Quantification of synapse formation and maintenance in vivo in the absence of synaptic release.

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.

Expression of stathmin and SCG10 proteins in the olfactory neurogenesis during development and after lesion in the adulthood.

Stathmin and SCG10 belong to a family of phosphoproteins associated to cell proliferation and differentiation. In the present study, we have analyzed immunocytochemically the distribution of these proteins during neurogenesis in the mouse olfactory system, from midgestation to adulthood. Data show that already at embryonic day 12, stathmin and SCG10 immunoreactivities were present in the olfactory and vomeronasal neurons, and their number increased greatly, colocalizing with neuronal specific tubulin, a marker of immature neurons. Later on up to adulthood, the distribution of stathmin and SCG10 became progressively restricted to a few immature receptor and chemosensory neurons. Significantly, in the olfactory epithelium, stathmin was seen in immature neurons and also in basal cells representing precursors of neuronal elements. Interestingly, before birth stathmin and SCG10 immunopositive cells were seen outside the olfactory epithelium, seemingly migrating toward the olfactory bulb. After regeneration in the adult following peripheral lesion of the olfactory epithelium, stathmin and SCG10 were again strongly expressed and generally colocalized with neuronal specific tubulin immunoreactivity. Overall these results indicate that stathmin and SCG10 are expressed in immature olfactory neurons as well as in the migrating cells generated from the olfactory epithelium, supporting the role of these proteins in neurogenesis and cell migration.

The cytosolic phosphoprotein stathmin is expressed in the olfactory system of the adult rat.

Stathmin is a cytosolic protein expressed particularly in the developing nervous system, whose phosphorylation is correlated with the action of multiple extracellular stimuli regulating cell proliferation and differentiation. In this study, we used an antibody that specifically recognizes the carboxyterminal region of stathmin to analyze the distribution of this protein in the olfactory system of adult rats, and found a high and selective immunoreactivity in immature olfactory receptors of the olfactory neuroepithelium and in cells of the rostral migratory stream. These results reveal an expression of stathmin in regions of the adult nervous system characterized by striking structural plasticity and cell renewal, suggesting that this protein could play a role in the differentiation of newly generated cell populations.