Release of glutamate by the embryonic spinal motoneurons of rat positively regulated by acetylcholine through the nicotinic and muscarinic receptors.

It has been shown that mature neurons in adult vertebrates can co-express glutamate and acetylcholine. Furthermore, interactions at the synaptic level have been demonstrated. In a previous study we found that also motoneurons at early embryonic stages, thus well prior to synapse formation, release acetylcholine, and that glutamate increases this release. We now report the existence of a glutamate release from embryonic motoneurons and the increase of glutamate release by acetylcholine. This effect is mediated by nicotinic and muscarinic cholinergic receptors present on embryonic motoneurons. Using conditions of partial or total depletion of calcium, we show that the glutamate release has two components: one is calcium-dependent and the other calcium-independent. Furthermore, we show that extracellular glutamate can be taken up by motoneurons, probably via the neuronal glutamate transporter EAAC1, which we find to be expressed at this stage. Monitoring of the glutamate release kinetics showed that extracellular glutamate concentration reached a steady-state level, strongly suggesting the establishment of equilibrium between glutamate release and uptake. Altogether, these results support the idea that glutamate can act as a neurotransmitter in embryonic motoneurons. We hypothesise that, glutamate acts as a regulator of motoneuron maturation and spinal cord development.

Short- and long-chain natural polyamines play specific roles in adult cricket neuroblast proliferation and neuron differentiation in vitro.

In the house cricket (Acheta domesticus) mushroom bodies, neurogenesis still occurs during adulthood. Using in vitro approaches, the respective roles of natural polyamines in neurogenesis were examined. Mushroom body neuroblast proliferation was assayed in organotypic culture using 5-bromo, 2'-deoxyuridine labeling. The number of labeled cells was significantly increased when putrescine was added to culture medium, whereas spermidine and spermine supplementation did not alter cell proliferation. Conversely, in vitro morphometric studies on mushroom body neurons cultured in a defined medium showed that putrescine addition failed to alter any morphological character of these interneurons, whereas addition of the long-chain polyamines, spermidine and spermine, stimulated neuron differentiation. These two polyamines significantly increased total neurite length; moreover, spermidine-treated cells exhibited more branches than the controls. The present data demonstrate that putrescine has a mitogenic effect on mushroom body neuronal precursors, and that spermidine and spermine, which failed to induce neuroblast proliferation, act on neuronal differentiation, inducing neurite outgrowth. Our results indicate that short- and long-chain polyamines play specific roles during neurogenesis, and provide a basis for further studies on neuronal precursor proliferation and differentiation.

Acetylcholine secretion enhanced by glutamate in rat embryonic spinal motoneurons: respective involvement of NMDA and AMPA receptors.

The spontaneous acetylcholine secretion and endogenous acetylcholine content were measured by means of chemiluminescent assay from isolated embryonic rat spinal motoneurons. The sensitivity of the detection allows to study the kinetics of the acetylcholine secretion with short time intervals. Following the demonstration of the presence of acetylcholine and glutamate in embryonic motoneurons, the aim of this work was to study the characteristics of acetylcholine secretion and the effect of glutamate in its modulation. The involvement of NMDA and AMPA glutamatergic receptors was mainly studied. Our data show that spontaneously acetylcholine secretion, is not calcium-dependent and is significantly enhanced by glutamate (1 mM). Pharmacological approaches show that glutamate effect on acetylcholine secretion is decreased in presence of APV (50 microM and 100 microM), or in presence of GYKI 53655 (10 microM), demonstrating that both NMDA and AMPA receptors are present at the membrane of embryonic spinal motoneurons and involved in the modulation of acetylcholine secretion. Presence of glutamate in the embryonic motoneuron and secretion may represent a mechanism of control of extracellular acetylcholine concentration, which was shown to control neuritic growth at early embryonic stage.

Dual effect of ecdysone on adult cricket mushroom bodies.

Mushroom bodies, which are the main integrative centre for insect sensorial information, play a critical role in associative olfactory learning and memory. This paired brain structure contains interneurons grouped in a cortex, sending their axons into organized neuropiles. In the house cricket (Acheta domesticus) brain, persistent neuroblasts proliferate throughout adult life. Juvenile hormone (JH) has been shown to stimulate this proliferation [Cayre, M., Strambi, C. & Strambi, A. (1994) Nature, 368, 57-59]. In the present study, the effect of morphogenetic hormones on mushroom body cells maintained in primary culture was examined. Whereas JH did not significantly affect neurite growth, ecdysone significantly stimulated neurite elongation. Moreover, ecdysone also acted on neuroblast proliferation, as demonstrated by the reduced number of cells labelled with 5-bromodeoxyuridine following ecdysone application. Heterospecific antibodies raised against ecdysone receptor protein and ultraspiracle protein, the two heterodimers of ecdysteroid receptors, showed positive immunoreactivity in nervous tissue extracts and in nuclei of mushroom body cells, indicating the occurrence of putative ecdysteroid receptors in cricket mushroom body cells. These data indicate a dual role for ecdysone in adult cricket mushroom bodies: this hormone inhibits neuroblast proliferation and stimulates interneuron differentiation. These results suggest that a constant remodelling of mushroom body structure could result from physiological changes in hormone titres during adult life.

Down-regulation of striatin, a neuronal calmodulin-binding protein, impairs rat locomotor activity.

Striatin, an intraneuronal, calmodulin-binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein-protein interactions, suggesting a role within a dendritic Ca(2+)-signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A-ODN) or mismatch ODN (M-ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A-ODN-treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A-ODN-treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down-regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A-ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines.

Influence of acetylcholinesterase on embryonic spinal rat motoneurones growth in culture: a quantitative morphometric study.

Rat spinal motoneurones sampled at day embryonic 15 were purified using a Nycodenz gradient and cultured in defined medium, during 7 days, on glass coverslips coated with poly-L-lysine and laminine. Purified acetylcholinesterase (AChE), ecothiopate, BW 284C51 and fasciculin II, inhibitors of either the catalytic or peripheral site of AChE, were added to the defined medium. Morphological changes of spinal motoneurones were measured using a statistical quantitative morphometric method, allowing the determination of various parameters such as the number of neurites and bifurcations, the length of neurites, the surface and spreading index. Presence of AChE in the medium (4 units/mL) increases the surface and the total length of neurites and axons without any change in the spreading index. When spinal motoneurones were cultured on AChE coated substrate, neurones rapidly migrate and form clusters. Addition of ecothiopate (10(-6) M) in the medium, which selectively blocks the catalytic site of AChE, leads to a slight increase in the number of primary neurite and a decrease of the spreading index during the three first days in culture. BW 284C51 (10(-5) M) which blocks the catalytic site but also affect the peripheral one, significantly reduces the number of primary neurites and increases the number of bifurcations. Fasciculin II, a potent blocker (10(-9)M) of the AChE peripheral site induces a decrease of both primary neurites and bifurcations with a significant increase of the length and growth velocity of the axon, giving a drastic enhancement of the spreading index. These phenomena are discussed in terms of catalytic and non-catalytic function of AChE, including the involvement of the enzyme in adhesive processes, occurring during growth and differentiation of spinal motoneurones.

An avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitro.

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37 degrees C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.

Predicted acetylcholine layer around embryonic motoneurones.

The secretion of acetylcholine (ACh) from cultures of dissociated rat purified embryonic motoneurones into the medium was measured at different times using a chemiluminescent assay. From these results it is proposed that a motoneurone is able to generate an extracellular ACh layer surrounding its membrane. This layer is expected to modulate the ACh concentration at the membrane in a range where ACh receptors can be sensitive, and thus to affect the response of the motoneurone. The influences of both membrane-bound and soluble-secreted acetylcholinesterase (AChE) were simulated and a local ACh membrane efflux was deduced. Since ACh usually exerts an inhibitory effect on neurite outgrowth, a possible autocrine influence of the ACh layer in motoneurone morphogenesis is discussed.

NCAM-fibronectin-type-III-domain substrata with and without a six-amino-acid-long proline-rich insert increase the dendritic and axonal arborization of spinal motoneurons.

The neural cell adhesion molecule (NCAM) is a modulator of neurite outgrowth in vitro and in vivo. To see if single or tandem extracellular NCAM domains can influence neurite outgrowth, motoneurons from embryonic rat spinal cord were cultured on several NCAM fusion protein substrata. Motoneurons growing on either of two fusion proteins comprising the combined two fibronectin type III homology domains of NCAM with or without a six-amino-acid-long, proline-rich insert (F3I,II+ and F3I,II, respectively) usually developed three or more neurites per cell. Motoneurons grown on NCAM-immunoglobulin domain I (IgI), by contrast, developed many unipolar and bipolar cells, a situation also seen when motoneurons were cultured on control substrata. The neuritic trees of motoneurons grown on F3I,II and F3I,II+ appeared broader and rounder than motoneurons cultured on either control or IgI substrata, and the spreading indices of motoneurons grown on F3I,II and F3I,II+ were significantly lower than when the other substrata were used. Neither of the NCAM-F3 fusion proteins stimulated the outgrowth of single neurites. By contrast, IgI substratum was able to stimulate neurite outgrowth over control substrata. Both NCAM-F3 substrata induced branches in axons and dendrites, whereas IgI substratum did not affect neurite branching significantly. These data indicated that neurite outgrowth and neurite branching on the chosen substrata were not closely linked to each other. Furthermore, the branching characteristics of motoneuron neurites potentially depend on their differentiation states and, possibly, on the conformation of the two NCAM-F3 domains.

Synthesis and release of an acetylcholine-like compound by human myoblasts and myotubes.

1. Exogenously applied acetylcholine (ACh) is a modulator of human myoblast fusion. Using a chemiluminescent method, we examined whether an endogenous ACh-like compound (ACh-lc) was present in, and released by, pure human myogenic cells. 2. Single, freshly isolated satellite cells and proliferating myoblasts contained 15 and 0.5 fmol ACh-lc, respectively. Cultured myotubes contained ACh-lc as well. Also, ACh-like immunoreactivity was detected in all myogenic cells. 3. Part of the ACh-lc was synthesized by choline acetyltransferase (ChAT), as indicated by the reduction of ACh-lc content when bromoACh was present in the culture medium, and by direct measurements of ChAT activity. Also, ChAT-like immunoreactivity was observed in all myogenic cells. 4. Myoblasts and myotubes released ACh-lc spontaneously by a partially Ca(2+)-dependent mechanism. 5. The application by microperfusion of medium conditioned beforehand by myoblasts (thus presumably containing ACh-lc) onto a voltage-clamped myotube induced inward currents resembling ACh-induced currents in their kinetics, reversal potential, and sensitivity to nicotinic antagonists. 6. In vitro, the spontaneously released ACh-lc promoted myoblast fusion but only in the presence of an anticholinesterase. 7. Our observations indicate that human myogenic cells synthesize and release an ACh-lc and thereby promote the fusion process that occurs in muscle during growth or regeneration.

Enhanced chemiluminescent assays for acetylcholine.

Acetylcholine and choline chemiluminescent assays have limitations when these compounds are detected in small areas of mammalian nervous tissue. Use of 7-dimethyl-aminonaphthalene-1,2-dicarbonic acid hydrazide (7-DMAN), instead of luminol, gives a threefold increase in emitted light in the chemiluminescent assay for acetylcholine based on the coupled choline oxidase-peroxidase reaction. Addition of light enhancers, such as para-iodophenol or D-luciferin, to luminol or 7-DMAN further increased the light emission. Under these conditions the detection limit for acetylcholine was 650 femtomoles. This enhanced chemiluminescent assay should be convenient for the detection of in vivo and in vitro acetylcholine release from mammalian neurons.

High-sensitive chemiluminescent assay for cholesterol.

Chemiluminescent measurement of cholesterol can be performed in various biological tissues and fluids. The method described in this study has a sensitivity of 54 pmol. The tissue samples used for the determination of cholesterol can be reduced to as little as 1 mg and assay can be performed on diluted biological fluids, allowing sampling of plasma or serum as little as 5 microliters. Cholesterol is solubilized in sodium cholate and aliquots are added to a reaction mixture containing cholesterol oxidase, luminol and peroxidase. Cholesterol oxidase, in the presence of cholesterol yields H2O2 which produces light in presence of luminol and peroxidase. Emitted light is quantified at a wavelength of 420 nm by means of a photomultiplier. Optimal conditions of the assay were determined and examples of cholesterol determinations, in blood plasma and nervous tissues, are presented.

Culture of hypoglossal cells, dissociated from foetal and new-born rats.

Primary cultures of dissociated cells from brainstem cranial nuclei have not been described in the literature. The present paper shows that dissociated rat posterior brainstem cells, as well as cells from the hypoglossal nucleus, taken from both foetal and postnatal animals, can be maintained in long-term culture. This can be achieved by using a DMEM/F-12 medium with defined supplements, with or without foetal calf serum. Under such conditions, the growth of neuritic processes as well as the formation of neural networks can be observed. The different cell types present in the cultures can be identified by immunohistochemistry with antibodies raised against neuron specific enolase, glial fibrillary acidic protein, galactocerebroside and choline-acetyl transferase. As previously demonstrated for other brain-dissociated neurones maintained in cultures, the use of molecules, involved in cell adhesive mechanisms, can modify the morphological properties of the growing cells. This was particularly observed when poly-L-lysine and laminin were used as substrata.

Influence of tongue myoblasts on rat dissociated hypoglossal motoneurons in culture.

Hypoglossal motoneurons of 1-2 day-old newborn rats were retrogradely labelled following an injection of fluorescent latex microspheres and carbocyanines. Motoneurons were identified among the cell population of the hypoglossal nuclei during the dissociation and culture procedures. DiI labelled motoneurons could be maintained in culture on poly-L-lysine coating and Dulbecco Minimum Essential Medium with Ham F12 complement, supplemented with additives and 3% fetal calf serum. Neuronal survival as well as extension of neurites, identified by their content in DiI or by the presence of choline acetyltransferase immunoreactivity, was increased in the presence of myoblastic satellite cells originating from tongue muscular explants. Co-cultures of dissociated hypoglossal cells with tongue myoblasts revealed the presence, after 10-15 days in culture, of structures morphologically similar to neuromuscular junctions. Such re-innervated muscular fibres exhibited muscular contractions which were blocked by curare and augmented by glutamate applications, demonstrating the functionality of the observed re-innervations.

Somatic Acetylcholine Release in Rabbit Nodose Ganglion.

In the rabbit, as in various other species, the presence of a cholinergic vagal afferent contingent has been demonstrated previously using biochemical and immunohistological approaches at the nodose ganglion level, where vagal afferent cell bodies are located. This structure is completely devoid of synaptic contacts. In the present study, somatic acetylcholine release is demonstrated on different types of in vitro rabbit nodose ganglion preparations (fragments of nodose tissue or isolated cell bodies) using chemiluminescent detection. Acetylcholine endogenous content was measured and was shown to be greater in the right nodose ganglion compared to the left. This difference was also observed when spontaneous and potassium chloride-evoked acetylcholine release was measured in extracellular fluid after a 15-min incubation of nodose ganglion fragments. Calcium removal totally blocked this somatic release. A kinetic study of acetylcholine release was also performed by placing the samples (nodose ganglion fragments or isolated cell bodies) directly in front of the photomultiplier, allowing the direct monitoring of (acetylcholine + choline) and choline effluxes. The net acetylcholine release was then deduced by subtraction. Identical kinetics was obtained with the two different nodose ganglion preparations used. This somatic release is calcium-dependent. The occurrence of acetylcholine release at the nodose ganglion level is discussed in comparison with the events occurring in the cholinergic nerve endings. These mechanisms could be implicated in the premodulation of the vagal afferent messages conveyed from the periphery to the central nervous system.

Dendritic processing: using microstructures to solve a hitherto intractable neurobiological problem.

In vivo, intracellular recordings of mammalian brain stem motoneurones, followed by peroxidase staining and tridimensional reconstruction, suggest that the shape of the dendritic tree plays an important role in the processing of neural information. To test this hypothesis attempts were made to guide, in culture, the growth of neuritic branches of neurones dissociated from the hypoglossal nucleus of rat brain stem. This was performed using topographical and adhesive microstructures which were designed to control the shape of the neuritic tree. Guidance of the neuritic processes can be observed with small grooves engraved on quartz and plastic substrates, and simple shapes with few processes and bifurcations on each neurite could be obtained using adhesive microstructures. These procedures, which allow the shape of a neurone to be controlled, are very promising in the study, by means of classical electrophysiological methods as well as optical recordings, of the involvement of dendritic architecture in the processing of neural information.

Substance P like-immunoreactivity release from enterochromaffin cells of rat caecum mucosa. Inhibition by serotonin and calcium-free medium.

Location, endogenous contents, and release of Substance P like-immunoreactivity were investigated in the rat caecum, using Immunohistochemistry and RadioImmunoAssay. Our immunohistochemical results indicate that Substance P was present both in the neuromuscular and mucosal compartments of this intestinal structure. However, detection of the peptide in the enterochromaffin cells of the mucosa remained very difficult. That may be explained by the very low endogenous contents of Substance P detected in the mucosa, using RIA. As we have already described a serotonin release from rat caecum mucosa, we show, now, that Substance P like-immunoreactivity may be released from the same structure. This release was stable, calcium-dependent, inhibited by serotonin, and not influenced by the chemical depolarization. Our data demonstrate an active release of Substance P like-immunoreactivity from intestinal mucosa, in the rat caecum. It seems that the endogenous pool of Substance P like-immunoreactivity is involved as a functional pool. The mechanisms responsible of this release seem to be different than that observed for the serotonin release. Substance P like-immunoreactivity may be released in precise physiological conditions, or even, in pathological conditions.

Choline uptake in cholinergic nodose cell bodies.

Isolated living cell bodies were obtained by mechanical and enzymatic dissociation from adult rabbit nodose ganglion followed by separation of fibres and cells using a Percoll gradient. A purification yield of 45% was measured. Based on previous results obtained in whole ganglion and showing the presence of cholinergic cell bodies among the afferent fibres of the vagus nerve, this preparation was used to study choline uptake by neuron cell somata. Cholinergic cells counted after choline acetyltransferase immunohistological staining showed a stained population of 2.9% among the isolated population. Two [3H]choline uptake mechanisms were detected at the cell body level. The first, with Km1 = 7 microM and Vm1 = 200 pmol/h per ganglion is sodium dependent, related to acetylcholine synthesis (43%) and has an IC50 with hemicholinium-3 equal to 50 microM. The second, with Km2 = 54 microM and Vm2 = 2235 pmol/h per ganglion is sodium independent, poorly associated to acetylcholine synthesis (12%) and exhibits an IC50 of 2 microM with hemicholinium-3. Except for their sensitivity to hemicholinium-3, the high and low affinity choline uptake mechanisms observed at the somatic level have, respectively, the same characteristics as the high and low affinity mechanisms described at the synaptic level. Their physiological role, their opposed sensitivity to hemicholinium-3 compared to the synaptic uptake systems and the relation between the somatic high affinity choline transport and an acetylcholine somatic release are discussed.

[Quantitative imaging of the heterogeneity of membrane activation of mammalian neurons and glial cells]. / Visualisation quantitative de l'hétérogénéité de l'activation membranaire des neurones et de la glie de mammifère.

By using quantitative imaging with an ultra-high sensitivity, it was possible to observe the simultaneous action of multiple patches unevenly distributed over the membranes of neurons and glial cells in culture. We used a voltage-sensitive probe to stain vitally the cells. The instrumentation consisted of a liquid-nitrogen cooled matrix of 222,530 photodetectors with a spatial resolution of 0.25 microns 2, a photodynamic range of 10(5), a detection level of a few tens of photons and a maximum time resolution of 500 microseconds. Electrical and pharmacological stimulations were applied to produce the activation of the cells which was accompanied by large variations of the level of fluorescence, giving a precise spatial localization of active domains over the soma-neuritic membranes. These images of fluorescent signals are interpreted as corresponding to the plasmalemmal localization of voltage-dependent channels. This finding, which had not been previously observed with voltage-sensitive probes in fluorescent dye imaging indicates the possibility of measuring the activity of independently functioning domains in single neurons.

Regulation of serotonin release from enterochromaffin cells of rat cecum mucosa.

The release of endogenous serotonin or previously taken up tritiated serotonin from isolated strips of rat cecum mucosa containing enterochromaffin cells was studied in vitro. Release of tritiated serotonin was increased by potassium depolarization and was decreased by tetrodotoxin, veratridine and the absence of calcium. Endogenous serotonin was released at a lower rate than tritiated serotonin; endogenous serotonin release was stimulated by potassium depolarization but was unaffected by tetrodotoxin, veratridine or the absence of calcium. Carbachol, norepinephrine, clonidine and isoproterenol decreased release of tritiated serotonin but had less or reverse effect on release of endogenous serotonin. The results suggest two different serotoninergic pools within the enterochromaffin cell population.