王歐力 助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University

1. Laboratory course: Model organism C. elegans Week 2: Sensory neurons Behavior Worm mating (crossing) 王歐力助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University Institute of Molecular & Cellular Biology College of Life Science

2. Features of the nervous system Neurons can be categorized in: interneurons, sensory neuronsand motorneurons interneuron sensory neuron • can be made visible • with a special dye: DiI/DiO motorneuron

3. Head neurons The nerve ring contains mostly sensory neurons and almost all interneurons Labialprocess bundles Amphid process bundles Ring ganglia UNC-104:: GFP

4. Posterior head Middle head Front head UNC-104:: GFP

5. Identifying amphids • Amphids: are a pair of laterally located sensilla in the head which are open to • outside at the base of the lips • These chemosensory organs can be stained with FITC or DiI • Some mutants fail to be stained => dyf = dye filling (mutant) • Each amphid is made up of 12 sensory neurons (ADF, ADL, ASH…) with • ciliated dendrites as well as one sheath and one socket cell • The axon of the amphid is located in the nerve ring

6. Reproduction • 99% of adult C. elegans are self-fertilizing hermaphrodites • This feature enables scientists to easily generate homozygous mutant stocks • Hermaphrodites are protandrous: the gonads produces germ cells which first differentiate as sperm (about 250 sperm cells) and then produces eggs • In C. elegans hermaphrodites reproducibility is determined by the sperm supply • C. elegans produce males to about 0.05% which are fully functional but produce • sperm only; the sperm is transferred to the hermaphrodites during mating => this feature enables scientists to transfer mutant alleles (to wildtypes or other mutants) spermatheca • Oocyte nuclei are produced by meiosis at the distal end • of the gonad and grow in a syncytium • Just before fertilization the single nuclei are enclosed by • a separate plasma membrane • Produced sperm is stored in the spermatheca • After fertilization the egg-shell is added: • => self-fertilization produces up to 300 eggs

7. General biology of C. elegans • Fertilization takes place by squeezing mature oocytes through the spermatheca • The eggs are laid through the vulva at a 40-cell stage • Adult hermaphrodites have about 10 mature eggs inside; the older eggs are laid as • fast as new eggs are generated A hermaphrodite produces up to 1300 eggs during its lifetime • Males cannot produce eggs but they can cross-fertilize hermaphrodites making • them very suitable in genetics to create genetic combinations (male-fertilizing) • The male sperm outcompetes the hermaphrodites sperm during cross-fertilizing • XO combination in male is a spontaneous loss of X chromosome: XX => XO

8. The C. elegans lifecycle • The 4 larval stages (“juveniles”) are common features of nematodes • The lifecycle takes about 2.5 days at 25°C, 3.5 days at 20°C and 5.5 days at 15°C • C. elegans also has an alternative L3 stage known as dauer (“enduring”) stage • The dauer stage is a metabolic diapause to survive extreme conditions (mainly lack • of food); in the wild, the dauer stage might serve to geographically disperse • The entry into the dauer stage is • determined by worm-crowding, high • temperature and lack of food • As a dauer, C. elegans can survive • for up to 3 month highly extending • its lifespan • On exposure to improved conditions (availability of food…) the L3 dauer exits and resumes development • Parasitic nematodes use the dauer to infect hosts

9. Recovered dauer larvae retain their transparent appearance and begin feeding with increasing motion Dauer larvae usually appear dark, thin, rigid and motionless

10. During dauer-formation the mouth closes • There is no aging at dauer state! Due to the dauer stage worms can live 10 times • longer than their normal lifespan! • Due to the mouth closure the worms are restricted from eating Dauer worm L3 worm Imagine a human that is supposed to become 90 years old might become 900 years old

11. During post-embryonic development the number • of somatic cell nuclei increases to 959 • Total life span under suitable living • conditions: 2 weeks (300 eggs every • 4 days) = 1300 eggs during a lifetime • Wildtype-worms: Bristol (most commonly • used strain) has been isolated from • mushroom compost in Bristol (England) = • N2 strain • Other strains isolated from soil and • moist environment are C. briggsae • and C. remanei about 1.3 mm in length and 80 µm in diameter

12. Behavior, learning and memory

13. Mechanosensing: Avoidance reflex circuits Mechanosensory neurons can sense different strength of touch: eyelash touch • “Mec response”: • touching the anterior side of the body results in a backward movement • touching the posterior side results in a forward movement

14. Mechanosensory neurons Gentle touch (eyelash) mechanosensory neurons Harsh touch mechanosensory neurons Nose touch and osmolarity sensors Texture sensing neurons

15. Simple behavior Thermotaxis Chemotaxis Traces of a worm which was allowed to freely move within one hour on a plate with an attractant (B = biotin) (right circle = buffer) • Traces of a worm in a radial thermal • gradient • Chooses an optimal temperature (similar to its cultivation temperature) • Circles in isotherms at that temp. • Can detect thermal gradients < 0.1°C

16. Habituation (non-associative learning) • If stimulus is applied less often (every 60 seconds) decrease of backing response is less abrupt (indicating habituation and not worm fatigue) • Also habituation can be rapidly abolished with an electroshock stimulus (dishabituation) • Further, worms recover from short-interval habituation faster compared to long-interval habituation • Tapping at the side of the petri dish stimulates backing response • Backing movement decreases if stimulus is continuously applied (every 10 seconds for total 30 minutes) Habituation occurrence can be stored for more than 1 hour in the nervous system

17. Na+ = food Cl- = no food (short incubation) 5 hours deprivation Classical conditioning (associative learning) • Non-associative learning (habituation): an animal alters its behavior to a single • stimulus • Associative learning: an animal learns to use a previously neutral stimulus to predict a second more significant stimulus • Example: after food deprivation, one ion is associated with food and the second • ion is associated with the absence of food => a conditioned animal will move to • the ion associated with food (even if no food is present) • lrn-1 and lrn-2 (lrn = learn) are genes involved in learning worms go to Na+

18. Male mating behavior

19. Male mating behavior WAmating.mov