MCB 186CIRCADIAN BIOLOGY

Slides Lecture 2 Basic Properties of Circadian Clocks

September 27, 2006

J. W. Hastings

DEFINITION of TERMS in CIRCADIAN BIOLOGY

GLOSSARY on COURSE WEB SITE and in TEXT BOOKS

CIRCADIAN RHYTHMS - KEY PROPERTIES

(1) RHYTHMS CONTINUE IN THE ABSENCE OF LIGHT/DARK

CYCLES WITH PERIODS CLOSE TO BUT NOT EXACTLY 24H

exact period length is a function of environmental conditions

(2) TEMPERATURE ALSO AFFECTS CIRCADIAN PERIOD BUT MUCH

LESS SO THAN FOR TYPICAL BIOCHEMICAL REACTIONS. Cellular

compensation is postulated to be responsible

(3) PHASE can be RESET by LIGHT: ENTRAINED or SYNCHRONIZED to

DAILY LIGHT / DARK or OTHER ENVIRONMENTAL CYCLES

resetting does not need cycles: single exposures or pulses suffice

RHYTHM IN HUMAN: LD & LL

Raster Plots:- Single, double, triple etc.- Modulo tau

Tau greater than 24h

CIRCADIAN CLOCKS MAY BE VERY PRECISE DECOURSEY, 1961

Activity of squirrelTau less than 24h

PERIOD (Tau) DEPENDS on LIGHT INTENSITY

PERIOD (Tau) of CIRCADIAN RHYTHM DEPENDS on PERIOD (T=20) OF PRIOR L/D CYCLE

MOUSEPITTENDRIGH & DAAN

DRUGS MAY ALSO HAVE an EFFECT ON PERIOD e.g., PROTEIN PHOSPHATASE INHIBITORS

TEMPERATURE-COMPENSATED CIRCADIAN PERIOD IN VARIOUS ORGANISMS

ENTRAINMENT OCCURS when ORGANISM is EXPOSED TO

LIGHT/DARK CYCLES

CAN BE CALLED SYNCHRONIZATION

HUMAN CIRCADIAN ENTRAINMENTCHANGING PHASE OF L/D CYCLE CHANGES PHASE of RHYTHM

ENTRAINMENT by L/D CYCLES in MONKEY DURING a FREE-RUN

MOORE-EDE ETAL

Schematic depiction of entrainment by light: T constant, Tau changes

GONYAULAX ENTRAINMENT by 14 HR LD CYCLES, then DD or LL

ENTRAINMENT by DIFFERENT LIGHT/DARK CYCLES and EFFECT of LIGHT INTENSITY on LIMITS of ENTRAINMENT

APPARENT ENTRAINMENT by TEMP CYCLE is a DIRECT EFFECT

FLYING SQUIRREL DE COURSEY

ENTRAINMENT is due to PHASE SHIFTS in CIRCADIAN RHYTHM

BUT PHASE SHIFTS do not REQUIRE FULL LIGHT/DARK CYCLES

A SINGLE “BRIEF” EXPOSURE TO LIGHT SUFFICES

GONYAULAX CELLS IN DD: PHASE SHIFT BY SINGLE LIGHT PULSESADVANCE OR DELAY DEPENDS ON TIME IN CYCLE

CONTROLIN DARK

LATE NIGHT PULSEPHASE ADVANCE

EARLY NIGHT PULSEPHASE DELAY

GONYAULAX LIGHT PHASE RESPONSE CURVE (PRC)

GONYAULAX LIGHT PHASE RESPONSE CURVE (the PRC)LIGHT PULSES GIVEN AT TIMES INDICATED

TIME 0 is theBEGINNING ofNIGHT PHASE

DEAD ZONEDAY PHASE

HOW DOES ONE DISTINGUISH DELAYS from

ADVANCES?

GIVE a STRONGER(BRIGHTER) EXPOSURE to LIGHT

THIS RESULTS in a GREATER PHASE SHIFT - but in WHICH DIRECTION??

GONYAULAX PHASE ADVANCES BY LIGHT PULSES

GONYAULAX ACTION SPECTRUM FOR PHASE SHIFTING BY 3 HOUR LIGHT EXPOSURES

PHASE RESPONSE CURVE DESCRIBED MOORE-EDE ETAL

Black is day phase

SCHEMATIC PHASE RESPONSE CURVES (PRCs)

PULSES of ANISOMYCIN (protein synthesis inhibitor) CAUSE PHASE SHIFTS in Gonyaulax

PHASE SHIFTS BY ANISOMYCIN 0.3 M, 1 HOUR

LONG DAYS (LIGHT PERIOD) VERSUS SHORT DAYS

LONG DAYS CAUSE BOTH DELAYS and ADVANCES

SHORT DAYS ONLY ONE or the OTHER

Phase angle (acrophase) differs with day length

LONG & SHORT DAY PHASE SHIFTS in an ORGANISM with a CIRCADIAN TAU OF 24.5 hr

ENTRAINMENT by T CYCLES with DIFFERENT PHOTOFRACTIONS

Short day

Long day

DROSOPHILA ENTRAINMENT TO FULL LD CYCLES BLACK DOTS INDICATE PEAK TIMES OF ECLOSIONPITTENDRIGH

SKELETON PHOTOPERIODS

TWO LIGHT EXPOSURES (e.g., 15 min, or 1 hr each) EVERY CYCLE

Longer dark interval (outside of 11-13) is self-selected as the night phase.

11-13 is the bistability region

Cycles can be different from 24 hrs.

LONGER INTERVAL is ALWAYS SELECTED as NIGHT PHASE DROSOPHILA ECLOSION ACROPHASES with DIFFERENT INTERVALS for TWO-PULSE SKELETON PHOTOPERIODS

Pittendrigh

BISTABILITY IN HAMSTER ACTIVITY RHYTHM ENTRAINMENT SKELETON PHOTOPERIOD 13.5:0.25:10:0.25 hrs

SINGLE PULSES EVERY 24hSometimes called T-cycles

A single light pulse (e.g., 15 min, 1hr) every cycle will entrain a circadian rhythm.

Cycle may be =24h, longer or shorter than 24

Organism self selects location of light pulse

If tau is greater than 24 it will fall at late night

If tau is less than 24 it will fall at early night

HAMSTER ACTIVITY RHYTHM is ENTRAINED by SINGLE LIGHT PULSES of CYCLE LENGTHS LONGER or SHORTER

THAN 24 h and PULSES ARE POSITIONED DIFFERENTLY

HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?

ALTER CONDITIONS (temperature, light cycle)

APPLY INHIBITORS OR DRUGS (what kinds?)

ISOLATE MUTANTS (select for what?)

DROSOPHILA CLOCK MUTANTS in the PERIOD (per) GENE

PERIOD MUTANTS OF CYANOBACTERIA (kai GENE) KONDO ET AL.1995

CIRCADIAN CLOCK GENES

1) DROSOPHILAper (PERIOD)tim (TIMELESS)

2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)

3) CYANOBACTERIAkai (CYCLE IN JAPANESE)

4) ARABIDOPSIStoc1 (TIMING OF CAB)

lhy (LATE ELONG HYPOCOTYL)

cca1 (CIRC CLOCK ASSOCIATED)

5) MOUSEclk (CLOCK)per1 (PERIOD)

6) HAMSTERtau (PERIOD)

POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION

ClockProtein

ClockProtein

| |P P

ClockGene

ClockmRNA

PositiveRegulators

Other ClockProteins

| |P P

ATP

Figure 1b

CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS