2/10/16 Lecture 8: Highlights a classic neurogenetic dissection . CIRCADIAN RHYTHM Combined use of genetics, molecular biology, ‘anatomy” and physiology to understand behavior. - what genes are required? (initial discovery that can be distant from understanding) - what are the molecular functions of the encoded protein? (a molecular mechanism”) -in which cells do these molecules work? (identifes a circuit element) - what do these cells do in the nervous system and how? (identifes circuit mechanisms/ computations involved in the behavior) -how model organism genetics contributes to neuroscience/ neurogenetics. Medical importance of circadian Circadian rhythms: general rhythms features • In Phase with the earth’s rotation. • Jet lag. • Sleep disorders. • Entrained and reset by environmental cues. • Seasonal affective disorders. • Temperature compensated. • Present in individual cells (cyanobacteria, protozoa) as well as complex multicellular creatures. 1 2/10/16 1. Screened 2000 mutant X chromosomes for flies with altered eclosion rhythms in a 12:12 LD cycle.. 2. Rescreened for defects in DD cycle. 3. Tested them for locomotor rhythm defects. The Classic LD Locomotor Activity Pattern of Drosophila 2 2/10/16 WILD-TYPE vs MUTANT (per-short) per GENE AND LOCATION OF BEHAVIOR (1971) THREE ORIGINAL MUTATIONS Rosbasch/Hall Young per GENE RESCUE Northern blot analysis of per RNA at different times of day per produces a cycling mRNA! 3 2/10/16 Model for circadian regulation of per gene expression Effects of Per: A model for circadian control (transcription)" 1. Per must activate its own repressor/ or represses its activator. Per mRNA, protein as molecular readouts for the circadian clock" 2. The proteins are turned over. 3. Light (Zeitgeber) must reset the clock - interfering with 1 or 2 Per promoter - as a centre of the circadian clock." Justify larger screens for circadian-cycle mutants. Circadian pacemaker: the negative transcriptional" feedback loop in flies CLK jrk CYC per/tim PER E-box PERTIM PERTIM TIM nucleus cytoplasm 4 2/10/16 PER TRANSCRIPTION IS VERY LOW IN THE CLK MUTANT Jrk Drosophila Clock Genes" (transcription feedback loop) luciferase E-box + Clock* + + Cycle* - - Period * - - Timeless * Drosophila Clock Genes" Protein turnover is essential for the clock (kinases) CLKCYC Dbt - cKIε* per/tim PER cKII* (affect stability of clock proteins) E-box Sgg – GSK3 PERTIM PERTIM TIM nucleus cytoplasm 5 2/10/16 Drosophila Clock Genes" Understanding circadian behavior (photoreceptor) Clock* PP2A, Vrille, Pdp1 PER Cycle* Pdf, To CLK CYC per PER PER Period * PER PER TIM TIM Timeless * TIM CLK CYC Cry * Mediates light-induced degradation of Tim tim TIM ? Dbt - cKIε* cKII* Sgg – GSK3 How does this light-entrained molecular oscillator work in an organism? Six Different Clock Cell Groups in the Adult Brain. cry pdf 6 2/10/16 The small and large LNvs Tissue-specific expression using the express the neuropeptide PDF GAL4/UAS system gal4 cell death gene pdf UAS X GAL4 cell death gene tissue specific cell death There is no morning peak without PDF or the LNvs ? M cells PDF E cells ? PDF Same effect in pdf-Gal4 X UAS hid. 7 2/10/16 Neurogenetic analysis of the central pacemaker What does this have to do with humans or mammals? 1. Pdf positive neurons are part of the central circadian 1. At a genetic level? pacemaker and Pdf is a neuropeptide used for circadian communication. 2. At a systems level? 2. Pdf (and Pdf-positive M cells) affect morning activity (and free-running rhythm) 3. Cry+ and Pdf- cells (E-cells) affect evening activity Natural Variation may occur through changes Different coupled oscillators are proposed to in genes identified in these analyses. orchestrate M and E peaks in mammals (Daan and Pittendrigh, 1976) 1. Hamster tau - wake up 2-3 hours early - mutations caused by defects in double-time (phosphorylates Per) 2. Advanced Sleep phase syndrome (ASPS) is caused by mutations in hper2 8 2/10/16 To illustrate the following principles. Circuit Dissection (and the Janelia Farm Project) a) Using genetics to address a fundamental problem in biology. 1. Identify / create sparse Gal4 lines 2. Document morphological connectivity of Gal4 labeled neurons b) How model organism genetics can contribute 3. Develop quantitative and “automated” behaviors 4. Study effects of conditional activation or conditional inhibition c) Te integration of genetics, molecular biology, ‘anatomy” and 5. Look for functional connectivity physiology in behavioral genetics. - Which genes? - Protein activity? - In which neurons? - How are the neurons wired and what do they do? Figure 3 - Detectable actions. “High throughput” automated behavioral screens Automated monitoring and analysis of social behavior in Drosophila. Heiko Dankert, Liming Wang, Eric D Hoopfer, David J Anderson & Pietro Perona. Nature Methods 6, 297 - 303 (2009). 9 2/10/16 10 .