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.

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.

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.

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.

[Results of the surgical treatment of colorectal cancer: analysis of recurrence and survival in 400 patients]. / Resultados del tratamiento quirúrgico del cáncer colorrectal: análisis de la recurrencia y sobrevida en 400 pacientes.

We analyzed retrospectively the long term survival and recurrence of 400 patients with colorectal cancer operated in a period of 13 years. Kaplan Meier curves were used for survival analysis and Cox's regression for multivariate analysis. Ninety eight percent of 377 surviving patients were followed for a mean period of 34 +/- 36 months (range 12-156). Global recurrence was 32% and higher for rectal than colon cancer. Sixty five percent of recurrences were distant. The main prognostic parameter for recurrence was peritumoral lymph node involvement. The initial tumoral stage was the main prognostic factor for survival. Five years survival probability was 94.4% in stage A, 81.3% in stage B, 63.8% in stage C1, 41.3% in stage C2 and 3.1% in stage D. Preoperative radiation therapy did not improve survival or recurrence. Postoperative radiation therapy prolonged the lapse between surgery and recurrence, without changing overall survival. The prolonged survival of some patients in stage D justifies palliative surgery in this stage.

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.

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.

Presence of cholinergic neurons in the vagal afferent system: biochemical and immunohistochemical approaches.

The presence of cholinergic fibers in the afferent vagal system of various species was shown using biochemical and immunohistochemical methods. Biochemical activity of choline acetyl transferase, the synthesizing enzyme for acetylcholine, was detected in the nodose ganglion of cat, rabbit, dog and sheep. Immunohistochemistry, using a monoclonal antibody raised against choline acetyl transferase, revealed labelled cell bodies in the nodose ganglion of the rabbit. Acetylcholine endogenous content, measured in nodose ganglia devoid of efferent fibers, was twice as high in the right ganglion as compared to the left. Enzyme transport and choline acetyl transferase activity analysis were each determined on separate peripheral vagus nerves. These results are discussed in terms of functional properties of the vagal afferent neurons, including the modulation of vagal afferent messages at the level of the nodose ganglion and the eventual control of peripheral intrinsic neurons by sensory vagal terminals.

Cholinergic neurons in the rat nodose ganglia.

Presence of acetylcholine (ACh) in the vagal afferent fibres of the rat was investigated. In the nodose ganglion, which contains the cell bodies of this sensitive contingent, a choline acetyltransferase (ChAT) activity, a choline (Ch) uptake and an endogenous content of acetylcholine were detected. These data were confirmed by ChAT immunohistological visualization.

Distribution and quantification of 5-HT nerve cell bodies in the nucleus raphe dorsalis area of C57BL and BALBc mice. Relationship between anatomy and biochemistry.

BALBc and C57BL mice have been shown to have a different 5-HT metabolism. The present study compares the number and the distribution of 5-HT cell bodies in the nucleus raphe dorsalis area (B7 + B6) of these strains. By using 5-HT immunohistochemistry, we found a higher number of 5-HT neurons in the most caudal part of NRD (B6) of BALBc mice compared to C57BL. This difference may be correlated with a higher level of endogenous 5-HT, a higher uptake capacity toward exogenous [3H]5-HT, and a lower release of the amine in this same area of BALBc mice compared to C57BL. It could also imply a significant participation of the nerve cell bodies in the regulation of 5-HT transmission inside 5-HT nuclei.

Simultaneous visualization of aortic and [3H]5-hydroxytryptamine-accumulating cell bodies in the nodose ganglion of the cat.

1. Single- and double-tracer experiments were performed in cats to investigate the relationship between the aortic cells and the cell bodies accumulating 5-hydroxytryptamine (5-HT) in the nodose ganglion. In one series of experiments, horseradish peroxidase (HRP) was applied to the central end of the aortic nerve, anterogradely transported and accumulated in ganglionar perikarya. The distribution of HRP-positive neurones was reconstructed in serial sections through the nodose ganglion. In a second series of experiments, the distribution of [(3)H]5-HT-accumulating cell bodies was assessed following incubation of the nodose ganglion in [(3)H]5-HT. The third series of experiments combined the treatments of the preceding ones: anterograde transport of HRP in the aortic nerve followed by incubation of the nodose ganglion in [(3)H]5-HT.2. The results from these experiments provide more information with regard to (i) the topographical relationship between the aortic and [(3)H]5-HT-accumulating cell bodies in the same ganglion and (ii) the distribution and number of double-labelled neurones, giving further indications about histochemical components of the aortic nerve.3. The HRP experiments demonstrated that HRP-positive cells show a preferential pattern of topographical organization. They were mostly located in the medial border of the ganglion where the laryngeal and aortic nerves enter. On the other hand, [(3)H]5-HT-accumulating neurones were scattered throughout the ganglion.4. In double-tracer experiments, three populations of labelled cell bodies were distinguished in the same nodose ganglion: (1) single HRP-cells; (2) single [(3)H]5-HT-accumulating cells and (3) double-labelled cells. The distribution of the latter population exhibited no preferential localization. Quantitative estimates indicated that double-labelled neurones constituted 65-85% of the population of HRP-positive cell bodies.5. These results show that most aortic neurones are able to take up exogenous serotonin and may be serotonergic neurones. They suggest that serotonin may be involved in physiological effects mediated via the aortic nerves.

Serotonin-like immunoreactivity in paraffin-sections of the nodose ganglia of the cat.

The presence of serotonin (5-HT)-like immunoreactivity has been detected in ganglionar cell bodies of the nodose ganglia of the cat, using the peroxidase-antiperoxidase immunohistochemical method, applied to sections of ganglia previously embedded in paraffin. The antibodies were raised in rabbits following injection of 5-HT conjugated to bovine serum albumin, with formaldehyde as the condensation reagent. Immunoreactive cell bodies are morphologically and topographically described. The ubiquitous distribution and non-uniform labelling suggest that these 5-HT-containing vagal neurones may give rise to several functionally different 5-HT types in vagal afferent fibres.

Efferent projections of the area postrema demonstrated by autoradiography.

The efferents connections of the area postrema (AP) have been studied autoradiographically following iontophoretic injections of 3H-glycine or 3H-leucine into the area postrema. Precise control of the diffusion of the labelled amino acids injected iontophorectically into the AP was made using the technique of sectioning with a cryostat. AP projects to a great number of structures. Projections to nucleus tractus solitarius (NFS), dorsal vagal nucleus, nucleus intercalatus, nucleus praepositus hypoglossi, nucleus hypoglossal, the mesencephalic nucleus of V nerve, locus coeruleus and superior and inferior colliculi are shown bilaterally. The density of the efferents was greatest to the NFS and the LC. Corelations are suggested with functional mechanisms of cardiovascular regulation.

Localization of aortic cells in the nodose ganglion by HRP retrograde transport in the cat.

Localization of the aortic cells in the nodose ganglion was attempted in the cat using retrograde axonal transport of horseradish peroxidase (HRP). Following dissection of the aortic nerve from the vago-aortic trunk, the cut ends of the aortic fibers were immersed in a HRP solution for 23--46 h. Labelled cells were found in the nodose ganglion and were located mainly in the medial border of the ganglion.