Current Biology, Volume 26

Supplemental Information

Reorganization of Sleep by Temperature

in Drosophila Requires Light,

the Homeostat, and the Circadian Clock

Katherine M. Parisky, José L. Agosto Rivera, Nathan C. Donelson, SejalKotecha, and Leslie C. Griffith

B

-80

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C Δ

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DayNight

A

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0 6 12 18 24slee

p (m

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ZT (hours)

25oC baseline29oC day 1

***

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**

*

n.s.

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Figure S1

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per01 ;; c

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Light PeriodD

ark Period

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Figure S2

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**

F

B

E

C

D

n.s.

n.s.

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******

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DayNight

A

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1015202530

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p (m

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ZT (hours) 25oC baseline29oC day 3

n.s.

Figure S3

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Slee

p (m

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29oC25oC

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60

day

time

slee

p (m

in) 180B *** ** ** **

ZT0-3 ZT4-6 ZT7-9 ZT10-12

25oC constant29oC ZT12-24

25oC25oC

-40

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20 ***

*

Figure S4

Δ m

ean

epis

ode

dura

tion

(min

s) Day

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10 n.s.

** Δ a

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Day

Night

A

B

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120 per01;;UAS-per

per01; pdfR-GAL4

per01; pdf-GAL4

per01; elav-GAL4

per01; c929-GAL4

per01;; UAS-per (male)Δ d

aytim

e sl

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(min

s)

Figure S5

All n.s.

A

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wildtype

per01; elav-GAL4;UAS-per

per01; c929-GAL4;UAS-per

per01; Clk4.1-GAL4/UAS-per (male)

per01

per01; pdfR-GAL4;UAS-per

per01; pdf-GAL4;UAS-per

Δ n

ight

time

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p (m

ins)

Δ

nig

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(min

s) per01;;UAS-per

per01; pdfR-GAL4

per01; pdf-GAL4

per01; elav-GAL4

per01; c929-GAL4

per01;; UAS-per (male)

***

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* *****

***

***

n.s.

**

*

n.s. **

**

LightSLEEP

Light + Clock

SLEEP

Dark

Light during thepreceding day

Homeostatic sleepdrive due to nighttime

sleep loss

-+

Figure S6

hTo hTo

Supplemental Figure Legends

Figure S1. Acute effects of temperature shift in male flies. (A) Baseline sleep per 30 min

comparing 25oC to 29oC after a temperature shift at ZT0 in wild type Canton S male flies (n =

91). (B-E) Changes from baseline in light and dark periods are shown for the following sleep

parameters: (B) Total sleep, (C) Mean sleep episode duration, (D) Total number of beam

breaks, and (E) Activity level while awake (beam breaks/min active). Data in panels B-E are

presented as means ± SEM and 25oC compared to 29oC using Type 2, 2-tail Student’s t-test,

P<0.05=*, P<0.005=**, P<0.0005=***, n.s.=not significant. Supports Figure 1.

Figure S2. Chronic effects of temperature on sleep after a ZT0 increase in temperature.

Data from wild type Canton S animals shown in Figure 1 from the third day after they were

shifted from 25oC to 29oC at ZT0. (A) Baseline sleep per 30 min comparing 25oC (black line) to

29oC (gray line) after a temperature shift at ZT0 in wild type Canton S flies (n = 35). (B-E) show

changes from 25oC baseline day in light and dark periods for the following sleep parameters: (B)

Total sleep, (C) Mean sleep episode duration, (D) Total number of beam breaks, and (E) Activity

level while awake (beam breaks/min active). Data in panels B-E are presented as means ± SEM

and 25oC compared to 29oC using Type 2, 2-tail Student’s t-test. (F) data from the genotypes

shown in Figure 2 from the third day after they were shifted from 25oC to 29oC. Changes in total

daytime (white bars) and nighttime (black bars) sleep, 25oC vs. 29oC, are shown. Data are

presented as means ± SEM and are compared within genotype (25oC vs. 29oC) using Student’s

t-test, P<0.05=*, P<0.005=**, P<0.0005=***, n.s.=not significant. Supports Figures 1 and 2.

Figure S3. Nighttime sleep loss causes a homeostatic increase in sleep the next day

independent of ambient daytime temperature. To address the issue of whether the increase

in sleep seen after a ZT12 temperature shift was actually homeostatic sleep or if still-high

temperature during the recovery period after temperature-induced sleep deprivation was

causing the observed increase in sleep, we subjected flies to a ZT12-ZT24 heat pulse of 29oC

with a shift back to 25oC at ZT0 when the lights come on. (A) Sleep plots comparing sleep at

baseline (25oC black line) to sleep after a shift to 29oC from ZT12-ZT24 (gray line) for wild type

flies. Shaded background area indicates dark period. (B) Comparison of sleep in 3-hour bins

during the day for wild type animals at 25oC continuously (black bars) or at 25oC after a ZT12-

ZT24 shift (gray bars). Data are presented as means ± SEM and compared using Student’s t-

test. P<0.05=*, P<0.005=**, P<0.0005=***, n.s.=not significant. Supports Figure 3.

Figure S4. ZT12 temperature shift is associated with changes in mean sleep episode

duration and changes in arousal. (A) Change in mean sleep episode duration in Canton S

wild type flies was calculated by subtracting mean episode duration during the baseline 25oC

period from the same parameter the day after a ZT12 shift to 29oC. (B) Change in activity of

Canton S wild type flies during wake periods (beam breaks/min during wake) after a ZT12 25°C

to 29°C shift is shown. Locomotor activity during the day, when sleep increases is unchanged

and locomotor activity at night, when sleep decreases is decreased. This indicates that changes

in arousal, not locomotor activity, underlie sleep alterations induced by temperature shift. Data

are presented as means ± SEM and comparison of baseline to shift days done with Student’s T

test, P<0.05=*, P<0.005=**, P<0.0005=***, n.s.=not significant. Supports Figure 3.

Figure S5. Daytime sleep changes in UAS and GAL4 controls and nighttime sleep in

rescue and control genotypes. (A) Change in total daytime sleep after a shift from 25oC to

29oC at ZT0 is shown for control genotypes: per01;pdfR2-GAL4/+ n = 26; per01;pdf-GAL4/+ n =

23; per01;elav-GAL4/+ n = 26; per01;c929-GAL4/+ n = 18; per01;;UAS-per16/+ n = 25 for female

and 8 for male. Data are presented as means ± SEM. Comparison of total sleep during baseline

day to the day after shift was done using Type 2, 2-tail, Student’s t-test, and there were no

significant differences for any genotype. (B-C) Nighttime sleep reduction was intact in all

genotypes. Change in total nighttime sleep of same animals shown in Figures 4 (B) and panel A

(C) after a shift from 25oC to 29oC at ZT0 is shown. Data are presented as means ± SEM.

Comparison of total sleep during baseline day to the day after shift was done using Type 2, 2-

tail, Student’s t-test, P<0.05=*, P<0.005=**, P<0.0005=***, n.s.=not significant. Supports Figure

4.

Figure S6. Integration of light, temperature and clock effects on sleep. Sleep is modeled as

an active on-going process that can occur both during the day and at night. Heat has an

intrinsically inhibitory effect on the generation of sleep that can be modulated by light and

circadian processes. Cartoon represents the 24 h day broken into light and dark periods (LD).

During the day in LD an increase in temperature at ZT0 increases sleep drive by suppressing

the intrinsic inhibitory pathway. This requires an intact molecular clock in a subset of neurons

(DN1ps or LNvs) in the fly clock circuit. If heat exposure persists, this clock-driven increase in

daytime sleep is augmented by homeostatic rebound sleep due to nighttime sleep loss.

Decreases in nighttime sleep do not require a clock, but are enhanced by the exposure to light

the previous day. Supports all Figures as a summary.