Project Summary/Abstract
The cholinergic system, using acetylcholine (ACh) as a neurotransmitter, shapes plasticity and cognitive
functions in the adult cortex by tuning cortical activity, and has been implicated in brain disorders such as epilepsy,
attention-deficit hyperactivity disorder, depression, and schizophrenia. However, the role of ACh signaling in
development of cortical circuits in normal and diseased conditions remains poorly understood. In particular, little
is known about whether and how ACh signaling regulates the wiring of inhibitory interneurons (INs), cellular
components critical for cortical computations. Cortical INs generally develop highly branched axons to establish
local, dense circuit modules. Despite representing a crucial event during the wiring of IN circuits, the cellular and
molecular mechanisms underlying IN axonal arborization remain elusive.
The objective of our proposal is to establish the role of ACh signaling in shaping IN axonal arbors
in vivo. We will also provide evidence that disease-relevant mutations in genes that are essential for ACh
signaling could impact IN axonal branching, and that the axonal phenotype could be ameliorated by
manipulating downstream components in ACh signaling. To achieve this goal, we will perform a series of
experiments using the chandelier cell (ChC), which exclusively innervates axon initial segments of pyramidal
neurons (PNs) and thus powerfully controls spike generation in PNs.
Because of its stereotypy of the axonal organization, the ChC serves as an ideal model to study IN axonal
morphogenesis. Our preliminary data has shown that (1) Axonal filopodia arising from varicosities serve as
precursors of branches in vivo, (2) Filopodia initiation as well as the basal Ca2+ levels in ChC axonal varicosities
are regulated by signaling from nicotinic AChRs (nAChRs) to T-type voltage dependent calcium channels (T-
VDCCs), independently of action potentials/network activity, (3) CRISPR/Cas9-mediated T-VDCC loss-of-
function (LOF) in single ChCs significantly reduces their axonal branching points, and (4) Systemic nicotine
administration to developing postnatal mice increases ChC axonal arbors. Based on these results, we propose
to test the hypothesis that the nAChR-T-VDCC signaling pathway shapes ChC axonal arborization, and disease-
relevant mutations in ACh signaling molecules cause wiring defects in ChCs. In Aim 1, we will elucidate the role
of the nAChR-T-VDCC signaling pathway in ChC axonal arborization in vivo. In Aim 2, we will determine the
effect of the epilepsy-related gain-of-function (GOF) mutation in nAChRs on ChC axonal arborization, and
elucidate whether manipulating T-VDCCs can be a way to revert the nAChR GOF phenotype.
Our study will provide first evidence for the developmental role of ACh signaling in the wiring of INs in the
normal and diseased cortices as well as a novel hint for developing strategies to prevent and ameliorate brain
disorders associated with ACh signaling.