Acute Dissociation of Lamprey Reticulospinal Axons to EnableRecording from the Release Face Membrane of Individual Functional PresynapticTerminals

摘要

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca²⁺ 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 Ca²⁺ currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca²⁺ and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca²⁺ entry has been characterized using imaging techniques and macroscopic current measurements – techniques that do not have sufficient temporal resolution to visualize Ca²⁺ 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 havesuccessfully addressed this problem in the giant reticulospinal synapse of thelamprey spinal cord by developing an acutely dissociated preparation of thespinal cord that yields isolated reticulospinal axons with functionalpresynaptic terminals devoid of postsynaptic structures. We can fluorescentlylabel and identify individual presynaptic terminals and target them forrecording. Using this preparation, we have characterized VGCCs directly at therelease face of individual presynaptic terminals using immunohistochemistry andelectrophysiology approaches. Ca²⁺ currents have been recordeddirectly at the release face membrane of individual presynaptic terminals, thefirst such recording to be carried out at central synapses.