| Xenopus nerve-muscle coculture is one of the few preparations where it is possible to simultaneously record neuronal somatic and terminal calcium current, as well as the associated postsynaptic response. The first part of my project utilized the voltage-clamp technique to identify and compare the calcium channel isoforms in Xenopus spinal neuron terminals to those in somas. Both compartments were found to express high voltage-activated (HVA) N- and R-type calcium channels. Low voltage-activated (LVA) channels were restricted to the somas while L-type HVA channels were only present at terminals. Additionally a calcium-dependent chloride (Cl Ca) channel type present in many somas was absent in terminals. Kinetic comparison between somatic and terminal calcium current revealed that functional requirements may dictate the distribution of channel isoforms. In particular, LVA channels and ClCa channels may be excluded from terminals on the basis of their slow kinetics that may not be compatible with fast neurotransmitter release.; In part 2, simultaneous pre- and postsynaptic recordings indicated that N-, L- and R-type calcium channel isoforms present in the presynaptic terminals can all support synaptic transmission. Unexpectedly, terminal L-type calcium currents were found to be sensitive to ω-conotoxin GVIA, a common N-type channel blocker. This finding may help explain why L-type channels' contribution to transmitter release was not documented in previous studies. Involvement of multiple calcium channel types in synaptic transmission at developing neuromuscular junctions may be a presynaptic mechanism of facilitating synaptic development.; In part 3 I tested whether postsynaptic AChRs played a role in establishing presynaptic release machinery. For this purpose I used optical measurements with FM 1–43 to monitor exocytosis from motoneuron terminals in intact zebrafish. Activity-dependent labeling of motoneuron terminals by FM 1–43 was similar in wild type fish and two mutant lines lacking muscle AChRs ( nic and sofa potato). Furthermore, exocytosis as determined by depolarization-induced FM 1–43 destaining was comparable in mutant and wild type fish. These findings suggest that early functional presynaptic development does not require the expression of postsynaptic receptors at zebrafish neuromuscular junctions. |
