Elsevier

Neuroscience

Volume 117, Issue 1, 17 March 2003, Pages 7-18
Neuroscience

Research paper
Developmental maturation of synaptic vesicle cycling as a distinctive feature of central glutamatergic synapses

https://doi.org/10.1016/S0306-4522(02)00835-7Get rights and content

Abstract

The formation of chemical synapses in the mammalian brain involves complex pre- and postsynaptic differentiation processes. Presynaptically, the progressive accumulation of synaptic vesicles is a hallmark of synapse maturation in the neocortex [J Neurocytol 12 (1983b) 697]. In this study, we analyzed the functional consequences of presynaptic vesicle-pool maturation at central glutamatergic and GABAergic synapses. Using (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) staining of recycling synaptic vesicles, we demonstrate a pronounced developmental increase in presynaptic vesicle accumulation during differentiation of neocortical neurons in culture. Using electrophysiological methods to study functional synaptic maturation, we found an improved recovery from hypertonic solution-induced depletion. As supported by the FM1-43 staining results, this change is most likely caused by a developmental increase in the number of reserve-pool vesicles. In addition, assuming a rapid reuse of freshly recycled vesicles, a developmental maturation of the endocytosis process may also contribute. The observed presynaptic maturation process occurred selectively at glutamatergic synapses, while GABAergic synapses did not show similar developmental alterations. Furthermore, we used high-frequency stimulation (HFS) of glutamatergic and GABAergic synapses to reveal the physiological consequences of reserve-pool maturation. As expected, recovery from HFS-induced depletion was incomplete at immature glutamatergic synapses and strongly improved during synapse maturation. Again, GABAergic synapses did not show similar developmental changes. Taken together, our study characterizes the functional consequences of a pronounced accumulation of reserve-pool vesicles occurring selectively at glutamatergic synapses.

Section snippets

Cell culture

Microisland cultures of neocortical neurons were prepared as described previously Segal 1991, Lessmann and Heumann 1997. In brief, the occipital neocortex was removed from Wistar rat pups at P0-P2, and the tissue was dissociated after trypsin (0.1%) treatment. Dissociated neurons were seeded on cultured glial cells at a density of 2.5×105 cells/dish. For attachment to glial islands, dissociated cells were incubated for 4–5 h in Dulbecco’s Minimal Essential Medium containing fetal calf serum

Developmental increase in the size of the cycling synaptic vesicle pool in cultured neocortical neurons

To characterize the developmental maturation of the presynaptic vesicle population, we visualized cycling synaptic vesicles with the styryl dye FM1-43 Betz and Bewick 1992, Cochilla et al 1999, Murthy 1999. Saturating stainings (incubation times >2 min) were performed using a depolarizing (50-mM K+) extracellular solution to stimulate synaptic vesicle cycling in the presence of FM1-43. This procedure led to a punctate labeling of functional presynaptic terminals (Fig. 1). To confirm that

Discussion

The characterization of the cellular and molecular mechanisms involved in formation and maturation of synapses is of crucial importance for the understanding of mammalian CNS development. In particular, differentiation of presynaptic terminals plays a major role in the establishment of mature functional properties at central synapses. Ultrastructural studies have well documented that synaptic vesicles are progressively accumulated inside developing presynaptic terminals (Blue and Parnavelas,

Acknowledgements

We want to thank Dr. P. Caroll for providing BDNF knock-out mice. We further thank Dr. M. Hartmann for help with FM1-43 imaging and H. Bartel for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft.

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    Present address: Department of Physiology and Pathophysiology, Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany.

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