Research report
Rab3A immunolocalization in the mammalian vestibular end-organs during development and comparison with synaptophysin expression

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Abstract

Rab proteins are essential for membrane vesicle docking and fusion and for transport vesicle formation at the presynaptic membrane, a step in the release of neurotransmitters. The vestibular sensory epithelia contain three types of synapses: afferent terminals, efferent endings and possible synaptic contacts between the apex of the afferent nerve calyces and the sensory cells. We report an immunocytochemical codetection of rab3A and synaptophysin in the vestibular end-organs of mouse, between fetal day 14 and adult, and of rat during the postnatal development. During mouse fetal development, rab3A appeared in afferent neurites on F16, and in sensory cells on F19. This was respectively two and five days later than the appearance of synaptophysin-IR in the same compartments. During the late postnatal development and in the adult sensory epithelia, rab3A and synaptophysin were strongly detected in nerve terminals of efferent and possibly afferent nature and in the upper part of the nerve calyces. The presence of rab3A in the nerve calyces is consistent with the putative secretory function of the calyx. In addition, rab3A immunostaining was also present in the sensory cells together with a faint synaptophysin-IR, that had not been described in previous reports [Scarfone, E., Demêmes, D. and Sans, A. J. Neurosci., 11 (1991) 1173–1181.]. The presence of these two proteins in the sensory cells supports the existence of a synaptic vesicle cycle in these cells. © Elsevier Science B.V. All rights reserved.

Introduction

Two major types of synaptic contacts have been described in the vestibular sensory epithelia: the afferent synapses transmit information between the sensory cells and the afferent fibers [41], and the efferent synapses transmit control messages from the efferent neurons to the sensory cells and afferent endings 15, 41. In addition, the activity of the type I hair cells in higher vertebrates may be controlled by the apical part of the afferent nerve calyces, via neurotransmitter release [32]. Three types of synapses are thus present in the mammalian vestibular sensory epithelia. It is not known whether these different synapses have the same molecular mechanisms of synaptic vesicle exo-endocytosis.

This issue can be investigated by identifying synaptic proteins and their distributions. The mature presynaptic terminal contains numerous proteins, each involved in a particular aspect of the synaptic vesicle cycle 16, 19, 27, 36, 37. Numerous studies have shown different localizations and/or expressions of these proteins in different presynaptic types 18, 25, 35, 39, suggesting that the protein composition of the synaptic vesicle cycle would correlate with the synapse type and localization in the neuron. In the adult mammalian vestibular end-organs, synapsin I and synaptophysin, two proteins associated with synaptic vesicle membrane [for review, 2, 3], are present in the apical part of the calyceal endings and efferent terminals, but not in the sensory cell presynaptic zones [32]. This suggests that it would be interesting to examine the distribution of a dynamic element of the synaptic vesicle cycle, rab3A, among the various presynaptic sites in the vestibular sensory epithelia.

Current models suggest that rab proteins catalyze vesicle docking and fusion to membranes, in their GTP-bound conformation, by interacting with the machinery supporting the docking of vesicles to the appropriate acceptor membranes, and would play a regulatory role 11, 14, 27, 37. Rab3A is a small GTP-binding protein of the rab family, highly expressed in the brain. Its expression is not uniform in all neurons, and the protein is localized in particular neuronal pathways where it is found exclusively associated with the membrane of synaptic vesicles (for review, see [34]). Rab3A is essential for the recruitment of synaptic vesicles for neurotransmitter exocytosis during repetitive stimulation [12]. During ontogenesis, it has been shown that the onset of rab3A presence at synapses is later than that of other synaptic vesicle proteins, and is therefore a late marker of the presynaptic element maturity [34]. Analysis of the progression of rab3A appearance during the development of the different presynaptic elements of the vestibular sensory epithelia may thus contribute to our understanding the characteristics of the different synaptic types.

In this study, we investigated the presence of rab3A in the vestibular end-organs of the mouse and rat, in the adult and during development, and compared its distribution with that of synaptophysin.

Section snippets

Materials and methods

Anti-rab3A polyclonal antibody was produced and characterized as described previously [34]. Synaptophysin was detected using a mouse monoclonal antibody (clone SY 38, Boehringer-Mannheim).

Rats (Sprague-Dawley) aged 5, 12, 17 postnatal days (P) and adult, and mice (CBA/C57), from fetal day (F) 14 to adult were used. F1 was defined as the first day on which a vaginal plug was observed in the dam. At least four animals were analyzed for each stage. All animals were housed and handled according to

Developmental stages

Fetal developmental stages were examined only in mouse. At F14, no rab3A immunoreactivity (IR) was observed in the mouse vestibular sensory epithelia (Fig. 1a1). By contrast, synaptophysin immunostaining was present, though faintly in a thin and loose plexus invading the whole thickness of the epithelium and in some sensory cells (Fig. 1a2). At F15, there was still no or extremely rare and faint rab3A staining in the epithelia, and synaptophysin staining began to concentrate at afferent endings

Immunolocalization of rab3A in the adult vestibular sensory epithelia

Rab3A was immunolocalized in three sensori-neuronal compartments: (1) the sensory cells, (2) the nerve endings at the base of the sensory cells, and (3) the afferent nerve calyces. The labeled nerve endings were probably mainly efferent nerve endings as previously observed by immunolocalization of synaptophysin and synapsin 32, 33. Rab3A is therefore present in two well characterized presynaptic compartments, the sensory cells and the efferent endings. In the third compartment, the nerve

Conclusion

In the adult rat and mouse peripheral vestibular system, rab3A-IR was observed in most vestibular hair cells and in nerve endings. In the sensory cells, synaptophysin staining was also faintly detected contrary to previous reports [32], the presence of these two proteins thus supporting the existence of a synaptic vesicle cycle in these cells. The immunolocalization of rab3A in the afferent calyces, with an intense staining of their apical part and a distribution pattern similar to that of

Acknowledgements

We thank D. Demêmes and J. Raymond for advice and comments on the manuscript, and A. Sahuquet for computer assistance. This study was partly supported by a grant from the Centre National d'Etudes Spatiales (CNES, France).

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