Diverse Patterns of Activity-Dependent Alternative Splicing

Project Summary/Abstract Neural circuits can change their properties under changes in activity. Hebbian plasticity acts as a positive regulator to increase or decrease synaptic strength and neuronal excitability. These destabilizing challenges are counterbalanced by homeostatic plasticity which maintains synaptic strength and neuronal excitability

within an optimal range, stabilizing circuit function. One way to regulate these two processes is by alternative splicing of ion channel genes, which changes channel properties to alter neuronal properties. Studies show that defects in alternative splicing of ion channels can impair circuit activity underlying neuropsychiatric

disorders. Evidence suggests that low and high neuronal activity regulates distinct patterns of alternative splicing of ion channel genes, supporting Hebbian or homeostatic plasticity, or both. However, the mechanism underlying these splicing patterns is unknown. My central hypothesis is that activity regulates alternative

splicing of ion channels through subcellular signaling mechanisms tailored to specific cell types. My proposal will focus on (1) how activity generally changes alternative splicing, (2) how these changes occur in different cell types, and (3) how cellular mechanisms control alternative splicing. I will assess these changes

under activity perturbation using tetrodotoxin (TTX) to chronically silence neurons and high potassium (40mM K+) to chronically depolarize neurons. In vitro investigations will determine the cellular mechanisms while ex vivo and in vivo investigations will determine the circuit mechanisms across cell types. Screening, assaying,

and manipulating alternatively spliced ion channels will be done using cutting-edge multiplexed methods. Aim 1 will identify examples of different patterns of alternative splicing in ion channels. Through an RNAseq screen and qPCR validation, this aim will characterize the prevalence of different alternative splicing patterns. I

predict I will characterize splicing patterns across multiple families of ion channels. Aim 2 will investigate how activity regulates alternative splicing in diverse excitatory and inhibitory cell types. I will combine neuroanatomical techniques with ex vivo organotypic slice cultures and after perturbations in vivo, to study

chronic silencing and depolarization in cell types in intact circuits and within a native environment in the animal. I predict that activity-dependent alternative splicing occurs in a cell type-specific manner. Aim 3 will determine how signaling from the soma or dendrites regulate alternative splicing. I will use an engineered somatic

Ca2+ blocking method and pharmacological agents to manipulate subcellular signaling in neuron cultures. I predict that somatic and dendritic signaling controls two distinct patterns of alternative splicing. The results of this study will clarify the regulation of activity-dependent alternative splicing from subcellular mechanisms and

from cell type-specific mechanisms, providing a multi-level understanding of how dysregulation of neuronal activity contributes to overall activity of the circuit.