Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish
- California Institute of Technology, United States
Figures
Figure 1 with 3 supplements
Prazosin treated larvae are less active and sleep more than vehicle treated controls.
Representative activity (A), waking activity (amount of locomotor activity while awake) (B) and sleep (C) traces of vehicle (blue) and prazosin (red) treated zebrafish larvae. Bar graphs of activity …
Figure 1—figure supplement 1
Prazosin night sleep dose–response curve.
Larvae were treated with prazosin over range of concentrations (n = 12 for each concentration) on the morning of day 5 of development. Sleep was measured during night 5. Mean ± s.e.m is shown for …
Figure 1—figure supplement 2
Clonidine treated larvae are less active and sleep more than vehicle treated controls during the day.
Representative activity (A), waking activity (B) and sleep (C) traces of vehicle (blue) and clonidine (red) treated zebrafish larvae. Bar graphs of activity (D), waking activity (E), sleep (F), …
Figure 1—figure supplement 3
Bopindolol treated larvae sleep more than vehicle treated controls.
Representative activity (A), waking activity (B) and sleep (C) traces of vehicle (blue) and bopindolol (red) treated zebrafish larvae. Bar graphs of activity (D), waking activity (E), sleep (F), …
Figure 2 with 2 supplements
dbh mutant larvae are less active and sleep more than sibling controls.
Representative activity (A), waking activity (B) and sleep (C) traces of dbh+/+ (blue) dbh+/− (cyan) and dbh−/− (red) zebrafish larvae. Bar graphs show mean ± s.e.m. activity (D), waking activity (E)…
Figure 2—figure supplement 1
Verification of zebrafish dbh mutant.
(A) Alignment of human, mouse, WT zebrafish and mutant zebrafish DBH amino acid sequences. The coding sequence of the mutant allele terminates before the conserved active site residue (asterisk). (B)…
Figure 2—figure supplement 2
Treatment of dbh−/− fish with prazosin does not alter sleep/wake behavior.
Representative activity (A), waking activity (B) and sleep (C) traces of vehicle (blue) and prazosin (red) treated zebrafish dbh−/− larvae. Bar graphs of activity (D), waking activity (E) and sleep …
Figure 3
dbh mutants and bopindolol-treated WT larave exhibit lower arousal threshold.
Stimulus-response curves generated by a tapping assay for dbh−/− and sibling control larvae (A), and bopindolol and DMSO treated WT larvae (B). Thirty trials were performed at each stimulus …
Figure 4 with 4 supplements
Reduced sleep at night due to Hcrt overexpression is suppressed in dbh mutants.
(A, C) Sleep traces from a single representative experiment; yellow region indicates time of heat shock (HS) during day 6. (B, D) Bar graphs show mean ± s.e.m. percentage change in sleep during …
Figure 4—figure supplement 1
Hcrt neurons project to dbh expressing cells.
Hcrt neurons (red) project to dbh expressing cells (green) in the LC. (A) Dorsal-ventral maximum intensity projection, (B) lateral maximal intensity projection (dorsal to the right). Boxed regions …
Figure 4—figure supplement 2
Reduced sleep at night following Hcrt overexpression requires the hcrtr.
(A, C) Sleep traces from a single representative experiment; yellow region indicates time of HS during day 6. (B, D) Bar graphs show mean ± s.e.m. percentage change in sleep during night 6 compared …
Figure 4—figure supplement 3
Reduced sleep at night due to Hcrt overexpression is suppressed by prazosin.
(A, C) Sleep traces from a single representative experiment; yellow region indicates time of HS during day 6. (B, D) Bar graphs show mean ± s.e.m. percentage change in sleep during night 6 compared …
Figure 4—figure supplement 4
The heat shock promoter is not suppressed in hcrtr or dbh mutants.
Representative images of brains from larvae that do not (A) or do (B) contain the hsp:Hcrt transgene, fixed 30 min after HS, and processed for ISH using a hcrt-specific probe. hcrt overexpression …
Figure 5 with 1 supplement
Optogenetic activation of Hcrt neurons increases locomotor activity.
(A) Representative images of Hcrt neurons co-labeled with EYFP or EGFP (green) and c-fos (red). Scale bar = 10 μm. (B) Mean ± s.e.m. percentage of EYFP- or EGFP-expressing neurons that also express c…
Figure 5—figure supplement 1
Specific expression of ChR2-EYFP in Hcrt neurons and temporal dynamics of hcrt:ChR2-EYFP induced locomotor activity.
(A) Representative image from a Tg(hcrt:ChR2-EYFP) larval brain showing Hcrt (red) and EYFP (green) immunoreactivity, and merged images (yellow). Arrow indicates a neuron that is shown at higher …
Figure 6 with 1 supplement
The hcrtr is required for Hcrt neuron-induced increased locomotor activity.
(A, C) Representative locomotor activity traces for hcrtr−/− and sibling controls without (A) and with (C) the hcrt:ChR2-EYFP transgene during blue light exposure. (B, D) Average locomotor activity …
Figure 6—figure supplement 1
Hcrt signaling is required for Hcrt neuron-induced increased locomotor activity.
In this experiment, larvae of all six genotypes were tested in the same behavioral plates. Tg(hcrt:ChR2-EYFP);hcrtr+/+ larvae (blue stripes) showed a significant increase in locomotor activity …
Figure 7
Optogenetic stimulation of Hcrt neurons activates LC neurons.
(A) Schematic representation of areas stimulated (Hcrt, yellow) and imaged (LC, green). a = anterior, p = posterior. (B, C) Representative images of a LC cell expressing GCaMP6s in a Tg(hcrt:EGFP) (B…
Figure 8
dbh is required for Hcrt neuron-induced increased locomotor activity.
(A, C) Representative locomotor activity traces for dbh−/− and sibling control larvae without (A) and with (C) the hcrt:ChR2-EYFP transgene during blue light exposure. (B, D) Average locomotor …
Videos
Video 1
Pulsed illumination of hcrt:EGFP neurons does not affect GCaMP6s fluorescence in LC neurons.
Two trials for a representative LC neuron are shown. Frames labeled ‘Stimulation’ indicate periods during which the soma of Hcrt neurons were illuminated by ten 0.3 s pulses of 488 nm light, during …
Video 2
Pulsed illumination of hcrt:ChR2-EYFP neurons increases GCaMP6s fluorescence in LC neurons.
Two trials for a representative LC neuron are shown. Frames labeled ‘Stimulation’ indicate periods during which the soma of Hcrt neurons were illuminated by ten 0.3 s pulses of 488 nm light, during …
Additional files
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Source code 1
Scripts used for analysis of behavioral data. sort_fish_sttime_192.pl is a Perl script (Prober et al., 2006) that converts data acquired by the Viewpoint videotracker system to a format that is useful for analysis using Matlab and removes notations that are not relevant to behavioral analysis. perl_batch_192well.m is a Matlab script that allows the sort_fish_sttime_192.pl script to run on the Matlab platform. TapAnalysis.m is a Matlab script that analyzes tapping assay data and generates a table that lists the number of larvae that moved during each tapping event. VT_analysis.m is a Matlab script (modified from Prober et al., 2006) that analyzes locomotion data collected by the Viewpoint videotracker system to quantify several metrics, including activity, waking activity, sleep, sleep architecture and sleep latency. Detailed instructions on the use of these scripts will be provided upon request.
- https://doi.org/10.7554/eLife.07000.024
