Young, Samuel M.

Helper-Dependent Adenoviruses (HdAd) are the ideal viral vector choice for
expression of Munc13 proteins due to the large packaging capacity needed (~9.8 kb) for
the triple expression cassettes of Munc13, iCre, and EGFP. Development of a HdAd
vector for the expression of Munc13 gene family proteins allows for the rapid expression
of molecules located at the synaptic active zone critical for neurotransmitter release. The Munc13 HdAd viral vector can be used to express proteins of the munc13 gene family in the calyx of Held. This will allow for the elucidation of the molecular mechanisms
employed by the calyx of Held in the process of encoding the onset and modulation of
sound. Production of an HdAd vector for the expression of Munc13 proteins occurs in
five parts; cloning, transfection, amplification, purification and titering. After production
of the HdAd vector, Munc13 protein expression was analyzed by western blot.

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.