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Richard TSIEN

How ion channel alternative splicing varies with neuronal activity and cell type

Y. Sun1, Y. Qin2, B. Li2, X. Chen3, R. W. Tsien1; 1Neuroscience Institute, New York University Grossman School of Medicine, New York, NY; 2Sun Yat-sen University, Guangdong, China; 3Allen Institute for Brain Science, Seattle, WA

Neurons can regulate their excitability and firing properties through two processes: Hebbian plasticity, which uses positive feedback to amplify responses to changes in activity, and homeostatic plasticity, which utilizes negative feedback to maintain functional stability. These seemingly opposing processes allow incorporation of new information while stabilizing overall network excitability. Balancing these two forms of plasticity involves changes in ion channels that determine firing properties. One way ion channels can be altered is by regulation of alternative splicing, but how patterns of splicing vary across genes, cell types, and changes in activity is understudied and unclear. To investigate the activity-dependence of ion channel splicing, we analyzed RNAseq and collected PCR confirmatory results of alternative splicing in different ion channels. We found that splicing is regulated in distinct ways. For example, the inclusion of an NTD of Cavβ4 increases under chronic inactivity and decreases under chronic depolarization. Expression of this splice variant has been shown to enhance current density of calcium channels, and its splicing changes suggest homeostatic regulation of neuronal excitability. On the other hand, Li et al. (2020) found that exclusion of an exon in BK channel is favored under both low and high activity, a non-monotonic pattern echoed by Nav1.2. For BK, exon exclusion widens the action potential, leading to homeostatic regulation under inactivity and Hebbian plasticity under depolarization. Thus, our results show exemplars of both monotonic and non-monotonic patterns of activity-dependent alternative splicing.

We next asked how the splicing changes we found might influence overall circuit function, a task that requires interrogation of individual cell types. Cell types play distinct roles in neural circuits: splicing of an ion channel that increases excitability will have opposite circuit effects depending on whether it occurs in an excitatory or inhibitory neuron. Detecting cell type-specific splice variants requires distinguishing diverse neuronal types in the brain and simultaneously probing splice variants in those same neurons. To do this, we capitalized on BARseq, a high-throughput in situ sequencing method that can resolve neuronal types in brain slices (Sun et al. 2021). Adapting BARseq for exon detection allowed us to characterize differential expression of splice variants across brain regions and cell types. This paves the way for assaying activity-dependent splicing in intact circuits and investigating cell type-specific modifications underlying circuit plasticity.