title: 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.
authors.affiliation: Respiratory and Neuroscience Research Groups, Faculty of Medicine, University of Calgary, 3330-Hospital Drive, NW, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Zhong-Ping
authors.initials: ZP
authors.lastname: Feng
authors.forename: Nikita
authors.initials: N
authors.lastname: Grigoriev
authors.forename: David
authors.initials: D
authors.lastname: Munno
authors.forename: Ken
authors.initials: K
authors.lastname: Lukowiak
authors.forename: Brian A
authors.initials: BA
authors.lastname: MacVicar
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Goldberg
authors.forename: Naweed I
authors.initials: NI
authors.lastname: Syed
mesh_terms: D000818:Animals; D001464:Barium / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D008195:Lymnaea / Q000502:physiology*; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D010539:Permeability; D017729:Presynaptic Terminals / Q000378:metabolism; D013569:Synapses / Q000502:physiology*
chemical_list: D015220:Calcium Channels; D001464:Barium; D002118:Calcium