Chapter 13

 Feeding and drinking  Patterns in metabolism  Migratory and reproductive cycles  Sleeping and waking cycles  Hibernation cycles  Periodic events with particular timing, like the onset of labor in humans is usually early morning or eclosion in insects  Internal alarm clock

 Epicycles = variable period length, ex. Lugworms feed every 6-8 min  Tidal = 12.4 hr period – ex. Crabs and bivalves – activity patterns are influenced by tides; Euglena in River Avon in Southern England  Lunar = 28 day period – ex. Grunion (marine fish) use moon as cue for spring spawning  Circadian = 24 hour period – ex. Activity cycles in many different animals. Active and inactive phases.  Circannual = 1 yr period– ex. Hibernation, reproduction, migration and reproduction in a wide range of animals  Intermittent cycles - variable interval, ex. Emergence in insects triggered by rain

1. Unaffected by changes in temperature, even in cold-blooded organisms. (Even though this affects chemical reaction rates) 2. Unaffected by metabolic poisons such as sodium cyanide that blocks biochemical pathways 3. They are self-sustaining even without environmental cues 4. They can be entrained to particular environmental cues or zeitgeber. That is the clock can be reset with exposure to particular cues.

 Thus animals seem to have an innate pacemaker or clock that triggers certain changes in activity.  This innate clock does not work in isolation.  It is adjusted by input from the environment.  Without input from the environment, the clock will tend to drift off course

Clock is never actually exact – without environmental cues, cycle will tend to drift  Aschoff’s rule

1. Differs in location among animals – but cellular mechanism is similar even in diverse animal systems. 2. Early work was done with insects: Ex. Saturniid moths – larva  pupa  adult ^ * this transition takes place at a specific time of day When ready to shed cocoon, insect waits; pealing cuticle away does not speed up process. At the right time, such a “pealed” moth goes through the motions of shedding the non-existent cocoon. Spreads wings and acts like an adult How is this adult behavior switched on at a particular time of day?

 Pupa developed normally and shed cocoon  But emergence was abnormal – some behavior was omitted, others out of sequence – emerged at all sorts of odd hours

 Animal emerged at the proper time of day!  Conclusion: brain was a neuroendocrine center and does not need to be connected by nerves to the body to regulate the clock

 Animals eclosed early  Conclusion: some hormone produced by the brain at the correct time for eclosion controls emergence time

 Times for eclosion were switched  Conclusion: Chemicals regulating eclosion time are not species specific, the cells in the brain act independently of the body.

 Animal emerged at correct time Exp. 6: Removed brain, placed loose brain in abdomen and exposed the abdomen and head areas to different L:D regimes.  Animals emerged at the correct time according to what the loose brain experienced

 Conclusion: The photoreceptor cells, which regulate the biological clock are found in the brain as is the clock itself. Cells secrete the molting hormone when stimulated by light- dark photoperiod to do so.  But how do cells in the brain “know” how long a day is and thus what time of the day it is?  How about circannual cycles?

 period (per) and timeless (tim) - discovered first.  Both show a daily cycle in gene expression, causing active protein production to fluctuate in a repeating 24 hour cycle.  Mutation for tim gene results in loss of circadian rhythm

 Transgenic flies – express luciferase (w/ luciferin) on commands from promoter of clock gene = “period” (also used GFP = green fluorescent protein in jelly fish)  Separated head, thorax, abdomen tissue – rhythmic bioluminescence periodicity could be changed by altering light and dark cycle  Found intense expression in antennae, mouthparts and ocelli  Cellular level expression in chemoreceptors on wings and legs  In constant dark – oscillations in bioluminescence decreased

 Independent oscillators – entrained in isolation  If all respond to light/dark cues independently, may free run independently- this may explain why rhythm is lost in constant dark  All tissue appears to be light sensitive

 5 genes regulate circadian feedback loop  Period (per)  Timeless (tim)  Drosophila Clock (dCLK)  Cycle (CYC)  Double-time (dbt)

Per and Tim form a dimer that enters nucleus at night PER-tim dimer binds with dCLK-CYC dimers and 1) Represses per-tim mRNA transcription and protein synthesis. Dbt breaks down Per protein. 2) Removes dCLK-CYC repression of dCLK transcription thus activates dCLK transcription By Mid-day: high levels of dCLK-CYC w/o Per-Tim, promotes transcription of Per and Tim mRNA dCLK-CYC represses dCLK transcription Per-Tim dimers accumulate and move to nucleus in evening.

Per DayNight Tim Per/Tim CYCdCLK dClk Per/Tim dClk Remove Cell cytoplasm Nucleus

 The hypothalamus connects and controls the master endocrine gland, the pituitary gland

 When this area is cut away, not only are circadian activity rhythms destroyed, but so are – heart rate, hormone secretion patterns, locomotory cycles, and feeding behavior cycles  Master clock of hypothalamus seems to be: SCN or Suprachiasmatic nucleus

 Destroy SCN, overt rhythmic behavior stops  Mutant – short circadian period ▪ Transplanted normal SCN into individual with own SCN destroyed - circadian rhythm restored – always that of donor tissue ▪ Basis = genetic – one gene locus – if heterozygous – circadian rhythm is 22 hr free running cycle ▪ homozygous = 20 hour free running cycle  SCN has a similar gene expression circadian clock to that described for fruit flies.  A similar clock has been described in other vertebrate groups as well.

 4 genes: CLOCK, BMAL, Period (Per), Cryptochrome (Cry)  CLOCK and BMAL form a dimer that regulates the expression of the dimer formed by Per and Cry, which in turn regulate the expression of the first two. Same as seen in fruit flies.  This molecular clock occurs in cells throughout the body – often in clusters of cells regulating activity in various organs

 As shown in hamsters, the brain has the master clock that regulates the other cells and their molecular clocks.  Expression in other genes are also linked to the circadian rhythm of CLOCK/BMAL dimer formation and breakdown  Ex. Liver: PPAR alpha increases lipid metabolism; PPAR gamma promotes fat production and storage. Energy regulated by turning on and off of these two genes.

 Tissues involved vary among species, but genes regulate circadian cycles by triggering on and off production of regulatory proteins  Genes are similar or the same across species.  Brain = source of hormones that tell target cells when to turn on or off, makes entrainment possible  Eyes are not needed in some organisms to entrain to LD cycles; characteristics of brain cells or even all cells.

 Daily Patterns or shorter  Maintains activity at appropriate times; metabolism  Hibernation/ Estivation  Prepare for dormancy before conditions change  Emergence from pupation; Birth process; Other  Migration  Long distance movement to avoid adverse conditions  Cyclical reproductive cycle  Reproduce in the correct place at the correct time

Many birds, mammals, and insects, etc. migrate on an annual cycle Exp. With the garden warbler indicate that it is due to seasonal changes in the birds’ internal physiological state induced by an internal circannual clock. ▪ Gwinner and Wiltschko hand reared 11 baby garden warblers ▪ Housed individually in cages and exposed to a constant environment L:D = 12:12; constant temperatures

 Birds molted and began to be restless at night with wing-whirring  Restlessness was repeated the following spring  Each time birds oriented and hopped in the appropriate direction (south in fall and north in spring)  This annual pattern is maintained up to 12 annual cycles = max lifespan in captivity Conclusions: birds knew when and in what direction to migrate w/o any external cues

 Many passerine birds migrate at night even though they are diurnal.  Entails major shift in physiology and behavior  Birds continue to have diurnal clock for activity  Second clock controls migratory behavior at night ▪ Separate – drifts separately from diurnal clock  Birds stop sleeping or do unihemispheric sleep ▪ Some birds can function with ½ brain awake and ½ of the brain sleeping. Not sure if this is what migrating birds do. Maybe they catnap while flying.  Flying at night, foraging by day, no sleep = extreme physiological strain.

 White crowned sparrows – reproductive organs grow and shrink during their reproductive cycle Winter flocking gonadsBehavior Cycle <1% max size Increased feeding Migration – south Physiological restless Cycle hormonehormone Restless dec inc Migration north gonads Establish territory Increased Feedingfull size summer Care of young

Exp. 1 Set clock L:D 8:16 no gonad stimulation Exp. 2 Set clock L:D 8:28 (lower light dark ratio) – testes grow Exp. 3 Set L:D = 8:16 + 2hr light hr after the start of “day” =>testes grow

Photosensitivity L:D 8:16 8:28 8:16 +2 no yes

 Daily cyclical change in sensitivity with a peak hr after start of light = entrainment – circadian rhythm  If dark during this period, hormones are not activated  If light, even occasionally, hypothalamus is stimulated which activates pituitary which stimulates testes - circannual rhythm  Significance with respect to their ecology: Their breeding grounds are in Alaska. Only there are days very long. Thus gonad development is not stimulated until males and females are at their breeding grounds.

 Some birds migrate around the equator or move from northern to southern hemispheres, so photoperiod isn’t a useful cue.  Such birds do have a circannual clock as shown with garden warblers that maintained seasonal migratory behavior despite D:L 12:12.  Mechanism is unclear at this point.