1. Infusion of EGF blocks locomotor activity in hamsters Running wheel assay Period of infusion; therefore it’s reversible

2. Mouse EGF Receptor mutant is defective in light response Together, these experiments suggest EGF is necessary and sufficient for activity/sleep cycle Red boxes represent light pulses

4. Sleep in worms EGF and EGF-R Target neuron - ALA Neuropeptides and receptors

5. CHROMOSOME_IIcctaagcctaagcctaaaatagtgactctggcagttctctaaaataagtgactctggcagttcaccaaaaattgtgactctgaccgttcaccaaaaatagtgactctgaccgttcaccaaaaatagtgactctgaccgttcacaaaaaatagtga ctctgaccgttcaccaaatatagtgactctgaccgttcaccaaaaattgtgacaatgaccgttcaccaaaaattgtgactctgaccgtcactatttttattgaactgccagagtcactatttttagtgaacttccagagtcacaatttttagtgaactgccagagtcactatttttag tgaactgccagagtcacttattttggtgcactggggtgggtcacgcccccagttctcagttatgggtactctgatccactcgggacccactttatcgtgttccccgtgcctcatttaccctagagcttcctcctttacctctcctctcgctatctctaacattccaatggaaactcc tatttgaattaccgccaccgatgtgcccgacgcgacttactgttagcccttgttttgcacaaatctgttggcttccatatttaaaagttaattaatgacccaatgttctttttttctctaaatctccacaagatgttctgttttccctactggacactatcgttcactgcgtctcaccaatt cacattgtctctactttaccttttttgtcatagtacacgttcgccaacggtgtcgacggccaaatgctttgggcagcgtttgctttttttataattagttttattttattaaaacaatagctctaaagtttacaagtcatttgttataggctaaatgagttatgtctaataagtaatttgaacta gatacttccgtgtaagtgacaatgtatcggaaaagtcctcaaagtgcgatgtagaagttcacatgtactttgtttggcatgttagtaaaagagccagtatgctgattcattttatattctatatactcatgtaatatgcccatgtaaggtttaattccaaaaatatgagcgtgttctatt ttataatattttactaaaatacctttcagttaattgcactcaaatttgttgttcttcattctctcgttatgatttaatcttattgcgtcaaggtcattattttaggtccattagttatcgatctgaaacatgttgttgtatttttctattcttgtgagctcaggacacctcatacaactccagagaa aatgtgtctcattattcttgtcttttttcaagatctaatcaattttctacattaacgacgtttttgtcgttctgcttctttttttcgttcgtttgtctcgtccatcagctgtccactcatttctctcccactcactaggcagtgctttgtttggttccgattggcagctggctgcagggcctgcat ctcttctatgtctctcatttacttgcattcttttcttcgttaatttttgttatgatatttaaacgggaagaagagtttgtggttcttctttttataatcactaaaacttttggataagtaacaattttctgataaaatattttcacggcgaagaaaaaagaaaaagaagagtagtttttgcacgt tttcatataattattttcgttgatcaaatgttcttctggagttttctaataaatttcttatcgactttttttcagaaatttttctcaacttgtcatgtcaatggtaagaaatgtatcaaatcagagcgaaaaattggaagtaagttctttataatttcatttatatactataagttttctcgatcaca ggagaaacaaaaacaacagacaacacaaaaaacaataaaacaatattgctctagtaatcaatagtgttgtaaagagggaagaaaattgttatctgtgtagcagtcaacgttgattgagatgttgtgtttgactatagagttgaaaataataacttcaaacttgcaagtcatga cttatcaaacactgccggaacttattctggatcaaaggaaagttgtccaactgtagagtcatgtttttcaaaagaaaacacaatttttaagtataaatattttgaaaaagtatgttttagaagtatgtcaaattaaaaaaaaaatccttggttaaaaaatgatttttttggatatatgtg tatttttaactaaaaatatatactttacatatatattttggcgcagttatttgatctataaatcaaactttttgatagacatttttttatatttacaacaactagggttgttatgaaaacgcctattattctacaaactaaattattttaatcatacattccccactatctaaaaactaatgcaattt tcagattttgtcatgtaaatgggtaggatgtctcaaatcaacagaagtgttcaaaacggttgaaaagttattagatcatgttacggctgatcatattccagaagttattgtaaacgatgacgggtcggaggaagtcgtttgtcagtgggattgctgcgaaatgggtgccagtc gtggaaatcttcaaaaaaaggtatttttaatttaatgtgcattttataatataaattcttcagaaagagtggatggagaatcacttcaaaacacgtcatgttcgcaaagcaaaaatattcaaatgcttaattgaggattgccctgtggtaaagtcaagtagtcaggaaattgaaac ccatctcagaataagtcatccaataaatccgaaaaaaggtattcacaatttgcatgatattgttataatctaattttcagagagactgaaagagtttaaaagttctaccgaccacatcgaacctactcaagctaatagagtatggacaattgtgaacggagaggttcaatgga agactccaccgcggtaagtgtgtttctttaaaaattacttccttttttcaattgtttgaaattaacaagaaacctgttggagcgtatttctgaacttttaaatcgaaaatatcatttgcaaaaaaacttgaaaattgagaaacttttttaaaagtggagtagcgtctgcgggtttttttgc cctaaatgacagaatacatacccaatataccgaatataaccgtaataaaattatgcgatttttatttttatttttcatgaatgttaggggcaaaaaacccacatgcgctactccgccttaagaagaatcagctgtgagcactatccactatacattggaaatttacaaataaaata gagattaagtaatataatttttaagggttaaaaaaaagactgtgatatactatgatgatgggccgaggtatgtatttccaacgggatgtgcgagatgcaactatgatagtgacgaatcagaactggaatcagatgagttttggtcagccacagagatgtcagataatgaaga gtacgttgttttgcaaattgattaaaagtggagtagcgtcagttaaaaactctaacatgtcttaggtttttcaaaagtttggtcaaagttttggcaaactgccaacttcttgaaaacttcgttaaaaaaattcttgaaatgatttgaaaatttgtattatgttattctcttatttctgcactat tctatatggcgctactatacttttaattgatttcttgaaagcagttcaataataattaattttagagtatatgtgaacttccgtggaatgaactgtatctcaacaggaaagtcggccagtatggtcccgagcaaacgaagaaattggccaaaaagagtgaagaaaaggctatcg acacaaagaaacaatcagaaaactattcgaccaccagagctgaataaaaataatatagagataaaagatatgaagtaagtcgaaattgacaaacagtggtttttgtttaagtttattgcgaaatattcaaaattagacatgttaaaattttgcgagataatctaaagattaggt atacagattttttcatgtaaagttacattcatcaaaatttttgtgttcaccaaattagacaaaaaatgttagttacacagtatatttattttttatatcaataaaacctttttcagctcaaataaccttgaagaacgcaacagagaagaatgcattcagcctgtttctgttgaaaagaaca tcctgcattttgaaaaattcaaatcaaatcaaatttgcattgttcgggaaaacaataaatttagagaaggaacgagaagacgcagaaagaattctggtgaatcggaagacttgaaaattcatgaaaactttactgaaaaacgaagacccattcgatcatgcaaacaaaata taagtttctatgaaatggacggggatatagaagaatttgaagtgtttttcgatactcccacaaaaagcaaaaaagtacttctggatatctacagtgcgaagaaaatgccaaaaattgaggttgaagattcattagttaataagtttcattcaaaacgtccatcaagagcatgtc gagttcttggaagtatggaagaagtaccatttgatgtggaaataggatattgattttataacgtgtaattgagttttggccaaaaaggtatggaaaggtggctgtttagttatatatttttctattatttatttgaaacatgcaaaattgaagtgaacaataagtgatgttcatggaaat ttaaactgttttatgatacttttttgagaaattgaaaaatctgttcattttagaaacaatgtccacatggttctaagagctaaaatttttattttcatccatttagagtactttctcttttagagtacggccccagagcgatgttagaaacctgagatcggtcaacacagaccgttaatttt gggaagttgagaaattcgctagtttctgacacgaatttcagctaataccaaataatgtgcaattgcattttgcatgtcagcattcagcattcatacaaaaatttcaaagagccaacttttcatacgtttatggtcaacactgtatgtgttacattgaacttttttaaattgtattatttcat atttgaatcatttccatgccattttcaaatctttttttaacaaaaatttagtttcaaagttttaaatttaggtgaaaacttgctacaaaataacacattctttagtcgtttcaattgctatctatccgcagactgcaattttgttttcccacaaccattcacacaataaataagggtataaagt tttgttcatataacacatttcaatactaacatttcaattttgaacaatttttctaaacttatttcccttcgcccaaacgtcattcaacattctttgtacaaaacattaccattatagaaaatctcatttttccactatttcatttatttttattgttccgcgataaatataaataaacatttacgtgtt ccgagttcaaagttttaccacgtttcagaataaaggaatcggaggggggggggggtgaaaaaatcatttcaacaaatcagaatttctcaaaatgtgagttttatcattttcattgttagaatcacgattagcttatattgaacaataacaataatttaatcccattcattccatatct tctcatgatgaaaaaataagcattattcgttttcttttgacgcgtttgatagggttctgccagcgccgaccaatattcttctcaattagattttccagaactgcactaactgaaaattgttttagtaaatagaacaaaactgactattagttcaatattataattttacttccctattttctg acagcattttgcaaatactctttacagtcattcttgtattttgacaacaaaattcgaatttaaaatttcatttttcttttaaaatatcagtacactcctggtagaacaaatattttatttcaatgtttatgtatgagtagaaaagttaaacagaactattatttggcatcaatttctgtttttttttt catctaaagtatggcatttattagcaaaaaatacatgcttgaacaggaataactattaatttcctatagcgacccaaaaacaaccaaaaattgttttaaataatttttttttggtcgacttccatagttatgagtggcaaaaactgagtaattgtcaccttttgacagtaaataaaga aattttcaaaaaatttttgaaaagttttattatgctattcgatcattttggcaccatgtaggctttaacaccccactggcgctactccgcatttaaaagtgatttataaaaagtgtaaggaatactttgctccaattttcgtctagtgcatggcgtacccatattttgttcatgttgtgtctc ttagcctctcctcggaggcgagtttccatagctacccacaaagaatttatttttctatgtatgcttcgttggctccaccttcattttctcagtcgtttctcagttttctcatgaattctctttttgttcgtttttagaatagctatcgttttaccacgaatgcgctttttcagttattaagaaaac agttttttgtagtacatttttcatagtctgaactttcagcatggaaatagtaaaaacaatcattccacacaaccgttcatacattgaaccgcccataccatctgccaccgagtggcttgcaagtaagttgttttgtctaaagacataatttctgaaagaaatctatccgatgagcgg aaaataaacagaagttgtttttaagaagactttgatgcaattacaaaaatatatggctatgtgcgtaaaaattagacgtgactcgtcataaagaccatataggtctacaaaggcgtttcgagtcttataaaagttcgttcaaactttttcggggtcttgttaaaagcacaccaaca atacgttaaaggatttataaaattgcaagtattcgtctcaattgcaatactttggaagccgaattttggcaagatattggtaaaacaggtacattacaatagctatttttggtaaaatgtactaggtatcttgtaatgagttgcgcttgcctcataggcatacatctaccgtatattctt tactagtgctgcaggcagcactaatttctaggcccttttttaatgcagtactattagagactgcagtactactggagatgcagcgctaatagagaatatacggtatgtagatgcctagaacgctaaggttgttttattcaaataaatttcttagaaaggctaatattatttttaaaat caagatttaagttaattaacacgacctcgtattctatatttcagatatgtcagttatgcatgtgtcatgtttaaccattgcttgtgtatttgttgctatcacatttttgtcatcatttttccatttattttttgtattgaaatatgtgtctaatgagaggatacgcaatgatatgtacgcattgata tttatgtttcccgtgagtttataaatggaattatgaagagtttggaaaatatattttagataaccacatttgcaagtttagtaggaatgttcataccaagagcggctattttcctttatgcagtttcccttgtgtaagtgtaccttaacaccacaaacagaaaaatgaactttattgattt gttccagatatttcatgtttaccctcttcataatggtcactcttttattcaacatttttggaggtcggcaagaaatgagtgcctacttgctccaaagaaatattcgagttaatttcactgttccaccactctgttttttcaagttcctgccaactgtagaaagtacagagtgagctcgaa aatcttatttttgtctttctaatatattttccagtcaaaacctacgtcgaatcgagtggttggtttttcaaactcctattatacgaactcttttagagttagtctcagtcgttgtgtctatggaacaagaaggaagacgagaaagcgtgtaagaccatgaaatgttttttgtgtgcgcaa cgtagtgcgaaaagttagttatttggagaggaagttatttgaaaagaatctttagaagatattgatattttatattctgtatgtaggcgctgtggcaaattctcgacttcacgggagaagcgcaaattttgtgctggtttccataatgcgctcgaaaaatgttgtatatttacattatt caaatctcaccgattaaagttgaatttcagttggttcgtattctctcaactcatggcactactctcgatgtgcattgccttttatggttgctacgttatggttcctcttggaagagaaaagcatgctccataccgattcgattttctatttcgtacatgtgacattgctcagtgtatttac acaattcaaaagtttgtctttgagtttgctgctgcagttggcttgatcacttcggatcgataccttccagctgctgcgaaggcattgtgtaagttcagaagtttaaatggagataggaatgtttcgattttcgtttataaaaacacctgttaatattaaattgttgacgtcatagctga ccattcatgcttctttgtctgattgtaattatataattgtaaaaatacaaacaagttaaccgaaaaagttctctgttacgtttgcggtaaattagaactttatgttgaaagatatcagcttaaagttaaaattttttgaaaattaaattttggctagaaattcaatttttctgataaaaagaa agtttggtgggaaatttaatttcattgatagaaaaactgaagtttgtgttttctgagaattaaaaaacagcgggcaatgaagattatgtctttgtaaatttcaatttaaaaatattccagggtgggcatcgtttatgtgcacctgggagatgatgctcctctctgctctttgctcatatt gtttgcgtccagccaagtgtaaattttttgatctttaccctggaaacgatatgccagctttatcggcaagagatggttcaaactcccgtgttccgtcattttctcgacgtctatctattgaatatgagccacgaattgctggagtgatgttggagccgccgtcaagaagttcactg agcattactcctagggataaaatagaagacccaacaaccgtatcgtattttgctgataattttgattctttatcgcaaatccaaggacaataattgaacttcctgccattatttcattgctcaatttacactattcttaactttttgacatgaattaaatctttttaaactgagtctaagcg tattattgtatcgtattttccccttctgatgtattcatttacttgtatttttgaaacccatcccagtgatacaactacccaattttcctgtgccatgtttcttgaaacaaatcaaatgtgat 5 C. elegans SLEEP: molecules & circuits graphic by Chris Grove [WormBase-Caltech] Elly Chow Julie Cho Cheryl Van Buskirk Erich Schwarz Daniel A. Lee, David Prober 302 neurons Paul Sternberg, Cal tech

6. SEM-5 Grb2 LET-341 SOS LET-60 RAS [IP 3 ] Vulval development PLC  [PIP 2 ] Ovulation ITR-1 IP 3 Receptor SLI-1 Cbl LFE-2 IP3K LET-23 EGF-R LIN-3 EGF [DAG] Y~P

7. Wild-type adult worms are almost constantly moving Cheryl Van Buskirk Van Buskirk & Sternberg Nat Neuro 2007

8. EGF expression severely inhibits locomotion (no movement unless perturbed) Cheryl Van Buskirk

9. EGF promotes quiescence during molting via ALA neuron Time (min) Velocity (mm/sec) 0 50 100 150 200 0.20 0.15 0.10 0.05 0 -.05 0.20 0.15 0.10 0.05 0 -.05 N2 let-23(sy12) plc-3(tm1340) ALA- Wild-type, intact Wild-type ALA-ablated EGF-receptor mutant PLC-  mutant Van Buskirk & Sternberg Nat Neuro 2007

10. The single ALA neuron is necessary for EGF-dependent sleep ~ALA-ablated animals are insomniac ~Expression of EGFR only in ALA is is sufficient for EGF-induced sleep ~ALA synapses not necessary for function Cheryl Van Buskirk

11. SEM-5 Grb2 LET-341 SOS LET-60 RAS [IP 3 ] Vulval development PLC  [PIP 2 ] Ovulation ITR-1 IP 3 Receptor SLI-1 Cbl LFE-2 IP3K LET-23 EGF-R LIN-3 EGF [DAG] Sleep Synaptic Release UNC-13, etc.

12. EGF signaling mediates stress-induced sleep Time (min) Velocity (mm/sec) 0 50 100 150 200 0.20 0.15 0.10 0.05 0 -.05 WT let-23(rf) lethargus behavior EGFEGFR EGF/EGFRSTRESS stress-induced sleep Van Buskirk Lab, Cal State Northridge

13. Micro-dissection & Transcriptional profiling of single ALA neurons L4 L4 Lethargus 4-5hr Single cell RT-PCR cDNA amplification 9 cells (2 pools) Igor Antoshechkin, Caltech Jacobs Genome Center Elly Chow Erich Schwarz, Miriam Goodman, Mihoko Kato Schwarz, Kato & Sternberg [PNAS 2012]

14. Erich Schwarz ALA enriched genes: 10 neuropeptide genes FRMFamide-Like Peptide Expression Level Enrichment 8110 protein coding genes detected in ALA

15. ALA-enriched neuropeptides (>12-fold over whole larvae)

16. FLP-24 FLP-7 FLP-13 VPSAGDMMVRF SPMQRSSMVRF TPMQRSSMVRF SPMERSAMVRF SPMDRSKMVRF AMDSPLIRF AADGAPLIRF APEASPFIRF ASPSAPLIRF SPSAVPLIRF ASSAPLIRF SAAAPLIRF VPSAGDMMVRFG VPSAGDMMVRF amide KRVPSAGDMMVRFGKR

17. FLP-13 NLP-8 FLP-24 FLP-7 NPR-7 NPR-22 sleep EGF ALA neuron neuropeptidesreceptors FRPR-3 ? STRESS EGFR Elly Chow

18. sleep neuropeptidesreceptors sleep neuropeptidesreceptors Elly Chow

19. 35°C, 30 min recover RT, 10 min score 4 neuropeptides necessary for EGF response Quiescence Time (min) WT

20. sleep neuropeptidesreceptors sleep neuropeptidesreceptors sleep neuropeptidesreceptors Elly Chow

21. Quiescence Time (min) Multiple receptors are necessary

22. sleep neuropeptidesreceptors sleep neuropeptidesreceptors sleep neuropeptidesreceptors sleep neuropeptidesreceptors Elly Chow

23. FLP-13 NLP-8 FLP-24 FLP-7 NPR-7 NPR-22 sleep EGF ALA neuron neuropeptidesreceptors FRPR-3 ? STRESS EGFR Elly Chow