Abstract
Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca(2+) into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca(2+) currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca(2+) and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca(2+) entry has been characterized using imaging techniques and macroscopic current measurements--techniques that do not have sufficient temporal resolution to visualize Ca(2+) entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca(2+) currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.
Highlights
Synaptic transmission is an extremely rapid and precise process
Action potential invasion of the presynaptic terminal leads to opening of voltagegated calcium channels (VGCCs) located in the release face membrane, the resulting increase in presynaptic Ca2+ acting as the trigger for vesicle fusion and neurotransmitter release[1]
Our dissociation protocol is significant by yielding isolated reticulospinal axons devoid of postsynaptic projections Figure 2f, but which retain functional presynaptic terminals Figure 4c and Figure 4d
Summary
Synaptic transmission is an extremely rapid and precise process. Action potential invasion of the presynaptic terminal leads to opening of VGCCs located in the release face membrane, the resulting increase in presynaptic Ca2+ acting as the trigger for vesicle fusion and neurotransmitter release[1]. In all other presynaptic terminals including the giant reticulospinal synapse in the lamprey spinal cord[13], the lack of access to the presynaptic release face membrane has necessitated the use of indirect approaches such as Ca2+ imaging to study presynaptic Ca2+ fluxes. Previous blind attempts at direct measurement of Ca2+ currents in reticulospinal axons in the intact lamprey spinal cord using cell-attached patch clamp technique have proven unsuccessful[13] due to lack of access to the presynaptic release face membrane owing to the opposing postsynaptic processes Figure 1b. We decided to use enzymatic treatment[17] followed by mechanical dissociation Using this approach, we have developed an acutely dissociated preparation of the lamprey spinal cord that yields viable isolated reticulospinal axons with functional presynaptic terminals devoid of any postsynaptic processes, thereby providing unrestricted access to individual presynaptic terminals. This is a significant breakthrough in the field of synaptic transmission since it is the first such recording to be carried out at central synapses
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