Low mobility of the Ca2+ buffers in axons of cultured Aplysia neurons.

Cellular Ca2+ buffers determine amplitude and diffusional spread of neuronal Ca2+ signals. Fixed Ca2+ buffers tend to retard the signal and to lower the apparent diffusion coefficient (D(app)) of Ca2+, whereas mobile buffers contribute to Ca2+ redistribution. To estimate the impact of the expression of specific Ca2+-binding proteins or the errors in Ca2+ measurement introduced by indicator dyes, the diffusion coefficient De and the Ca2+-binding ratio kappa(e) of endogenous Ca2+ buffers must be known. In this study, we obtain upper bounds to these quantities (De < 16 microm2/s; kappa(e) < 60) for axoplasm of metacerebral cells of Aplysia california. Due to these very low values, even minute concentrations of indicator dyes will interfere with the spatiotemporal pattern of Ca2+ signals and will conceal changes in the expression of specific Ca2+-binding proteins, which in the native neuron are expected to have significant effects on Ca2+ signals.

Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro.

The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.