Infusion of EGF blocks locomotor activity in hamsters · Sleep in worms! EGF and EGF-R! Target neuron - ALA! Neuropeptides and receptors!

Infusion of EGF blocks locomotor activity in hamsters

Running wheel assay

Period of infusion; therefore it’s reversible

TGFα, an EGF like factor, disrupts sleep-wake behavior

Mouse EGF Receptor mutant is defective in light response

Together, these experiments suggest EGF is necessary and sufficient for activity/sleep cycle

Sleep in worms

EGF and EGF-R

Target neuron - ALA

Neuropeptides and receptors

CHROMOSOME_IIcctaagcctaagcctaaaatagtgactctggcagttctctaaaataagtgactctggcagttcaccaaaaattgtgactctgaccgttcaccaaaaatagtgactctgaccgttcaccaaaaatagtgactctgaccgttcacaaaaaatagtgactctgaccgttcaccaaatatagtgactctgaccgttcaccaaaaattgtgacaatgaccgttcaccaaaaattgtgactctgaccgtcactatttttattgaactgccagagtcactatttttagtgaacttccagagtcacaatttttagtgaactgccagagtcactatttttagtgaactgccagagtcacttattttggtgcactggggtgggtcacgcccccagttctcagttatgggtactctgatccactcgggacccactttatcgtgttccccgtgcctcatttaccctagagcttcctcctttacctctcctctcgctatctctaacattccaatggaaactcctatttgaattaccgccaccgatgtgcccgacgcgacttactgttagcccttgttttgcacaaatctgttggcttccatatttaaaagttaattaatgacccaatgttctttttttctctaaatctccacaagatgttctgttttccctactggacactatcgttcactgcgtctcaccaattcacattgtctctactttaccttttttgtcatagtacacgttcgccaacggtgtcgacggccaaatgctttgggcagcgtttgctttttttataattagttttattttattaaaacaatagctctaaagtttacaagtcatttgttataggctaaatgagttatgtctaataagtaatttgaactagatacttccgtgtaagtgacaatgtatcggaaaagtcctcaaagtgcgatgtagaagttcacatgtactttgtttggcatgttagtaaaagagccagtatgctgattcattttatattctatatactcatgtaatatgcccatgtaaggtttaattccaaaaatatgagcgtgttctattttataatattttactaaaatacctttcagttaattgcactcaaatttgttgttcttcattctctcgttatgatttaatcttattgcgtcaaggtcattattttaggtccattagttatcgatctgaaacatgttgttgtatttttctattcttgtgagctcaggacacctcatacaactccagagaaaatgtgtctcattattcttgtcttttttcaagatctaatcaattttctacattaacgacgtttttgtcgttctgcttctttttttcgttcgtttgtctcgtccatcagctgtccactcatttctctcccactcactaggcagtgctttgtttggttccgattggcagctggctgcagggcctgcatctcttctatgtctctcatttacttgcattcttttcttcgttaatttttgttatgatatttaaacgggaagaagagtttgtggttcttctttttataatcactaaaacttttggataagtaacaattttctgataaaatattttcacggcgaagaaaaaagaaaaagaagagtagtttttgcacgttttcatataattattttcgttgatcaaatgttcttctggagttttctaataaatttcttatcgactttttttcagaaatttttctcaacttgtcatgtcaatggtaagaaatgtatcaaatcagagcgaaaaattggaagtaagttctttataatttcatttatatactataagttttctcgatcacaggagaaacaaaaacaacagacaacacaaaaaacaataaaacaatattgctctagtaatcaatagtgttgtaaagagggaagaaaattgttatctgtgtagcagtcaacgttgattgagatgttgtgtttgactatagagttgaaaataataacttcaaacttgcaagtcatgacttatcaaacactgccggaacttattctggatcaaaggaaagttgtccaactgtagagtcatgtttttcaaaagaaaacacaatttttaagtataaatattttgaaaaagtatgttttagaagtatgtcaaattaaaaaaaaaatccttggttaaaaaatgatttttttggatatatgtgtatttttaactaaaaatatatactttacatatatattttggcgcagttatttgatctataaatcaaactttttgatagacatttttttatatttacaacaactagggttgttatgaaaacgcctattattctacaaactaaattattttaatcatacattccccactatctaaaaactaatgcaattttcagattttgtcatgtaaatgggtaggatgtctcaaatcaacagaagtgttcaaaacggttgaaaagttattagatcatgttacggctgatcatattccagaagttattgtaaacgatgacgggtcggaggaagtcgtttgtcagtgggattgctgcgaaatgggtgccagtcgtggaaatcttcaaaaaaaggtatttttaatttaatgtgcattttataatataaattcttcagaaagagtggatggagaatcacttcaaaacacgtcatgttcgcaaagcaaaaatattcaaatgcttaattgaggattgccctgtggtaaagtcaagtagtcaggaaattgaaacccatctcagaataagtcatccaataaatccgaaaaaaggtattcacaatttgcatgatattgttataatctaattttcagagagactgaaagagtttaaaagttctaccgaccacatcgaacctactcaagctaatagagtatggacaattgtgaacggagaggttcaatggaagactccaccgcggtaagtgtgtttctttaaaaattacttccttttttcaattgtttgaaattaacaagaaacctgttggagcgtatttctgaacttttaaatcgaaaatatcatttgcaaaaaaacttgaaaattgagaaacttttttaaaagtggagtagcgtctgcgggtttttttgccctaaatgacagaatacatacccaatataccgaatataaccgtaataaaattatgcgatttttatttttatttttcatgaatgttaggggcaaaaaacccacatgcgctactccgccttaagaagaatcagctgtgagcactatccactatacattggaaatttacaaataaaatagagattaagtaatataatttttaagggttaaaaaaaagactgtgatatactatgatgatgggccgaggtatgtatttccaacgggatgtgcgagatgcaactatgatagtgacgaatcagaactggaatcagatgagttttggtcagccacagagatgtcagataatgaagagtacgttgttttgcaaattgattaaaagtggagtagcgtcagttaaaaactctaacatgtcttaggtttttcaaaagtttggtcaaagttttggcaaactgccaacttcttgaaaacttcgttaaaaaaattcttgaaatgatttgaaaatttgtattatgttattctcttatttctgcactattctatatggcgctactatacttttaattgatttcttgaaagcagttcaataataattaattttagagtatatgtgaacttccgtggaatgaactgtatctcaacaggaaagtcggccagtatggtcccgagcaaacgaagaaattggccaaaaagagtgaagaaaaggctatcgacacaaagaaacaatcagaaaactattcgaccaccagagctgaataaaaataatatagagataaaagatatgaagtaagtcgaaattgacaaacagtggtttttgtttaagtttattgcgaaatattcaaaattagacatgttaaaattttgcgagataatctaaagattaggtatacagattttttcatgtaaagttacattcatcaaaatttttgtgttcaccaaattagacaaaaaatgttagttacacagtatatttattttttatatcaataaaacctttttcagctcaaataaccttgaagaacgcaacagagaagaatgcattcagcctgtttctgttgaaaagaacatcctgcattttgaaaaattcaaatcaaatcaaatttgcattgttcgggaaaacaataaatttagagaaggaacgagaagacgcagaaagaattctggtgaatcggaagacttgaaaattcatgaaaactttactgaaaaacgaagacccattcgatcatgcaaacaaaatataagtttctatgaaatggacggggatatagaagaatttgaagtgtttttcgatactcccacaaaaagcaaaaaagtacttctggatatctacagtgcgaagaaaatgccaaaaattgaggttgaagattcattagttaataagtttcattcaaaacgtccatcaagagcatgtcgagttcttggaagtatggaagaagtaccatttgatgtggaaataggatattgattttataacgtgtaattgagttttggccaaaaaggtatggaaaggtggctgtttagttatatatttttctattatttatttgaaacatgcaaaattgaagtgaacaataagtgatgttcatggaaatttaaactgttttatgatacttttttgagaaattgaaaaatctgttcattttagaaacaatgtccacatggttctaagagctaaaatttttattttcatccatttagagtactttctcttttagagtacggccccagagcgatgttagaaacctgagatcggtcaacacagaccgttaattttgggaagttgagaaattcgctagtttctgacacgaatttcagctaataccaaataatgtgcaattgcattttgcatgtcagcattcagcattcatacaaaaatttcaaagagccaacttttcatacgtttatggtcaacactgtatgtgttacattgaacttttttaaattgtattatttcatatttgaatcatttccatgccattttcaaatctttttttaacaaaaatttagtttcaaagttttaaatttaggtgaaaacttgctacaaaataacacattctttagtcgtttcaattgctatctatccgcagactgcaattttgttttcccacaaccattcacacaataaataagggtataaagttttgttcatataacacatttcaatactaacatttcaattttgaacaatttttctaaacttatttcccttcgcccaaacgtcattcaacattctttgtacaaaacattaccattatagaaaatctcatttttccactatttcatttatttttattgttccgcgataaatataaataaacatttacgtgttccgagttcaaagttttaccacgtttcagaataaaggaatcggaggggggggggggtgaaaaaatcatttcaacaaatcagaatttctcaaaatgtgagttttatcattttcattgttagaatcacgattagcttatattgaacaataacaataatttaatcccattcattccatatcttctcatgatgaaaaaataagcattattcgttttcttttgacgcgtttgatagggttctgccagcgccgaccaatattcttctcaattagattttccagaactgcactaactgaaaattgttttagtaaatagaacaaaactgactattagttcaatattataattttacttccctattttctgacagcattttgcaaatactctttacagtcattcttgtattttgacaacaaaattcgaatttaaaatttcatttttcttttaaaatatcagtacactcctggtagaacaaatattttatttcaatgtttatgtatgagtagaaaagttaaacagaactattatttggcatcaatttctgttttttttttcatctaaagtatggcatttattagcaaaaaatacatgcttgaacaggaataactattaatttcctatagcgacccaaaaacaaccaaaaattgttttaaataatttttttttggtcgacttccatagttatgagtggcaaaaactgagtaattgtcaccttttgacagtaaataaagaaattttcaaaaaatttttgaaaagttttattatgctattcgatcattttggcaccatgtaggctttaacaccccactggcgctactccgcatttaaaagtgatttataaaaagtgtaaggaatactttgctccaattttcgtctagtgcatggcgtacccatattttgttcatgttgtgtctcttagcctctcctcggaggcgagtttccatagctacccacaaagaatttatttttctatgtatgcttcgttggctccaccttcattttctcagtcgtttctcagttttctcatgaattctctttttgttcgtttttagaatagctatcgttttaccacgaatgcgctttttcagttattaagaaaacagttttttgtagtacatttttcatagtctgaactttcagcatggaaatagtaaaaacaatcattccacacaaccgttcatacattgaaccgcccataccatctgccaccgagtggcttgcaagtaagttgttttgtctaaagacataatttctgaaagaaatctatccgatgagcggaaaataaacagaagttgtttttaagaagactttgatgcaattacaaaaatatatggctatgtgcgtaaaaattagacgtgactcgtcataaagaccatataggtctacaaaggcgtttcgagtcttataaaagttcgttcaaactttttcggggtcttgttaaaagcacaccaacaatacgttaaaggatttataaaattgcaagtattcgtctcaattgcaatactttggaagccgaattttggcaagatattggtaaaacaggtacattacaatagctatttttggtaaaatgtactaggtatcttgtaatgagttgcgcttgcctcataggcatacatctaccgtatattctttactagtgctgcaggcagcactaatttctaggcccttttttaatgcagtactattagagactgcagtactactggagatgcagcgctaatagagaatatacggtatgtagatgcctagaacgctaaggttgttttattcaaataaatttcttagaaaggctaatattatttttaaaatcaagatttaagttaattaacacgacctcgtattctatatttcagatatgtcagttatgcatgtgtcatgtttaaccattgcttgtgtatttgttgctatcacatttttgtcatcatttttccatttattttttgtattgaaatatgtgtctaatgagaggatacgcaatgatatgtacgcattgatatttatgtttcccgtgagtttataaatggaattatgaagagtttggaaaatatattttagataaccacatttgcaagtttagtaggaatgttcataccaagagcggctattttcctttatgcagtttcccttgtgtaagtgtaccttaacaccacaaacagaaaaatgaactttattgatttgttccagatatttcatgtttaccctcttcataatggtcactcttttattcaacatttttggaggtcggcaagaaatgagtgcctacttgctccaaagaaatattcgagttaatttcactgttccaccactctgttttttcaagttcctgccaactgtagaaagtacagagtgagctcgaaaatcttatttttgtctttctaatatattttccagtcaaaacctacgtcgaatcgagtggttggtttttcaaactcctattatacgaactcttttagagttagtctcagtcgttgtgtctatggaacaagaaggaagacgagaaagcgtgtaagaccatgaaatgttttttgtgtgcgcaacgtagtgcgaaaagttagttatttggagaggaagttatttgaaaagaatctttagaagatattgatattttatattctgtatgtaggcgctgtggcaaattctcgacttcacgggagaagcgcaaattttgtgctggtttccataatgcgctcgaaaaatgttgtatatttacattattcaaatctcaccgattaaagttgaatttcagttggttcgtattctctcaactcatggcactactctcgatgtgcattgccttttatggttgctacgttatggttcctcttggaagagaaaagcatgctccataccgattcgattttctatttcgtacatgtgacattgctcagtgtatttacacaattcaaaagtttgtctttgagtttgctgctgcagttggcttgatcacttcggatcgataccttccagctgctgcgaaggcattgtgtaagttcagaagtttaaatggagataggaatgtttcgattttcgtttataaaaacacctgttaatattaaattgttgacgtcatagctgaccattcatgcttctttgtctgattgtaattatataattgtaaaaatacaaacaagttaaccgaaaaagttctctgttacgtttgcggtaaattagaactttatgttgaaagatatcagcttaaagttaaaattttttgaaaattaaattttggctagaaattcaatttttctgataaaaagaaagtttggtgggaaatttaatttcattgatagaaaaactgaagtttgtgttttctgagaattaaaaaacagcgggcaatgaagattatgtctttgtaaatttcaatttaaaaatattccagggtgggcatcgtttatgtgcacctgggagatgatgctcctctctgctctttgctcatattgtttgcgtccagccaagtgtaaattttttgatctttaccctggaaacgatatgccagctttatcggcaagagatggttcaaactcccgtgttccgtcattttctcgacgtctatctattgaatatgagccacgaattgctggagtgatgttggagccgccgtcaagaagttcactgagcattactcctagggataaaatagaagacccaacaaccgtatcgtattttgctgataattttgattctttatcgcaaatccaaggacaataattgaacttcctgccattatttcattgctcaatttacactattcttaactttttgacatgaattaaatctttttaaactgagtctaagcgtattattgtatcgtattttccccttctgatgtattcatttacttgtatttttgaaacccatcccagtgatacaactacccaattttcctgtgccatgtttcttgaaacaaatcaaatgtgat!

6

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

Neuropeptide signaling network promotes EGF-dependent sleep

Sleep dampens sensory response by

multilevel modulation of a sensory-motor circuit

1

2

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Vulval development

PLCγ [PIP2]

Ovulation

ITR-1 IP3 Receptor

SLI-1

Cbl LFE-2 IP3K

LET-23 EGF-R

LIN-3 EGF

[DAG]

Y~P Y~P

Wild-type adult worms are almost constantly moving

Cheryl Van Buskirk Van Buskirk & Sternberg Nat Neuro 2007

EGF expression severely inhibits locomotion

(no movement unless perturbed)!Cheryl Van Buskirk

WT lethargus hs:EGF adult

unperturbed perturbed

Octanol latency

hs:EGF induces sleep-like behavior outside of lethargus

cessaQon of feeding and locomoQon

reduced sensory responsiveness

quick reversibility

hatch!

embryogenesis L1 ! L2 ! L3 ! L4 ! Adult!

molt molt molt molt

Cheryl Van Buskirk

second

s unQ

l respo

nse

EGF promotes quiescence during molting via ALA neuron

Time (min)

Velocity (m

m/sec)

0 50 100 150 200 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-‐γ mutant

Van Buskirk & Sternberg Nat Neuro 2007

EGF promotes quiescence during molting via ALA neuron

Time (min)

Velocity (m

m/sec)

0 50 100 150 200 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-‐γ mutant

Van Buskirk & Sternberg Nat Neuro 2007

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Vulval development

PLCγ [PIP2]

Ovulation

ITR-1 IP3 Receptor

SLI-1

Cbl LFE-2 IP3K

LET-23 EGF-R

LIN-3 EGF

[DAG]

Sleep

Synaptic Release UNC-13, etc.

EGF signaling mediates stress-induced sleep

Time (min)

Velocity (m

m/sec)

0 50 100 150 200 0 50 100 150 200

0.20 0.15

0.10

0.05

0

-‐.05

WT let-‐23(rf)

lethargus behavior EGF/EGFR

STRESS

stress-‐induced sleep Van Buskirk Lab, Cal State Northridge

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 funcQon

Cheryl Van Buskirk

Micro-dissection & Transcriptional profiling of single ALA neurons

L4

L4 Lethargus 4-‐5hr

Single cell RT-‐PCR cDNA amplificaQon 9 cells (2 pools)

Igor Antoshechkin, Caltech Jacobs Genome Center

Elly Chow Erich Schwarz, Miriam Goodman, Mihoko Kato Schwarz, Kato & Sternberg [PNAS 2012]

Erich Schwarz

ALA enriched genes: 10 neuropeptide genes

FRMFamide-Like Peptide

Ex

pre

ssio

n L

eve

l

Enrichment

8110 protein coding genes detected in ALA

ALA-‐enriched neuropepQdes (>12-‐fold over whole larvae)

Validation of expression in ALA neuron using enhancers predicted from “ALA motif”

FLP-24

FLP-7

FLP-13

VPSAGDMMVRF!!

SPMQRSSMVRF!TPMQRSSMVRF!SPMERSAMVRF!SPMDRSKMVRF!

!

AMDSPLIRF!AADGAPLIRF!APEASPFIRF!ASPSAPLIRF!SPSAVPLIRF!ASSAPLIRF!SAAAPLIRF!

VPSAGDMMVRFG!

VPSAGDMMVRFamide!

KRVPSAGDMMVRFGKR!

FLP-13

NLP-8

FLP-24

FLP-7

NPR-7

NPR-22

sleep EGF

ALA neuron

neuropeptides receptors

FRPR-3

?

STRESS

EGFR

Elly Chow

sleep neuropeptides receptors


Elly Chow

35°C, 30 min recover RT, 10 min

score

4 neuropeptides necessary for EGF response

Qui

esce

nce

Time (min)

WT




Elly Chow

Qui

esce

nce

Time (min)

Multiple receptors are necessary





Elly Chow

FLP peptides increase response latency






Elly Chow

receptor mutant block peptide overexpression

FLP-13

NLP-8

FLP-24

FLP-7

NPR-7

NPR-22

sleep EGF

ALA neuron

neuropeptides receptors

FRPR-3

?

STRESS

EGFR

Elly Chow

Documents

Infusion of EGF blocks locomotor activity in hamsters · Sleep in worms! EGF and EGF-R! Target neuron - ALA! Neuropeptides and receptors!