Genetic Ablation of Hypocretin Neurons Alters Behavioral State Transitions in Zebrafish

Abstract

Sleep is an essential biological need of all animals studied to date. The sleep disorder narcolepsy is characterized by excessive daytime sleepiness, fragmentation of nighttime sleep, and cataplexy. Narcolepsy is caused by selective degeneration of hypothalamic hypocretin/ orexin (HCRT) neurons. In mammals, HCRT neurons primarily regulate the sleep/wake cycle, feeding, reward-seeking, and addiction. The role of HCRT neurons in zebrafish is implicated in both sleep and wake regulation. We established a transgenic zebrafish model enabling inducible ablation of HCRT neurons and used these animals to understand the function of HCRT neurons and narcolepsy. Loss of HCRT neurons increased the expression of the HCRT receptor (hcrtr). Behavioral assays revealed that HCRT neuron-ablated larvae had normal locomotor activity, but demonstrated an increase in sleep time during the day and an increased number of sleep/wake transitions during both day and night. Mild sleep disturbance reduced sleep and increased c-fos expression in HCRT neuron-ablated larvae. Furthermore, ablation of HCRT neurons altered the behavioral response to external stimuli. Exposure to light during the night decreased locomotor activity of wild-type siblings, but induced an opposite response in HCRT neuron-ablated larvae. Sound stimulus during the day reduced the locomotor activity of wild-type sibling larvae, while HCRT neuron-ablated larvae demonstrated a hyposensitive response. This study establishes zebrafish as a model for narcolepsy, and indicating a role of HCRT neurons in regulation of sleep/wake transitions during both day and night. Our results further suggest a key role of HCRT neurons in mediating behavioral state transitions in response to external stimuli.

Altered Behavioral Performance and Live Imaging of Circuit-Specific Neural Deficiencies in a Zebrafish Model for Psychomotor Retardation

“…To test the response of mct8−/− larvae to light and dark stimuli, we exposed the larvae to three cycles of alternating 30 min periods of light and darkness during the day (ZT3–ZT6). Both mct8−/− (n = 139) and WT siblings (n = 139) responded to light and dark stimuli with robust changes in locomotor activity, as previously shown [51], [53]. Confirming our finding of reduced activity in mct8−/− larvae (Fig.…”

“…Embryos were produced by natural spawning and raised in egg-water containing methylene blue (0.3 ppm) in a light-controlled incubator at 28±0.5°C, as previously described [51]. All animal protocols were reviewed and approved by the Bar-Ilan University Bioethics Committee.…”

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9

“…We characterized these ectopic Hcrt neurons at 120 hpf, when the Hcrt neuronal circuit is functional (Prober et al, 2006;Elbaz et al, 2012), and confirmed that all zebrafish Hcrt neurons in the hypothalamus and hindbrain express pdyn and vglut1, two markers of mammalian Hcrt neurons (Chou et al, 2001;Rosin et al, 2003). Unlike previous studies (Appelbaum et al, 2009;Rosin et al, 2003), we did not observe expression of vglut2a or vglut2b in hypothalamic or hindbrain Hcrt neurons, indicating that Hcrt neurons in larval zebrafish express different VGLUT family genes than adult zebrafish or rats.…”

Zebrafish as a Model for Monocarboxyl Transporter 8-Deficiency

“…Embryos were generated by natural spawning and raised in egg water in a 28 Ϯ 0.5 ºC, light-controlled incubator, as previously described (23). All animal protocols were reviewed and approved by the Bar-Ilan University Bioethics Committee.…”

“…Triplicate first-strand cDNA aliquots from each sample served as templates in real-time PCR. The relative quantification of dio1, dio2, dio3, and tsh␤ mRNA expression levels were normalized against ␤-actin mRNA expression levels and subjected to the ⌬⌬C T method (23). Statistical significance was determined using one-way analysis of variance.…”