318 (1987). Gallien, L. and Durocher, M.: Table chronologique du developpement chez Pleurodeless waltlii Michah. Bull. Biol. Fr. Belg. 91, 97-114 (1957). Garcia-Arraras, J.E., Fauquet, M., Chanconie, M. and Smith, J.: Coexpression of somatostatin-like immunoreactivity and catecholaminergic properties in neural crest derivatives: comodulation of peptidergic and adrenergic differentiation in cultured neural crest. Dev. Biol. 114, 247-257 (1986). Huang, S., Saint-Jeannet, J.P., Kan, P. and Duprat, A.M.: Extracellular matrix: an immunological and biochemical (CAT and TOH activity) survey of in vitro differentiation of isolated amphibian neuroblasts. Cell Differ. Dev. 30, 219-233 (1990). Huff, R., Furst, A. and Mahowald, A.P.: Drosophila embryonic neuroblasts in culture: autonomous differentiation of specific neurotransmitters. Dev. Biol. 134, 146-157 (1989). Joh, T.H., Geghman, C. and Reis, D.S.: Immunochemical demonstration of increased accumulation of tyrosine hy-
droxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine. Proc. Natl. Acad. Sci. USA 70, 2767-2771 (1973). Kosaka, T., Hataguchi, Y., Hama, K., Nagatsu, I. and Wu, J.Y.: Coexistence of immunoreactivities for glutamate decarboxylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: possible coexistence of y-aminobutyric acid (GABA) and dopamine. Brain Res. 343, 166-171 (1985). Kosaka, T., Tauchi, M. and Dahl, J.L.: Choline& neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivites in various brain regions of the rat. Exp. Brain Res. 70, 605-617 (1988). Leblanc, G.G., Trimmer, B.A. and Landis, S.C.: Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with radioactive intestinal peptide and choline acetyltransferase. Proc. Nat]. Acad. Sci. USA 84, 3511-3515 (1987). Lundberg, J., Bylock, A., Goldstein, M., Hansson, H.A. and Dahlstrom, A.: Ultrastructural localization of dopaminefi-hydroxylase in nerve terminals of the rat brain. Brain Res. 120: 549-552 (1977). Maxwell, G.D. and Sietz, P.D.: Development of cells containing catecholamines and somatostatin-like immunoreactivity in neural crest cultures: relationships of DNA synthesis to phenotypic expression. Dev. Biol. 108, 203-209 (1985). Maxwell, G.D., Sietz, P.D. and Jean, S.: Somatostatin-like immunoreactivity is expressed in neural crest cultures. Dev. Biol. 101, 357-366 (1984). Melander, T., Hokfelt, T., Rokaeus, A. and Cuello, A.C.: Coexistence of galanin-like immunoreactivity with catecholamines, S-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J. NeuroSci. 6, 3640-3653 (1986). Mize, R.R.: Enkephalin-like immunoreactivity in the cat Superior coiiiculw distribution, ultrastructure and colocalization with GABA. J. Comp. Neurol. 285, 133-155 (1989). Moriss, J.Y. and Gibbins, H.: Neuronal colocalization of peptides, catecholamines, and catecholamine-synthesizing enzymes in guinea pig paracervical ganglia. J. NeuroSci. 7, 3117-3130 (1987). Ottersen, O.P., Storm-Mathisen, J. and Somogyll, P.: Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res. 450, 342353 (1988). Pearson, J.C. and Jennes, L.: Localization of serotoninand substance P-like immunofluorescence in the caudal spinal trigemical nucleus of the rat. NeuroSci. Lett. 88, 151-156 (1988). Pituello, F., Deruntz, P., Pradayrol, L. and Duprat, A.M.: Peptidergic properties expressed in vitro by embryonic neuroblasts after neural induction. Development 105, 529-540 (1989a). Pituello, F., Kan, P., Geffard, M. and Duprat, A.M: Initial GABAergic expression in embryonic amphibian neuroblasts after neural induction. hit. J. Dev. Biol. 33, 445-453 (1989b).
81 Potter, D.D., Landis, S.C., Matsumoto, S.G. and Furshpan, E.J.: Synaptic functions in rat sympathetic neurons in microcultures. II. Adrenergic/cholinergic dual status and plasticity. J. NeuroSci. 6, 1080-1098 (1986). Saint-Jeannet, J.P., Foulquier, F., Goridis, C. and Duprat, A.M.: Expression of N-CAM precedes neural induction in Pleurodeles walfl (Urodele, Amphibian). Development 106, 675-683 (1989a). Saint-Jeannet, J.P., Huang, S. and Duprat, A.M.: Target cell contacts and neural commitment in Pleurodeles waltlii. Cell Differ. Dev. 27 (suppl.), 52 (1989b). Saint-Jeannet, J.P., Huang, S. and Duprat, A.M.: Modulation of neural commitment by changes in target cell contacts in Pleurodeles w&iii. Dev. Biol. 141, 93-103 (1990).
Saland, L.C., Wallace, J.A., Samora, A. and Gutierrez, L.: Colocalization of tyrosine hydroxylase (TH)- and serotonin (5-HT)- immunoreactive innervation in the rat pituitary gland. NeuroSci. Lett. 94, 39-45 (1988). Schultzberg, M., Hokfelt, T. and Lundberg, J.M.: Coexistence of classical transmitters and peptides in the central and peripheral nervous system. Br. Med. Bull. 38, 309 (1982). Xue, Z.G., Smith, J. and Le Douarin, N.: Developmental capacities of avian embryonic dorsal root ganglion cells: neuropeptides and tyrosine hydroxylase in dissociated cell cultures. Dev. Brain Res. 34, 99-109 (1987). Yazulla, S. and Yang, C.Y.: Colocalization of GABA and glycine immunoreactivities in a subset of retinal neurons in tiger salamander. NeuroSci. Lett. 95, 37-41 (1988).
