MECHANISMS OF SYNAPTIC TRANSMISSION IN THE RETINA

The long-term objective of this project is the elucidation and molecular
understanding of the cascade of reactions that couples the synaptic
receptor and channel on retinal depolarizing bipolar cells (DBCs). The
postsynaptic action of glutamate, the transmitter that is released onto
DBCs, is unusual for an excitatory amino because it produces a
hyperpolarization by closing synaptic channels. these channels are
believed to be gated by a diffusible intracellular messenger. Our
specific aims are to identify the intracellular messenger that opens the
synaptic channel in DBCs of tiger salamander, and to determine the
mechanism by which glutamate controls the levels of this intracellular
messenger. We also wish to learn if Ca2+ can modulate any step in the
cascade and what consequence such modulation might have for synaptic
transmission between photoreceptors and DBCs. (I) The candidacy of cGMP
as the intracellular messenger will be accessed. We will attempt to
measure the ability of cyclic nucleotides (cAMP and cGMP) to open
directly synaptic channels by applying cyclic nucleotides to patches
excised from DBCs. The ion selectivity and cyclic nucleotide affinity
of the channels will be determined. We will also attempt to impose jumps
in the intracellular concentration of cGMP and measure the response.
Inactive "caged" cGMP will be dialyzed into DBCs and then activated by
flash photolysis. (II) Glutamate is thought to lower the levels of cGMP
by activating a cAMP-phosphodiesterase (PDE). We will test this
hypothesis by dialyzing poorly hydrolyzed analogs of cGMP into intact
DBCs. If hydrolysis of cGMP by PDE is an obligatory step in the cascade,
then these analogs should prevent glutamate from closing channels. (III)
We will use both excised patches and intact isolated cells to measure the
permeability of Ca2+ through the glutamate channels. We will also
monitor the levels of Ca2+ in intact cells with fura-2 while recording
responses to glutamate in order to determine whether Ca2+ levels change
during glutamate stimulation. (IV) We will measure the effects of
changing Ca2+ levels on the synaptic current. DBCs will be dialyzed with
one of two different "caged" Ca2+ chelators. One chelator binds Ca2+
after uncaging, and the other binds Ca2+ prior to uncaging. The
chelators will be uncaged by light and their effects on the synaptic
current will be accessed.