Neural transmission.

Oxidative stress causes neural damage and inhibits essential cellular
processes, such as synaptic transmission. Despite this knowledge, currently
available pharmaceutical agents cannot effectively protect neural cells from acute
oxidative stress elicited by strokes, heart attacks, and traumatic brain injuries in a
real life clinical setting. Our lab has developed an electrophysiology protocol to
identify novel drugs that protect an essential cellular process (neurotransmission)
from acute oxidative stress-induced damage. Through this doctoral dissertation,
we have identified three new drugs, including a Big K+ (BK) K+ channel blocker
(iberiotoxin), resveratrol, and a custom made resveratrol-like compound (fly2) that
protect synaptic function from oxidative stress-induced insults. Further developing
these drugs as neuroprotective agents may prove transformative in protecting the
human brain from acute oxidative stress elicited by strokes, heart attacks, and
traumatic brain injuries. Inhibiting the protein kinase G (PKG) pathway protects neurotransmission
from acute oxidative stress. This dissertation has expanded upon these findings
by determining that the PKG pathway and BK K+ channels function through
independent biochemical pathways to protect neurotransmission from acute
oxidative stress. Taken together, this dissertation has identified two classes of
compounds that protect neurotransmission from acute oxidative stress, including
resveratrol-like compounds (resveratrol, fly2) and a BK K+ channel inhibitor
(iberiotoxin). Further developing these drugs in clinical trials may finally lead to the
development of an effective neuroprotective agent.

Neuronal circuit output is dependent on the embedded synapses’ precise
regulation of Ca2+ mediated release of neurotransmitter filled synaptic vesicles (SVs) in
response to action potential (AP) depolarizations. A key determinant of SV release is the
specific expression, organization, and abundance of voltage gated calcium channel
(VGCC) subtypes at presynaptic active zones (AZs). In particular, the relative distance
that SVs are coupled to VGCCs at AZs results in two different modes of SV release that
dramatically impacts synapse release probability and ultimately the neuronal circuit
output. They are: “Ca2+ microdomain,” SV release due to cooperative action of many
loosely coupled VGCCs to SVs, or “Ca2+ nanodomain,” SV release due to fewer more
tightly coupled VGCCs to SVs. VGCCs are multi-subunit complexes with the pore
forming a1 subunit (Cav2.1), the critical determinant of the VGCC subtype kinetics,
abundance, and organization at the AZ. Although in central synapses Cav2.2 and Cav2.1 mediate synchronous transmitter release, neurons express multiple VGCC subtypes with
differential expression patterns between the cell body and the pre-synapse. The calyx of
Held, a giant axosomatic glutamatergic presynaptic terminal that arises from the globular
bushy cells (GBC) in the cochlear nucleus, exclusively uses Cav2.1 VGCCs to support
the early stages of auditory processing. Due to its experimental accessibility the calyx
provides unparalleled opportunities to gain mechanistic insights into Cav2.1 expression,
organization, and SV release modes at the presynaptic terminal. Although many
molecules are implicated in mediating Cav2.1 expression and SV to VGCC coupling
through multiple binding domains on the C-terminus of the Cav2.1 a1 subunit, the
underlying fundamental molecular mechanisms remain poorly defined. Here, using viral
vector mediated approaches in combination with Cav2.1 conditional knock out transgenic
mice, we demonstrate that that there a two independent pathways that control Cav2.1
expression and SV to VGCC coupling at the calyx of Held. These implications for the
regulation of synaptic transmission in CNS synapses are discussed.