In vivo cell type-specific CRISPR knockdown of dopamine beta\xa0hydroxylase reduces locus coeruleus evoked wakefulness

Hiroshi Yamaguchi,F Woodward Hopf,Luis De Lecea,Shi-Bin Li

doi:10.1038/s41467-018-07566-3

Hiroshi Yamaguchi, F Woodward Hopf + Show 2 more

Open Access

https://doi.org/10.1038/s41467-018-07566-3

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Abstract

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. Optogenetic stimulation of LC-NE neurons induces immediate sleep-to-wake transitions. However, LC neurons also secrete other neurotransmitters in addition to NE. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas9 technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Unilateral dbh gene disruption abolished immediate arousal following optogenetic stimulation of LC. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors.

Highlights

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal
We show that the LC-specific disruption of dbh reduces NE concentration in the LC and projection areas including the prefrontal cortex and prevents immediate arousal following LC photostimulation
The current responses to hyperpolarizing voltage were not different between sgControl and sgDbh-expressing LC cells (Fig. 2b, n = 4 sgControl, n = 5 sgDbh). These results indicated that dbh gene disruption by CRISPR/Cas[9] did not affect basic excitability of LC cells

Summary

Introduction

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas[9] technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors. Conclude that NE is crucial to maintain and induce wakefulness

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