Presynaptic GABA receptors mediate temporal ...

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.

Research Article: New Research | Sensory and Motor Systems

Presynaptic GABA receptors mediate temporal contrast enhancement in Drosophila olfactory sensory neurons and modulate odor-driven behavioral kinetics Temporal contrast enhancement in the Drosophila antennal lobe Davide Raccuglia1, Li Yan McCurdy1,2, Mahmut Demir3, Srinivas Gorur-Shandilya2, Michael Kunst1, Thierry Emonet3,4 and Michael N. Nitabach1,5,6 1

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA 2

Interdepartmental Neuroscience Program, Yale University, New Haven, CT 06520, USA

3

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA

4

Department of Physics, Yale University, New Haven, CT 06520, USA

5

Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA

6

Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06520, USA DOI: 10.1523/ENEURO.0080-16.2016 Received: 13 April 2016 Revised: 4 July 2016 Accepted: 21 July 2016 Published: 25 July 2016

Author contributions: D.R., L.Y.M., M.D., S.G.-S., M.K., T.E., and M.N.N. designed research; D.R., L.Y.M., and M.D. performed research; D.R., L.Y.M., M.D., and S.G.-S. analyzed data; D.R. and M.N.N. wrote the paper; S.G.-S., M.K., and T.E. contributed unpublished reagents/analytic tools. Conflict of Interest: Authors report no conflict of interest. Work in the laboratory of M.N.N. was supported in part by the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH) (R01NS055035, R01NS056443, R01NS091070), National Institute of General Medical Sciences, NIH (R01GM098931), and the Kavli Institute for Neuroscience. L.Y.M was supported by a Gruber Science Fellowship and NIGMS, NIH (T32GM007223). Correspondence should be addressed to: Michael N. Nitabach. E-mail: [email protected] Cite as: eNeuro 2016; 10.1523/ENEURO.0080-16.2016 Alerts: Sign up at eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published.

Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Copyright © 2016 Society for Neuroscience

1

7LWOH3DJH

2



3

7LWOH

4

3UHV\QDSWLF*$%$UHFHSWRUVPHGLDWHWHPSRUDOFRQWUDVWHQKDQFHPHQWLQDrosophilaROIDFWRU\

5

VHQVRU\QHXURQVDQGPRGXODWHRGRUGULYHQEHKDYLRUDONLQHWLFV

6

$EEUHYLDWHGWLWOH

7

7HPSRUDOFRQWUDVWHQKDQFHPHQWLQWKHDrosophilaDQWHQQDOOREH

8



9

$XWKRUQDPHVDQGDIILOLDWLRQV

10

'DYLGH5DFFXJOLD/L¨)) 0D[¨)) VSRVWSXOVH@¨)) 0D[7KLVIRUPXOD

293

UHSUHVHQWVWKHUHODWLYHGLIIHUHQFHRIQHXURQDODFWLYLW\EHWZHHQDWLPHSRLQWGXULQJVWLPXODWLRQ

294

DQGDIWHUZDUGVV $VKDUSQHVVFRHIILFLHQWRIUHSUHVHQWVWHPSRUDOFRQWUDVWRI

295

QHXURQDODFWLYLW\ZKLOHUHSUHVHQWVQRWHPSRUDOFRQWUDVW7KHVKDUSQHVVFRHIILFLHQWRI

,QRUGHUWRTXDQWLI\WHPSRUDOFRQWUDVWHQKDQFHPHQWZHHPSOR\HGDVKDUSQHVV

13

296

SHULSKHUDOHOHFWULFDOUHVSRQVHVGHFUHDVHVZLWKLQFUHDVLQJRGRULQWHQVLW\)LJ+ ,Q

297

FRQWUDVWSUHV\QDSWLFHOHFWULFDOUHVSRQVHVUHPDLQHTXDOO\VKDUSDFURVVWKHHQWLUHUDQJHRI

298

WHVWHGRGRULQWHQVLWLHVDQGDUHVLJQLILFDQWO\VKDUSHUWKDQSHULSKHUDOUHVSRQVHVWRDQG

299

(ESXOVHV)LJ+ +RZHYHUDGLUHFWFRPSDULVRQEHWZHHQSHULSKHUDODQGSUHV\QDSWLF

300

$UF/LJKWVLJQDOLVFRPSOLFDWHGE\WKHSRVVLELOLW\WKDWSHULSKHUDO$UF/LJKWVLJQDOPD\UHIOHFW

301

ILULQJIUHTXHQF\DQGUHFHSWRUSRWHQWLDOZKLOHVSLNHVPD\WUDYHOPRUHHIIHFWLYHO\WRWKH

302

SUHV\QDSWLFWHUPLQDOVWKDQVORZHUFKDQJHVLQPHPEUDQHSRWHQWLDO:HWKHUHIRUHGLUHFWO\

303

FRPSDUHWKHVKDUSQHVVRIWKHSHULSKHUDOILULQJUDWHZLWKWKHSUHV\QDSWLF$UF/LJKWVLJQDO)LJ

304

, 7RDFFRXQWIRUWKHSURQRXQFHGVHQVRU\DGDSWDWLRQZHQRZXVHDVKDUSQHVVFRHIILFLHQW

305

ZKLFKLVEDVHGRQWKHQHXURQDODFWLYLW\DWRGRURIIVHWVKDUSQHVVRIIVHW¨)) RIIVHW¨)) VSRVW

306

SXOVH@¨)) RIIVHW 7KLVVKRZVWKDWSUHV\QDSWLFHOHFWULFDOUHVSRQVHVWRDQGDUH

307

VLJQLILFDQWO\VKDUSHUWKDQWKHSHULSKHUDOILULQJUDWH)LJ, ZKLFKLQGLFDWHVWKDWWKHVXVWDLQHG

308

SHULSKHUDOUHVSRQVHVREVHUYHGLQ2UDH[SUHVVLQJQHXURQVDUHWHPSRUDOO\VKDUSHQHGLQ

309

WKHLUSUHV\QDSWLFWHUPLQDOVLQWKH$/$WVXVWDLQHGQHXURQDODFWLYLW\LVDOVRPHDVXUDEOHLQ

310

SUHV\QDSWLFWHUPLQDOVZKLFKFRXOGLQGLFDWHWKDWWHPSRUDOVKDUSHQLQJFDQRQO\EHDFKLHYHGXS

311

WRDVSHFLILFRGRULQWHQVLW\7RWHVWZKHWKHUVKDUSHQHGSUHV\QDSWLFYROWDJHUHVSRQVHVDWKLJK

312

RGRUFRQFHQWUDWLRQVDUHIRXQGLQRWKHUJORPHUXOLZHPHDVXUHG25EH[SUHVVLQJ261V

313

ZKRVHWHUPLQDOVSURMHFWWRWKH'0JORPHUXOXV)LJ-1 :KLOHSHULSKHUDOYROWDJH

314

UHVSRQVHVVKRZVXVWDLQHGQHXURQDODFWLYLW\DWRGRUGLOXWLRQVDQG)LJ. 

315

SUHV\QDSWLFYROWDJHUHVSRQVHVDUHVLJQLILFDQWO\VKDUSHQHGDWWKHVHFRQFHQWUDWLRQV)LJ0

316

1 7KHREVHUYHGSUHV\QDSWLFVKDUSHQLQJFRXOGEHDJHQHUDOPHFKDQLVPIRUWHPSRUDO

317

FRQWUDVWHQKDQFHPHQWWRLPSURYHHGJHGHWHFWLRQDQGSOXPHJXLGHGQDYLJDWLRQ7RVWXG\WKLV

318

SXWDWLYHPHFKDQLVPRIWHPSRUDOFRQWUDVWHQKDQFHPHQWZHIXUWKHUIRFXVHGRQRSWLFDO

319

HOHFWURSK\VLRORJ\DVZHOODVSKDUPDFRORJLFDODQGJHQHWLFPDQLSXODWLRQVWRGLUHFWO\YLVXDOL]H

320

SUHV\QDSWLFVKDUSHQLQJLQWKHSUHV\QDSWLFWHUPLQDOV

321

7HPSRUDOFRQWUDVWHQKDQFHPHQWLVPHGLDWHGE\SUHV\QDSWLF*$%$UHFHSWRUV

14

322

,WKDVUHFHQWO\EHHQVKRZQWKDWSUHV\QDSWLFLQKLELWLRQRIDrosophila261VSURPRWHV

323

EURDGEDQGV\QDSWLFWUDQVPLVVLRQRIROIDFWRU\VWLPXOLE\RYHUFRPLQJIUHTXHQF\UHVWULFWLRQV

324

LPSRVHGE\VKRUWWHUPGHSUHVVLRQ1DJHOHWDO 0RUHRYHUWKLVVWXG\VKRZVWKDW

325

SUHV\QDSWLFLQKLELWLRQVKDUSHQV31UHVSRQVHVWRVSDUVHVWLPXOL7RLQYHVWLJDWHWKHUROHRI

326

*$%$$DQG*$%$%UHFHSWRUVRQWKHWHPSRUDOVKDUSHQLQJRISUHV\QDSWLFYROWDJHUHVSRQVHV

327

ZHILUVWFRQGXFWHGSKDUPDFRORJLFDOPDQLSXODWLRQVDQGPHDVXUHGYROWDJHUHVSRQVHVGLUHFWO\

328

LQWKHSUHV\QDSWLFWHUPLQDOV:KLOHSKDUPDFRORJLFDOLQKLELWLRQRI*$%$%UHFHSWRUV&*3

329

—0 VKRZVQRHIIHFWRQSUHV\QDSWLFYROWDJHUHVSRQVHVWRD(ESXOVH)LJ$ 

330

LQKLELWLRQRI*$%$$UHFHSWRUV37;—0 DSSHDUVWRLQFUHDVHUHVSRQVHPDJQLWXGH)LJ

331

% $QH[SODQDWLRQIRUWKHGLIIHUHQFHZLWKSUHYLRXVVWXGLHVGHPRQVWUDWLQJWKDW&*3

332

LQFUHDVHVQHXURQDODFWLYLW\LQSUHV\QDSWLFWHUPLQDOV2OVHQDQG:LOVRQ5RRWHWDO

333

 FRXOGEHWKDWWKHSKDUPDFRORJLFDOLQKLELWLRQRI*$%$%UHFHSWRUVPLJKWKDYHDELJJHU

334

HIIHFWRQSUHV\QDSWLFFDOFLXPDQGV\QDSWLFWUDQVPLVVLRQWKDQRQSUHV\QDSWLFYROWDJH

335

,QWHUHVWLQJO\VLPXOWDQHRXVSKDUPDFRORJLFDOLQKLELWLRQRI*$%$$DQG*$%$%UHFHSWRUV

336

LQGXFHVSURORQJHGSUHV\QDSWLFYROWDJHUHVSRQVHVWRDQG(ESXOVHV)LJ&( ZKLOH

337

RGRUNLQHWLFVDUHXQDOWHUHGEHWZHHQWKHPHDVXUHPHQWV)LJ') 4XDQWLILFDWLRQRISHDN

338

UHVSRQVHPDJQLWXGHDQGVKDUSQHVVLQGLFDWHVWKDWRQO\VLPXOWDQHRXVSKDUPDFRORJLFDO

339

LQKLELWLRQRI*$%$$DQG*$%$%UHFHSWRUVVLJQLILFDQWO\LQFUHDVHVDPSOLWXGH)LJ* DQG

340

UHGXFHVVKDUSQHVVRI261SUHV\QDSWLFYROWDJHUHVSRQVHV)LJ+ ZKLFKDOVR

341

GHPRQVWUDWHVWKHHIIHFWLYHQHVVRIERWKGUXJV&*3DQG37;7KLVLVFRQVLVWHQWZLWK

342

WKHSUHYLRXVILQGLQJWKDWGLVLQKLELWLRQLQWKH$/LVSRRUO\DFKLHYHGE\HLWKHU&*3RU

343

37;DORQHEXWIXOO\DFKLHYHGE\VLPXOWDQHRXVDSSOLFDWLRQRIWKHVHGUXJV2OVHQDQG:LOVRQ

344

 ,QJHQHUDOWKHFRQFOXVLRQRIWKLVH[SHULPHQWLVFRQVLVWHQWZLWKWKHILQGLQJWKDW

345

SUHV\QDSWLFLQKLELWLRQLQ261WHUPLQDOVLVPHGLDWHGE\*$%$$DQG*$%$%UHFHSWRUV2OVHQ

346

DQG:LOVRQ 7KHPRUHVHYHUHGLVUXSWLRQRISUHV\QDSWLFVKDUSHQLQJIRU(ESXOVHV

347

WKDQIRU)LJ+ LVFRQVLVWHQWZLWKWKHFRQFHQWUDWLRQGHSHQGHQWVKDUSQHVVRISHULSKHUDO

348

UHVSRQVHV)LJ) 6XVWDLQHGDFWLYLW\DIWHUEORFNLQJRISUHV\QDSWLFLQKLELWLRQLVKLJKHUWKDQ

349

RQHZRXOGH[SHFWEDVHGRQWKHVXVWDLQHGSHULSKHUDOILULQJUDWHV)LJ 7KLVPLJKWEH 15

350

EHFDXVHORZIUHTXHQF\ILULQJLVEHWWHUUHIOHFWHGE\$UF/LJKWLQFRPSDULVRQWRKLJKIUHTXHQF\

351

ILULQJ,WFRXOGDOVREHWKDWLWLVQRWRQO\VSLNLQJDFWLYLW\EXWDOVRVORZFKDQJHVLQPHPEUDQH

352

SRWHQWLDOWKDWWUDYHOWRWKHSUHV\QDSWLFWHUPLQDOVDQGXQGHUOLHSUHV\QDSWLFLQKLELWLRQ7KLV

353

K\SRWKHVLVLVVXSSRUWHGE\WKHVXEVWDQWLDOO\SURORQJHGFDOFLXPNLQHWLFVZHREVHUYHLQWKH

354

SUHV\QDSWLFWHUPLQDOV2XUSKDUPDFRORJLFDOVWXGLHVVXJJHVWWKDWERWK*$%$$DQG*$%$%

355

UHFHSWRUVPHGLDWHSUHV\QDSWLFLQKLELWLRQRI261VWRLPSOHPHQWWHPSRUDOFRQWUDVW

356

HQKDQFHPHQWRIVXVWDLQHGSHULSKHUDOUHVSRQVHV7RWHVWZKHWKHUWKHFRPELQHGDFWLYLW\RI

357

*$%$$DQG*$%$%UHFHSWRUVFDQPHGLDWHWHPSRUDOFRQWUDVWHQKDQFHPHQWLQRWKHUJORPHUXOL

358

ZHPHDVXUHGSUHV\QDSWLFYROWDJHUHVSRQVHVRI25DH[SUHVVLQJ261VZKLFKUHVLGHLQWKH

359

PD[LOODU\SDOSVDQGZKRVHWHUPLQDOVSURMHFWWRWKH90JORPHUXOXV)LJ, )RU90ODWHUDO

360

LQKLELWLRQFDQEHUHGXFHGE\UHPRYDORIWKHDQWHQQDHZKLFKLQFUHDVHVRGRUUHVSRQVHVLQ

361

9031V2OVHQDQG:LOVRQ 7KLVGLVLQKLELWLRQFDQRQO\EHPLPLFNHGE\VLPXOWDQHRXV

362

EORFNDJHRI*$%$$DQG*$%$%UHFHSWRUV2OVHQDQG:LOVRQ VXJJHVWLQJWKDWERWK

363

UHFHSWRUVDUHSUHVHQWDWWKHSUHV\QDSWLFWHUPLQDOVRI25DH[SUHVVLQJ261V:HILQGWKDW

364

UHPRYDORIWKHDQWHQQDHLQFUHDVHVSUHV\QDSWLFYROWDJHUHVSRQVHVWR(EDQGDOVR

365

UHGXFHVSUHV\QDSWLFVKDUSHQLQJ)LJ, 7KLVVXJJHVWVWKDWWKHLPSOHPHQWDWLRQRIWHPSRUDO

366

FRQWUDVWHQKDQFHPHQWYLDWKHFRPELQHGDFWLYLW\RISUHV\QDSWLF*$%$$DQG*$%$%UHFHSWRUV

367

LVDJHQHUDOSKHQRPHQRQLQWKH'URVRSKLOD$/+RZHYHURXUILQGLQJVGRQRWUXOHRXWWKH

368

SRVVLELOLW\WKDW*$%$$DQGRU*$%$%PHGLDWHGLQKLELWLRQRIRWKHUQHXURQVLQWKH$/ROIDFWRU\

369

QHWZRUNDUHLQYROYHG

370

7RDGGUHVVWKLVLVVXHZHJHQHWLFDOO\VXSSUHVVHG*$%$UHFHSWRUH[SUHVVLRQLQ261V

371

LQQHUYDWLQJDVLQJOHJORPHUXOXVE\H[SUHVVLQJ*$%$UHFHSWRUGLUHFWHG51$LKDLUSLQ

372

FRQVWUXFWVXVLQJOR-GAL4GULYHUVDQGPHDVXUHGHIIHFWVRQRGRULQGXFHGSUHV\QDSWLF

373

HOHFWULFDOUHVSRQVHVRI25DH[SUHVVLQJ261V)LJ $VDFRQWUROIRUSRVVLEOHQRQ

374

VSHFLILF51$LHIIHFWVZHH[SUHVVHG51$LGLUHFWHGDJDLQVWWKHQHXURSHSWLGH3LJPHQW

375

'LVSHUVLQJ)DFWRU3') ZKLFKLVQRWH[SUHVVHGLQ261V7RDFKLHYH51$L±PHGLDWHG

376

NQRFNGRZQRI*$%$$RU*$%$%UHFHSWRUVZHLQGLYLGXDOO\H[SUHVVHGHLWKHU*$%$$±51$L

377

* /LXHWDO/LXHWDO RU*$%$%551$L5RRWHWDO UHVSHFWLYHO\LQ 16

378

25DH[SUHVVLQJ261V(DFKRIWKHVH51$LOLQHVKDYHSUHYLRXVO\EHHQHVWDEOLVKHGWR

379

HIIHFWLYHO\GRZQUHJXODWHWKHLUFRUUHVSRQGLQJ*$%$UHFHSWRUVXEW\SHV/LXHWDO5RRW

380

HWDO/LXHWDO ,QGLYLGXDODQGVLPXOWDQHRXVNQRFNGRZQRI*$%$$RU*$%$%

381

UHFHSWRUVLQFUHDVHVWKHSHDNPDJQLWXGHRISUHV\QDSWLFYROWDJHUHVSRQVHV)LJ$ 

382

&RQVLVWHQWZLWKRXUSKDUPDFRORJLFDOUHVXOWVRQO\VLPXOWDQHRXVNQRFNGRZQRI*$%$$DQG

383

*$%$%UHFHSWRUVUHGXFHVVKDUSQHVVRISUHV\QDSWLFYROWDJHUHVSRQVHV)LJ% 7R

384

LQYHVWLJDWHDWZKDWWLPHDIWHUWKHRGRURIIVHWNQRFNGRZQRI*$%$UHFHSWRUVDIIHFW

385

SUHV\QDSWLFFRQWUDVWHQKDQFHPHQWZHSHUIRUPHGDWLPHGHSHQGHQWDQDO\VLVRIWKH

386

VKDUSQHVVFRHIILFLHQWVKDUSQHVVRIIVHW)LJ&- ,QWHUHVWLQJO\NQRFNGRZQRI*$%$$

387

UHFHSWRUVLQFUHDVHVVKDUSQHVVGXULQJWKHLPPHGLDWHSRVWSXOVHK\SHUSRODUL]DWLRQSKDVH

388

)LJ')+ 7KLVFRXOGEHDUHVXOWRILQFUHDVHG*$%$%UHFHSWRUDFWLYLW\ZKLFKFRXOGEH

389

GXHWRKRPHRVWDWLFSURFHVVHVWULJJHUHGE\WKHGRZQUHJXODWLRQRI*$%$$UHFHSWRUV7KLV

390

K\SRWKHVLVLVVXSSRUWHGE\WKHILQGLQJWKDWNQRFNGRZQRI*$%$%UHFHSWRUVDQG

391

VLPXOWDQHRXVNQRFNGRZQRI*$%$$DQG*$%$%UHFHSWRUVVLJQLILFDQWO\UHGXFHVKDUSQHVV

392

GXULQJWKHLPPHGLDWHSRVWSXOVHK\SHUSRODUL]DWLRQSKDVH)LJXUH' 6KDUSQHVVRI

393

VXVWDLQHGQHXURQDODFWLYLW\ZKLFKRFFXUVRQO\DWKLJKHULQWHQVLWLHVLVUHGXFHGRQO\E\WKH

394

VLPXOWDQHRXVNQRFNGRZQRI*$%$$DQG*$%$%UHFHSWRUV)LJXUH)+- )RUDQG

395

VKDUSQHVVLVUHGXFHGGXULQJDYHU\QDUURZWLPHZLQGRZRIDQGVDIWHUWKHRGRU

396

RIIVHW)LJ)+ )RUWKHDOUHDG\SURORQJHGQHXURQDODFWLYLW\LQWKHFRQWUROLVIXUWKHU

397

LQFUHDVHGZLWKVLPXOWDQHRXVNQRFNGRZQRI*$%$$RU*$%$%UHFHSWRUVDQGOHDGVWRUHGXFHG

398

VKDUSQHVVVDIWHURGRURIIVHW)LJXUH, 7KHVHFHOOVSHFLILFJHQHWLFPDQLSXODWLRQV

399

HVWDEOLVKWKDWWKHDFWLYLW\RIERWK*$%$$DQG*$%$%UHFHSWRUVH[SUHVVHGE\WKH261V

400

LQQHUYDWLQJDVLQJOHJORPHUXOXVLQFUHDVHWKHWHPSRUDOFRQWUDVWRISUHV\QDSWLFUHVSRQVHVWR

401

KLJKLQWHQVLW\RGRUVWLPXOL

402

3UHV\QDSWLFLQKLELWLRQRI261VDFFHOHUDWHVEHKDYLRUDOUHVSRQVHVWRRGRURIIVHW

403 404

:HQH[WVRXJKWWRGHWHUPLQHZKHWKHUSUHV\QDSWLFLQKLELWLRQRI261VLQIOXHQFHVWKH WLPHFRXUVHRILQQDWHEHKDYLRUDOUHVSRQVHVWRWLPH±YDU\LQJROIDFWRU\VWLPXOL:HXVHG 17

405

DXWRPDWHGIO\WUDFNLQJVRIWZDUHDGDSWHGIURPRSHQVRXUFHFRGHVHH([SHULPHQWDO

406

3URFHGXUHV WRWUDFNWKHORFRPRWRUUHVSRQVHVRIZDONLQJIOLHVWR(ESXOVHVGHOLYHUHGIURPDQ

407

RGRUSRUWLQWRDFLUFXODUDUHQDFRQWDLQLQJIOLHV7RSURSHUO\FRPSDUHWKHEHKDYLRUDO

408

H[SHULPHQWVZLWKWKHSK\VLRORJLFDOH[SHULPHQWVLQ)LJXUHZHXVHGWKHVDPHFRQWURO

409

H[SUHVVLQJ3')51$LZLWKRXW$UF/LJKW 7KLVLVDGYDQWDJHRXVRYHUXVLQJLQEUHGSDUHQWDO

410

OLQHVDVLQEUHHGLQJFDQDIIHFWORFRPRWRUDFWLYLW\0DQHQWLHWDO $OWKRXJKZHXVHWKH

411

VDPHRGRUGLOXWLRQVDVLQWKHSK\VLRORJLFDOH[SHULPHQWVWKHRGRULQWHQVLWLHVWKHIOLHV

412

H[SHULHQFHFRXOGEHTXLWHGLIIHUHQWGXHWRGLVWDQFHIURPWKHRGRUSRUWDQGWKHIDFWWKDWWKH

413

EHKDYLRUDODUHQDLVFORVHG7RTXDQWLI\DWWUDFWLYHRUDYHUVLYHUHVSRQVHVZHFDOFXODWHGWKH

414

GLVWDQFHRIHDFKIO\IURPWKHRGRUSRUWRYHUWLPHUHODWLYHWRLWVLQLWLDOSRVLWLRQDWWKHEHJLQQLQJ

415

RIHDFKWULDOVSXOVHVRI(ELQGXFHDWWUDFWLRQZLWKIOLHVPRYLQJFORVHUWRWKHRGRU

416

SRUW)LJ$ ,QFRQWUDVWVSXOVHVRI(ELQGXFHDYRLGDQFHZLWKIOLHVPRYLQJDZD\

417

IURPWKHRGRUSRUW)LJ% 7KLVLVFRQVLVWHQWZLWKWKHSUHYLRXVREVHUYDWLRQWKDWORZ

418

FRQFHQWUDWLRQVRI(EVROHO\DFWLYDWH261VPHGLDWLQJLQQDWHDWWUDFWLRQZKLOHKLJKHU

419

FRQFHQWUDWLRQVUHFUXLWDGGLWLRQDO261VPHGLDWLQJLQQDWHDYRLGDQFH6HPPHOKDFNDQG:DQJ

420

 &RQWUROIOLHVH[SUHVVLQJ3')51$LLQ25DH[SUHVVLQJ261VLQQHUYDWLQJ'0DUH

421

DWWUDFWHGWRSXOVHVRIDQG(E)LJ&' )OLHVH[SUHVVLQJ*$%$$51$L* 

422

DORQHRUVLPXOWDQHRXVO\H[SUHVVLQJ*$%$$51$L* DQG*$%$%51$LH[KLELWUHGXFHG

423

DWWUDFWLRQ)LJ&' &RQWUROIOLHVDUHQHLWKHUDWWUDFWHGQRUUHSHOOHGE\SXOVHVRI(E

424

)LJ( FRQVLVWHQWZLWKWKHLQWHUSUHWDWLRQWKDWWKLVLQWHQVLW\RI(EVWLPXODWHV261V

425

PHGLDWLQJDWWUDFWLRQDQGDYRLGDQFHWRDUHODWLYHH[WHQWWKDWFRXQWHUEDODQFHVEHKDYLRUDO

426

UHVSRQVHV,QVWDUNFRQWUDVWWRFRQWUROIOLHV*$%$$51$L* DQG*$%$$51$L* 

427

*$%$%51$LIOLHVDUHUHSHOOHGE\(ESXOVHV)LJ( $OOIOLHVUHJDUGOHVVRIJHQRW\SH

428

DUHVWURQJO\UHSHOOHGE\SXOVHVRI(EZLWKLQFUHDVHGDYRLGDQFHLQ*$%$$51$L* 

429

*$%$%51$LIOLHV)LJ) $YRLGDQFHWRDQGLVHYHQVWURQJHULQIOLHVH[SUHVVLQJ

430

'LFHUWRJHWKHUZLWK*$%$$51$L* *$%$%51$LZKLFKXQGHUOLQHVWKHVSHFLILFLW\RI

431

WKHEHKDYLRUDOFKDQJHV6WDWLVWLFDODQDO\VLVVXSSRUWLQJWKHVHFRQFOXVLRQVLVVKRZQLQ)LJ

432

*([SUHVVLRQRIDGLIIHUHQW*$%$$51$L( /LXHWDO LQ25DH[SUHVVLQJ 18

433

261VDOVRLQFUHDVHVDYRLGDQFH)LJ+ VXSSRUWLQJWKHVSHFLILFLW\RIWKHVH*$%$$51$L

434

HIIHFWV,QOLJKWRI6HPPHOKDFNDQG:DQJ ZKRGRQ¶WREVHUYHDQ\FKDQJHVLQYDOHQFH

435

DIWHUVLOHQFLQJ'0RXUILQGLQJVRILQFUHDVHGSUHV\QDSWLFHOHFWULFDODFWLYLW\LQ'0OHDGLQJWR

436

LQFUHDVHGDYRLGDQFHFRXOGEHH[SODLQHGE\DOWHUHGQHWZRUNDFWLYLW\,QFUHDVHGDFWLYLW\LQ

437

'0PHGLDWHGE\NQRFNGRZQRI*$%$$UHFHSWRUV)LJ FRXOGIRUH[DPSOHLQFUHDVHODWHUDO

438

LQKLELWLRQZKLFKFRXOGDIIHFWRWKHUJORPHUXOLOHDGLQJWRLQFUHDVHGDYRLGDQFH 7KHVHVXVWDLQHGDWWUDFWLYHDQGDYHUVLYHUHVSRQVHVWRVRGRUSXOVHVOLNHO\UHIOHFW

439 440

FKHPRWDFWLFUHVSRQVHVWRVWHDG\VWDWHRGRUJUDGLHQWVLQWKHROIDFWRU\DUHQD7RSUREHWKH

441

HIIHFWVRISUHV\QDSWLF261LQKLELWLRQRQSHUFHSWLRQRIDWLPHYDU\LQJVWLPXOXVZHGHOLYHUHG

442

VRGRUSXOVHVDQGIRFXVHGRXUDWWHQWLRQRQEHKDYLRUDOUHVSRQVHVGXULQJDQGDIWHUWHUPLQDWLRQ

443

RIWKHSXOVH)LJ)LJ 5HVSRQVHVRILQGLYLGXDOIOLHVWREULHIRGRUSXOVHVGHSHQGKHDYLO\

444

RQRGRUGLOXWLRQDQGWKHLQLWLDOGLVWDQFHIURPWKHRGRUSRUWDWWKHWLPHRIRGRURQVHW)LJ$

445

% )OLHVWKDWDUHLQLWLDOO\FORVHWRWKHRGRUSRUWDYRLGLWZKLOHIOLHVWKDWDUHLQLWLDOO\IDUDZD\GR

446

QRWUHVSRQG:HIRFXVHGRQWKHKLJKRGRULQWHQVLWLHVDQGXVHGWKH3,'WRPHDVXUHRGRU

447

G\QDPLFVZLWKLQWKHEHKDYLRUDODUHQD)LJ&' ,QWHUHVWLQJO\ZLWKLQFPRIWKHRGRUSRUW

448

IOLHVH[SHULHQFHRGRUG\QDPLFVVLPLODUWRHQWHULQJDQGH[LWLQJRIDSOXPHZLWKDUDSLG

449

LQFUHDVHRIRGRULQWHQVLW\DQGDIDVWGHFOLQH0RUHWKDQFPDZD\IURPWKHRGRUSRUW

450

FKDQJHVLQRGRULQWHQVLW\DUHPRUHJUDGXDO7RYLVXDOL]HWKLVZHFDOFXODWHGWDXZKLFK

451

UHSUHVHQWVWKHWLPHWDNHQIURPWKHSHDN3,'YDOXHWRRIWKH3,'YDOXH)LJ() 7R

452

VWXG\WKHIOLHV¶EHKDYLRUXSRQH[LWLQJSOXPHOLNHRGRUG\QDPLFVZHIRFXVHGRQWKRVHIOLHV

453

ZKRVHLQLWLDOSRVLWLRQZDVZLWKLQFPIURPWKHRGRUSRUW)LJ$' 

454



455

* RU*$%$$51$L* *$%$%51$L'LFHU LQ25DH[SUHVVLQJ261V

456

LQQHUYDWLQJ'0LQFUHDVHVYHORFLW\DZD\IURPWKHRGRUSRUW)LJ$& 7KLVOLNHO\UHIOHFWV

457

LQFUHDVHGDYRLGDQFHZKLFKZDVDOVRVKRZQIRUWKHVRGRUSXOVH)LJ* 7KHYHORFLW\

458

EHWZHHQVDIWHUWHUPLQDWLRQRIWKHRGRUSXOVHUHPDLQVVLJQLILFDQWO\KLJKHURQO\LQIOLHV

459

H[SUHVVLQJ*$%$$51$L* DQG*$%$%51$LIOLHV)LJ& &RQWUROIOLHVH[KLELW

,PPHGLDWHO\DIWHUDVRGRUSXOVHVSRVWSXOVHWLPH RI(E*$%$$51$L

19

460

VWURQJHUDYRLGDQFHUHVSRQVHWRSXOVHVRIWKDQ(EDQGLQWHUHVWLQJO\WKLVDYRLGDQFH

461

WHUPLQDWHVUDSLGO\XSRQWHUPLQDWLRQRIWKHSXOVH)LJ%' $VWKHQHXURQDODFWLYLW\RI'0

462

SUHV\QDSWLFWHUPLQDOVWRRGRUSXOVHVZRXOGVXJJHVWDQHYHQPRUHSURORQJHGDYRLGDQFH

463

WKLVFRXOGGHPRQVWUDWHKRZWKHDFWLYLW\RIPXOWLSOHJORPHUXOLLVXVHGWREDODQFHDQGVZLWFK

464

LQQDWHEHKDYLRUEHWZHHQDWWUDFWLRQDQGDYRLGDQFH+RZHYHUZKLOH*$%$UHFHSWRU

465

NQRFNGRZQGRHVQRWDIIHFWDYRLGDQFHGXULQJRULPPHGLDWHO\DIWHUWKH(ESXOVH

466

VLPXOWDQHRXVNQRFNGRZQRI*$%$$DQG*$%$%UHFHSWRUVSURORQJVDYRLGDQFHEHWZHHQV

467

DIWHUWHUPLQDWLRQRIWKHRGRUSXOVH)LJ' ,QFRQWUDVW*$%$UHFHSWRUNQRFNGRZQGLGQRW

468

DIIHFWIOLHVWKDWDUHPRUHWKDQFPDZD\IURPWKHRGRUSRUWDQGH[SHULHQFHDPRUHJUDGXDO

469

FKDQJHLQRGRUG\QDPLFV)LJ() 7KLVVXJJHVWVWKDWWKHFRPELQHGDFWLYLW\RISUHV\QDSWLF

470

*$%$$DQG*$%$%UHFHSWRUVZKLFKPHGLDWHVJDLQFRQWURODQGWHPSRUDOFRQWUDVW

471

HQKDQFHPHQWLQROIDFWRU\VHQVRU\QHXURQVHQKDQFHVGHWHFWLRQRIWHUPLQDWLRQRIWKHRGRU

472

SXOVH

473

'LVFXVVLRQ

474

/DWHUDOLQKLELWLRQLQWKHYLVXDOV\VWHPLPSURYHVHQYLURQPHQWDOSHUFHSWLRQE\

475

HQKDQFLQJFRQWUDVWYLVLRQWRHQDEOHDFFXUDWHVSDWLDOHGJHGHWHFWLRQ.XIIOHU%XOG\UHY

476

DQG7D\ORU 7HPSRUDOHGJHGHWHFWLRQLQWKHDXGLWRU\V\VWHPLPSURYHVVRXQG

477

ORFDOL]DWLRQ&KDLWHWDO +HUHZHDGGUHVVZKHWKHUVLPLODUPHFKDQLVPVH[LVWLQWKH

478

ROIDFWRU\V\VWHPWKDWLPSURYHRGRUHGJHGHWHFWLRQVHH)LJIRUVXPPDU\ :HILQGWKDW

479

KLJKLQWHQVLW\RGRUSXOVHVLQGXFHVXVWDLQHGSHULSKHUDOUHVSRQVHVLQ261V)LJ :H

480

XVHRSWLFDOHOHFWURSK\VLRORJ\WRYLVXDOL]HWKDWVXVWDLQHGSHULSKHUDOUHVSRQVHVXQGHUJR

481

FRQWUDVWHQKDQFHPHQWE\SUHV\QDSWLF*$%$UHFHSWRUVWRJHQHUDWHVKDUSHUUHVSRQVHVLQ

482

261SUHV\QDSWLFD[RQWHUPLQDOVLQWKH$/)LJ )XUWKHUPRUHWKHFRPELQHGDFWLYLW\

483

RISUHV\QDSWLF*$%$$DQG*$%$%UHFHSWRUVPRGXODWHVWKHNLQHWLFVRILQQDWHROIDFWRU\

484

EHKDYLRUDIWHUWHUPLQDWLRQRIDQRGRUSXOVH)LJ 

485 486

2XUYROWDJHDQG&DPHDVXUHPHQWVUHYHDOFRQWUDVWHQKDQFHPHQWRISUHV\QDSWLF 261HOHFWULFDODFWLYLW\EXWQRWSUHV\QDSWLFLQWUDFHOOXODU&D)LJ ,WLVSRVVLEOHWKDWKLJK 20

487

RGRULQWHQVLWLHVLQGXFH&DUHOHDVHIURPLQWHUQDOVWRUHVUHVXOWLQJLQVXVWDLQHGSUHV\QDSWLF

488

&DLQFUHDVHV0XUPXHWDO0XUPXHWDO $OWHUQDWLYHO\VXVWDLQHGSHULSKHUDO

489

UHVSRQVHVFRXOGDFWLYDWHYROWDJHJDWHG&DFKDQQHOVDORQJWKHD[RQVRIWKH261V0XUPX

490

HWDO ZLWKVXVWDLQHGSUHV\QDSWLF&DLQFUHDVHVPLUURULQJVXVWDLQHGSHULSKHUDO

491

UHVSRQVHV$VHOHFWULFDOUHFRUGLQJVIURPVLQJOH31VLQGLFDWHWKDWPHPEUDQHGHSRODUL]DWLRQLV

492

WLJKWO\FRXSOHGWRQHXURWUDQVPLWWHUUHOHDVH1DJHOHWDO LWLVOLNHO\WKDWVKDUSHQHG

493

SUHV\QDSWLFYROWDJHUHVSRQVHVDUHIDLWKIXOO\SURSDJDWHGHYHQZKHQSUHV\QDSWLFLQWUDFHOOXODU

494

&DUHPDLQVKLJK7KLVFRXOGEHEHFDXVH&DLQGLFDWRUVUHSRUWEXONF\WRSODVPLF&DLQWKH

495

Q0WR—0UDQJHDQGQRWWKHVXEVWDQWLDOO\KLJKHU&DWUDQVLHQWVLQWKH&DFKDQQHO

496

DVVRFLDWHGPLFURGRPDLQVWKDWGULYHV\QDSWLFYHVLFOHUHOHDVH/OLQDVHWDO2KHLPHWDO

497

0DWNRYLFHWDO :KLOHEXONF\WRSODVPLF&DFRXOGUHPDLQHOHYDWHGDIWHUDWUDLQ

498

RIDFWLRQSRWHQWLDOVLQYDGHWKHSUHV\QDSWLFWHUPLQDOVWKHPLFURGRPDLQFRQFHQWUDWLRQDW

499

V\QDSWLFUHOHDVHVLWHVPD\KDYHDOUHDG\GHFOLQHGEHORZWKHWKUHVKROGIRUWULJJHULQJUHOHDVH

500

&RQVLVWHQWZLWKWKLVLQWHUSUHWDWLRQRIRXUREVHUYDWLRQVRGRUVWLPXOLHOLFLWLQJVXVWDLQHG&D

501

LQFUHDVHVLQ261SUHV\QDSWLFWHUPLQDOVLQGXFHVXEVWDQWLDOO\PRUHDEEUHYLDWHG&D

502

LQFUHDVHVLQSRVWV\QDSWLF31GHQGULWHV$VDKLQDHWDO +RZHYHULWLVDOVRSRVVLEOH

503

WKDWSRVWV\QDSWLFLQKLELWLRQFRQWULEXWHVWRWHPSRUDOVKDUSHQLQJRI31UHVSRQVHV:LOVRQDQG

504

/DXUHQW)XMLZDUDHWDO 

505

/DWHUDOLQKLELWLRQLQWKH$/KDVH[WHQVLYHO\EHHQVWXGLHGLQGLIIHUHQWLQVHFWVSHFLHV,Q

506

/RFXVW*$%$HUJLFORFDOLQWHUQHXURQVKDYHVKRZQWRV\QFKURQL]HRVFLOODWLRQVEHWZHHQRGRU

507

FRGLQJQHXUDODVVHPEOLHVLQWKH$/0DF/HRGDQG/DXUHQW ,QWKHKRQH\EHHWKHVH

508

V\QFKURQL]HGRVFLOODWLRQVKDYHEHHQVKRZQWREHHVVHQWLDOIRUWKHGLVFULPLQDWLRQRI

509

PROHFXODUO\VLPLODURGRUDQWV6WRSIHUHWDO 0RUHRYHUFDOFLXPLPDJLQJVWXGLHVLQWKH

510

KRQH\EHHKDYHGHPRQVWUDWHGWKDWORFDOLQWHUQHXURQVPHGLDWHJOREDOLQKLELWLRQLQWKH$/WR

511

HQKDQFHVSDWLDOFRQWUDVWEHWZHHQJORPHUXOL6DFKVHDQG*DOL]LD ,QWKHKDZNPRWK

512

Manduca sextaEORFNLQJLQKLELWLRQLQWKH$/LPSDLUVWKHORFDOL]DWLRQRIRGRUVRXUFHVE\

513

DIIHFWLQJWKHWHPSRUDOILULQJSDWWHUQLQ31V/HLHWDO ,QDrosophila LWKDVEHFRPH

514

HYLGHQWWKDWSUHV\QDSWLF*$%$UHFHSWRUVSOD\DFUXFLDOUROHLQPHGLDWLQJODWHUDOLQKLELWLRQLQ 21

515

WKH$/2OVHQDQG:LOVRQ5RRWHWDO :KLOHSUHV\QDSWLF*$%$UHFHSWRUVKDYH

516

VKRZQWREHHVVHQWLDOIRURGRUREMHFWORFDOL]DWLRQ5RRWHWDO ZHVKRZWKDW

517

SUHV\QDSWLF*$%$UHFHSWRUVDOVRHQKDQFHWHPSRUDOFRQWUDVWZLWKLQJORPHUXOLWRLPSURYHWKH

518

GHWHFWLRQRIWHPSRUDOVWUXFWXUHVRIRGRUSOXPHV

519

Drosophila melanogasterOLYHIHHGDQGUHSURGXFHRQIHUPHQWLQJIUXLWV7RORFDWH

520

IHUPHQWLQJIUXLWDrosophila QDYLJDWHYLDSOXPHVRIRGRUV*DXGU\HWDOYDQ%UHXJHO

521

DQG'LFNLQVRQ *DLQFRQWUROPHGLDWHGE\SUHV\QDSWLFLQKLELWLRQLVDQLPSRUWDQW

522

PHFKDQLVPIRUPDLQWDLQLQJVHQVLWLYLW\WRDZLGHUDQJHRIH[SHULHQFHGRGRULQWHQVLWLHV2OVHQ

523

DQG:LOVRQ5RRWHWDO :KLOH*$%$%PHGLDWHGSUHV\QDSWLFJDLQFRQWUROLV

524

NQRZQWREHLPSRUWDQWIRUORFDOL]LQJSKHURPRQHHPLWWLQJREMHFWV5RRWHWDO QR

525

EHKDYLRUDOUROHIRUSUHV\QDSWLF*$%$$UHFHSWRUVKDVSUHYLRXVO\EHHQUHSRUWHG+HUHZH

526

VKRZWKDWSUHV\QDSWLF261*$%$$UHFHSWRUVPRGXODWHLQQDWHEHKDYLRUDOUHVSRQVHVWRWKH

527

IUXLWUHODWHGRGRU(E)LJ :KLOHEORFNLQJV\QDSWLFRXWSXWLQ'0ZDVSUHYLRXVO\

528

UHSRUWHGWRKDYHQREHKDYLRUDOFRQVHTXHQFH6HPPHOKDFNDQG:DQJ RXU51$L

529

PHGLDWHG*$%$$NQRFNGRZQUHVXOWVLQGLFDWHWKDWLQFUHDVHGQHXURQDODFWLYLW\LQ'0OHDGVWR

530

LQFUHDVHGDYRLGDQFH)LJ $SRVVLEOHH[SODQDWLRQFRXOGEHWKDWLQFUHDVHGDFWLYLW\LQ

531

'0DOWHUVQHWZRUNDFWLYLW\LQWKH$/DIIHFWLQJRWKHUJORPHUXOLWKDWPHGLDWHDWWUDFWLRQRU

532

DYHUVLRQ,QWHUHVWLQJO\51$LPHGLDWHGSUHV\QDSWLFNQRFNGRZQRI*$%$%UHFHSWRUV

533

LQGLYLGXDOO\KDGQRVWDWLVWLFDOO\GLVWLQJXLVKDEOHHIIHFWRQEHKDYLRUDOUHVSRQVHVWR(E)LJ

534

 :KLOH*$%$%UHFHSWRUVSOD\DQLPSRUWDQWUROHLQVXVWDLQHGSKHURPRQHUHODWHGEHKDYLRUV

535

DQGDUHGLIIHUHQWLDOO\H[SUHVVHGDFURVVJORPHUXOL5RRWHWDO *$%$$UHFHSWRUVPLJKW

536

EHPRUHLPSRUWDQWIRUSURFHVVLQJWUDQVLHQWIUXLWUHODWHGRGRUVWLPXOL+RZHYHUIXWXUH

537

LPPXQRKLVWRORJLFDOVWXGLHVQHHGWRVKRZWKHSUHVHQFHRISUHV\QDSWLF*$%$$UHFHSWRUVDQG

538

WKHLUGLVWULEXWLRQDFURVVGLIIHUHQWJORPHUXOL,WFRXOGDOVREHWKDW*$%$%UHFHSWRUVDIIHFWWKH

539

$/QHWZRUNDFWLYLW\LQDGLIIHUHQWZD\WKDQ*$%$$UHFHSWRUV$WWKHKLJKHVWRGRULQWHQVLW\

540

WHVWHGRQO\VLPXOWDQHRXVNQRFNGRZQRI*$%$$DQG*$%$%UHFHSWRUVLQ'0LQFUHDVHG

541

DYHUVLYHEHKDYLRU)LJ)* 7KLVGHPRQVWUDWHVDFRPELQHGUROHIRU*$%$$DQG*$%$%

542

UHFHSWRUVLQPHGLDWLQJSUHV\QDSWLFLQKLELWLRQDVKDVSUHYLRXVO\EHHQREVHUYHGLQ 22

543

SK\VLRORJLFDOVWXGLHV2OVHQDQG:LOVRQ 0RUHRYHUZHDOVRYLVXDOL]HWKHFRPELQHG

544

UROHIRU*$%$$DQG*$%$%UHFHSWRUVLQPHGLDWLQJWHPSRUDOFRQWUDVWHQKDQFHPHQWRI

545

SUHV\QDSWLFHOHFWULFDOUHVSRQVHV)RUWKHILUVWWLPHZHOLQNSUHV\QDSWLFLQKLELWLRQPHGLDWLQJ

546

JDLQFRQWURODQGWHPSRUDOFRQWUDVWHQKDQFHPHQW)LJ WREHKDYLRUDOUHVSRQVHVDIWHU

547

RGRUSXOVHWHUPLQDWLRQ)LJ 7KLVVXJJHVWVWKDWWKHFRPELQHGSDUWLFLSDWLRQRI*$%$$DQG

548

*$%$%UHFHSWRUVFRXOGEHDQDGYDQWDJHIRUDQLPDOVWKDWHQFRXQWHUDYHU\ZLGHG\QDPLF

549

UDQJHRIRGRUVWLPXOL

550

7HPSRUDOVKDUSHQLQJRIROIDFWRU\LQIRUPDWLRQFRXOGDOVRSOD\DQLPSRUWDQWUROHLQ

551

DVVRFLDWLYHOHDUQLQJ'XULQJDVVRFLDWLYHOHDUQLQJLQPDPPDOVDQGLQVHFWVWHPSRUDOO\OLPLWHG

552

ROIDFWRU\VWLPXOLGHWHUPLQHDFULWLFDOWLPHZLQGRZIRUWKHLQWHJUDWLRQRIRWKHUVHQVRU\

553

LQIRUPDWLRQVXFKDVVXJDUDVDUHZDUGRUHOHFWULFVKRFNDVSXQLVKPHQW7XOO\DQG4XLQQ

554

+DPPHUDQG0HQ]HO'HODPDWHUHWDO ,QVLQJOHQHXURQVLPSOLFDWHGLQ

555

OHDUQLQJ*$%$HUJLFLQKLELWLRQKDVEHHQVKRZQWRWUXQFDWHQHXURQDODFWLYLW\DQGWKXVKDV

556

EHHQK\SRWKHVL]HGWRGHILQHWKHWLPHZLQGRZIRUFRLQFLGHQFHGHWHFWLRQ3RXLOOHDQG

557

6FDQ]LDQL0LWWPDQQHWDO5DFFXJOLDDQG0XHOOHU ,QIDFWDOWHULQJWKH

558

GHJUHHRI*$%$HUJLFLQKLELWLRQRUDUWLILFLDOO\DFWLYDWLQJ*$%$UHFHSWRUVGXULQJOHDUQLQJ

559

LQWHUIHUHVZLWKWKHIRUPDWLRQRIDVVRFLDWLYHPHPRULHVLQLQVHFWV/LXHWDO/LXDQG

560

'DYLV5DFFXJOLDDQG0XHOOHU 7KH*$%$HUJLFSUHV\QDSWLFWHPSRUDOFRQWUDVW

561

HQKDQFHPHQWZHUHYHDOKHUHFRXOGSOD\DUROHLQGHWHUPLQLQJDFRQFHQWUDWLRQLQYDULDQW

562

FULWLFDOWLPHZLQGRZIRUHQKDQFLQJWKHWHPSRUDODFFXUDF\RIDVVRFLDWLYHPHPRULHV

563

+RZFRXOGSUHV\QDSWLFLQKLELWLRQXQGHUOLHWHPSRUDOFRQWUDVWHQKDQFHPHQW"3UHDQG

564

SRVWV\QDSWLFLQKLELWLRQLQWKH$/DUHPHGLDWHGE\PXOWLSOH*$%$HUJLF/1VZKLFKUHFHLYH

565

H[FLWDWRU\RGRULQGXFHGLQSXWVIURPPXOWLSOHJORPHUXOLDQGWKHQLQKLELW261SUHV\QDSWLF

566

WHUPLQDOVDQG31SRVWV\QDSWLFGHQGULWHV6WRFNHUHWDO1JHWDO 0RVW

567

LQGLYLGXDO/1VLQQHUYDWHDODUJHQXPEHURIJORPHUXOL&KRXHWDO6HNLHWDO 

568

261SUHV\QDSWLFDFWLYLW\FRXOGEHPRGXODWHGE\VXVWDLQHGDFWLYLW\RI/1V:LOVRQHWDO

569

:LOVRQDQG/DXUHQW&KRXHWDO1DJHOHWDO RUDWUDQVLHQW 23

570

LQFUHDVHLQ/1DFWLYLW\XSRQDVKDUSGHFOLQHLQRGRULQWHQVLW\1DJHOHWDO $UHFHQW

571

VWXG\KDVVKRZQWKHSUHVHQFHRI*$%$HUJLF/1VZKLFKUHVSRQGWRRGRURIIVHWV1DJHODQG

572

:LOVRQ DQGZRXOGEHSHUIHFWO\VXLWHGWRPHGLDWHWHPSRUDOFRQWUDVWHQKDQFHPHQW,WLV

573

DOVRSRVVLEOHWKDWVORZNLQHWLFVRIPHWDERWURSLF*$%$%UHFHSWRUVPHGLDWHVVXVWDLQHG

574

LQKLELWLRQDIWHUDUDSLGGHFOLQHLQRGRULQWHQVLW\:LOVRQDQG/DXUHQW *LYHQWKH

575

FRPELQHGUROHRI*$%$$DQG*$%$%UHFHSWRUVLQWHPSRUDOFRQWUDVWHQKDQFHPHQWLWLVOLNHO\

576

WKDWDFRPELQDWLRQRIWKHLQWULQVLFSK\VLRORJLFDOSURSHUWLHVRI/1VDQGWKHNLQHWLFVRI*$%$

577

UHFHSWRUDFWLYDWLRQFRQWULEXWHWRVKDSLQJWKHWLPHFRXUVHRISUHV\QDSWLFLQKLELWLRQWKDW

578

XQGHUOLHVWHPSRUDOFRQWUDVWHQKDQFHPHQW)XWXUHVWXGLHVDUHUHTXLUHGWRSUREHWKHUROHRI

579

WKLVPHFKDQLVPLQDFWXDOSOXPHJXLGHGQDYLJDWLRQ



24

580

5HIHUHQFHV

581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629

Ache BW, Young JM (2005) Olfaction: diverse species, conserved principles. Neuron 48:417-430. Anderson JS, Carandini M, Ferster D (2000) Orientation tuning of input conductance, excitation, and inhibition in cat primary visual cortex. J Neurophysiol 84:909-926. Asahina K, Louis M, Piccinotti S, Vosshall LB (2009) A circuit supporting concentration-invariant odor perception in Drosophila. J Biol 8:9. Buldyrev I, Taylor WR (2013) Inhibitory mechanisms that generate centre and surround properties in ON and OFF brisk-sustained ganglion cells in the rabbit retina. J Physiol 591:303-325. Cao G, Platisa J, Pieribone VA, Raccuglia D, Kunst M, Nitabach MN (2013) Genetically targeted optical electrophysiology in intact neural circuits. Cell 154:904-913. Celani A, Villermaux E, Vergassola M (2014) Odor Landscapes in Turbulent Environments. Physical Review X 4:041015. Chait M, Poeppel D, Simon JZ (2008) Auditory temporal edge detection in human auditory cortex. Brain Res 1213:78-90. Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295-300. Chou YH, Spletter ML, Yaksi E, Leong JC, Wilson RI, Luo L (2010) Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe. Nat Neurosci 13:439-449. Clark DA, Fitzgerald JE, Ales JM, Gohl DM, Silies MA, Norcia AM, Clandinin TR (2014) Flies and humans share a motion estimation strategy that exploits natural scene statistics. Nat Neurosci 17:296-303. Couto A, Alenius M, Dickson BJ (2005) Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15:1535-1547. Cury KM, Uchida N (2010) Robust odor coding via inhalation-coupled transient activity in the mammalian olfactory bulb. Neuron 68:570-585. de Bruyne M, Foster K, Carlson JR (2001) Odor coding in the Drosophila antenna. Neuron 30:537-552. Delamater AR, Desouza A, Rivkin Y, Derman R (2014) Associative and temporal processes: a dual process approach. Behav Processes 101:38-48. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S, Couto A, Marra V, Keleman K, Dickson BJ (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151-156. Fiala A, Spall T (2003) In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression. Sci STKE 2003:PL6.

25

630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680

Fujiwara T, Kazawa T, Sakurai T, Fukushima R, Uchino K, Yamagata T, Namiki S, Haupt SS, Kanzaki R (2014) Odorant concentration differentiator for intermittent olfactory signals. J Neurosci 34:16581-16593. Gaudry Q, Nagel KI, Wilson RI (2012) Smelling on the fly: sensory cues and strategies for olfactory navigation in Drosophila. Curr Opin Neurobiol 22:216-222. Hallem EA, Carlson JR (2006) Coding of odors by a receptor repertoire. Cell 125:143-160. Hammer M, Menzel R (1995) Learning and memory in the honeybee. J Neurosci 15:1617-1630. Jin L, Han Z, Platisa J, Wooltorton JR, Cohen LB, Pieribone VA (2012) Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75:779-785. Kaupp UB (2010) Olfactory signalling in vertebrates and insects: differences and commonalities. Nat Rev Neurosci 11:188-200. Kuffler SW (1953) Discharge patterns and functional organization of mammalian retina. J Neurophysiol 16:37-68. Lei H, Riffell JA, Gage SL, Hildebrand JG (2009) Contrast enhancement of stimulus intermittency in a primary olfactory network and its behavioral significance. J Biol 8:21. Liu X, Davis RL (2009) The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning. Nat Neurosci 12:53-59. Liu X, Krause WC, Davis RL (2007) GABAA receptor RDL inhibits Drosophila olfactory associative learning. Neuron 56:1090-1102. Liu X, Buchanan ME, Han KA, Davis RL (2009) The GABAA receptor RDL suppresses the conditioned stimulus pathway for olfactory learning. J Neurosci 29:1573-1579. Lledo PM, Saghatelyan A, Lemasson M (2004) Inhibitory interneurons in the olfactory bulb: from development to function. Neuroscientist 10:292-303. Llinas R, Sugimori M, Silver RB (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256:677-679. MacLeod K, Laurent G (1996) Distinct mechanisms for synchronization and temporal patterning of odor-encoding neural assemblies. Science 274:976-979. Manenti T, Pertoldi C, Moghadam NN, Schou MF, Kjaersgaard A, Cavicchi S, Loeschcke V (2015) Inbreeding affects locomotor activity in Drosophila melanogaster at different ages. Behav Genet 45:127-134. Martelli C, Carlson JR, Emonet T (2013) Intensity invariant dynamics and odor-specific latencies in olfactory receptor neuron response. J Neurosci 33:6285-6297. Matkovic T, Siebert M, Knoche E, Depner H, Mertel S, Owald D, Schmidt M, Thomas U, Sickmann A, Kamin D, Hell SW, Burger J, Hollmann C, Mielke T, Wichmann C, Sigrist SJ (2013) The 26

681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731

Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles. J Cell Biol 202:667-683. McGann JP, Pirez N, Gainey MA, Muratore C, Elias AS, Wachowiak M (2005) Odorant representations are modulated by intra- but not interglomerular presynaptic inhibition of olfactory sensory neurons. Neuron 48:1039-1053. Mittmann W, Koch U, Hausser M (2005) Feed-forward inhibition shapes the spike output of cerebellar Purkinje cells. J Physiol 563:369-378. Murmu MS, Stinnakre J, Martin JR (2010) Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons. J Exp Biol 213:4163-4173. Murmu MS, Stinnakre J, Real E, Martin JR (2011) Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila. BMC Neurosci 12:105. Mylne KR, Mason PJ (1991) Concentration Fluctuation Measurements in a Dispersing Plume at a Range of up to 1000-M. Quarterly Journal of the Royal Meteorological Society 117:177-206. Nagel KI, Wilson RI (2011) Biophysical mechanisms underlying olfactory receptor neuron dynamics. Nat Neurosci 14:208-216. Nagel KI, Wilson RI (2016) Mechanisms Underlying Population Response Dynamics in Inhibitory Interneurons of the Drosophila Antennal Lobe. J Neurosci 36:4325-4338. Nagel KI, Hong EJ, Wilson RI (2015) Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics. Nat Neurosci 18:56-65. Ng M, Roorda RD, Lima SQ, Zemelman BV, Morcillo P, Miesenbock G (2002) Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36:463-474. Ni JQ, Liu LP, Binari R, Hardy R, Shim HS, Cavallaro A, Booker M, Pfeiffer BD, Markstein M, Wang H, Villalta C, Laverty TR, Perkins LA, Perrimon N (2009) A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182:1089-1100. Oheim M, Kirchhoff F, Stuhmer W (2006) Calcium microdomains in regulated exocytosis. Cell Calcium 40:423-439. Olsen SR, Wilson RI (2008) Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452:956-960. Pirez N, Wachowiak M (2008) In vivo modulation of sensory input to the olfactory bulb by tonic and activity-dependent presynaptic inhibition of receptor neurons. J Neurosci 28:6360-6371. Poo C, Isaacson JS (2009) Odor representations in olfactory cortex: "sparse" coding, global inhibition, and oscillations. Neuron 62:850-861. Pouille F, Scanziani M (2001) Enforcement of temporal fidelity in pyramidal cells by somatic feedforward inhibition. Science 293:1159-1163.

27

732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783

Raccuglia D, Mueller U (2013) Focal uncaging of GABA reveals a temporally defined role for GABAergic inhibition during appetitive associative olfactory conditioning in honeybees. Learn Mem 20:410-416. Raccuglia D, Mueller U (2014) Temporal integration of cholinergic and GABAergic inputs in isolated insect mushroom body neurons exposes pairing-specific signal processing. J Neurosci 34:16086-16092. Root CM, Masuyama K, Green DS, Enell LE, Nassel DR, Lee CH, Wang JW (2008) A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59:311-321. Sachse S, Galizia CG (2002) Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study. J Neurophysiol 87:1106-1117. Seki Y, Rybak J, Wicher D, Sachse S, Hansson BS (2010) Physiological and morphological characterization of local interneurons in the Drosophila antennal lobe. J Neurophysiol 104:1007-1019. Semmelhack JL, Wang JW (2009) Select Drosophila glomeruli mediate innate olfactory attraction and aversion. Nature 459:218-223. Shusterman R, Smear MC, Koulakov AA, Rinberg D (2011) Precise olfactory responses tile the sniff cycle. Nat Neurosci 14:1039-1044. Silbering AF, Galizia CG (2007) Processing of odor mixtures in the Drosophila antennal lobe reveals both global inhibition and glomerulus-specific interactions. J Neurosci 27:11966-11977. Stocker RF, Lienhard MC, Borst A, Fischbach KF (1990) Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Res 262:9-34. Stopfer M, Bhagavan S, Smith BH, Laurent G (1997) Impaired odour discrimination on desynchronization of odour-encoding neural assemblies. Nature 390:70-74. Tully T, Quinn WG (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster. J Comp Physiol A 157:263-277. Urban NN (2002) Lateral inhibition in the olfactory bulb and in olfaction. Physiol Behav 77:607-612. van Breugel F, Dickinson MH (2014) Plume-tracking behavior of flying Drosophila emerges from a set of distinct sensory-motor reflexes. Curr Biol 24:274-286. Vosshall LB, Wong AM, Axel R (2000) An olfactory sensory map in the fly brain. Cell 102:147-159. Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R (1999) A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96:725-736. Wallace DG, Gorny B, Whishaw IQ (2002) Rats can track odors, other rats, and themselves: implications for the study of spatial behavior. Behav Brain Res 131:185-192. Wehr M, Zador AM (2003) Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex. Nature 426:442-446. 28

784 785 786 787 788 789 790 791

Wilson RI, Laurent G (2005) Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. J Neurosci 25:9069-9079. Wilson RI, Turner GC, Laurent G (2004) Transformation of olfactory representations in the Drosophila antennal lobe. Science 303:366-370. Zaninovich OA, Kim SM, Root CR, Green DS, Ko KI, Wang JW (2013) A single-fly assay for foraging behavior in Drosophila. J Vis Exp:e50801.

792 793



794





29

795

)LJXUH/HJHQGV

796

)LJXUH,QFUHDVLQJRGRUFRQFHQWUDWLRQVLQGXFHVXVWDLQHGSHULSKHUDO261UHVSRQVHV

797

$RIDQ261GHSLFWLQJWKHGHQGULWHVDQGFHOOERG\LQWKHDQWHQQDDQGWKHSUHV\QDSWLFD[RQ

798

WHUPLQDOVLQWKHDQWHQQDOOREH7KHFLUFOHLQGLFDWHVIRFXVRQRGRUHOLFLWHGDFWLYLW\LQWKH

799

GHQGULWHVDQGFHOOERG\

800

%([WUDFHOOXODUVLQJOHVHQVLOOXPUHFRUGLQJV665 RIDFWLRQSRWHQWLDOVLQDQDEVHQVLOOXP

801

ZKLFKFRQWDLQV25DH[SUHVVLQJ261VVHFSXOVHVRIHWK\OEXW\UDWH(E RIWKHLQGLFDWHG

802

JDVSKDVHGLOXWLRQVZHUHGHOLYHUHGGXULQJWKHLQGLFDWHGLQWHUYDO\HOORZER[ 5HFRUGLQJVDUH

803

UHSUHVHQWDWLYHRIWKRVHREWDLQHGIURPIOLHVDQGVHQVLOODSHUFRQFHQWUDWLRQ

804

&&RPSDULVRQEHWZHHQVSRQWDQHRXVDQGSRVWSXOVHILULQJUDWHDWVDIWHURGRUSXOVH

805

RIIVHWVKRZVVXVWDLQHGILULQJIRURGRULQWHQVLWLHVDQGKLJKHU0HDQ6(0Q 

806

6WDWLVWLFDODQDO\VLVRQHZD\$129$IRUUHSHDWHGPHDVXUHPHQWVZLWK'XQQ¶VSRVWKRFWHVW

807

VSRQWDQHRXVILULQJDVFRQWURO  S

808

'0HDQILULQJUDWHVQ IOLHV RIWKHDQHXURQUHFRUGHGLQDEVHQVLOODRI$UF/LJKW

809

H[SUHVVLQJIOLHV

810

(0HDQILULQJUDWHVQ IOLHV RIWKHDQHXURQUHFRUGHGLQDEVHQVLOODRI&DQWRQ6IOLHV

811

)5HSUHVHQWDWLYHRGRUVLJQDOVPHDVXUHGDWWKHRXWOHWRIWKHRGRUGHOLYHU\V\VWHPXVLQJD

812

SKRWRLRQL]DWLRQGHWHFWRU3,' 

813

*25D261VH[SUHVVLQJ$UF/LJKWGLVSOD\UHGXFHGVSRQWDQHRXVDQGRGRULQGXFHGSHDN

814

ILULQJUDWHVFRPSDUHGWRZLOGW\SH25D261V&DQWRQ6 'DWDIURP'DQG(ZDV

815

DYHUDJHGDFURVVFRQFHQWUDWLRQV6WDWLVWLFDODQDO\VLVXQSDLUHGWWHVW S

S

816

+0HDQORFDOILHOGSRWHQWLDO/)3Q  RIDEVHQVLOODLQZIOLHVVKRZLQJVXVWDLQHG

817

QHXURQDODFWLYLW\

818

 30

819

)LJXUH2SWLFDOHOHFWURSK\VLRORJ\UHYHDOVVXVWDLQHGSHULSKHUDO261UHVSRQVHV

820

$&RPELQHGIOXRUHVFHQWDQGWUDQVPLWWHGOLJKWLPDJHRIWKHDQWHQQDRIDIO\H[SUHVVLQJ

821

$UF/LJKWLQ25DH[SUHVVLQJ261V6FDOH —P

822

%(6LPXOWDQHRXV665RIDEDQGYROWDJHLPDJLQJRIWKHDQWHQQDRIIOLHVH[SUHVVLQJ

823

$UF/LJKWLQ25DH[SUHVVLQJ261V5HSUHVHQWDWLYH3,'VLJQDOVDUHVKRZQDQGZHUH

824

PHDVXUHGaFPEHKLQGWKHIO\ArcLight

C

ethyl butyrate

¿ULQJUDWH>+]@

1:5 1:3 1:1

200 150

E

50

1:3 1:5 VSRVWSXOVH

1:1

200 150 100 50 0

0

2

4

6 8 WLPH>V@

10

12

14

VSRQWDQHRXV ¿ULQJUDWH+]

PID 0

2

4

6 8 WLPH>V@

10

12

14

LFP w 1118 P9

0

2

4

6 8 WLPH>V@

0

G

OR22a>ArcLight 9

H

10

V 250 Canton S

100

0

F

1:25 1:5 1:3 1:1

*

20

10

12

14

16

2

4

**

12 8 4 0

$UF/LJKW &DQWRQ6

6 8 WLPH>V@ SHDN¿ULQJUDWH+]

¿ULQJUDWH>+]@

D 250 OR22a>ArcLight

*

*

30

0 EHIRUH 1:25 SXOVH

¿ULQJUDWH>+]@

DM2

OR22a>ArcLight 40

200

10

12

14

*

100

0

$UF/LJKW &DQWRQ6

Figure 4 '0 GABABDQWDJRQLVW before CGP54626 2% —0 ¨))

A

B

1:5 1

'0 GABA$+GABABDQWDJRQLVWV 2% ¨))

D

before CGP54626+PTX

1:5 5

P9

1:5

'0 GABA$+GABABDQWDJRQLVWV

E

before PTX —0

2% ¨))

1:5 1

C

'0 GABAADQWDJRQLVW

2% ¨))

F

before G 4 CGP54626+PTX

P9

1:3 3 1:3 0 12

VKDUSQHVVPD[ #WVSRVWSXOVH

3 QV

4 0 WLPH>V@

8

1

2

3

***

***

1.2

1:5

1:5

QV

QV

1:5

4

before CGP54626 PTX CGP54626 37;

4 1:3

** ***

0.8

***

0.4 0

1:5 1:5 1:5 RGRUGLOXWLRQ 90 DQWHQQDHUHPRYHG 12 before 2% DQWHQQDH UHPRYHG

¨))

*

8 4

1:5 0

1:3

PD[¨))

I

2

QV

0

H

1

1

2 WLPH>V@

3

4

0

1:5

VKDUSQHVVPD[ #WVSRVWSXOVH

PD[¨))

G

1.2 0.8

*

0.4 0

RGRUGLOXWLRQ

1:5

Figure 5 A PD[¨))

12

*** ** *

** *

* ** **

8

*$%$$51$L *$%$$*$%$%51$L

** ** **

*** *** ***

1.2

*

**

***

0.8

4 0

B

sharpnessmax @ t 1.5 s post pulse

FRQWURO3')51$L *$%$%51$L

0.4

1:125

1:25 1:3 1:5 odor dilution

0

1:1

C

1:125

1:25

1:3 1:5 odor dilution

1:1

D sharpnessoffset @ t x s post pulse

2% ¨)) 1s 1:25

3 2 1

* 0.5-0.85 * 0.23-1.1

0 0

0.5

F E sharpnessoffset @ t x s post pulse

3

1:5

* 0.17-0.42

* 1.09-1.63

0 0.5

0

sharpnessoffset @ t x s post pulse 1

2 time [s]

3

4

1 1.5 post pulse time [s]

2 1:3

1

* 1.02-1.84

0 0

0.5

I

1:1

1:5

* 0.21-0.4

sharpnessoffset @ t x s post pulse

J

2

1

H

1:3

1 1.5 post pulse time [s]

2

0

G

1:25

* 0.12-0.46

1 1.5 post pulse time [s]

2 1:1

1

0

* 1.3-2 -1 0

0.5

1 1.5 post pulse time [s]

2

Figure 6 A

B 1:1

odor port

1:125

odor port

10

6 4 2 0 GABAA+GABAB-RNAi GABAA+ GABAB-RNAi + Dicer

D 1:25 8

¨Gt [mm]

¨Gt [mm]

control (PDF-RNAi) GABAB-RNAi GABAA-RNAi C 1:125 8

4

0

0 0 1:5

30

F

0

-10

-20

0

20 10 time [s]

*** *** ***

10 ¨G10 [mm]

20 10 time [s]

30

0

20 10 time [s]

30

0

-10

G

0

30

10

*** ***

1:125

-20

H

*** * ***

-10 -20

0 1:1 10

¨Gt [mm]

¨Gt [mm]

10

20 10 time [s]

1:25 1:5 odor dilution

1:1

¨G10 [mm]

E

4

0

-10 -20

*** ***

*** ***

control (PDF-RNAi) GABAA-RNAi (8-10G) GABAA-RNAi (2-7E2) 1:1 1:125 1:25 1:5 odor dilution

time [s]

8

Figure 7 A FRQWUROÀLHV3')51$L

B FRQWUROÀLHV3')51$L 5

0

1:125 1:25 1:5 1:1

-4

-8

0-2

C

*

2-4 4-6 6-8 initial distance from port [cm]

0 cm 1 cm 2 cm

¨Gt [mm]

velocity [mm/s]

4

0

-5

-10

8-10

0

1

'

2 time [s]

6 cm 7 cm 8 cm

3 cm 4 cm 5 cm

3

4

1V 1s 1V

1s 1:5

1:1

F 2500

2500

2000

2000

1500

1500

tau [ms]

tau [ms]

E

1000

500

500 0

1000

0

1

2

3

4 5 dfp [cm]

6

7

8

0

0

1

2

3

4 5 dfp [cm]

6

7

8

Figure 8 A

B

ÀLHV#GISFP

ÀLHV#GISFP

¨Gt [mm]

0

0 -10

-10 control (PDF-RNAi) GABAB-RNAi GABAA-RNAi GABAA + GABAB-RNAi GABAA + GABAB-RNAi + Dicer

-20 -30 -1

C

0

1

2 3 time [s]

-20 -30

4

5

6

5

5 ÀLHV#GISFP

velocity [mm/s]

-2.5

** ** **

** **

velocity [mm/s]

0

*

2 3 time [s]

4

5

6

ÀLHV#GISFP

** **

* *

**

-5

-2 - -1 -1-0 0-1 1-2 2-3 SRVWSXOVHWLPH>V@ 4 ÀLHV#GIS!FP

3-4

4-5

-7.5

-2 - -1 -1-0 0-1 1-2 2-3 SRVWSXOVHWLPH>V@ F 4 ÀLHV#GIS!FP

2

2

0

0

-2

1

-2.5

-5 -7.5

E

0

2.5

2.5 0

-1

D

-2 - -1 -1-0 0-1 1-2 2-3 SRVWSXOVHWLPH>V@

3-4

4-5

-2

-2 - -1 -1-0 0-1 1-2 2-3 SRVWSXOVHWLPH>V@

3-4

4-5

3-4

4-5

Figure 9 high concentration ethyl butyrate odor pulse OR22a

SHULSKHUDO¿ULQJ

peripheral voltage ORN

Network interaction sharpened presynaptic voltage

GABAergic LN

GABAB

GABAA

blocking presynaptic GABA receptors (A+B) PNs temporally contrast enhanced behavioral response avoidance