Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Abstract

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca 2+ into the presynaptic terminal, through voltagegated 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. Video LinkThe video component of this article can be found at

Single Calcium Channel Nanodomains Drive Presynaptic Calcium Entry at Lamprey Reticulospinal Presynaptic Terminals

“…300000, Sigma-Aldrich) coated coverslips by applying mechanical tension along the rostral-caudal axis, until the reticulospinal axons were isolated from the spinal cord interior (Fig. 1B; Ramachandran and Alford, 2014).…”

“…Dissociations were conducted on poly-D-lysine coated coverslips, in Ringer's solution, in a custom designed low-noise recording chamber, maintained at 10°C (Ramachandran and Alford, 2014). Dissociated axons were allowed to recover for 1 h and presynaptic terminals were labeled with FM1-43 (5 mM) during 30 mM KCl (perfused in Ringer's solution) evoked stimulation.…”

“…Using a similar approach to accessing the presynaptic VGCCs, we developed an acute dissociation of the lamprey spinal cord (Ramachandran and Alford, 2014), by applying a combination of enzymatic digestion of spinal cord tissue and acute mechanical dissociation, enabling isolation of reticulospinal axons with functional presynaptic terminals without any postsynaptic processes (Fig. 1B).…”

“…We recorded Ca 21 currents at the release face membrane of individual fluorescently labeled AZs, with 10 mM [Ca 21 ] external in patch pipette (Fig. 3A, n = 5 patches), using custom low-noise single channel cell-attached patch clamp recordings (Ramachandran and Alford, 2014). Single AZs were patched in cell-attached configuration, and the holding potential was set to À88 mV (adjusted from the known intracellular membrane potential determined from intracellular sharp electrode recording).…”