Development of Ca²⁺ hotspots between Lymnaea neurons during synaptogenesis

Abstract

Calcium (Ca2+) channel clustering at specific presynaptic sites is a hallmark of mature synapses. However, the spatial distribution patterns of Ca2+ channels at newly formed synapses have not yet been demonstrated. Similarly, it is unclear whether Ca2+‘hotspots’ often observed at the presynaptic sites are indeed target cell contact specific and represent a specialized mechanism by which Ca2+ channels are targeted to select synaptic sites. Utilizing both soma–soma paired (synapsed) and single neurons from the mollusk Lymnaea, we have tested the hypothesis that differential gradients of voltage‐dependent Ca2+ signals develop in presynaptic neuron at its contact point with the postsynaptic neuron; and that these Ca2+ hotspots are target cell contact specific. Fura‐2 imaging, or two‐photon laser scanning microscopy of Calcium Green, was coupled with electrophysiological techniques to demonstrate that voltage‐induced Ca2+ gradients (hotspots) develop in the presynaptic cell at its contact point with the postsynaptic neuron, but not in unpaired single cells. The incidence of Ca2+ hotspots coincided with the appearance of synaptic transmission between the paired cells, and these gradients were target cell contact specific. In contrast, the voltage‐induced Ca2+ signal in unpaired neurons was uniformly distributed throughout the somata; a similar pattern of Ca2+ gradient was observed in the presynaptic neuron when it was soma–soma paired with a non‐synaptic partner cell. Moreover, voltage clamp recording techniques, in conjunction with a fast, optical differential perfusion system, were used to demonstrate that the total whole‐cell Ca2+ (or Ba2+) current density in single and paired cells was not significantly different. However, the amplitude of Ba2+ current was significantly higher in the presynaptic cell at its contact side with the postsynaptic neurons, compared with non‐contacted regions. In summary, this study demonstrates that voltage‐induced Ca2+ hotspots develop in the presynaptic cell, concomitant with the appearance of synaptic transmission between the soma–soma paired cells. The appearance of Ca2+ gradients in presynaptic neurons is target cell contact specific and is probably due to a spatial redistribution of existing channels during synaptogenesis.

Expression and Modulation of an Invertebrate Presynaptic Calcium Channel α1 Subunit Homolog

“…Finally, identified synapses between Lymnaea neurons are highly amenable for analysis using molecular and electrophysiological approaches and optical imaging (21,(65)(66)(67). A combined approach using in vitro expression and hypothesis testing in native invertebrate neurons may, thus, permit us to dissect the interplay between presynaptic calcium channels, second messenger systems, and synaptic proteins.…”

Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

“…Intracellular Ca 2+ ( [Ca] i ) was measured as described previously using a fura-2 ratiometric Ca 2+ imaging system (Feng et al, 2002). Cells were incubated for 30 minutes with 4 M Fura-2 AM (Molecular Probes), and then washed with snail saline three times before imaging.…”

“…(B) Calibration curve of Fura-2 signal against free [Ca 2+ ] i . From the Ca 2+ -imaging data (Figs 4 and 6), a calibration curve was constructed as described previously (Feng et al, 2002;Grynkiewicz et al, 1985). -binding sites (red), EF3 and EF4, are adjacent to each other and the C-terminus.…”

Peripheral oxygen‐sensing cells directly modulate the output of an identified respiratory central pattern generating neuron

“…In experiments where we intended to prevent chemical transmission between cells, zero calcium saline (in m m : 42.8 NaCl, 1.7 KCl and 14.0 MgCl 2 ) was used. Various solutions were delivered to the target cells via a fast perfusion system (see Feng et al. , 2002 for details).…”