Hypocretin neuron-specific transcriptome profiling identifies the sleep modulator Kcnh4a

Abstract

Sleep has been conserved throughout evolution; however, the molecular and neuronal mechanisms of sleep are largely unknown. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate sleep\wake states, feeding, stress, and reward. To elucidate the mechanism that enables these various functions and to identify sleep regulators, we combined fluorescence cell sorting and RNA-seq in hcrt:EGFP zebrafish. Dozens of Hcrt-neuron–specific transcripts were identified and comprehensive high-resolution imaging revealed gene-specific localization in all or subsets of Hcrt neurons. Clusters of Hcrt-neuron–specific genes are predicted to be regulated by shared transcription factors. These findings show that Hcrt neurons are heterogeneous and that integrative molecular mechanisms orchestrate their diverse functions. The voltage-gated potassium channel Kcnh4a, which is expressed in all Hcrt neurons, was silenced by the CRISPR-mediated gene inactivation system. The mutant kcnh4a (kcnh4a-/-) larvae showed reduced sleep time and consolidation, specifically during the night, suggesting that Kcnh4a regulates sleep.DOI: http://dx.doi.org/10.7554/eLife.08638.001

Ancestral Circuits for the Coordinated Modulation of Brain State

“…Secondary antibodies used were donkey anti-mouse Alexa647 (Jackson Immuno Research, 715-606-151), donkey anti-rabbit Alexa 594 (Jackson Immuno Research, 711-586-152), donkey anti-rat DyLight405 (Jackson Immuno Research, 712-475-153), donkey anti-rat Alexa647 (Jackson Immuno Research, 712-606-153), donkey anti-goat DyLight405 (Jackson Immuno Research, 705-475-147), and donkey anti-goat Alexa647 (Jackson Immuno Research, 705-606-147). The labeling pattern observed with these antibodies closely resembled expression patterns of genes encoding these proteins or other neurotransmitter-related proteins, as previously observed with in situ hybridization (antibody target, gene ): hypocretin- hcrt (Prober et al, 2006), cocaine- and amphetamine-regulated transcript (CART)- cart2 (Thisse and Thisse, 2004), galanin- gal (Podlasz et al, 2012), neuropeptide Y- npy (Mathieu et al, 2002), neuropeptide VF- npvf (Yelin-Bekerman et al, 2015), somatostatin- sst1.1 (Herget and Ryu, 2015), serotonin- slc6a4a/b (Norton et al, 2008), tyrosine hydroxylase- th1 (Ryu et al, 2006; Filippi et al, 2010), dopamine beta-hydroxylase- dbh (Holzschuh et al, 2003; Filippi et al, 2010), and choline acetyltransferase- vachta (Hong et al, 2013). …”

“…Afterward, protocols were followed as detailed in Choi et al (2016), using the hybridization chain reaction (HCR) technique. Twelve DNA 20-mer probes were designed against hypocretin ( hcrt ) mRNA (Yelin-Bekerman et al, 2015) using Stellaris software (LGC Biosearch Technologies), with added ‘B1’ amplifiers to the 5′ and 3′ ends of each probe, and B1-linked Alexa 647 hairpins (Molecular Instruments, Caltech). Fish were then re-mounted in agarose on a Petri dish lid (in SSCT), and imaged again under the two-photon microscope.…”

Pharmacological treatment and BBB-targeted genetic therapy for MCT8-dependent hypomyelination in zebrafish

“…In order to study AHDS and hypomyelination, we used the zebrafish model, which combines invertebrate-like genetics with vertebrate brain structures (Elbaz et al, 2015; Levitas-Djerbi et al, 2015; Yelin-Bekerman et al, 2015), and its transparency allows real-time imaging of myelination in a live animal (Kirby et al, 2006; Buckley et al, 2008). In addition, zebrafish larvae have emerged as an attractive model for genetic manipulations and high-throughput therapeutic drug screens (Kaufman et al, 2009; MacRae and Peterson, 2015; Tsuji et al, 2014).…”

Genome-wide association analyses of sleep disturbance traits identify new loci and highlight shared genetics with neuropsychiatric and metabolic traits

“…The leading associations overlap interesting candidate genes enriched in murine/zebrafish hypocretin expressing neurons 37,38 , differentially expressed in sleep-deprived rats 39 , and/or regulate sleep in Drosophila 40 . Credible set analyses 41 highlighted a number of potential causal variants at each locus (Table 1) and future experimental studies will be necessary.…”