.4 '3‘ 35 2 6) I . . . . . . . . I . . . . . .. .. . - a . . i. . :1. .. . . £7.05... sung; -. .8 .V . .... .. . . : J ......i - V... ...... .03.. .3... .5... . 06.2.; 0.. 10.20. ..0 a . .9. . . . .1 1...-.. . .... 0. . ... .10..-.3. ...} ..... .00.. . ....00 . . ..00... 9““. .0. .. .....0 4 . 0 .000» ‘8... .009... n: . ... . 005. . , : . . . . . .. .... 0. .. .0 .0 0 o. 7.0.0... 7 .. .0... - .... _. .0.. . .80. a 1. 00 . . , . .. .p . .. . i3) 0 ... 0. .. . . . .. :60... .. . 0 ...l 0 ..0. .. 0.0. .u .090 .0 . . . :11. .10 .. V . 4 _ . . .. . .... . ........:. :0 .. . . .... .1. ...? 2......0. 10.. . 3 .0 ...; ...... .. ... a. 0 . . .... .. . .. . .. 0.3..“ ...0 .. . . 8000.... ...-0.0... 01‘) 0...... ...! u .5; 0 . . 4 . M . . 00. 1 ... . v ,. . . . . . . .... ... .. . . .... 0. 0 0 . . 01000.0. . ... ... .. ..f .....0. .. . ... ... . . . . .. ...: . (0:0 0.... 0.. ..00 0 0| 0 .0000. 1:0Vfi‘... J: 0 3.0... 0000 ...—0.... .0. .0. .... . ..1..:.80¢.0... 000 00., M00 , . . . . . 0 Y0 . 00 . . 0. 9.0 0. 00. . 0.10:0 .. .0. 0.0 .. .0. ...0 0 0... ..000 .0 .. .v. . 0.0.Fuh1‘. ..“30000 Lift-0.0. 0 . o . .... 0. . J 0 00m 0.... ...u .. 0.0 . 30““ . .. . . . . . .. . .00.)..00' .0... ... ....u .-. ...0 0.5. u 0.. ... .. . . 0 ... . .0. 01...... 0 .. 0 o ...:30. 0.0 0...... 00000.... 00.0. . . . ... . .1. .00 ..ll . 0 3.00 . 1.0090 ...? 0 0 0 00 00 ...; I0. ..I . 0.0! _. . _ .. o a. £000... 9: Ely”. 1.. 0 0 . . .‘v .. 0;... 0 .. 0.. . . 0 ... .. n 10.: . 0 . 0000.: .0 ... <. 0 000. .0. .00 . 0 :0 .003 ..0 0 . 00.. . . 00.00.. 0 .. . . .00 . 00- 0.5.00 ...0 .021) ‘0‘,, , 101.00....510. . v.00 .. 0. . 0 .0 . an. 9...... . , . . . 0 0|: ... 000 0.0.! .. .. ... 0.0. 0030. 00000.. . .. 00 ... 00 . 00.. . I .. 0.10200. L00 00.0.00 0.00... ,. 03 ”J0... 00“,.‘300, .0 000.0 ": . 00.0001 . ....t «.00. . 1 . . ..u .0 v 0 J.- 00? .. .00.- .. .. 21‘: . . . .. . . .00.. ...: 2 0.. .0... 0... ... .0. . ...... . .00... 0.0.00. . . .03.... ~70 , . .. .. 0. 40.0 0. 0 .\ 0000 . . . . 1.000 0 0 w .000. I.‘ ..0 . . .. . 0 0. . . fl. 0.0.4.. . _ .. _ .90......nu..~0.».ll 0...... . ...... u... ...n ...-.1300... "Rita“? . . . . . . . .u; .0... .... .. \0 .0 .... .....x. . 0.3.9.0. .. . .. 0 I... . . . . . . . ...: 0 . ...-.... $0.00 . .. o . . . .. . u . . .h .0. p .00 .... . . . . . . .. . . .. . o 0 0. . .p .. .. .. .30 .. . . .. . . . .V . .. .. _ . . . . . . . . . . . ..0. 2:0... ..0 H r. . , wh.ww.wuwfl.00. 0 :0) \.-0 000.054.0000 \0“.x“..0 ..n...au%n..0w.v .. . . .. . . .000. 0 I. . .0 00.. u 0 ‘00.. 0.0 001.000.0302.. .0 ...J . . . . . . O0. JUNO” 0n. {.0 0.?‘20 9. ...» 0 ‘% ”W0 ~ 0 .0 0 . ... 0 0 . . . . . ... . .0: ..0.. . 00 0.00.0000. . 0... . . . ., ‘000’4‘ .. ..V 0 0 30"W00”."..H:..w. H .90.“ 0.«.u03§u.0.hu. . 0 .0 “0.0....u0‘.0. . .000 .0. 10" .00...00. .. :00qu 0 0.0-..‘0. .n10 u. - 0.. 0 0000 ‘0 w..0._.nv“.0..0 "0".1W00' ...-00‘21. ‘ .... 0.“n“h0 0 .. . . 0‘ .00 0 0 000-0 .. 0. .. V 0. . . v . . A”... k ...,4 0 . ,. .. .n. . x310... ... .... x. M. .. t... :10 ...... 1 .87... . ... 14...... .- .0. 0 .2. 0...... ...... ... .....0 0.0....uuuu.. .04 0 . . ... . .fi...fi.t..0..s.u..x u: . . .0 0000..."...0-0 . .0 . 000.0...00 40 0.300 .- 0 . . 9 0400 .0, .. ..0. 0 0 0| .0 10 .001 000000 A . 0 0O”. . l 0 .0 0.0. . ‘0 00 II . _ l .0 .0. fl .. .0 0‘ 01 . 00‘0- ”. ‘0 0 0 I0 'aa-08 «‘07’2!“ . I .0. ... . 0 .. ... .02..— h... ... .u.. 0. 0.... v ..."...unwnvonn...9.0;0.h. ” . W00l..0...>.00. v! H 3.0.0.0009...” ......“ “0000.....030. 09003.. . ...01 . {03.0. . .0000 . .... . . . .03 ... 0 . 0.0 00.000 .00 .... 0 I .0... \0 0 _ ..u ... ..0. . .... ... . “0.0.0070 . aha . . T 4. 0 . . . . . .. v .. .. .0.', .. .0 .. ...: . : .. .0 . .00 . o . . ., . . .. _ \L 1’0 , ..fihucvn... . 0. ..0 . .. ...\........L. . & .... ... .02 .03.. ... . . 3 . ... 211.4020. 3: .. . 0. -. 8......0. ....l....u0.9=; .... .....h. . . 0.. .5...” ...... .h...”»...00.l1:.a... 3.0- 0.2.0 ..n«.c...- . . . ...!.0.~.\.0...«.“05..u . ..000 ..b. _. 0 ...: 00 0.0.0000 . . . ... - HLV "noun.“ ...... . . . . 0 .. ._ to. | 0.. 9.00. 0.. . ... 0 .0 .. ..00 .0 ... .p’. 00010, ... 0.0 . , . a 0 00 . .000... .0 0:100 .9 0 . . . . .'. 00.00 00 70.0.023'.‘ . . . . . #000010 a, . ... 000.0“... ”....0 u 0.1.0.; g‘hfi .. n u 0.’ . .0 . . V . . 0. (I. «.0 000,0.00000 0.0 2000:. 0.0.0.. 0.. - 00 I . .0 0 .K.. 0.00. 0 ._ .. .05.... 00 .. ..0 ...-0.0 0 I. .0 000 . 00.0 .‘00 010 0%.‘500 ..n. .0... 00&0§0\‘ ‘0. h. . : . 2.30.00.....:. . 00 . v .0. 0 0 0 .0... .0 . 0. . .0 o. . .030 .00 .... 30.0.0 0.. .0...0. . 0 . .... :0. . .530... 0... 00.. a. *0... 00.0.7. .00..i.v.0.f.0.0 . 0 .001'V “.000... $11.". 0. 00". 0“...)‘0 0.01 .0. . n.) 040-00. .0].- .. .. n 0.. . 4 . . . 300.0000. ~00 0.". 00. I tic“, . . .. . r u .- .... 000...... . v .. 3 .0 0.0 , . ., _ . . . . 0:00 0.40 0...... 009000. 0.. 0 0000 2 . . .. . .0. .0003" 0 v .0 0 00. . .0 .. .0! 040000 . . . . . . . _ . . . ... .003; ..u0.”0.v0 03.0 00.. 00 0?..0w0r 000.4 0‘ ..0 0.000.. . . . .. .0.- v. . . . . .. .000. .... . . - .00 .52.. .....0. .0. )0}... 00020.00. . . . . . . . . . . _ . 0 .7 .... {000.03.03.05. ...-0094,00. . 03.40. ...! .01.... ..30. .... . , . . . . . . . . .00....3:0.... 0.2!. ..l... . 0 . . .....- 0. . . . . . . . . .0. a... .1 . ,. .0 0....k0.) .’09..... 0. u“. . . .0. I. .0137. . . . . . . . . . .. . . . _ : 4 :0.»- 0.,.0a.!..0. ..- ..000 0.0 . 0.00. 00‘ ’ 0 .. .0? : . . . . . . . 0.i303.0.r0og “000.. ,0 0.0 . . . .. . 0 . . . . , . . . 0 ... '00. 00. 0 i0 .0. 0. . 0 .. . .. . . . . . , 1.20.”.3... . . . . . . . . . . .. . . . . . . .1151 80...... .. .0..u...:..l.. .08 ”.0.“ .u.. ...0. ......003 ...: .0.00..0....0.....0.¢ 0 .0 ....0 0 08. ..V . h . . . . . . . . .7 0 00.. 0 0 0 .00 00.. .033 -000 . .. . . .. 0. .- 40.01.060.30: . . . o. . .... .... 0. . . .00". ...... 0» .. 0;... .... £233.80 7 ..0 U .0..r..0..0.. .0 . n .0..... 0 0....‘0; . .l 0 d. 00. 0.0 .. . .00 .0...0 00.. .u ,0. 00000 0- .0 ...-00.0.0.0 0.0 Q. ...00: 00... 07.0000 .00. 0.0 0.40 0.. 00 30.00.00" . .. .000 ....0 0.09.0130. .. v. 0. . ..3..'0\.0. .0... - aw. A0 . . o... . ..0 .o 0 0. 0.0 00.00000 a. .03“ “40.900” 00.0... . .0001. . ... 0 .00.... 0 . . .00...0 ......) . . 000 . .00 0 00" .....0 I .000 0.0 00.0.. 0.. 0.00%00I 0.. 9 0.10 0.0 ‘0‘ 0 . 00.. .....33: 0.0.0000. 0.00 2.0.0... 0.0.0. 01.00.... 0.0. .003“. .90).. _ l3...0.n0.0)..$_f... .0 ”......00303433. . . .. 0 04 ... 0 I. ,3 . . . a lo. 0.... .030 0. ... 0.... 23-0.2: .00. . . 00 ...... .9. 00. - anuh .300?" 00.. a" 0 .3030“. 0..“ ...0‘. .... I . «I . .u a. . 0.0. . 0.00.0 .. . 00. 0 ... 0.. 3.00 ....0 .10..-. .. 0 ..000 0 0 001'. . 00.0.0... . .00? a ....t .... ..... ......a .33... . . 4 ...2....L.n......ua 52.... ........ 5.... 2...... 4- 51.5.... .... ......:......2........... .....9......:..P.. . . ., 4 .-.. . .3.... .00.. .00... 3.0... . 0, .. . 0 .0 .... ...-.... 0' 0.00 0. .. 510.330: .0.0.\00. .. .0 0 .0 ...000 ..0'00‘000 , . 00.. IP00-000 0 .0 ‘00.". . "undo-0.0" "u. 0 .0. J30n0.”0.”unu.h . ...-D .... ..0. . 2 00. 000 . 0. 0.!8000030-00! 0.0. .0 H. 50.10... 0....0...000.....I‘. 0&10M.3n 0.0.... 00000000020 00’: 0!" 0. .0 I. . 1 ....II 00. 0. '10 .0. ... .0 0..0 . $200.0... .33...." 10-0 :0 000000. ..00 .00’. . ..u 00.010. 00".-..0s .0. 0. 10000.0 0 0. . 290.0" 0000. 0:, .000... . 0.0.0 . 000 0. 0.000 . .00.. 0.0. 300’ 0 0 ... ... .00... . . 0..) 0 {00. 0. . .0 000 of . .0 . 0 . 00.0 . no . . .00.... . 0| . 0 . 3.. I... O .... 00.. . . 20.10000 .\ .... . . .1...0...0 .. . ..0 0'S.-‘.0 .. 0 .0 .0... .17..0000000. .‘00 . ... .0 ‘00 7 .0. 0:0... 000 o 0.0... .0. 0 .0 .. 0.01.2.0. la...) 01‘. 0 A 00 .0 ...!- , . . , . .. 2 . . 00 . . . .. 0 .0303? .5: .0 ‘0‘.“0: 0.. . rou‘an0.un0.ouu00 3,4 .. .0 0 an” 00.0 o .0. 0.“? ...00 0... .1 ....00 .30u003 .0 .01 . 0.. 4.001. ...». ‘00: .10?) £50,100 ”’033. .! ...... .0304. 0.0. .0..0..00,.~0:.. . ..l 0 . I 0‘0 . 0 . . 0. 80... ..0... w”... 00.031“. iamw. 3gmn‘ . . t. . a. _ 0. .. . .. 01.0.... 2 (.0 3.0.100 . . 30.3.5.) 0 0.. 0.70.0000 ’3 ..0. 000 0. . x... ....S’....0..0 . .... . . . . .0. 0. ..rcl. 0 ....uuflliakrl. .L. A {0.004.300 27......0... p 0.. 01.91... .0 . L: .. 0 :80? 60:00.... .0 v . .. ... .... ...- ...... a. .1 o. .2 . .. 0. .0 0a.: 0. . - J... 3.3.. 0.. ..0 .00 .00t . 2.0;...“ 300.93%. 108' ... . . . . .. . 00.00 0.700020 50... 1 00 .. . 0 .0300 .00 0-0... . 0 00 .0.0.0..‘30..’c...00£0.00...- 0. 20.110 0 070.910.0000. 0. ‘1 . . .000 .. u n. . ..V . ....00 ...... ..-. X , .0 . . 40.0.... 0 . 9.. .. . .. .3! 0. .... $0000.. . 0 00... 02.3020 0.. ... 0... a... . c I). 0.0, . 30.0.. .02... .. .0. . t fifi...§.0.0ol.0. : . I. 3:4. .. 1...... 1.. ..0 ... ., . , . ... 0.00. 0.....0 20.10.12... .0... 0.0.0 . ... ¢ . . . .. J .0 00.. 0.0 . 332.01.990.10017 0. :0. .0 3.0.0070 .3010: 0.0.0 30.”. .0... .1 .31. v... .0000... Q . 00. 00.. . . . . . . . . . . .00}... 0 0.10.0 . 0 . 0. l 0000. .0.-00.,000‘. ..0. 0 . no 0.... .00 0 0 $0 0 . . .‘o'fi.0’ 00400100 0’ 00.0.)..502 " 9.00.00 1. 00r 0 .00.. .. ....0. 0 Is 00. , 00. . . . 00000.0..0..’.0000 00 ...} 3.000.. 0..‘-0\0.0.0..00.. 000.0. 0.. 0.0000 00.013300 .0100)... 000 $0 .0 000 0.2.... 00030"- ..0,.0 ‘ . .. ...... o 0.0 0 . . . . v ... 000 O 00 00. 0.00. 000: .30., 0-0 0 0T... 000 000.0,. .. 0 . . .0 ‘0..0 '0. ..0. .1900. 0 0. 0'.00“0.00¢ . 0;. 00 .09.?0 0’ 0.00‘0Q‘I0 ...-(.0 .00; o 0 3022:]. ....00 ...... 0y . , o 0 3... 00-. . .... . .. ......0030 ...... .l... u. o ..0‘ 0.3.0.3.... .0 03.0100 .’00000.0...00 0 .. 000. {tr-0. 0 13‘. 00.4.0.3...- 0.J004~.00..}1....0 0.- 0C0 '00 ... 0 I ...-... [’3 . . . . . . . . . . 0 .v . 0. .00- 0:0...018300. 0.00 . ... . 0.... 0000.. . 0 .0 0,00. 3.0 0.0.0... 0 .00....0. 00.00.00.000... 00. .6 .0. 0 00.0000. 0‘0”?! 010.0 J 00.- .00. 0 J. 0. 0. w 000” , , . 0“,. .300 . I... .. .....0. V .. V . .. . . . .. 0 u... 20.“... 0.. . ......u... 8,3“. . . . .15.... .... .00.“... . ......r0 . 2...... 3.3.5.... 0 ...-... .. . ...“... 0108.00 .. . : .. .... 0 ... . . .u 1.0. .... 0.1.0.050 «31... 0.... 0.. .... a .4 3.0.0.0.... 21. .0 19.00;... 05.4.. 00.00. I r. 001. .30. t .. .0 ..r .. . 0. . .. . ... :3. .... ..0 0. . ... . 0 3.70.3.0. 0. 0.0. . .00.... . 000.00 0 0 0! 0 . ..- 0 .h ....0000 00'00I00.P.o 00. .Jgiph‘ 00.00 . 0 0.900. 0. 0 . . . .. ‘0 0.. .. u. .0 .0. 0. 0. 200.0 0.00 .0 0.00.0 .00 00.... 0 0. . .0... .00 l .0 0 00000.... 0000. 0,. 000.00; 0.0. 3. 00000.01... 30“) .0000... .0 00 .‘3 r03u 10.0 s 0 0. . 0... . . ,. .. . .0 ... . . . ..I‘ .0000.0000-.l00. . .1 3,000.90 00' ...0‘.. '- 0.0 ...L 000000.00“ 0.3.0.0 0. ’0. 0“ a 0.00 0, 0 '. ’.01 I 0 d 0 .0... :.00.‘.0 0 .0.‘ 0 0.. .3010: a” 00- .... ... 00:... .0393. . . . 2 . . . . . ..0! .....00 ..«L.mfl.uv.fi0.0.uu. flunk.) 3......Juln .0 . 3.2.5.... .. . ...! ... int... 5...... p... ....Pdf .00.. .530...- 0.’ 8...... 30.0.... ..K.....q!_04u_.r.r; .....0. ...-20. ....00. .. . . . .. . .. . . . . . . , . .. 2 . . . : .. , . . - .. .. .0 . ... .. .... . 0. S . .. . ...n .v 0 . . . . .. . _ . . 0.....0 . ......000-3. . . .20..» 3.3.. .0.- ....0.1 ..00038 ..000 n .0. 0.. .00. .03. . . . . . 00.00. 00... ..0 00.0. "unnuo...0.u.00r.. (0000 01nu0..0. «9000‘ 0...Nnm.0.0'. .ufi "1...”..0. 0 0310.300 .u” 0‘3..." ‘0 _ . . . . . ... .. .. . .....71...4.w.. . 0.40.0... .. . .... 0. . ...: .0 . .9 ......t ...: ...... ...... ... 3.0 .. 4 ... 1... . 0.30.. ........4.0. 3.3.1306 0.. . ...... ...... 0:0 . 32.01.33. 3&1020! 03300 c. f .. . ... . . - .. .....s -. 0.. ... .. 0. .... 0 ... f... ...-0 000 1.2.3.922... .01 ... ..0 .. .... .0... ...... 52.330.000.002?!“ 0“unit-... I: 0.. 300...... 080......- _. . . 0. ; . .. . . . . .... . 00.. «...... 2.0.0.“)..03. ...... .0. 000000.. .00. . .3... ...0 00 030...... 05.0.3.0 .100330‘0‘140..00001 200‘: 00‘.) 0000......000 - .36“. V... .. o . J .. .... 00.00 _ 4 . .000 00 . . 00 0. 0. .2. .... 00020.. .0 .. ~00 ... I... 23.. .00 0.0.0? .. 0.. .. 0000‘ . 0.00.00 10.3 . 00’.L00.20.000.-.0.. “0004000001000 .. 0 40 . . .. . ... y. . . .,. 1.... 0.0 o. 0000.“ ...a. 0 0.. .... 000-0.... T 000... .030 ,.00..... \ 0.0.2... .50.J.00.‘).040ld. >20.0¢‘I..§:I .01. .. .320; ...! .... .0... ..V 0.. o ‘ 0 . 0 0 ..0 . L 0. . . 0 . 03.9.039100-00. 1000.0’. .00.0h.0 .. 00 0,000. 0 . ‘04. 0. 00030.04 0.000303). 0 .0.. . .0 . . ... .0. .. 0. 000 .. .4 l . . .. .00... .01.... 0.0.. 00. I.0.0.0000..0000.. 00. ...!007 ...-c .§”.‘0.. 1101".” .0030- J: 0 .1 .03.-..20. (.0003 D . L 30.20.... 00000 .0 500... . .0 .00 .03 0. . . ...... 00 I50 00 00. 00 . 00 .0 00: 0.0.00 .50.. 00.. . 0.0... . 0.0.0000. 0.0.,0. 00.0”00000 . 01.0‘.0 .0 I 0000-»: 0 000.....000 0%..“ . II. . ‘0 0o O 0.0.. .- . 00200-1 .0 .00... 0 Si. {’10. 0:00.... .30.. .0..- .0’0. .80.; .0 .130000303120 .430.¥50nn'2 ”Mr? ,‘.. 0 I ’ 0...... 0 O... 0.0.x. r.‘l0’ I. . .0... .00.. 0’0... (00.00000) 0.0.0. .000 .10. 3535.30 0.. .00.:00’ .' 00000 00.0 00000000 0.... IQ. . 00 0 .0000... 0 0.‘.O.5Il.l.000.00. 000000. 0.0.9.00 ..’0 0.000).... . .. . 2 32.4.0.0... . 10.0. 40.91.... ...80 3.1.3.390. ... .... .n . . r00“! . . . .. . . . . . 30.0.1. 0.... 3.0.0.003000 0'..00....0. ...f. 0.. .0.“ «.0. .20. ...0 2...... 3.00.3.0... .00. ’0. q .09 00.0. . .. . .. . . . V . . . . . . . . .0 .20... .. 00... . ~.0000..\.Il.p00 $.00. . 00.0 ‘ .0M?. .00.. 0000 0000300. . . . . . . . . . . . . . . . . . . .. . .. . Y . . . . . 0....000‘050. .0031)...‘ 0000 0 000000.! 760.00.. 00 5 ..RI. 0 ..t”.? ....0... 0.. . . . .0. .00. ... 2.0.. .00300030. 0 , . .. . . . . . . . . . . . . . . . . . ..00 ... . . .0 . 0.0.5... 00. . . . . .. . .. . . . . 0. . . . ...0. 00. .0 I 0. 00 .0. .00‘...0.0..0.000 . ..0 . “0.0000 000 0 . 00 {00. .3000 a 000 ...00 .hv. 0.: .00u01000‘... 0.. 0.0.....&0 000000000 00 . 010, 000.00 0 00 . . r000 . .0.0 0.03 0000.0 {0.0.0.0 . 0 . 0‘. . 31.0 .0 0y ..0 0.1000. 00.0 0. .0 30010000000. 10.000.33.30‘I’020I 00‘ 0.... “a”; 08.x“ . . . .... . .0 9.0 0. .... 0.. .... ..0.. :0 0.40.1200}..- 00:30...- . 3... 1.00.0.2... 4...00...0.P100. .0‘.00.....0' J'J.0093....~0!..0 I, . 0 .10 00.. .0 .. . u. . u . . , . . . . .. . . 0 '00.. 0 0' 3000100 .000 05.0 0:003: 00 0.40330 0002:000‘000‘0.00¢ ‘0 v - 00.01000? .01.. 0000000,...g‘j . .0" 00" 1‘ 00‘ ‘1‘ Q0 ‘ 5.0» 0 . . . . . . . . . . .. . . . . . «.... i... . .00» _. {00.03.00.- .00..00 .1. ’00. .0000. 0.0.1.0.. .030} t .. n 00 703.000‘ 0.200.105.0090. ... 0r i ’ 1 . .. .... .. . . . . : . .4 . . 0...... . . .. . _ .....x . .... ...-.... . _ . ...]. 0. ...: 10:70.02}...0....000....0....J.. ._(.)..0..)0.0.00.0..0..).0 00.0.. 3.5330000. .. . .. . . . . . . . . . . ... 0 ... . _ . 0.0. . .1100... 00.00.. 2‘”); .. :0 2.0.3}... . . :5... 00.0.); 0.00... 0000 .... . 01.0.0 . . . . . .30 ..0... .. . . .. .. . . . .. . . . .0 0.00.0 .0000. ... O .03. . .003...“ 1... 10.10.090.030} 01 ...-0 ...-.0 .-I‘.000)‘0. '0‘ 0.... 00 ’00 .1. .. ...: .0 0.0 .. .. . .. . . . .. . .. . . ... . , . . . . .. . . .. 0 0.00.....0...0:}0.0.... .. 0.. 0 .300 1.6.0.0000. $90.00.. 0. t. ... 100..)‘3. ... 3,40 .00. 00...!) a, .0...- .. ... .... ...0. ....00. .00 0 . ... . . . . . . .. . . .. . . . f..000.. .00 .300 ...: ...... :90 0 .. 000.00%: 7.00 0.0.000... 0|. .0". «.05 .10. 0.1.0.010000 0.0. q _ . . . .. . . , .V . . . . . . . . . 3.000000 0 . 00......0....l0 . ... 0. ”.0...- 0 .0 .....‘ 6..,\.0 30004.00}. .00- 1. 9.00.9“! I: a .. : . . . . . . .. , , . . . .0 .. .0303 .0303. ... .... 0.. .1000... $00.13“. 0:210:12} 0" 00.0.3.0 73.0. .10.. . . . . . . . . . . 00.....0 1. 00.. 00.7.0... .00 .0 .0. 022.010.. .0. ‘1 .390’0‘0:36‘00u.0005 ...00 0 .. . .. . . . . . . 00......) 00 0 ...... .000..0-. 0 .0002.- 0. .. ..0..030.\010.0.00...0 00.00 0....000000...0 50000003....01000. .0. .00- .000 .0... 3000 .0..0 .1090000..1h~000l.dr .0’d 0. .00,""". r". . . . . . . . . .00.- . .20 .00. 00.020.03.0’0000‘ 10 ...-3.000- . . . . . . $.30... 0"? 0" .. . . . . . . . . 1.00.009000.‘ . . .. . . . . . . .. . h . 0.0 00. a .10 0‘ 00' f‘ .3. 0:0 ‘0”..90‘00‘00.o..0‘00000"0 5.. . .. .... .o .....2... 0.352..- . . . .. .. . . . . . _ . .. . .. 12....00 l: .. 21.02.0039... 0000 .02.... . . . . . _ . , . . . . i .0,- 0 Or0 . .3... .00. 00.. {0.00.3.0 0....- . . .. 20.. 0‘0: 0 0 0 .. . , . . _. . '0 0 D 03.00.. 0 .4000. 00... .0. .40. 0. 0 . \ v: ‘O}. .‘.0 0 000 I I 0.. .... 0.0 . . . . U . . y . .00 0 0... . ..l . 1 0.0 .. . .00“ 0. .00 O. . 0. ‘00 0.00. .. . . . ..00. .0 ...0. ...!nil. .00 .0? . . . ...! 5100 0 0 . . , I.n'.0.D. .0‘0‘00300.00.0 .0 .00.; 0 0. 000....18. .....0000...I.I...0 . .8.é0..0,.’10‘00.0.02 0.. ..Ia‘..~‘b.0‘z..l..0|3 . 0.0.00.0..u‘...) $000.00..0 10.. {000.010.03.153}; . 000 . : ..0’ .00. . , . .- ....000....0..0.0..0.00..l000 ..00. {(0300.00)}. 000.103.000.030- .. .00 . . . 00.00 9.... 0 ....00...0J0..01....0 300 0. 0. 000 . ‘0... 3.0.. . 00.0.0. 03.0.0 0....030J0.))00..§ .00 00000“ .lziflu0‘0f .0.“ I} . .. 0 . . ...0. 00 0.. I 0.0 0.03.. . I . .3..710..0000 .0330... .00. 3%....0.0..0x.0l..0... . ...-0.0.30. 00000.. .0.‘ 000.0... ...00 .0.. 0.0.0.13 00.50.... 0. . he . .... .. ......000...i., . . .... 0. ...00 ... ..J0 . i w} ...; ...: 00...... ... ... 0.000500...0...00 00080 00.0 050...}, ....» .. Alimonii... 0.“ 0.000: .0fii‘ A . I... ...}... .02.}... .. . . .....03. :0 7.0.... 0.. .. 2...... . 0 3:05.......: ..0 .08....0... ... .0200. . v . 0 5:01.150 .000..00..0.00.0.0. II. 000,000.04... . 0., 00.. 03.0.... .. 00.0..01 00. ”0.3.7.01- ... 0 c (-1.00% . 000 .0 0000.3. 0.0.00 0.0 0..}...... 00 ... 0000.000 000 0.090.010.0000. . .04 0-.0....00..... ’00.0r.0..,000..l .0). 000: .30.).‘0!\.i$3, . u . z... 1.000 u) .u ... .. . . . .0. 0 0.0.0.. 0.. . 0.. .0... ......l-Ll‘olnit (3300.... r... .0 ...; ”oval! (01"...Lon 0.000004... 00:. u. .. 001.0 .3330). 3000.931 A .... ...... . . L . . . . . .. 0 0.3.4003? 8...... 2 .. ......200...‘ .....ifv..: 0...... .250. .10 000010100. 0 800.033-: . “0.00%.!” :0. . .0... 7‘! .... .. . #0 00000.0 .. . . .0.- . .. . . 30.00. 0 0 0 .0... 00 51-10.10... 0 000?? 00.00. 30.00.0020. .00.. .0050 00’ 0.) ‘00.!00.u(0100.00.00‘00000f‘0.’ 'IS" . 000. V 0’0. x V :3 . 0 . v. ab. .00 . .. .... L... . . 2 . . . . . ... 0. ......3 ...0 ... ...: ..0‘ 10.0 00 00.0.2.0. .... 0......35 .0 .00... 00.. 0...! ...0 h; r ...... ...-0.00.10.00.00... . . ..0. 0... . . . q ... 6.0.3... .... 0......0. .00 . .. 1.0!. 00... V . . . . . .. . . . 2.1.0.0392: u... 0. 0.. 3.....‘302003 ...-..IJI.0$)..I~. . 000.. :00. 0— h . . . . .0. 0 n0 ‘0‘.h0 « .000000 ...0.0..0.O\0 0 . .0. . . . . . . .. .00‘0000—00 0..0.000.u.".0a.00. :0." .0 .00 0.0.0 00000 0, ‘0‘..d.5.0..' I. . .. . 0 . 0:0. . .0 4 .0 . 0.00 0000.30 .0 0000.0. 0 .0. 00. . u . . . . 0-00001, .0._0‘.00. 0.0.00 0. . 0 0 '0 0 0 0 ... . 0 0 0.0 D .... .0 0.0. 033. .0, 0... 000 I I. . . . . u. . . . 0.0.0 .. 0 . .. 0. 0 . . . . . . 0 . . 0 . a ..000‘........‘ 0.0... 0.0 0 .0 00.. 00000. .00.0 0. 00 0000- 000 0 ..... .00.. . ..0... 0“; .0 010-0 00" 0 .. 00.00 0.00{dv.000 ‘0 0 0. 0.00060'.. 01000000 0 .000 .100 0.0 ..0. y . . ..0 0.0.. .00 J 0 ..00. . . . . 000.. 0.. 0000.0 0 30.. 0- 00.. 0000 3000.0 00“. ‘0 V 2.0 0 . .0 00. 43.3.0.3... 00.. . 0. .00. .000... .... 0 90030.0 . 0 . 010...!‘9. ..J. 000 ..00 0.0.30.1. 00 0.0.4300I30' .0.00J...¢00000 .00 .0 ....00 ...”...0... ”0.00. 0. ... . . . .. . . ......00... .10. .30.. :00... 0.0"“ . .. . . .... . . .0 .00. 0 . .. . .. . .../0. . .0 .. ... 00.0000 . 0.00 .000. 00 ..0. 000... .. 000;..0 . . 0120...... 0.0. 0.0 ... . .. 0.0....0. .0... .00.~.*...0 ...»: Tuna}; .0 .02...) .00“ . 0...”: 105.01.001.00 .0. ...-00' l 0.. . . . ... . . ‘00.. .10.‘ . .00 0 . , .00‘0. 0.. .000 30000.0. 1 0 0-. 000...}. 0... - w I. 0 0. 00 0, .0. .I. . 00..‘.‘.00..0§0‘§ 3‘00... .000.- 00.. .. .0 0 1 ...:0000 . . . 0 I! V. .... . .0... .0 0. . . . - . . 000 .0400... ”0” ...... 0. 0. o‘ .0 00000000 . .0 ....00. .000. 00. .0 . 0‘ 0.00 . . . . .000- 00.0 I 00 0, l D o. 0 .001. 09.000..- :0. ., . m. 0.) 0 7 h . 0.3.0 . . 00 .0000. 0.0.0:. .000 . 0 . .040 0 . 00 0 ...000...! . V 0 . . . . 0... I. 0 000 0.0.0.. 4 0 . 000. . 0‘.- .. ..00 I 070.. 0. 0. 0 $000. . 4 00.00. 000'. :o 0 00.00. 0 0 . . . .6 0.0.0. . . . . _ 00 0.0.. . . 0 00 l... ....00 .0 .. 0.4. .0...“- .0 . .‘ "000, 0-.“010HQ00 A)“ .u" 0. ...“..ULH. 0 . . W - I 0 ’1!)»’0’”K v00 ‘£"I’ I ... . . . . . ., . . . . ... nu «V- 4? . . .0 0 0.0L. . 0 . .. . . .. ..00 0.”. 0 . 0.0.0 0.0” H." . vii-00. .0“. 0.! 0 — 0 0.00.... 0 0.... . . . . . 0.000.. 3,»..0 . .- .... $000000 . D0. 0.0..~..0. . . . . . . ‘00. .00.0.l.0..’0‘" O00‘7000‘..)F.00.£J.$0L0‘h0fi§0 0,0.“- .2301‘4 . o . 0 00 . . T .. . .... .00....1..._ 0.00 cl. . . 07” 00. .. . 0 0. f . 0o 000:0. 0 a ‘0 . 0.. 0 0 .03.. 12000.0 0 . . .. .. . .. . .0003! '30. 0000000 £3.50.) 0.100.100.0007, 0 .-;‘020" 3.0!. . 42.....0 .. .. . .... .....x. 0 .. «nuns-1h...” . 1.0.... ......Jhn. . . : .0 0.. . . . a... t... . .0. ..0.........010$s40.0.0.&. -10. 000... 00:2,"..931 wijnufirtfig 000. . u o . . o . 01.... 0 ... ‘. 0 0 .0. 0. 50V: 0.. 0‘.‘ ’00.... 00.. a : .i000 .10., . . ... 00.0 0.00,... 3.00.71 0. . l ... . 1‘0 . 000003.00.’000..u0o..0.0 ..0 ..000§1§..0’.«"0..Z0.:”.3..ériégigzodz. 0.0000 ....0 .0. 00.. . V. . 0 . . 0 . . , _ ....0.0 30.00. ... ...0: . . 00.0 . 000.00.....)0.0. 40... 0.0000 .23. .0... ..0 0.0.4.9....03010‘0 . 0 0.0 .0 .0...00..)00..0I0..0‘.t)h"0g Mo“: 0 . 0 0 0. .0}, 00 00. ...le {00.0.}. 00 0000.0 . . . . 0.7.0 (‘37-. 0000 0‘0“. 00 I” '0.‘§00II.0 ..‘00000'00‘07. “h",liilf 0’...‘ v. 0.. . 0 0 - .0 I . .00. 0.0. ..00 0.00 ..0.. .. )0.- .. . , . . 40.10.. 0. 3.40.. 0‘00:- 000 .0. 3.0.0 .0 00.00 ...-.0 000.... 0 $§jt0¢l¢i a. $;.. 9.0 0.0 00. ...... 4’10. .0 000 .0 0.0 s. . 0“. . ‘00.; $4.00). I~.f..‘00..z.r. 0..Io J0).000.010’0o\0000’00‘:00§3. ,l.0l . H.033. -. 0.0 0 .3. . -. .. -1 0 ....0 ... Ml y n. . (if. .00....) .0. 0. 0:0. 0 30......90: 0.00.0250. ....0 0.0.130;.0013.0..:0.H0 0.0.9.0003?!“ . ..0 . ... .10.... . .0 a. . .00 0.0 30.00.. {00. 0 u .000 (.0 00000.0..(‘05'Il‘r06‘3 .000. 000.05.... 00 ....rlzg-v 00!. 200.0530... . 0 .1... 0 . . . . . . . . . . 0... ......9 00.00. ..0 ... ......0 ... ......0 .......1 . . . 0 ,0 300.000.. .0... 0...: ....,m0:0.1000..0.0)000.3i gaze-31.003.09.900. _ _ . 0.0 .1020 . . 0.. . . 0.. 0. . . , . . .0...» .. 00 3.00 In. 0 . . 000.000 00 3‘ 000 00023000. . .. ”00- 0 .01.! 000000000.- .00.‘00.0.§007300l. 0.6;..0000‘ Q... I. . . .0 .0 00......0 .. . ... .0 00.0.0000 5000. ... . 0.0.0.00 . . . . . . . . . 30... ..0.Yn’00.0300,’-. ......0 0.1.0gtaigt‘ 1.0.00... 0... 0.000. . 0.00.3300 .. . 0.... 0. ..0 . 000 . .0. ..0. .0: . . . I . . . . 0'3‘000: 3 ’03.0t0"0.‘.0 .0'."0I,0¢,l...00‘"‘~00 00.. i .. 0 .0. .0 01. .. -. .... ...I 0.- 0.. 0.0 0..- .g. .0 (00...... . 0.00.0 0.00... 0.0.0.0000! «4.00.003’R0o. 1.00:0!" . . ..V. i . .....00. . v00 .0 4 . .. .f.I. . ... .00.. «n0 .... .. 0r .1...) 003.0. «'00.. n 0 70.00.00.000" 3100-1100300, . . . ...... . ... . . . . t - .00.... 00.0 . 0 ... 0 0 0.1.0.1. . .00 00 . ‘0 .30. 1M0’r0éfn000. £843.00 73:00.00 0000.00 0, 00. ..0.... 0. . ..0 4 0,00. .i‘. ..0... 0 ....0... 0.0 0 ... . .- ... .0. 00 4000.0.. .000.1!.00.?. .‘.6§.3"§3d .. .... 0.0 ..h ,. . . . . . . .. 000...: . ... 50.. . . . 0.0. s“... .. _ 5.0.0.09 .0l . 000000.030..¢0v0\00 00.200000 0 00.! . . . 00 . .... . .. 0| ... 0.; . .0. 00 .0 ..0. ............0. .. . . .tl.u€l. ...-00.0.. 2-2....) I000L..00.0.I.0)0..0100 zi.i§§ . . 90.. a m I,“ 00 “.0... 5.3.0.. u 00“ ... ... . "......h’." ...00....nr.0 3000 u...» 0.0 .... P) . . . . . . 0t 3.. .S 0.000 ,10 .0 .1000" .wyflfifinalfc 00 h‘. Mlyir‘ez.”)g,:l . . . . . 0 . 0 i 0... n .00 . 00.10 0 ... N -...0 .0. . . .. . . . I .. ‘0‘ .010 I: I I .- l 0 .00.. 0 . - a” ..n . .00... 0 .0 0.0””..E0... ..0 0 0000.000 0 .00“. . 0.v0. .00 ‘01.! .0 .20.. 0 . .00. 0 . 0 . . “4000““. 0 .0 . ‘ 0V .0. 00.00.0000... 0'00, .0. .0000 N. .«0, “do-00.00000 .. 00.00.00,:010 of:.0‘30-.)0..0§-0000000‘»I0000v‘0‘30‘03‘ cai.‘..i ... . . . ... 0... .0 -0 .... . . . . . . . . £000.. ......00 . ... ...-......0... 0.0.00... . 2.7.3.8.. 0.0.0.. .4040! 0!..0 Jog.300.000000..l J00 00.00... l..0.0“«.l10....00..0\.0). .0‘00030000‘3.’ 00 1.0 000 .000... 0. 0 . . .. . . .. .. . . . . . 0.0000. 0000'! . 00’:- ‘0 . :33..ng 0. . 0 .00) 000 .000.1.00.0-0000$0000000( 300.004. .‘0’7..00000.0I0'.l.00 ..100‘0‘.“ 0...".s‘l . . . .... 000... . ... .. . .. . .... . . ...)... . (0:08.393: ......" 011.00..) ...-.9000000‘005103030980.‘0I§.I 050000.]; L... . n . ... . . . . . .. 0'. 2.0 . 00.... 0000...... I 10.00.00. .0000000..00..0.0053 00010... 000.00.03.00: 0'000000 ...-0.000000110090’00; I... . .0 .. . . .... .u . .. . . . . . . .. . .. . . . . . 3.00.... 00 000’ .0000. 0.00.13.30.00 4.000}... 3.1.0.00. . ‘00-'13 0.0.00.5.000000‘tdv 0,0400'00590: 0.. . .0, .0 . . _ 00 ... .0 00 . . . . . . . . . .4 ..0. (.00031 0.1.2000}: {01.00.2001} . 073......- ..000. .. 3.0.0.4.L0400302001.‘ 0.00.) 0.2000 00000000000000 . ...... 0.0 0 0.). . u... . 0.0.... .. . .. . . . .. . . . . . . .. . . .. $020...33.0.9320....!.0l...000..l$0.«0000.1090.30I.\Iu00‘0 I, J0.‘.00.0.\0.Q.000\.‘0.0.0010000.00§ . 3 . 0.. .. 0 .0.. 00.80.00. 00.n000 ...0 . 0 . . . . . p 04.0.. 00... ..0. . 000 000.... ...-.0. 0.0000... «\- 0.‘ 0900... .0100“ 0'000..00.‘l"0 0030000000003’0HJJ0N00W'0P (.0000 a; . . . ... .. .. .. .- . .7 0.0 .00...... .\ 0.. 3.0100 00.0000 ti .9. 0.0.0.0...". 00301000009302.0003130 0:1 20.. 000. 02 . . 2020.00 .... “0.00.03 .00 .0 .o u . .. ...... p. .040... . . . ... 10.0.. 0. .3001. 3.0.. 0. . 00.. 30.00.05.130... ....4 ...uuusv 90025.0 . . 1... ...!‘0102102500. 000. 0i§of§.§.3'§§." . . .. .. 0.0.0. . . V. . 0 .0 . .0:0_.J.0 .300? . 0.0.0.0.. 0. 0. a I.) 000...? ..0.......9..0.0..\. .. . 000.0: . . . ..0’000 ..00......00..1.00000’.:0 003...’.$1,...000)00 II.) 0.“ . .0 I ... . . J . s. ... .. . . 4.0 . 0 00 .0. :1. ..t 000. I 000 .00. .\ 0.. 0000. ... «0.0.0.0 . 000.....00050. 0.. 3 s....-‘.0§. §}00‘0§l‘0..’{.10 0.0" 0. .000 . .. . .. fl. . . . . . .. ..V); ... . . 000. x 3 . ... ... ...... 0... 0.. 0: v.0) . 4 .0032... 3.0.0. ..I 050.70.. 3.1093010. ... 10 30.00.000.080. 0, . . ... .1. r0... . . .0 .. 0... .. . 2 0.. 0. 0... .v: ... .0. 0 . . . .. 01...! ... I 0... .0... .. ..r . 0...; .0. 0000.02.03} 0 .030. . 000. . 101000....0: ...100JR0‘..JA.70.3 0 0 . .. 00 .... ..l . 0 . .0.“ 0. 0| . .. . .00. I000! 0.10.0 .. l .5 .0 . 1'00. ..0 0 00. .0 0.05.0200. 0. 0.....‘0 {...-... ..., 23$. 50000007000035...00.0-.!.0..00-I.’00. 1.00-000000010. 10000. ’07.!4100 '0 ‘3. . . .....f» .. 0 00. 0.. . u . .0 . . ‘0 u. ’00. :0. . . ... 0... ..0. 3.0 0..0f.0 . 0.-.. .. 0 ..‘ 00. .... .0. . 0..0£0..S.....)! 0 . . 0.0....000....0)l.0 .0. ..00...:000.000.000 9000,0303)"; .03 ‘00., I. .0 . 0 . ..u..:.0. 0.0. . . o. . .0 .4. . .‘0 .0... . . . 4. . . . . .0). 1 ..0.0.0~l0 . . .. . .. .. 1 L040 .102... . 00000.0. .0 . . . . 0000. . 00‘: 000. 0.0 0. A0?,Jaa 0000-..000.1 ...0000. .. 0.I\.g...0.02000 0.0"!!0\A00‘v\..0000 t . 02:. . , ....0 ...: .. ... ..r. .~ . n... .. . . . .. . . ..a .00. n. .. 0.. .0 0 ...0. ‘. . ...; 0...... 0.. . .0000 0.. . .10. 0.. . .0 .00.. 0. 0.!0010‘00000. 0 >5 0 0.. 0.03.0000. 00-0, .100" . .0 . . a. 0.u.. .. . . . 0 .. : . .3 .. .. .I 1...... 0. .s .1 . .0. . ... .. 2.9.0 ... ...r . [1:0 . . 0:00 0. . .00 . $320.30... .0... 0000.. . . .02..)0000. 013.3).(700..§ . . .... .Pa. 0.. L.... ....0... .0 00 -... . . I .. .. ...-0.. ~ 0.0 . .0 ... .... ..0 1..) . . . . : a n 0 ..13000031.) 30.00... .I.........0. 000.01.... 0010.20.01... 2020.03.90.34 7. 0.0800S 0.33.000 . .. ... . .. .. . .... . . . ._ . . .0. . . ... ...... 0 .....000 S .I..!....... 35.0... I . .01.. .fl: .0532... .3 00 ,(0: .0 .500. L 3.3.0.! 0.0....0...0..\00.0.2‘ 1.0 00.00.00.010} .0 . . . 0 ..44 ... . . 00 . . . . t 0.. r0... . ....l... 0 ...... 0. . . .00.... 0...! . 3 .0 0. .1- . V .l .0. . 5.00300 .0»). 7.9.0.00 at... fi’oo..‘..0 £05..0.£I00.0709030.0£I‘ 300000 .. . "0.0.00 . 0. ' 0 . ...0... u. .. 0 . c c. . .000 000. 0 0 0..Q00..\ . 0. 000. o. 0 Clint-0'00 10.... 0.0... 00 . 0000.00... . 30 ,‘00g.’.0. 00.0.00 ’0‘ ‘0. 0 3?.0000.’p’rt 000.0“ 0 ’0‘.“ 0. 0 . \0‘0 . .0 . .0 0.0. C .0. ‘ . 0.0....- 0. .. ..‘0... . .. .0 00 u . 00. . 0.000 00 0 000.0 . 0 t .0 0 0 O.’ L00-VQI0 .’. 0.. 00'0.’I {0000030.- 02 00] .00‘ 000. . . . 0-50 Q 0“; I 0 ’00 . . .1 . 0 .0. . . 1 l4 . 0 ...: . ......4 0.. .05. 0. 0. . .000; d«...... ....0..000 . 0...... {.00. 00.0.0.0... .. . ... 0 00.. 00... ... 0.0 .. 0.31—0.00... .00 ...00 40. ......00 ...p 0000..’. ’0l0zi’0fdaltpl‘yzl0.i$§3\uz!‘ "a . . 0 . .0 .. . .. ... .. . ...0 ..0. . 0 o . . .1 . T .0... .00 . .. ., . .....A 0 0 ..0 .. .0 0 .0. .00.... .... .- .... 0 .0: . 0. 0. ... C ..000..100(0...00...\....0 0:0. 0.0:...0... 00.0 .00... ‘20 .g0gd0 .00.. .. . . .. ... 0.. 0 .... .0” ..p : . ..0. ., o. 0.0 . .. ... . . .. . ...: ..0. 0. . . . . v . .0. 000 .110 .0323. 2.360.000.2031 .. 0 0 00.... ‘33 .3. J30}! . 2.0000 0000.900 1;..‘0 0“}, C‘ . 0 ..0. .. 0 ”0000.0 ... .003“. 0'- v I 0 . .0 . 0 )0 0 0.. 0 ll ‘._0\ . .40‘ . .. v 0 I... ...-30.0.. 0 g .0 .\ all’au‘ 0100100.}; 0. . ...0 ..0.1 . 0‘ q 0 .\00'0...,. . 0 30.. . .0 . . 00 0.....00o.000|.40..0 . ..l 0. .... :0. 0. I .0 .. f3 200 ... v0 0. 0.. . 0v 0 004.0030, ..00. . 0.0;,flezgluu0’0n30zl0éll‘ ..00... .0... 0.00 .. .. . .. 0000 0.2.”. u 6. 0 .. . . 0 0 400.. ‘0 0k‘0 \‘0 .u0.-0 .0 1000‘ 000009000 . ‘00 0000}..0.§ ’P'_.ll‘i. 00,. I0! 0.. .0 ... . ....0 . .. . N J . n. . w a. . 4 . - .X 0 .... ..... . ... . . . u. . . . v .0 1.02.. 0.0.... 0.. ... . . .. .I:0..l:. L .01.... 0.0.. 0.5.70.0. 00010010005003} 4 . 0 . . . ... 0 0. .0. . . . 0.0. . . 00.... 00 . ...... .. . . 0 ... 10.00. . 1 . . u . - .... 70.50., [.0 ..0. 0000!0.0.30.0§.00.0. 0.1‘0'00 . 20:3,ng . 0 .. . . 1. x . .. ... . t . . ... .. . ..0 .. .. .. .0. . 0- .. .. 20.0.3.0... 0.3.0:... 10... a 001 0 0 .. .10.0.:0!l.00.0.0$00.000§3) a..." . . ~0 ..0 . . . ..u 0.. . 0.0.0. . .... 0 0.100 . ... .. .0 .... ..0. .. 0 r ..0 ...‘...0.\ .0 0200,: . ...-... .5! 0-! 0000.005\« is... ..0 10‘. 73.000000. 05000 0.080 .., .0 "..V. . .l . .. .. . ..V .. .00. . . .0 . n .. k. . ,...1. -... . i .- L. J .0. .....v $00000. 01.. . v0. 0 . .... 0.00 00.0...0I.” 00.00 $00.0 001.000.... Ti . .020 .. .... .. . .. .a 0 00.. . . ... 0 .0 . 00 0.. .0 a......v0¢00.0 . 0 .u.l00 0. 0]....0.... 005.000... .0. 00.0.0000- . . ...“.000n0000 ...00013‘0‘12 0. . 0 .. .0 0 . .. . .0. a . r0. . ..00 .. .. 0.7-1....0 . 00. 1 . . 0. 0 . .. I 0....... ...).010 ......0). 0 00 . 0 i. .0’.‘ 0...... 00 0.0000 ..00..00.0.1f..00...-00‘\‘004§l:y00.1 .E. "0’00 .00 0. . .. ... o o f l .0 . . ...... ..- ...I . ..... . . , .... .00....0.. . . ... ._ . ... ... .. f 00 0... fl .0. .... .0 0 00...... . ... 0.0. 3,0.35... .. I «30. .00.”!(0 00900000003000.0800. . : 0 . ... . 0 . ... uh.- .a. 0 .m . . . h.0. . .. I... 0 . 0.0 ..00 00. 0 . 04000. 0 0'... 3‘. .0 .-..100 )0. .00. .02.."00'000‘."000. 0002”“ 0.. Viiuflfl1 0 1‘0 0 . 0%.. O 0.0 . 0 . 0 . 0.. 0.. a 000 .. . .. s 0. v0 .0..0‘J0..¢..0.. 00 J 0 .00 4 ......r5. 0:!“ s. 0.. . . . .. ‘20..“0 .ll.‘.\.0‘;‘ . _ I. 0 0. 0-' .o .5 0 A 10 ..W .0 . 0 n. .0... .0 . .. .. . . 000.0. 0.. 00 . .. ...-000 0.00.. J -..-0. . 01.0.. . 02.)...f00: 00 .0.500..0..Q. . 00:01.00. ’.Y}.‘sr.ll §. . 00 . .2. . .0 o . 0 . 0 . o :0. .o ... ... 0.. .. . . . .. . . . p. .01.. ,. ....0 . ... 0. u. .0 001.00. . 0.00. 00. 00...... ...."00’0 io’iu‘liiig . .0 0 . . . . . .0 u. .. .0 . . .-.. . .. .... .0 .3...Jv. 2:000 00'..00 0.0.0004...J). 000i. .. . .. £0.i\‘..4 AI”. . 0 0 . .0 C .0 n 0 _ ..0 0a. . h... . . .. . . ... . 00000 .0 00000. . . _ . .0 0.0.0... 0.0.00 o I‘d.0.\...00 .5....0\0). 0.00.0. 0.. 00. 3‘00... . 0 . g0 . ' 1 0 0.0 0 0 .0. 0 I 0. 0.. . . . . . I :01 .. . . . . . . .0. . 0:0. . 0) £30. ’0‘ . 9,)§00..0.%00I 0“ v0.0.000000l . . :5 0 0 0.. .. .I 70 . .0 I ... - . . .. ..0 .. .. .. . . . . . .. ...0.0 .00. .. . . . 0... ......u... 00.000. .0. . .00. ..0‘..llax0 ... ‘0 D0 ..0.) 0031., 0 '001100'.).I.0.\‘ ’0‘}. .{00 0‘-.‘ 0010“"; .0 . 0 o .0 4 0 . . -13.... .. . . . . . . . . .. . ... .2 . . . . . . 3.5.0.... 0.’., .0: c ....I 0.00 00.000.00.000 .0 00.030903100400300000’3’3 \l 0“ . 0 .. . . .. ... . .0. . . .H0. . . . . . . 0 0. 0 . .0..... 0 0.. 030.14 6 ...-..0 . ... 0 . 0:510. ‘00 ’nllvz... ,I‘... 000 0. 10‘0i‘04033 . v . I. 0.. .0 . .000 . - . . . .00. 0 04.0000. .0 o . 0 .0 000. 5040‘ 0 0 0. 00. I0 0% v. .00. . 1. .. .0 0! ..I'I...‘ .000. 00.040 0 00.00. a ..0 0.}.i.’ . 0 .. . 0 0 . ..0. 0 . .. . . . .. . . .1. .. «MHZ-s. 0:0 .00 . 0 .0 ..00 0. . ...... l u .. .0....000.000'0.J‘0 .0010. 0.’\\.0.0.1%£4.00 ’00 . - 0 ...0. ‘0 . .. . p .. . l 0 ‘ .0 00 O . \l. 0.. 0. 0.00 000. . . .. .1. .¢§.3 0. .3 o . .0 00 020 0 3 00“ 0 0 .0.v "rl' ‘.".51 30.1" ,0‘0130 0} . .‘ 0 . 0 I . .. .00. . .. 00, 00 (I 10.0). x 0-. .0..." :\ ... .0 0.0 ..0 . 0 0 A -.\g000- 0*.0Pi’.’é0\fl . 00030000.- .. .. c . : n .0. .. . .. . . .0. 0.00.0. . . .. . ..l . 0 . . 0 . . . \....J.0.... 0.0 w ..00 0 .00 00.00.1302! .00’ib. .../0.0.00.0, ... ..V . . .0 0 o . .. . u .0 . :0... a. 010. . . . \0 I: . 0. . ...04. . 30.0.10 . .... . 0 .014 0 u. 00. .Kk‘0 110010040 0.010.020.3’030 15’ . .. . . .. l ... .. .. 0o... 00. p... : 0 . ... ... . 4 . . I- .0. .. u 0.0... 0.0 .. 0.0...0. 03.00-100.331? 50.20. .0-000001'1’0' .0 . “on . u _ 7’0 . _ 00 . 0 .. . 0 0 00.0 . . . ¢ 0 7 . 0 . 0 I )Q 0 .100: 00 . 70900.. 1‘!- ..00. . . 000‘ 0 ‘00..."f 0.0/91: 000 5‘0 20‘- ‘ 0 . .0. .. J0 .. . z . 0. .. 2.020 ... . ... . -..! .... . t. . .00... . 03 :1..0 .. .\.‘ I: .... .0..f000.!l?03 03000100.!0xrls 0,0. 0‘t .200" 0 I 0 . . r. .0 A .0 . .0 .. 0.0. ... I. . "0.0 q l. 0.0. :0 ..000...0f 0 1010. 00. 0 . 0.0. I 0 . ‘f‘. 0.00 . 00RQ00K‘... 0001-01. .000000- z‘ 0 . 0 0 0 0. 0. 0... 0 00.00 . . y ..0. 00 .0000 0v 0. _ 0.001.000..0l... 0 ‘04..“ 0 .l‘. 'Izl.ll ’("0.’0fl.\‘i‘ . . .... . . . 0 . ... . .. . .... 0 V . 0. .... .. ...0 .... ..0. 3.002... ... .. 000 0......‘0I00100i ....0 010.0 .. 0'0500 0”}? 0f . .0. 0 . 0 10 x c I 0. r1 0 I .... o. .0 0 ... ..00 0.. ..0L0\0.' 20.700.2‘0.‘ .000".n0 ‘200. 62‘; ... ..0. . . . y. . . . . 0 . 0 .. . . . ... 0 ... t .. .70.... It. . 0.... 0:1 0... .4 00 01.0..~‘-\I0 0000.00.00." 0‘ “.0003“. 40‘!" 0 . A00 00. 0 o O 0.. 0 .0 0 1.0.0 ...... ..00 . ... 0 0... .. .... .0.0 . I .. .. . 1 0 95.)..., ’00.0.00\0... l‘t‘ .00 1: 0 02"... ..0 ... - ... 0 . I... . 0.100 . ...: 0. . \l.\. .000: z... .0 .0 .. £50‘il‘0801‘0 '00»).0O000l'0 .. 0 0 000 0 0 . 0. . l 00 . 0 0 0 .0 10.00.. 0. F. 0.‘.’0¢ {000000991 00.]. .0100 ‘02.??0I" a . .... . . - . . 0 .4 .. ..0 0... .... .0 .0 0.: 0\\ 0‘.0QLI.0 02‘00'0.’0"00(\000090-. . ~ .4) 0 0 0 .. . .. I . ,0 0 0000 0 0 I0 0 I- "0Cl00 \ 0 7 £000.00 L. 3 I '0 0 0000 . .. 0 00 0 .0 u . I . l. I 000 00 ‘/(uu.006,.0. -.‘000 ‘00 0 i , . t n . . . , . . .f Ouolb Inf..- 4.00.- 00 c .05000 ".0 .... 0. . 0.. .0 . . . . 4.. ... .00.... . . ..0 0.0. . .0. .. - .0 , . . . . 0. ,.. . .00 6 . 0 I . .. . . , . . . . 0.4 . . I ... 000.0 .0w.wu(..0..,0 .0 00 ‘0 «w 0 .0. ..00 00. .33.)! 3:00 0 0 . . .....0“ 0. I. 0. . 00.0. 0.0- 0010 ...h.“ 00 ”Writinn‘fipt 00”... a... J. .—0‘h.3 “l ...‘fiu0- Id .34 ‘3 0 “at .4? . .. . . .. . .. . . .,..0.. 1‘ ..., -. 0 . ‘3‘.” 0.0. o. 0 ... .. .. ... 0 0 ...I0 \0. 00. o . 00.0... 3000...? 3‘00 ”:4 3 P-S ‘. ‘1. J > . . y a“ ’0 0 0 .00.? ..00 00 10 .0 . . ,....... L..V1.0..00040.0 ..|_ .000. 00...; .0 0. “an“ ..m . 0 o .00 ....3 :0 09.01.. F000... «.0... 0 0....0 .1. \000 . .0»... 0.0 0~§ . 4‘ 0 ‘Vc.fl.um‘$ 0‘06!“ 03.00.”..00davi0 u. \ .... "0...... . . “0...... ..30 . 00“.. ~“i.“0.0-o fun. a. ’0? 0 0'0 . . . . .. . . . .. .. H {1.00mn.01100r.v.0.0~ .. . . . .0... . ...: 0.0 l .. ... - 20.1.... 2.00. .- .... . . . an. . . . 1 . . ._ 3 . . . 1 V .00. ... 0. l0 0. 1 2.0. . 00.... . ... (000.. 0.0 0 . _ , . . . . a . . . . .0 . . his .a . .0: 0a... 0. 0.... pm. .—-‘.0J.. . 3.97:4”; 0.. 0.. ...-004.0. 00.0.. ......1: ...! . .... o. . .80 0 ... .0. . . 0.. .. 000. _ .... 0.....0. , . . . . . . 0...... . 0 0.0210.- ‘. .0 '0... . . f I[ ' I- r LN ‘Q This is to certify that the dissertation entitled MOLECULAR AND FUNCTIONAL ANALYSIS OF A NOVEL CATION CHANNEL IN DROSOPH/LA MELANOGASTER presented by TIANXIANG ZHANG has been accepted towards fulfillment of the requirements for the Ph.D. degree in Department of Microbiology and Molecular Genetics a: :1. , Major Professor’s Signature 8/257 20 /0 Date MSU is an Affirmative Action/Equal Opportunity Employer LIBRARY Michigan State University '——— ..—--n-.-.-—»-.-.—.--.— .....-....—.-.-.-.-.-.-._._.-.-.-._.-._._.-.-.-._t-.-._.-.-.—.-.-.-.-.-.-.- 4.4—...__,—.—p———-—-—— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProi/Acc&Pres/ClRC/DateDue.rndd MOLECULAR AND FUNCTIONAL ANALYSIS OF A NOVEL CATION CHANNEL IN DROSOPHILA MELANOGASTER By Tianxiang Zhang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Microbiology and Molecular Genetics 2010 ABSTRACT MOLECULAR AND FUNCTIONAL ANALYSIS OF A NOVEL CATION CHANNEL IN DROSOPHILA MELANOGASTER By Tianxiang Zhang Voltage—gated ion channels (VGICs) play essential roles in regulating neuronal activities, such as initiation and propagation of action potentials and synaptic transmission. Different families of VGICs play different roles in modulating these activities and in turn differentially contribute to the neuronal excitability and function. The Drosophila Sodium Channel 1 (DSC1) gene was identified almost two decades ago. Based on the deduced amino acid sequence, DSC1 was predicted to encode the first voltage-gated sodium channel in insects. An in situ RNA hybridization study of DSC1 transcripts showed that DSC1 is expressed mainly in the nervous system of pupae and adult flies. An immunohistochemical study indicated that the DSC1 protein is present in both central and peripheral nervous systems of adult flies. Contrary to the prediction of a sodium channel, electrophysiological characterization in Xenopus oocytes revealed that a DSC1 orthologue in cockroach, BSC1, encodes a novel voltage-gated cation channel that is more permeable to Ca2+ ions than to Na+. It is not known whether the DSC1 channel shares the novel ion selectivity of the 8301 channel, nor is the specific role of the DSC1 channel in the insect nervous system clear. My dissertation research focuses on the molecular and functional characterization of the DSC1 gene. i found that the DSC1 transcript undergoes alternative splicing and potential RNA editing. Several alternative exons contain a premature stop codon that results in truncated proteins. Similar to the 8801 channel, the DSC1 channel is also a voltage-gated cation channel which is permeable to Na+, Ca2+, and Ba2+ ions. Analyses of two DSC1 mutant lines, generated by precise gene knockout via homologous recombination, showed an important role of the DSC1 channel in modulating insect behavior in response to environmental stresses. Specifically, the locomotor activities of the DSC1 knockout flies exhibit hypersensitivity to heat shock and starvation. These phenotypes are associated with altered electrophysiological properties of the giant fiber system (GFS). Pharrnacologically, DSC1 knockout flies also show enhanced susceptibility to a class of sodium channel activating insecticides, pyrethroids, but not to a sodium channel blocker insecticide N-decarbomethoxyllated JW062 (DCJW), or a v-aminobutyric acid (GABA)-gated chloride channel inhibitor (fipronil). The enhanced susceptibility of DSC1 knockout flies is also associated with altered electrophysiological properties of the GFS. Taken together, these results provide evidence for an important role of the 0801 channel in modulating insect neuronal excitability under environmental stresses. ACKNOWLEDGMENTS in my five years of study at Michigan State University, I was supported and assisted by a lot of wann-hearted people. Without their kind help and guidance, I might have ended in “nowhere” along my scientific journey. There are no words sufficient to express my gratitude to them. First of all, I would like to thank my mentor, Dr. Ke Dong, who supported me most during my research. I was attracted to Ke’s research when I attended a seminar during the first month after I came to MSU. Then I talked with Ke and set up my first rotation in her lab. As I worked and communicated with Ke, l was deeply impressed by Ke’s profound knowledge and optimistic, dauntless spirit. I was encouraged by others in her lab as well. So, after I finished my rotations, I decided to join Ke’s team. Ke has given me tremendous guidance and training and helped me overcome one obstacle after another in my project. She also polished the English of my manuscripts and taught me plenty of skills in scientific writing and presentation, which will benefit my whole career. With this experience, I am now more open-minded and self-confident, which are critical to be a scientist. Besides Ke, l have four other committee members who have provided indispensable encouragement and support for my study. Dr. Suzanne Thiem gave a lot of suggestions on my molecular biology work. In addition, she also kindly helped me understand American culture. Dr. David Arnosti and Dr. Donna Koslowsky provide many suggestions on the molecular biology and genetics portions of my research. The classes they instructed opened up broad and deep iv views of molecular biology, biochemistry and genetics to me. Dr. William Atchison helped me a lot on my understanding of basic neurobiology. Here, I would like to thank them and I believe the passion and perseverance of all my committee members in scientific research will keep influencing me for the rest of my life. It is my fortune to work with so many talented and wamr-hearted people in our lab. Lingxin Wang, Mike Chumbley, and Becky Aslakson are the ones I have been working closely with in obtaining exciting experimental results and maintaining all the D. melanogaster stocks. Dr. Zhiqi Liu cloned the DSC1 cDNA and made the donor construct for the 0801 knockout experiments. Dr. Ningguan Luo generated the DSC1 knockout founder lines. Their work laid the foundation for my project. Dr. Weizhong Song, Dr. Yuzhe Du, and Dr. Zhaonong Hu taught me how to do electrophysiological recording and provided heart-warming assistance during my setting up the recording apparatus. Yoshiko Nomura helped me with my molecular cloning and Rachel Olson helped me with my English. Dr. Sebastien Hayoz, Dr. Rong Gao, and Dr. Eugenio Oliveira gave me many suggestions during my writing manuscripts and thesis. I also would like to thank our collaborators, Dr. Chun-Fang Wu and his lab members, especially Zhe Wang, at the University of Iowa. They taught me basic techniques of recording and analyses of the GF system. Besides these people, I also thank the Department of Microbiology and Molecular Genetics and especially Dr. Richard Schwartz and Dr. Robert Hausinger for providing me with the opportunity to study abroad and assisting me along the way. The last but not the least, I would like to say thanks to my parents, my sister and brother, and my wife, Fengrui Zhang. They care about me even more than they care about themselves. They are the source of my energy, my heart and soul. Without them, I would not have gone this far. vi TABLE OF CONTENTS LIST OF TABLES .................................................................................. x LIST OF FIGURES ................................................................................ xii LIST OF ABBREVIATIONS ..................................................................... xiv CHAPTER 1 GENERAL INTRODUCTION ................................................................................ 1 1.1 Voltage-gated sodium channels ...................................................................... 3 1.1.1 Structure and gating properties of voltage-gated sodium channels .......... 3 1.1.2 The family of mammalian sodium channel a subunit encoding genes ...... 8 1.1.3 The sodium channel 8 subunit. ............................................................... 10 1.1.4 The Drosophila voltage-gated sodium channel, Para. ............................ 11 1.2 The Drosophila Sodium Channel 1 (DSC1) .................................................. 13 1.2.1 Molecular biology of the DSC1 ............................................................... 13 1.2.2 Tissue distribution studies of DSC1 transcripts and protein .................... 14 1.2.3 Functional studies of DSC1 channels ..................................................... 16 1.3 Pyrethroid insecticides act on the voltage-gated sodium channel ................. 19 1.3.1 Pyrethrum and pyrethroids ...................................................................... 19 1.3.2 Classification based on chemical structure and symptoms ..................... 20 1.3.3 Mode of action ........................................................................................ 20 1.4 The giant fiber (GF) system .......................................................................... 24 1.4.1 The cellular composition and function of the GF system ......................... 24 1.4.2 The synaptic connectivity among identified neurons of the GF system...25 1.4.3 The giant fiber system recording ............................................................. 25 CHAPTER 2 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF TWENTY DSC1 cDNA CLONES ................................................................................................... 37 2.1 Abstract ......................................................................................................... 38 2.2 Introduction ................................................................................................... 38 2.3 Materials and methods .................................................................................. 41 2.3.1 Amplification and cloning of the coding region of the DSC1 gene by RT-PCR ........................................................................................................... 41 2.3.2 Sequencing of Twenty DSC1 cDNA clones ............................................. 43 2.3.3 Expression of DSC1 channels in Xenopus Oocytes ............................... 43 2.3.4 Electrophysiological recording and data analysis ................................... 44 2.3.5 TTX sensitivity assay .............................................................................. 45 2.4 Results .......................................................................................................... 45 2.4.1 Sequence comparison of 20 DSC1 full length cDNA clones ................... 45 2.4.2 Functional analysis of DSC1 channels in Xenopus oocytes ................... 48 2.4.3 DSC1 channels are insensitive to TTX ................................................... 49 2.5 Discussion ..................................................................................................... 50 vii CHAPTER 3 BEHAVIORAL AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF DSC1 KNOCKOUT MUTANTS ........................................................................... 66 3.1 Abstract ......................................................................................................... 67 3.2 Introduction ................................................................................................... 68 3.3 Materials and Methods .................................................................................. 69 3.3.1 Generation of DSC1 knockout lines ........................................................ 69 3.3.2 Climbing assay ....................................................................................... 71 3.3.3 Heat shock assay ................................................................................... 72 3.3.4 Recovery assay ...................................................................................... 72 3.3.5 Starvation assay ..................................................................................... 73 3.3.6 Giant fiber system (GFS) recording ........................................................ 73 3.3.7 Statistics .................................................................................................. 75 3.4 Results .......................................................................................................... 76 3.4.1 DSC1 knockout flies exhibit an abnormal jumping response during heat shock ............................................................................................................... 76 3.4.2 DSC1 knockout flies showed a defect on recovery from heat shock ...... 76 3.4.3 Abnormal jump response and shortened life span of DSC1 knockout flies during starvation .............................................................................................. 77 3.4.4 Reduced long latency refractory period (LLRP) in response to heat shock in DSC1 knockout flies ..................................................................................... 77 3.4.5 The heat-induced reduction of LLRP was rapidly reversed for w’"8 files, but not for DSCfa flies ..................................................................................... 79 3.4.6 The long latency refractory period (LLRP) of DSC1 knockout flies was reduced by starvation ...................................................................................... 80 3.5 Discussion ..................................................................................................... 81 CHAPTER 4 DSC1 KNOCKOUT MUTANTS ARE MORE SUSCEPTIBLE TO PYRETHROID INSECTICIDES ................................................................................................. 106 4.1 Abstract ....................................................................................................... 107 4.2 Introduction ................................................................................................. 108 4.3 Materials and Methods ................................................................................ 110 4.3.1 Fly stocks .............................................................................................. 110 4.3.2 Contact bioassay .................................................................................. 110 4.3.3 Topical bioassay .................................................................................... 111 4.3.4 Giant fiber recording ............................................................................. 112 4.3.5 Statistics ................................................................................................ 112 4.4 Results ........................................................................................................ 113 4.4.1 DSC1 knockout flies are more sensitive to pyrethroids, but not to DCJW and fipronil ..................................................................................................... 113 4.4.2 DSC1 knockout flies are more sensitive to knockdown by deltamethrin113 4.4.3 Pyrethroids destabilized the GFS of DSC1 knockout flies to a greater extent ............................................................................................................. 115 4.5 Discussion ................................................................................................... 117 viii CHAPTER 5 SUMMARY AND CONCLUSIONS .................................................................... 139 5.1 Summary and conclusions ....................................................................... 140 5.2 Future studies .......................................................................................... 141 BIBLIOGRAPHY ............................................................................................... 145 ix LIST OF TABLES Table 1-1 Tissue distribution and gating properties of nine mammalian voltage-gated sodium channel 0 subunits ......................................................... 27 Table 1-2 Size and tissue distribution of four [5 subunits ................................... 29 Table 2-1 DSC1 cDNA sequencing primers ...................................................... 54 Table 3-1 Response latencies (ms) of the short latency pathway of W1118 and 08018 flies measured at different time points of heat shock process ................ 86 Table 3-2 Refractory periods (ms) of the short latency pathway of W1118 and DSC1a flies measured at different time points of heat shock process ................ 86 Table 3-3 Response latencies (ms) of the long latency pathway of w1118 and DSC1a flies measured at different time points of heat shock process ................. 87 Table 3-4 Refractory periods (ms) of the long latency pathway of W1118 and 08018 flies measured at different time points of heat shock process ................. 87 Table 3-5 Response latencies (ms) of the short latency pathway of W1118 and 08018 flies measured at different time points of recovery process .................... 88 Table 3-6 Refractory periods (ms) of the short latency pathway of W1118 and DSC1a flies measured at different time points of recovery process .................... 88 Table 3-7 Response latencies (ms) of the long latency pathway of W1118 and DSC1a flies measured at different time points of recovery process .................... 89 Table 3-8 Refractory periods (ms) of the long latency pathway of W1118 and DSC1a flies measured at different time points of recovery process .................... 89 Table 3-9 Response latencies and refractory periods of W1"8 and DSC1a flies after 72 hours starvation ..................................................................................... 90 Table 4-1 Susceptibility of W1118 and DSC1 knockout flies to perrnethrin (contact bioassay) .......................................................................................................... 121 Table 4-2 Susceptibility ofw1118 and DSC1 knockout flies to bioresmethrin (contact bioassay) ............................................................................................. 121 Table 4-3 Susceptibility of wma and DSC1 knockout flies to deltamethrin (contact bioassay) .......................................................................................................... 122 Table 4-4 Susceptibility of W1118 and DSC1 knockout flies to fenvalerate (contact bioassay) .......................................................................................................... 122 Table 4-5 Susceptibility ofw1118 and osc1a flies to DCJW (contact bioassay) .......................................................................................................................... 123 Table 4-6 Susceptibility of wma and DSC1a flies to fipronil (contact bioassay) .......................................................................................................................... 123 Table 4-7 E050 of wma and DSC1a flies to knockdown phenotype ............... 124 Table 4-8 ED50 of W1118 and DSC1a flies to abdomen elongation phenotype .......................................................................................................................... 124 Table 4-9 Response latencies and refractory periods of male w1118 and 08018 flies recorded before and after exposure to 4ng/fly bioresmethrin for 15 minutes .......................................................................................................................... 125 Table 4-10 Response latencies and refractory periods of male W1118 and DSC1a flies recorded before and after exposure to 0.4ngl‘fly deltamethrin for 15 minutes .......................................................................................................................... 126 xi LIST OF FIGURES IMAGES IN THIS DISSERTATION ARE PRESENTED IN COLOR Figure 1-1 Two-dimensional molecular map of the a-subunit and B-subunit of the voltage-gated sodium channel .......................................................................... 30 Figure 1-2 Distribution of alternative exons on para transcripts ........................ 32 Figure 1-3 Structure of commercial type I and type II pyrethroids ....................... 33 Figure 1-4 The Drosophila giant fiber system ..................................................... 35 Figure 2—1 Molecular characterization of 20 DSC1 full-length cDNA clones ...... 55 Figure 2-2 Alignment of amino acid sequences of DSC1-1.1, B801 and Para proteins ............................................................................................................. 57 Figure 2-3 DSC1 currents recorded from Xenopus oocytes expressing DSC1-1.1 channels ............................................................................................................ 64 Figure 3-1 Generation of DSC1 knockout D. melanogaster lines ...................... 91 Figure 3-2 Schematic illustration of the giant fiber pathway ................................ 92 Figure 3-3 DSC1 knockout files have normal locomotor activity but are more jumpy than w’m flies. ......................................................................................... 93 gigure 3-4 DSC1 knockout flies recover more slowly from heat shock than W111:5 les. .................................................................................................................... Figure 3.5 DSC1 knockout flies jump more and die earlier during starvation ....96 11 Figure 3-6 Giant fiber recording carried out on w 18 and DSC1a flies at different time points during heat shock ............................................................................. 98 Figure 3-7 Giant fiber recording carried out on Wm8 and DSC1a flies at different time points during a recovery process after heat shock .................................... 101 Figure 3-8 Giant fiber recording in wma and DSC1a flies after 72 hours of starvation .......................................................................................................... 104 Figure 4-1 Deltamethrin dose-response relation to knockdown phenotype of W1118 and DSC1a flies .............................................................................................. 127 xii Figure 4-2 Abdomen elongation phenotype of W1118 and DSC1a flies ............. 128 Figure 4-3 Time-response curves of knockdown phenotype of wma and DSC1a flies .................................................................................................................. 1 30 Figure 4-4 The GFSs of W1118 and DSC1a flies respond differently to bioresmethrin .................................................................................................. 1 31 Figure 4-5 The GFSs of w1118 and 03013 flies respond differently to deltamethim .......................................................................................................................... 1 34 Figure 4-6 Pyrethroids destabilize the GFS of DSC1 knockout flies to a greater extent ................................................................................................................ 137 xiii LIST OF ABBREVIATIONS ANOVA- analysis of variance AP - action potential CNS - central nervous system DCJW - N—decarbomethoxyllated JW062 DLM - dorsal longitudinal muscles DSC1- Drosophila Sodium Channel 1 GABA - y-aminobutyric acid GF - giant fiber GFS — giant fiber system HRP - horseradish peroxidase LL - long latency LLRP - long latency refractory period PNS - peripheral nervous system PSI - peripherally synapsing intemeuron SCBIs - sodium channel blocker insecticides SL - short latency SLRP - short latency refractory period 'l'l'M - tergotrochanteral muscle TTX - tetrodotoxin UAS - upstream activation sequences VGIC - voltage-gated ion channels xiv HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid L050 - median lethal concentration ED5o - median effective dose XV CHAPTER 1 GENERAL INTRODUCTION To communicate with the outer world and to maintain the homeostasis of the inner environment, higher metazoans develop a neural system to receive stimulations and make corresponding reactions. Although neurons from different parts of the neural system may have different conformations and functions, they do have a common basic character — they use action potentials to propagate signals. An axon action potential is a brief, spike-like depolarization that propagates as an electrical wave along dendrites or axons of a neuron. During 1940s and 19505, Alan Hodgkin and Andrew Huxley performed a series of experiments and demonstrated that action potentials are the result of different ions moving through the cell membrane. They also proposed that the movement of ions is mediated by a channel-like structure on the cell membrane by means of activating (open) and inactivating (close) in response to changes of the membrane potential (Hodgkin and Huxley, 1952). The channel like structures were later found to be transmembrane proteins, termed voltage-gated ion channels (VGIC). These channel proteins are located not only on neurons, but also on muscle and endocrine cells. In response to changes in membrane potential, these channels mediate rapid ion flux through a highly selective pore. Based on the ion selectivity, voltage-gated ion channels can, be grouped as sodium channels, potassium channels, calcium channels, chloride channels. Or they can be just named as voltage-gated cation or anion channels if the channel is permeable to more than one type of cation or anion (Catterall, 1992; Catterall et al., 2005). In this dissertation, research was conducted to study the molecular biology, electrophysiology, and neurobiology of a novel cation channel, Drosophila Sodium Channel 1 (DSC1), in Drosophila melanogaster. Because of the sequence similarity between the sodium channel and the DSC1 channel, this chapter will start with the literature on voltage—gated sodium channels. 1.1 Voltage-gated sodium channels 1.1.1 Structure and gating properties of voltage-gated sodium channels In Hodgkin and Huxley’s studies, they demonstrated that the movement of sodium ions through the cell membrane is essential for the generation and propagation of action potentials. They also characterized three key features of the structure mediating sodium currents: 1) voltage-dependent activation; 2) quick inactivation; 3) selective conductance of sodium ions (Hodgkin and Huxley, 1952). Subsequently, many labs tried to isolate the voltage-gated sodium channel. But, for a long time, the sodium channel protein could not be isolated and the topology of the channel protein remained unclear until 1980. By using photo labeling technique, Beneski et al. first isolated a large polypeptide (260 kDa) and a smaller polypeptide (36 kDa) from synaptosomal membranes of the rat brain with a photoactivable derivative of scorpion toxin, a sodium channel specific neurotoxin (Beneski and Catterall, 1980). At the same time, another group also isolated a polypeptide of the channel protein with similar size as the larger one mentioned above from the electric organ of the eel, Electmphorus electricus, by using radioactive-labeled tetrodotoxin (TTX), a high affinity blocker of sodium channels (Agnew et al., 1980). The larger peptide was denoted as a subunit while the small 3 one was denoted as [5 subunit. The understanding of voltage-gated sodium channel advanced a big step in 1984 when Noda et al. (Noda et al., 1984) screened the expression library of the eel, Electrophorus electricus, with rabbit antiserum to purified El. electricus sodium channel peptides and isolated the full-length cDNA encoding the sodium channel a subunit. The deduced amino acid sequence indicated that the a subunit is a transmembrane protein that consists of 1820 amino acids folded into four domains (DI — DIV) with six transmembrane segments (S1 - S6) in each domain (Fig. 1-1) (Catterall, 2000). In segment four of every domain, there are five to eight positively charged amino acids which are considered to sense the change of the membrane potential and guide the opening and closing of the whole channel. These results also support the hypothesis that the a subunit is the pore forming subunit. Later studies on the a subunit of the rat brain sodium channel showed that the a subunit was sufficient to mediate sodium current when expressed in Xenopus oocytes indicating that the a subunit is the pore-fonning subunit while the [3 subunit works as a auxiliary subunit (Noda et al., 1986). Depending upon the membrane potential, voltage gated sodium channels have three functional states: closed (ready to open), activated (opened), and inactivated. At resting membrane potential, sodium channels are closed. When the membrane potential is depolarized to certain level, sodium channels open through a series of conformational changes which is defined as activation. As soon as sodium channel opens, sodium ions flow from the outside of cell membrane to the inside due to the concentration gradient of sodium ions. The kinetics of the sodium current passing through open channels is very rapid, reaching its peak in less than one millisecond and declining to baseline within a few milliseconds due to a second conformational change defined as fast inactivation. In the past several decades, an extensive effort has been made to understand the molecular structure and the mechanism of gating of the pore-forming a subunit. Several substructures such as the selectivity filter, voltage sensor, and inactivation gate that were identified are essential for the channel function (Fig. 1-1). Facilitated by site-directed point mutagenesis and crystal structure studies of a voltage-gated potassium channel, it is a consensus now that all the four domains contribute to form the pore structure of the channel. The $5, $6, and the P-region connecting them from each domain form the inner surface of the channel, while the 81-3 of each domain form the supportive structure and outer surface of the channel. Currently, the inner surface is divided into two structures, outer pore and inner pore, which are facing the extracellular and intracellular sides, respectively. It is well established that the ion selectivity of voltage-gated sodium channels is determined by the amino acids D, E, K, and A in the pore positions of domains I, II, III, and IV, respectively (i.e., the DEKA motif) (Catterall, 2000) (Fig. 1-1). Four other amino acids E, E, M, and D, which are also located in the pore position of domain l-IV, determine the ion selectivity of voltage-gate sodium channel as well (the EEMD motif, Fig. 1-1) (Terlau et al., 1991). These eight amino residues are highly conserved in sodium channels of different species (Catterall, 2000). Substitution of DEKA with EEEE in a rat brain sodium channel changes the selectivity of a sodium channel to a calcium channel (Heinemann et al., 1992 ). The amino acids composing the selectivity filter are also the primary binding sites of TTX (Fig. 1-1) (Noda et al., 1989; Du et al., 2009a). 84 segments of each domain are critical for voltage sensing. The positively charged arginine or lysine residues in S4 of each domain work as voltage sensors responding to the change of the membrane potential (Catterall, 1986; Guy and Seetharamulu, 1986). Mutation of the voltage sensor can profoundly change the activation of sodium channels (Stuhmer et al., 1989; Kontis et al., 1997). 81-84 are coupled with S6 via the 84-85 linker helix. This coupling enables channel opening and closing upon changes in the membrane potential. It is the nature of the coupling between the $6 and the voltage-sensor domains that decides if the channel will open or not at a certain voltage level (Mannikko et al., 2002). Three comparable models have been proposed to explain the coupling of voltage sensor with surrounding residues and the movement of 84 during activation; the helical screw model, the transporter model, and the paddle model (Borjesson and Elinder, 2008). However, none of the models explain all the experimental results. Currently, the helical screw model has received the most proponents. This model assumes that the positive residues of S4 pair with negatively charged residues in the neighboring segments. When the membrane is depolarized, this pairing is disrupted and the S4 segments move outward causing a conformational change, which results in opening of the channel pore (Catterall, 1992). Inactivation is a very important gating property of all voltage gated ion channels; it closes the channel and prevents it from reopening until there has been sufficient time for recovery. During this time, which is also termed as refractory period, the channel will not open no matter how membrane potential alters. This has two functions. The first one is to determine the frequency of action potential firing. The other one is to prevent a breakdown of ionic gradients and cell death during a long depolarization. Many toxins, such as insecticides, and clinically used drugs function by affecting sodium channel inactivation. For voltage gated sodium channels, normally, inactivation kinetics can be divided into two categories, which are named as fast inactivation and slow inactivation respectively, and each of these can be modulated by cellular factors or accessory subunits. The mechanism of slow inactivation is still controversial, but there is a widely accepted ‘ball-and-chain’ or ‘hinged lid’ model for fast inactivation model, in which a cytoplasmic region (the inactivating particle) occludes the pore by binding to a region nearby (the docking site) at certain membrane potential level. The short intracellular linker connecting Dlll and DIV is where the ‘ball’ and the ‘chain’ locate. Three contiguous hydrophobic amino acid residues, isoleucine, phenylalanine, and methionine (IFM) positioned in the middle of the linker are the central structure of the ‘ball’ (West et al., 1992). Recently, a fourth amino acid, threonine, was suggested to be important for fast inactivation and redefined the inactivation particle as IFMT motif (Rohl et al., 1999). The docking sites for the inactivating particle include the 84-85 linkers of Bill and DIV along with the cytoplasmic portion of DIVS6 (Goldin, 2003). The docking sites are buried in the channel protein when channels are closed. It is hypothesized that the conformational change brought up by the movement of S4 exposes the hydrophobic residues of docking site to the IFMT motif to lock it in and occlude the channel. The conformational change underlining the folding the ‘chain’ was elucidated by studying the bacteria sodium channel (Zhao et al., 2004). Based on this study, a glycine residue flanking the inactivation particle is suggested to serve as a ‘hinge’ that facilitates the folding of the ‘chain’ and binding of to the docking sites with IFMT motif. 1.1.2 The family of mammalian sodium channel a subunit encoding genes In mammals, the sodium channel is composed of one a subunit and one or two [3 subunit(s) (Fig. 1-1). A variety of different isoforrns of the mammalian voltage—gated sodium channel a subunit have been identified. Based on their amino acid sequences, these isoforrns are grouped into three families, NaVI-Nav3. Nav1 family has 10 members while Nav2 and Nav3 have 3 and 1 members respectively. To date, sodium currents have not been recorded from members of Nav2 and Nav3. However, nine of ten members of the Nav1 family have been functionally expressed and named as Nav1.1-Nav1.9. These nine mammalian sodium channel isoforrns share more than 50% amino acid sequence similarity. They vary in the tissue and developmental distribution, biophysical properties and sensitivity to neurotoxins (Goldin, 2001). Nav1.1, Nav1.2, and Nav1.3 are expressed in the central nervous system (CNS) and are inhibited by nanomolar concentrations of TTX. Recently, Nav1.1 was also identified in the rat peripheral 8 nervous system (PNS). Nav1.4 is expressed in skeletal muscles while Nav1.5 is expressed in all striated muscles. Nav1.6 can be detected on both CNS and PNS while Nav1.7, Nav1.8, and Nav1.9 are detected only in PNS. In addition to variable tissue distribution, Nav1 isoforms have been verified to have unique developmental profiles. Nav1.1, Nav1.2, and Nav1.6 are present at high levels in the adult CNS, but their presence in embryonic stages and neonatal stages are different. Nav1.1 becomes detectable shortly after birth, whereas Nav1.2 becomes detectable during embryonic stages. Both of them reach maximal levels during the adult stage. Nav1.6 is the most abundant isofom'l in the adult CNS, but the highest level is in late embryonic and early postnatal periods. Nav1.3 can only be detected during embryonic and neonatal stages. Transcripts of Nav1.5 are detectable in neonatal skeletal muscles but are replaced by Nav1.4 in adult skeletal muscles (Goldin, 2001). The nine isoforms in Nav1.1 family exhibit significant differences in functional properties. The voltage required for activation and inactivation of these isoforms is listed in Table 1-1. The combination of channel subtypes in individual cells is a key determinant of the membrane excitability, conduction velocity, and in some tissues the resting membrane potential. Alterations in expression or activity of sodium channel subtypes are associated with several disease states such as epilepsy, stroke, and pain (Wood et al., 2004; Stafstrom, 2007). 1.1.3 The sodium channel B subunit. Compared with the pore-forming a subunit, the B subunit works as an auxiliary subunit and is not required for the in vitro expression of sodium channels. Currently, four B subunit (B1-B4) encoding genes have been identified (Goldin, 2001; Yu et al., 2003). The size and tissue distribution of the four B subunits are listed in Table 1-2. The function of B subunits has two aspects. First, B subunits modulate the channel gating and in turn modulate the excitability of neurons. The sodium current mediated by a subunit alone does not exhibit the “fast-gating” kinetic parameters observed in neurons. Coexpression with B1 and B2 subunits increases the rate of both activation and inactivation (lsom et al., 1992; lsom et al., 1995). Interaction between the a subunit and B subunits also regulates the persistent sodium current (Aman et al., 2009). Second, B subunits are likely responsible for targeting the sodium channel complex to the node of Ranvier by interacting with other cell adhesion molecules (Ratcliffe et al., 2001). The B subunit is composed of an extracellular immunoglobulin-like fold, a transmembrane o-helix, and a short intracellular tail. The effect of the B subunit is mediated entirely by the extracellular immunoglobulin-Iike fold. In neurons, the interaction between the a subunit and the B subunits is either covalent or noncovalent. The noncovalently linked B subunits include B1 and B3 (lsom et al., 1992; Morgan et al., 2000). The loop on the extracellular side of transmembrane segment DIVS6 was suggested as an interaction point of B1 subunit. On the other hand, the B2 and B4 subunits covalently link to the 0 subunits via disulfide bonds IO (lsom et al., 1995; Yu et al., 2003). 1.1.4 The Drosophila voltage-gated sodium channel, Para. In contrast to the multiple sodium channel genes in mammals, there is only one functional sodium channel a subunit encoding gene identified in insects (Dong, 2007), such as para from Drosophila (Loughney et al., 1989). Similar to mammalian counterparts, the insect sodium channel a subunit has four homologous domains, each containing 6 transmembrane segments. In Drosophila, para is the only gene that encodes a functional sodium channel. In 1971, Suzuki et al. (Suzuki et al., 1971) isolated a temperature- sensitive paralysis mutant, parats. The phenotype of this mutation is immediate paralysis when exposed to restrictive temperatures (37°C). This phenotype is due to a temperature-dependent blockage in the propagation of nerve action potentials (Siddiqi and Benzer, 1976). In 1989, Loughney et al. (Loughney et al., 1989) mapped the parats locus to 1406-01 of the X chromosome and cloned the para cDNA and genomic DNA as well. The full-length genomic sequence of the para gene is about 65 kb whereas the para cDNA is 6.8 kb after splicing. In 1997, Wannke et al. confirmed that para encodes a voltage gated sodium channel and has all of the expected channel functions and electrophysiological properties when expressed in Xenopus oocytes (Wannke et al., 1997). The ortholog of B subunit encoding gene has not been identified in Drosophila. However, a structurally different subunit, TlpE, has been identified to have a function similar to that of the B subunit (Feng et al., 1995). TlpE increases the 11 Para sodium current in Xenopus oocytes when coexpressed with the Para protein. Recently, four tipE like genes, termed as TEH1-4, were identified (Derst et al., 2006). TEH1 is CNS-specific while TEH2, TEH3 and TEH4 are more widely expressed. TEH1 substantially increases the sodium current amplitude, while TEH2 and TEH3 increase the sodium current amplitude to a medium extent. THE4 has no effect on the sodium current amplitude. The fast inactivation and recovery from fast inactivation can be altered by coexpression of TEH1, indicating that TEH1 works as an auxiliary subunit. Although para is the only gene that encodes functional sodium channels in Drosophila, electrophysiological studies showed that the properties of sodium currents detected in various isolated insect neurons are not exactly the same (O'Dowd et al., 1995; Wicher et al., 2001; Defaix and Lapied, 2005). How do insects achieve the diversity of sodium channels? Recent research from both our lab and others demonstrate that the para and other insect sodium channel transcripts undergo extensive alternative splicing and RNA editing. To date, seven alternative exons, a, b, e, f, h, i, j plus four mutually exclusive exons, c/d, k/l, have been identified in para transcripts (Loughney et al., 1989; Thackeray and Ganetzky, 1994; O’Dowd et al., 1995; Thackeray and Ganetzky, 1995; Olson et al., 2008). Detailed study of the para transcripts in embryonic and adult stages indicated that alternative splicing of para transcripts is developmentally regulated and varies extensively, resulting in a variety of sodium channel isoforms with different gating properties (Thackeray and Ganetzky, 1994; Lin et al., 2009). 12 RNA editing is another post-transcriptional mechanism to increase the diversity of sodium channels in insects (Liu et al., 2004; Song et al., 2004; Olson et al., 2008). For example, a U-to-C editing event resulting in an F/S1950 amino acid substitution on the Para channel generates a sodium channel variant with a unique persistent current. This RNA editing event and its functional consequence are conserved in the German cockroach sodium channel transcript (Liu et al., 2004). 1.2 The Drosophila Sodium Channel 1 (DSC1) 1.2.1 Molecular biology of the 0801 The DSC1 gene was first discovered in the 1980s by probing a Drosophila genomic DNA phage library with an eel sodium channel cDNA (Salkoff et al., 1987; Ramaswami and Tanouye, 1989). Sequence analysis predicted that DSC1 encodes an ion channel that has more than 50% similarity to the vertebrate sodium channel protein. The DSC1 channel protein has four homologous domains each containing six membrane-spanning segments connected by extracellular or intracellular linkers of various sizes. Sequences that are presumed to be critical for channel function, such as the voltage sensor, 85 and S6, which form the inner surface of channels, and the intracellular linker connecting D3 and D4, show high evolutionary conservation compared to the channels identified in rat and eel. Because of its overall similarity in deduced amino acid sequence with mammalian sodium channel, DSC1 was proposed to encode a putative sodium channel protein. In situ hybridization showed that DSC1 locates within a single I3 site at region 60E on chromosome 2R. 1.2.2 Tissue distribution studies of DSC1 transcripts and protein In 1994, Chang-Sock Hong and Barry Ganetzky (Hong and Ganetzky, 1994) examined the spatial and temporal expression patterns of both para and DSC1 transcripts in 12th-, 13th-, 15th- and 17th -stage embryos, 3rd instar lavae, 25 and 72 hr old pupae, and the adult. ln embryos, the 5’ un-translated region of para gene was detected during late stage 12, suggesting the initiation of para transcription. The transcripts were detected in neurons but not in neuroblasts of both CNS and PNS during early stage 13 and the following stages. DSC1 transcripts were detected in cells clustered along the ventral nerve cord at early stage 12 prior to germ band retraction. In PNS, several cells appearing in the dorsal wall of the foregut are DSC1 transcripts positive. The position of these cells indicates that they may belong to the stomatogastric nervous system. By stage 15, DSC1 is expressed in a regular pattern in some cells located along the longitudinal commissures of the CNS. In the PNS of the same stage, DSC1 transcripts are expressed prominently in the frontal ganglionic region. Also at this stage, there are several cells in the CNS labeled by DSC1 probe, but they may not be neurons since they could not be labeled by a neuron-specific anti-HRP antibody. In third instar larvae, para transcripts were detected in the CNS including larval brain, ventral ganglia, and the PNS, such as eye discs and leg discs. In the CNS, para expression was limited to mature neurons that regulated the behavior of the larva. The para expression pattern in ventral ganglia reflects l4 the continued expression of para in embryonically derived neurons. In the PNS, para transcripts detected in photoreceptor cells in eye discs and some sensory neurons in leg discs. However, in this stage, DSC1 transcripts were identified only in the laminar region of the optic lobes. Interestingly, the expression pattern of both para and DSC1 in the CNS were completely overlapped in the pupal and adult stages. Transcripts of these two genes were present in brain, thoracic ganglion, visual system, and antennal neurons. However, the expression of para and DSC1 was distinctive in the developing pupal wing. At 25 hours after pupariation, only transcripts of para, but not DSC1, were detected in some sensory neurons along the anterior margin of the wing and in campaniforrn sensilla along the third wing vein. Castella et al. (Castella et al., 2001) used a DSC1 polyclonal antibody and a neuronal specific monoclonal antibody, 22010, to study the tissue distribution of DSC1 channels in the nervous systems of adult D. melanogaster. DSC1 expression was detected exclusively in neurons, but not in other excitable cells such as muscle cells. In the CNS, DSC1 is mainly distributed in synaptic regions and axonal tracts in the brain and in thoracic ganglions. However, DSC1 was not detected in the cell body of neurons, which is a main difference from Para. This is not consistent with the in situ hybridization study by Hong and Ganetzky (Hong and Ganetzky, 1994), which showed that the DSC1 transcripts were localized in the cortical cell bodies. In the PNS, the distribution of the DSC1 protein is strikingly wide. DSC1 signal was very weak in compound eyes but strong in the antennal sensilla and nerves. DSC1 was also detected on sensory neurons and 15 motor neurons of the proboscis, thoracic muscles, legs, anal plate, and the penis apparatus. 1.2.3 Functional studies of DSC1 channels To study the function of the DSC1 channel Gerrneraad et al. (Genneraad et al., 1992) examined sodium currents from primary cultured embryonic neurons of a DSC1 homozygous deficiency line using the voltage-clamp technique. Their results suggested that these DSC1 absent neurons expressed sodium currents similar to those from the wild type neurons, indicating that para is the major sodium channel encoding gene in embryonic neurons. One study suggested a functional role of the DSC1 channel in olfaction (Kulkami et al., 2002). A P-element insertion line, smi60E, exhibits a three-fold shift in the dose response for avoidance of benzaldehyde, a repellent odorant to Drosophila, toward higher odorant concentrations. The P-element insertion was mapped to the second intron of the DSC1 gene. To verify the olfactory defect phenotype is due to the P—element insertion, smi60E was crossed with 6 EP insertion lines, which are mapped 58 bp downstream of the P-element insertion site at the same intron of the DSC1 gene. The heterozygous offspring failed to complement the olfactory defect, indicating the association of the P-element insertion and the olfactory defect. There is another gene, L41, at the insertion site. Southern blot analysis showed a 66% reduction in the DSC1 transcripts and 41% reduction in the L41 transcripts. When the P—element was excised, the olfactory l6 impairment could be rescued even though the transcription level of L41 was not restored, suggesting that the olfactory defect is not associated with L41. Although 0801 was isolated two decades ago, the function of the DSC1 channel remains undetermined. Liu et al. (Liu et al., 2001) cloned a DSC1 ortholog, BSC1, from Blattella gennanica. The amino acid sequences between the predicted DSC1 and BSC1 proteins share 81%, 78%, 80%, and 88% identities in the transmembrane domains l-IV, respectively. Furthermore, BSC1 transcripts are found to be alternatively spliced in a tissue-specific and developmental stage-specific manner. The BSC1 channel was successfully expressed in Xenopus oocytes and was found to function as a cation channel that is permeable to Na+, K+, Ca2+, and Ba2+ (Zhou et al., 2004). Interestingly, both Co2+ and cc2+ block the BSC1 channel, similar to their effects on voltage-gated calcium channels (Hille, 1992). The BSC1 channel is permeable to monovalent cations (i.e., Na+, K) in the absence of external Ca2+. The permeability ratios were PBa/PK a 30 and PBa/PNa = 22 for the BSC1 channel, indicating that the BSC1 channel is more permeable to Ca2+lBa2+ than to either Na+ or K. That is much lower than the permeability ratio for L-type Ca2+ channels (PBaIPNa :2 470 and PCa/PNa z 1170) and much higher than the ratio for sodium channels (PCa/PNa = 0.1) (Hille, 1992). In addition to the unique ion selectivity, the BSC1 channel is also different from voltage-gated sodium channels with respect to the kinetics of activation, deactivation, and inactivation. The BSC1 channel is a high-voltage-activated 17 channel with a half-maximal activation voltage of 50 :I: 8 mV. Both activation and inactivation of the BSC1 channel are slower than those of sodium channels, resulting in a large tail current upon repolarization. It took a 40-ms depolarization to fully activate the channel, and there was no significant inactivation observed when BSC1 currents were recorded with 40-ms depolarization pulses from -50 to 80 mV from the holding potential of -100 mV. However, complete inactivation was evident when a long depolarization pulse (500 ms) was applied. As described in Chapter 1.1.1, single amino acids located in the P-regions of four domains determine the ion selectivity of sodium channels and calcium channels. These amino acids are D, E, K, and A in sodium channels and E, E, E, and E in calcium channels, respectively. Comparison of the sequence of BSC1 with voltage-gated sodium and calcium channels shows that the BSC1 channel has D, E, E, and A at the same positions, respectively. The permeability to Ba2+ of the BSC1 channel is reduced when the second E was substituted with K. Moreover, the permeability to Na+ was enhanced with the same amino acid substitution, indicating that the E residue in domain I" is important for the selectivity of the BSC1 channel toward Ba2+ or Ca2+. Phylogenetic analysis of the relationship between the BSC1 channel and other voltage-gated sodium and calcium channels suggests that BSC1 and DSC1 channels belong to a novel family of ion channels that are closely related to calcium channels as they are to sodium channels (Zhou et al., 2004). 18 1.3 Pyrethroid insecticides act on the voltage-gated sodium channel 1.3.1 Pyrethrum and pyrethroids Pyrethrins are a series of natural compounds found in pyrethrum extract from Chrysanthemum flowers. The insecticidal properties of pyrethrins were discovered two centuries ago (McLaughlin, 1973; Casida, 1980). They are esters of chrysanthemic acid (Fig. 1-3). However, because of its instability under the sun, the pyrethrum extract and pyrethrins are mainly used in control of household insect pests, such as mosquitoes and houseflies, not used as an agricultural insecticide. With an increasing demand for organically grown products, use of pyrethrum extract has also increased. The synthetic analogs of the pyrethrins, termed as pyrethroids, are a group of environmentally friendly, highly effective and selective insecticides which are used globally for pest control. The first several pyrethroids were discovered from 1940 to 1970 (Elliott and Janes, 1978; Elliott, 1980). A few of these compounds, such as allethrin, tetramethrin, and resmethrin, exhibited excellent insecticidal activity. However, similar to pyrethrins, they are not very stable in the environment. The successful synthesis of pennethrin in 1973 by Elliott and coworkers (Elliott et al., 1973) announced the first potent and photostable pyrethroid, opening a new era of synthetic pyrethroid research and development. Following perrnethrin, several pyrethroids, such as cyperrnethrin and deltamethrin, were synthesized and proven to be highly potent and effective for pest control (Hall, 1978). Nowadays, numerous photostable pyrethroids have emerged that exhibit an extreme efficiency as agricultural insecticides (Elliott and Janes, 1978; Elliott, 1980) (Fig. 19 1-3). 1.3.2 Classification based on chemical structure and symptoms Based on whether there is an o-cyano-3-phenoxybenzyl alcohol structure in the molecule, pyrethroids are grouped into two groups, named as type I and type II pyrethroids. (Fig. 1-3). Type I pyrethroids do not contain the o-cyano-3-phenoxyenzyl alcohol structure while type II pyrethroids do. Interestingly, type I and type II pyrethroids can cause different poisoning symptoms (Gray and Soderlund, 1985; Soderlund and Bloomquist, 1989; Bloomquist, 1993). At doses toxic to mammals, type I pyrethroids cause a whole-body tremor, or T syndrome (Verschoyle and Aldridge, 1980) while type II pyrethroids produce choreoathetosis (sinuous writhing) and profuse salivation, Indicating an intoxication to the CNS (Verschoyle and Aldridge, 1980). Differential poisoning syndromes caused by type I and type II pyrethroids also occur in insects, but they are less distinct than those observed in mammals (Soderlund and Bloomquist, 1989; Bloomquist, 1993). Actually, the type I and type II classifications are not absolute in either insects or mammals, and certain compounds show effects intermediate between the two classes (Soderlund and Bloomquist, 1989). 1.3.3 Mode of action The symptoms of pyrethroids intoxication suggest a disruption of the nervous system. The earliest electrophysiological study on the mechanisms of pyrethrum 20 insecticides was carried out using extracellular electrodes to record compound nerve action potentials in insect and crayfish ventral nerve cord preparations (Lowenstein, 1942; Welsh and Gordon, 1947). Isolated nerve fibers exposed to pyrethrins generate repetitive discharges in response to a single stimulus. These findings were confirmed in the first intracellular recording studies (Narahashi, 1962b, a), in which low concentrations of allethrin prolonged the falling phase of the nerve action potential in cockroach giant fiber preparations and induced repetitive discharges, whereas high concentrations of the same compound reduced the amplitude of the action potential, eventually blocking nerve conduction. These results were also the first to indicate that the voltage-gated sodium channel is one of the primary targets of allethrin in modulating the nerve action potential. Following studies on invertebrate giant axons (Clements and May, 1977; Lund and Narahashi, 1983) and frog sciatic nerve (Vljverberg et al., 1982) have shown that type I compounds produce repetitive discharges similar to those described for pyrethrins and allethrin, whereas type II compounds do not produce repetitive discharges, but lead to stimulus-dependent nerve depolarization and blockage of the action potential. Voltage clamp technique brought the research on the mode of action of pyrethroids to a new level. With this technique, studies using various nerve preparations have shown that the repetitive firing and depolarization caused by pyrethroids resulted from prolonged opening of sodium channels (Soderlund and Bloomquist, 1989; Vljverberg and van der Bercken, 1990; Narahashi, 1992; Bloomquist, 1993). Pyrethroids slowed or delayed the inactivation of sodium 2] channels resulting in a prolonged open state. Pyrethroids also produced a slowly decaying sodium current, known as tail current, that continues to flow after the membrane is repolarized. At the cellular level, the delayed shutting of pyrethroid-modified sodium channels leads to a persistent inward current to flow after an action potential, resulting in repetitive firing (type I pyrethroids) and depolarization the nerve membrane (type II pyrethroids) (Soderlund and Bloomquist, 1989; \fijverberg and van der Bercken, 1990; Narahashi, 1992; Bloomquist, 1993). Voltage-clamp experiments also showed different decay kinetics of the tail current induced by type | and type II pyrethroids. The decay of tail currents induced by type II pyrethroids is at least one order of magnitude slower than those induced by type I pyrethroids. These quantitative differences in tail-current decay kinetics between type I and type II pyrethroids may account for their different actions on the nervous system (Dong, 2007). The patch-clamp technique is another powerful tool for studies of the mode of action of pyrethroids. This method allows more detailed analysis of pyrethroid modification of sodium currents at the single-channel level. Analysis of single sodium channel currents in the presence of pyrethroids revealed a population of channels with slowed kinetic transitions between different channel states (Narahashi, 1992; Bloomquist, 1993). Channels modified by pyrethroids display normal single-channel conductance but exhibit a prolonged open state with altered activation kinetics (Narahashi, 1992; Bloomquist, 1993). Single-channel and tail-current analysis indicate that the open state of the sodium channel is more apt to be modified by pyrethroids than the closed state due to lower affinity 22 for this state (Narahashi, 1992). The higher affinity for open channel could, at least in part, explain the stimulus-dependent effects of the pyrethroids (Narahashi, 1992) VVlde use of pyrethroids in pest control stems not only from their high efficiency, but also their relatively lower toxicity to mammals. Pyrethroids exhibit a highly selective toxicity to insects over mammals. Potency evaluation in vitro showed that allethrin was 1000-fold more potent on cockroach sodium channels than rat TTX- sensitive sodium channels (Narahashi et al., 2007). Another important factor affecting pyrethroid activity is temperature. Pyrethroids are much more potent at low temperatures than at high temperatures. The enhanced sensitivity of sodium channel to pyrethroids is due to increase in the time constant and the amount of charge during the tail current (Song and Narahashi, 1996; Motomura and Narahashi, 2000). This temperature dependence becomes an important factor for pyrethroids safety because there is a 10°C difference in body temperature between mammals and insects. Voltage-gated sodium channels are the primary target of pyrethroids. Nevertheless, there are several lines of evidences suggesting that pyrethroids also affect other ion channels (Ray and Fry, 2006), such as voltage-gated calcium channels (Shafer and Meyer, 2004), and chloride channels (Burr and Ray, 2004). However, it is still premature to draw a solid conclusion that these channels are also important to the acute neurotoxicity of pyrethroids. 23 1.4 The giant fiber (GF) system 1.4.1 The cellular composition and function of the GF system Giant nerve fibers are a group of neurons, which are associated with animal escape response in many invertebrates and some lower vertebrates, including D. melanogaster (T rimarchi and Schneiderman, 1995c; Trimarchi and Schneiderman, 1995b; Trimarchi and Schneiderman, 1995a). The classic case is when there is a predator approaching; the shadow of the predator stimulates the visual system of the insect and initiates a powerful jump with the middle legs. Once the fly is airborne, flight usually follows (T rimarchi and Schneiderman, 19950). The entire circuit, including giant fiber neurons, intemeurons, motor neurons, and muscle cells, is termed as the GF system and is schematized in Fig. 1-4. In Drosophila, the somas of giant fiber neurons are located in the brain with their large axons extending to the thorax, where the terminals of the giant fiber axon form synaptic connections with two different neurons: a large motorneuron that innervates the tergotrochanteral muscle (IT M) and a peripherally synapsing intemeuron (PSI). The PSI axon crosses the midline and synapses with motor neurons, which innervate the dorsal longitudinal muscle (DLM). The GF neurons on each side are connected by the giant commissural intemeurons (GCls) that ensure the right and left sides respond simultaneously. Three groups of thoracic muscles, the TTM, DLMs, and dorsoventral muscles (DVMs) (not shown in Fig. 1-4), provide the major forces for jump and flight. Visually evoked jump-flight behavior is mediated by the GF system (T rimarchl and Schneiderman, 1995b; Trimarchi and Schneiderman, 1995a) and is assumed to be an escape reflex. 24 1.4.2 The synaptic connectivity among identified neurons of the GF system As illustrated in Fig. 1-4 B, the GF-TTMn and GF-PSI synapses are mixed electrochemical synapses. However, the neurotransmitter of these mixed-synapses is not very clear. The neurotransmitter of the synapse between the PSI and DLM motor neurons is acetylcholine (Gorczyca and Hall, 1984), while glutamate is the transmitter at neuromuscular junctions in the GFS (Kosaka and lkeda, 1983; Koenig and lkeda, 2005). 1.4.3 The giant fiber system recording In the laboratory, the GF pathway can be triggered from different neurons by giving electronic stimuli through electrodes poking into either compound eyes or thorax (Fig. 1-4 C). The activity of the GFS can be reflected by recording muscle potentials from either the TI'M or DLM (Tanouye and Wyman, 1980). The time interval between the stimulus and the first muscle potential is termed the response latency. It reflects the time required for the stimulation signal to conduct from activated neurons to the innervated muscle where the recording electrode is located. Based on the length of latency, the responses can be grouped into two classes, long latency (LL) and short latency (SL) responses. When a relatively low-strength stimulus (i.e., low voltage) is applied, a muscle potential with longer response latency is usually elicited. As the strength of the stimulus becomes higher, the muscle response with shorter response latency is triggered. The difference between these distinct response latencies is due to the involvement of afferent neurons presynaptic to the giant fiber neuron (Fig. 1-4 C). The afferent 25 neurons are involved in the long latency response, but will be bypassed when higher strength stimuli are applied. Therefore, the giant fiber neurons are activated directly resulting in a shorter response latency. Based on the difference, the GFS can be referred as long latency and short latency pathways. The lowest voltage by which the long latency response is elicited is termed the long latency threshold and the lowest voltage to elicit the short latency response is called the short latency threshold. When a pair of identical pulses separated by a variable interval is delivered, the longest interval at which a muscle potential is elicited by the first pulse but not by the second one is termed the refractory period, which is an indication of the stability of the GF circuit. Both long and short latency pathways exhibit distinct refractory period. Usually, the long latency refractory period (LLRP) is around 40 ms and has big variations (around 10 ms or more), while the short latency refractory period (SLRP) is less than 10 ms and varies much less than the LLRP. 26 Table 1-1. Tissue distribution and gating properties of nine mammalian voltage-gated sodium channel 0 subunits (Goldin, 2001 ; Catterall et al., 2005). lsoforrn Species Primary Tissue Activation (mV) Inactivation Distribution (mV) Nav1.1 Rat CNS, PNS -33 -72 Guinea pig Nav1.2 Rat CNS -24 -53 Human Nav1.3 Rat CNS -23 to ~26 ~65 to -69 Nav1.4 Rat Skeletal -3oa or-26b -50.1cor-56d Human muscle Nav1.5 Rat Denervated -47 -84 Human skeletal mucle, _569 4009 heart muscle _27f -61f Nav1.6 Rat CNSrPNS -8.89 -55k Human 17It 51l Mouse ' i ' m Guinea pig -26 _ -97.6 417.7J Nav1.7 fiat PNS -31" -65p uman o q Rabbit 45 '78 r -60.5 -39.6s Nav1.8 Rat PNS -16to-21t ~-3ot Mouse Dog Nav1.9 Rat PNS .47 to -54t -44to-54t Mouse Human a rat a subunit in Xenopus oocytes b human 0 subunit in CHO cells c human 0 subunit in Xenopus oocytes with 200-ms depolarizations using macropatch voltage-clamp d human 0 subunit in CHO cells with 500-ms depolarizations (D with phenylalanine as the major anion in the intracellular solution fwith aspartate as the major anion in the intracellular solution 9 mouse a subunit in xenopus oocytes with cut-open oocyte voltage-clamp 27 Table 1-1 (continued) h mouse a subunit with B1 and B2 in xenopus oocytes with cut-open oocyte voltage-clamp mouse a subunit with inactivation removed and b1 and b2 in xenopus oocytes with cut-open oocyte voltage-clamp rat a subunit in Xenopus oocytes with macropatch voltage-clamp mouse a subunitin Xenopus oocytes with 500-ms depolarizations using two-electrode voltage-clamp mouse a subunit with B1 and B2 in Xenopus oocytes with 500-ms depolarizations using two-electrode voltage-clamp rat a subunit in Xenopus oocytes with 5-s depolarizations using macropatch voltage-clamp rat a subunit in Xenopus oocytes with macropatch TTX-sensitive current in DRG neurons TTX-sensitive current in DRG neurons with 50-ms to 1-s depolarizations using whole-cell patch clamp rat a subunit in Xenopus oocytes with 10-s depolarizations using two-electrode voltage-clamp human 0 subunit in HEK cells with 2-s depolarizations using whole-cell patch clamp human 0 subunit with _1 subunit in HEK cells with 2-s depolarizations using whole-cell patch clamp rat DRG neurons 28 Table 1-2. Size and tissue distribution of four B subunits (Goldin, 2001; Yu et al., 2003). Name Species Tissue Size Genbank (33) access number NavB1.1 Rat CNS 218 M91808 Human L10338 Rabbit U35382 NavB12 Rat CNS 215 U37026 U37147 Human AF007783 NavB1 .3 Rat CNS 215 AJ243395 Human AJ243396 NaVB1.4 Human, CNS, PNS 228 BK001031.1 mouse, BK001030.1 rat AY149967.1 29 Figure 1-1. Two-dimensional molecular map of the a-subunit and B-subunit of the voltage-gated sodium channel showing how they fold and cross the plasma membrane. The d-subunit is composed of four homologous domains (I to IV), each of which contains six transmembrane segments (1 to 6). Cylinders represent o-helical transmembrane segments. Linkers and connections are illustrated as solid lines, roughly in proportion to their length in the amino-acid sequence of the sodium channel. Positive charged amino acid residues are indicated (+) on the S4 transmembrane segments, which serve as voltage sensors. The S5 and S6 segments and the P-region between them together form the walls of the sodium-ion-conducting pore. Circles indicate the positions of amino acids DEKA and EEDD which are important for ion conductance and selectivity (‘+’ positively charge , ‘-’ negatively charged) that are important for ion conductance and selectivity. Solid gray circle indicates the inactivation particle, IFMT (isoleucine, phenylalanine, methionine, threonine), which is thought to fold into and block the ion-conducting pore. The B subunit is composed of an extracellular immunoglobulin-like fold, a transmembrane d-helix, and a short intracellular tail. One a subunit can combine with one to three different B subunits to form the sodium channel complex. (Modified from Catterall, 2000) 30 22th 95:3 co=m>=om£ omm=0> E zf+ =mo some. 955822 w _ \ _ cacao cofiocd €38 e 31 Figure 1-2. Distribution of alternative exons on para transcripts. A schematic diagram of the Para protein topology labeled with names and locations of 11 alternative exons identified in para transcripts. Exons a, b, e, f, h, i, andj are optional exons. Exons b/c and UK are two pairs of mutually exclusive exons. (Adapted from Olson et al., 2008) I II III IV A )-——k C . C C i-—II l-——li lia bd/cefhl/k 32 Figure 1-3. Structure of commercial type I (A) and type II (B) pyrethroids. The cyano group in the structure of type II pyrethroids is marked. Pyrethrin l R=CH3 Pyrethrin ll R=COOCH3 Allethrin O N \ \ 0\/ O O Tetramethrin O %.m 0 Bioresmethrin 33 Figure 1-3 (continued) kayo/o Perrnethrin B \ O CI 0 cu* Cyperrnethrin OWDO/E: 0 cu* CI Fenvalerate Br 00 \ 0 Br 0 CN* Deltamethrin 34 Figure 1-4. The Drosophila giant fiber system. A. Schematic display of the position of the Drosophila CNS and the neurons and muscles of the giant fiber (GF) system. B. The nature of the synaptic connections in the GF system. In the brain, the giant fibers (GF) form electrical synapses with the giant commissural interneurons (GCls). In the mesothoracic neuromere, the GFS form mixed electrochemical synapses with the motorneurons (TT Mn) of the tergotrochanteral muscle (TTM; left) and with the peripherally synapsing intemeuron (PSI). The PSI forms chemical synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs; right). The neuromuscular junctions are chemical synapses. Note that in A only one of the TTMn axons is shown exiting the CNS and contacting the muscle on the left side and one set of DLMns and the corresponding neuromuscularjunctions are shown on the right side; in B only one side of the bilateral pathway and two of the DLMns and DLMs are shown (modified from Allen, 2006). C. Schematic illustration of the giant fiber pathway (one side shown). High-voltage stimulation (high) activates the cervical giant fiber (CGF) to induce a short-latency response, whereas low voltage stimulation (low) excites brain afferent neurons to trigger a long-latency response. (TT M) Tergotrochanteral muscle; (TTMn) TTM motoneuron; (DLM) dorsal longitudinal muscle; (DLMn) DLM motoneuron; (PSI) peripherally synapsing intemeuron. (Adapted from Engel et al. 2000) A—----------1b-----—----— Thorax .‘J—QDLMS TTM DLMns \. .. Electrical Mixed synapse Electrical-chemical Q. Chemical synapse synapse 35 Figure 14 (continued) stim. V: low 5. electrochemical -4 chemical 36 CHAPTER 2 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF TWENTY DSC1 cDNA CLONES 37 2.1 Abstract Drosophila Sodium Channel 1 (DSC1) was predicted to encode a sodium channel due to a high sequence similarity with vertebrate and invertebrate sodium channel genes. However, BSC1, a DSC1 ortholog in Blattella gennanica. was recently shown to encode a cation channel with ion selectivity towards Ca2+ in Xenopus oocytes. To functionally characterize the DSC1 channel, we isolated 20 cDNA clones that covered the entire coding region of the DSC1 gene from adults of D. melanogaster. The 20 clones can be grouped into nine splice types. Sequence analysis revealed ten optional exons, four of which contain in-frame stop codons. Optional exons containing premature stop codons were found in seven clones. Only three variants generated DSCI currents when expressed in Xenopus oocytes. Like the BSC1 channel, all three functional DSC1 channels are permeable to Ca2+ and Ba2+, and also to Na+ in the absence of external Ca2+. Furthermore, the DSC1 channel is insensitive to tetrodotoxin (TTX), a potent and specific sodium channel blocker. Our study shows that DSC1 undergoes extensive alternative splicing and encodes a voltage-gated cation channel similar to the BSC1 channel in B. gennanica. 2.2 Introduction Voltage-gated ion channels are a diverse group of integral transmembrane proteins that are essential for electrical signaling in neurons, muscle and 38 endocrine cells. In response to changes in membrane potential, these channels mediate rapid ion flux through a highly selective pore. Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium ions, respectively, during electrical signaling in cell membranes. Both sodium and calcium channels consist of a large o-subunit and a variable number of smaller subunits (Yu et al., 2005). The o-subunit forms the ion conducting pore while the associated subunits modulate channel expression and gating. Although the amino acid sequences of the pore-forming d-subunits of sodium and calcium channels are quite different, the overall topology is similar, which consists of four repeated homologous domains (l-IV), each having six membrane spanning segments (S1-S6). The S1-S4 segments serve as the voltage sensing module and the $5 and S6 segments and their connecting P loop serve as a pore-forming module (Catterall, 2000). Analysis of the genome of Drosophila melanogaster revealed only two sodium channel-like sequences, para, and DSC1 (Littleton and Ganetzky, 2000). It is well-established that para and para-orthologs in other insect species encode functional sodium channels (Warmke et al., 1997). Extensive alternative splicing and RNA editing of the transcripts of para and para-orthologs generate molecular and functional diversity of sodium channels in insects (Dong, 2007; Olson et al., 2008; Lin et al., 2009). DSC1 (Drosophila Sodium Channel 1) was first discovered two decades ago by probing a Dmsophila genomic DNA library with an eel sodium channel cDNA (Salkoff et al., 1987). It was predicted to encode a sodium channel based on its overall similarity in deduced amino acid sequence 39 and domain organization with eel and mammalian sodium channels. However, the function of the DSC1 channel remains largely unknown. A P-element insertion in the second intron of DSC1 in a smell-impaired (smi) mutant, smi60E, reduced DSC1 transcript level by two-fold and results in a three-fold decrease in olfactory response to benzaldehyde (Kulkami et al., 2002), suggesting that the DSC1 channel plays an important role in olfaction. Sodium currents in embryonic neurons from homozygotes for a chromosome deficiency at 60E5/6, where the DSC1 gene is located, were similar to those from wild-type neurons, indicating that para, riot DSC1, is the primary sodium channel gene in embryonic neurons (Genneraad et al., 1992). Comparative studies revealed different spatial and temporal expression patterns of DSC1 and para transcripts and proteins, particularly in the adult peripheral nervous system, suggesting different functions of DSC1 and Para channels in the nervous system. For example, in adults, expression patterns of DSC1 and para transcripts overlap in the CNS based on the RNA in situ hybridization study (Hong and Ganetzky, 1994). However, an immunohistochemical study indicated that DSC1 channels were only distributed in synaptic regions and axonal tracts while Para channels were also localized on cell bodies in the PNS. Only weak DSC1 signal could be detected from compound eyes where the expression level of Para channels is high. The dense localization of the DSC1 protein was detected in the nerve endings of motor neurons in the thorax, leg muscles and also in the proboscis, tibia and tarsi, where the mechanoreceptors or chemosensory organs are located (Castella et al., 2001) 40 DSC1 orthologs have been identified in other insect species including Blattella gennanica, and Heliothis virescens (Park et al., 1999; Liu et al., 2001). Tissue-specific alternative splicing of BSC1 has been reported (Liu et al., 2001). More significantly, BSC1 was confirmed to encode a cation channel with ion selectivity toward Ca2+ more than Na+ (Zhou et al., 2004). Although the complete mRNA sequence of DSC1 (NM_166696.2) has been deduced from the corresponding genomic sequence (NT_033778.3) and also from the RT-PCR analysis of partial cDNAs (Kulkami et al., 2002), no alternative splicing and functional analysis has been reported for DSC1. In this study, we isolated 20 cDNA clones covering the entire coding region of the DSC1 gene from adults of D. melanogaster. Sequence analysis revealed that DSC1 transcripts undergo extensive alternative splicing. Functional analysis in Xenopus oocytes confirmed that, like BSC1, DSC1 encoded a cation channel. Furthermore, the DSC1 channel was insensitive to tetrodotoxin (TTX), a potent and specific sodium channel blocker. 2.3 Materials and methods 2.3.1 Amplification and cloning of the coding region of the DSC1 gene by RT-PCR Five to seven day-old W1118 adults were used to isolate total RNA. First-strand cDNA was synthesized using Oligo dT primers (lnvitrogen, Carlsbad, CA), and the SuperScript® ll reverse transcription kit (Invitrogen, Carlsbad, CA). 41 Conditions for the first-strand cDNA synthesis reaction were: 42°C for 2 min followed by a 60 min incubation at 48°C. RNA was removed by a 20 min incubation with RNaseH at 37°C. The PCR primers for amplification of the cDNA were, 5’- CCGCTCGAGGCCACCATGGGTGATGATCAAGCGACG -3’, and 5’- TACCCCTAGGAAATATCAGATAGAAAGTTC -3’. To improve the expression efficiency, a Kozak sequence (GCCACCATGGGT) was added to the forward primer and the first nucleotide of the second codon was substituted from A to G to meet the sequence requirement. The Xho I and Avr ll restriction enzyme sites (underlined) were added to the fonlvard and reverse primer, respectively, to facilitate the cloning of the PCR product into a modified pGH19 vector in which an Xho I restriction site was removed by site-directed mutagenesis. The original pGH19 (a Xenopus expression vector containing the 3’ and 5’ untranslated regions of the Xenopus B-globin gene at the 3’ end of T7 promoter and 5’ end of SP6 promoter, respectively) was kindly provided by Barry Ganetzky, University of Wisconsin, Madison, WI. The PCR reaction conditions were the same as described preciously (Zhou et al., 2004). The reaction mixture (50 ml) contained 0.5 ml cDNA, 50 pmol each primer, 200 mM each dNTP, 1 U eLONGase (lnvitrogen), 1.5mM MgClz, and 1xPCR reaction buffer. PCR was carried out as follows: one cycle of 94°C for 1 min; 33 cycles of 94°C for 30 s, 58°C for 30 s, 68°C for 8 min; and one cycle of 68°C for 15 min. The PCR products were purified using the QIAEX ll Gel Extraction Kit® (QIAGEN Sciences, MD) and cloned into the modified pGH19. MAX Efficiency Stbl2—competent cells (lnvitrogen) were used as host cells. 42 2.3.2 Sequencing of Twenty DSC1 cDNA clones The inserts of DSC1 cDNA clones were sequenced by moderate throughput sequencing (ABI 3730xl, Research Technology Support Facility, Michigan State University). The sequences of the sequencing primers are listed in Table 2-1. Sequence data were analyzed using software Lasergene 6® (DNASTAR, Inc., Madison, WI). 2.3.3 Expression of DSC1 channels in Xenopus Oocytes Oocytes were obtained surgically from healthy female Xenopus Iaevis (Xenopus l, Ann Arbor, MI) and incubated with 1 mg/ml type IA collagenase (Sigma Inc. St. Louis, MO) in a Ca 2”I-free ND-96 medium, which contains 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5. Follicle cells remaining on the surface of oocytes were removed manually with forceps. Isolated oocytes were incubated in N096 culture medium containing 1.8 mM CaClz supplemented with 50 pg/ml gentamicin, 5 mM pyruvate, and 0.5 mM theophylline (Goldin, 1992). Healthy stage V—Vl oocytes were picked and used for cRNA injection. To prepare DSC1 cRNA, plasmid DNA containing full-length DSC1 cDNA was linearized with Notl (New England Biolab, lnc., Ipswich, MA), which does not cut the insert, followed by in vitro transcription with T7 polymerase using the mMESSAGE mMACHlNE Kit (Ambion, Inc., Austin, TX). Procedures for oocyte preparation and cRNA injection were identical to those described by 43 Tan et al. (Tan et al., 2002). Each oocyte was injected with 3 ng DSC1 cRNA. After injection, oocytes were incubated in ND-96 culture solution containing 96 mM NaCl, 2 mM KCI, 1.8 mM CaClz, 1 mM MgClz, 5 mM HEPES, 2.5 mM Na-pyruvate, 0.5 mM Theophyline for 24-72 hours before recording. 2.3.4 Electrophysiological recording and data analysis Methods for electrophysiological recording and data analysis are similar to those described previously (Zhou et al., 2004). Currents were recorded using standard two-electrode voltage clamp technique. The borosilicate glass electrodes were filled with filtered 3 M KCI in 0.5% agarose and had resistance at 4-6 Mn. Currents were measured using the oocyte clamp instrument OC725C (Warner Instrument Corp., Hamden, CT), Digidata 1200A interface (Axon Instrument, Inc., Foster City, CA), and pCLMP 8.2 software (Axon Instrument, lnc., Foster City, CA). Three recording solutions, sodium, calcium and barium solution were used to check the current mediated by the DSC1 channel. The sodium recording solution consisted of 50 mM NaOH, 45 mM TEAOH, 10 mM HEDTA, and 10 mM HEPES. The calcium solution consisted of 50 mM Ca(OH)2. 55 mM TEAOH, and 10 mM HEPES. The barium solution consisted of 50 mM Ba(OH)2, 55 mM TEAOH, and 5 mM HEPES. All recording solutions were adjusted to pH 7.0 with methanesulfonic acid. The recording protocol consisted of a 15-ms depolarization from -100 mV to 80 mV followed by repolarization to -100 mV. All experiments were performed at room temperature (22°C-25 °C). 44 2.3.5 TTX sensitivity assay For the application of TTX, the disposable perfusion system developed by Tatebayashi and Narahashi (Tatebayashi and Narahashi, 1994) was used. The test solution was transferred into a Petri dish placed on a support stand. Two glass capillary tubes (10 cm in length) connected together with a short length of Tygon tubing were used to aid solution flow from the Petri dish to the recording chamber. The solution flow was controlled by hydrostatic force created by adjusting the level of the Petri dish relative to the recording chamber. Disposable recording chambers (1-1.5 ml volume) were made with glue dams in the Petri dish. To avoid cross contamination, recording chambers, perfusion system, and the glass agarose bridges connecting the oocyte chamber with the ground electrode chamber were all discarded after a single use. The TTX working solutions were diluted from a stock solution (2 mM) using the external recording solution immediately before use. Effects of TI'X on peak currents reached a steady-state level within 5 min after perfusion. 2.4 Results 2.4.1 Sequence comparison of 20 DSC1 full length cDNA clones For functional characterization of the DSC1 channel in Xenopus oocytes and detection of possible alternative splicing variants, we isolated cDNA clones that covered the entire coding region of the DSC1 gene by RT-PCR from total RNA isolated from adults of D. melanogaster. A total of 20 DSC1 cDNA clones were isolated and sequenced. Sequence comparison of the 20 cDNA clones with the DSC1 genomic sequence (NT_033778.3) revealed 20 exons, named 1 to 20 (Fig. 45 2-1A). In addition, 10 optional exons, named exon 4A, 6A, 11A, 11B, 11C, 16A, 17A, 17B, 17C, and 19A respectively, were also found (Fig. 2-1 A, B). Exon 6A encodes a sequence in the N-terrninal region, but is found in the middle of exon 6 which encodes the linker connecting domain I and II, suggesting a trans-splicing event. Exon 4A encodes part of the loop sequence connecting $5 and S6 in domain I. Exons 11A, 11B, and 110 are three tandem exons that encode part of the linker connecting domains II and Ill. Exons 17A, 17B, and 17C are another group of tandem exons encoding part of the linker connecting domains Ill and IV as well as part of IVS1. Exon 16A encodes part of the loop connecting S5 and S6 in domain Ill. Finally, exon 19A encodes a sequence in the C-terminal region. Exons 4A, 11C, 16A, 17A, 17B, 17C, and 19A have the consensus GT and AG sequences at the 5’ donor and 3’ acceptor sites, respectively, whereas exon 6A lacks the consensus GT and AG sequences. The donor sites of exons 11A and 11 B do not have the GT sequence. Based on optional exon usage, the 20 cDNA clones can be grouped into nine unique splice types, named DSC1-1 to DSC1-9 (Fig. 2-1 C). These cDNA clones belonging to the same splice type contain additional scattered amino acid differences (see below) and are named according to the splice type. For example, the six cDNA clones in splice type 1 are named DSC1-1.1 to DSC1-1.6. The nucleotide sequence of DSC1-1.1 has been deposited in GenBank (DQ466888.1) and its deduced amino acid sequence is presented in Figure 2-2. The most common optional exons (17B and 19A) are present in splice type 1 variants (Fig. 2-1 C). Exons 6A and 11B are 19 bp 46 (5’-CTCAACGTAAAAAAACTAA-3’) and 15 bp (5’-A'l'l"l'TCAAGAAGAAG-3”) long, respectively. The nucleotide sequences of other longer optional exons are deposited in GenBank (HM 348600-348607). lntriguingly, exons 4A, 11A, 16A, and 17A contain in-frame stop codons. Inclusion of these exons in DSC1 transcripts create truncated proteins. Type 1 and 2 splice variants, the two most abundant types, do not include these exons and are predicted to produce full-size DSC1 proteins. However, seven other splice types are predicted to encode truncated proteins containing only domain I, domains I and II or the first three domains, because of inclusion of one or two of stop-codon-containing exons (Fig. 2-1 C). In addition, although exon 6A contains 19 nucleotides without a stop codon, inclusion of this exon, as in DSC1-7.1 causes a frame shift in the downstream sequence, resulting in a premature stop codon. The deduced amino acid sequence of DSC1-1.1 was compared with those of BSC1 and para (Fig. 2-2). The DSC1-1.1 protein shares 52% identity in the overall sequence with BSC1, and 29% with Para. The highest sequence identities are found in the transmemebrane domains with 83%—90% between DSC1-1.1 and BSC1 proteins, and 48% -54% between DSC1-1.1 and Para proteins. The sequence identities of the first intracellular linker connecting domains I and II and the second linker connecting domains II and III are less than 30% between DSC1-1.1 and BSC1 proteins. It is worth mentioning that the second linker of the DSC1-1.1 channel is much longer (838 amino acids) than those of BSC1 and Para, which have 330 and 257 amino acids, respectively. Interestingly, the linker connecting domains Ill and IV is highly conserved 47 between DSC1-1.1 and BSC1 (90% identity) or Para (52% identity). It is well established that this linker, particularly the IFM (mammals) or MFM (insects) motif in the middle of the linker, is critical for fast inactivation of sodium channels (Catterall, 2000). Corresponding to the MFM motif in Para, the DSC1-1.1 and BSC1 channels have “VFL” and “MFL,” respectively. In comparing the deduced amino acid sequence of our 20 cDNA clones with the deduced amino acid sequence of DSC1 in GenBank (NM_166696.2), we found 9 to 27 scattered amino acid changes in each cDNA clone. An A-to-l editing site in the linker connecting domains Ill and IV was previously reported in DSC1 transcripts (Hoopengardner et al., 2003). This editing event occurred in 13 clones, including DSC1-1.1 resulting in a M2027V change in the VFL motif (Fig. 2-1). The other seven clones contain the conventional MFL motif. Further molecular analysis is required to determine whether other nucleotide differences in these clones are the result of RNA editing or errors introduced during RT—PCR. In additional, nucleotide substitutions introduced premature stop codons in four clones, two of which also contain stop-codon-containing exons. A six nucleotide sequence, CCGTCT, was found in all 20 cDNA clones, but not in the genomic DNA sequence (NT_033778.3). This difference might be caused by sequence polymorphisms between fly lines used in the genome sequence project (y, cn bw sp; +; +) and our study (wms). 2.4.2 Functional analysis of DSC1 channels in Xenopus oocytes To determine whether the DSC1 channel is also a cation channel with gating 48 properties reported for the BSC1 channel (Zhou et al., 2004), I examined the 20 DSC1 variants in Xenopus oocytes using two-electrode voltage clamp technique. To elicit DSC1 currents, membrane potential was depolarized to 80 mV for 15 ms from a holding potential of -100 mV and then repolarized back to -100 mV (Fig. 2-3 A). Only three variants, DSC1-1.1, DSC1-1.2 and DSC1-5.1 produced detectable currents (Fig. 2-3 B). None of the 10 variants that encode truncated proteins produced currents using this recording protocol. I also did not observe detectable currents from seven DSC1 clones that do not contain a premature stop codon. Using Ba2+-containing recording solution, I detected a small outward current during depolarization followed by a large tail current associated with repolarization (Fig. 2-3 B). When Ca2+- or Na+-containing recording solution was used, the amplitude of tail current was smaller. 2.4.3 DSC1 channels are insensitive to TI'X The sodium channel blocker TTX inhibits insect sodium channels at nanomolar concentrations (Catterall, 2000). To determine whether TTX also inhibits DSC1 channels, we recorded DSC1 currents in Ba2+-containing recording solution in the absence or presence of TTX using the recording protocol described above. Even at 10 BM, TI'X did not reduce the amplitude of either the outward current or the tail current, indicating that DSC1-1.1 channel is TTX-insensitive (Fig. 2-3 C). 49 2.5 Discussion In this study, my sequencing analysis of 20 cDNA clones reveals extensive alternative splicing of DSC1 transcripts. A total of 10 optional exons were identified, four of which contain in-frame stop codons. Surprisingly, none of these optional exons is homologous to those identified in the BSC1 gene (Liu et al., 2001). The only alternative splicing site conserved between the two genes results in optional exons 11A-C in DSC1 and optional exons at splicing site B in BSC1 (Liu et al., 2001). Like alternative exon 11A in DSC1, the two optional exons at the splicing site B in BSC1 also contain in-frame stop codons, generating truncated proteins containing the first two domains. Furthermore, transcripts containing these BSC1 optional exons were detected only in leg muscle, suggesting a possibly tissue-specific role of these truncated proteins (Liu et al., 2001). The conservation of this splicing site in two phylogenetically distant species raises the possibility that two-domain truncated proteins might play a role in insect neurophysiology. Interestingly, premature stop codon-containing alternative exons are also detected in voltage-gated sodium and calcium channel genes. For example, transcripts containing two mutually exclusive exons, 18A and 18N, encoding lllS3-S4, are found in mouse Nav1.6 (Plummer et al., 1997). Exon 18N contains a stop codon and transcripts containing it are detected in fetal brain and non-neuronal cells (Plummer et al., 1997). Moreover, this stop-codon containing exon is conserved in sodium channel genes of human, pufferfish, and the German cockroach (Tan et al., 2002). Other sodium channel genes also 50 produce premature stop-codon-containing alternative exons, such as exon 17A in Nav1.2 and Nav1.3, exon 173 in Nav1.3, and exon 16A in Nav1.7 in mouse and human (Kerr et al., 2008). Like exon 18N, the inclusion of exon 17A is tissue- or developmental stage-specific (Kerr et al., 2008). In my experiments, only three of the 20 clones produced detectable currents. While it is not surprising that the 10 truncated DSC1 proteins did not produce any current, it is not clear why 7 clones that contain no premature stop codon did not produce currents. We previously encountered a similar problem with functional expression of BSC1 variants in Xenopus oocytes. While one BSC1 variant gave robust currents (Zhou et al., 2004), no currents were detected from other two BSC1 clones that do not contain premature stop codon (Chung, Liu and Dong, unpublished data). It is likely that unidentified accessory subunits or other chaperone proteins are required for the expression of these variants in Xenopus oocytes. It is also possible that the lack of channel expression from these clones is caused by sequence differences that are independent of alternative exons. I found that the DSC1 channel is permeable to Na+, Ca2+, and Ba2+. These results are similar to those recorded from BSC1 channels (Zhou et al., 2004), suggesting that, like BSC1, DSC1 encodes a voltage-gated cation channel that is more permeable to bivalent cations. Other gating properties, such as voltage dependence of activation and inactivation, of the DSC1 channel remain to be investigated. It is well established that the ion selectivity of voltage—gated sodium channels is determined by the amino acids D, E, K, and A in the pore positions of domains I, 51 II, III, and N, respectively (i.e., the selectivity-filter motif “DEKA”) (Catterall, 2000). In contrast, four glutamic acids (EEEE) at the corresponding positions determine the ion selectivity for voltage-gated calcium channels (Catterall, 2000). It has been shown that a K to E substitution in the DEKA motif in a mammalian sodium channel altered the sodium selectivity to more toward calcium (Heinemann et al., 1992 ). Both the DSC1 and BSC1 channels contain the DEEA motif instead of DEKA or EEEE motifs. Substitution of the second E with K in the DEEA motif reduced the ion selectivity of the BSC1 channel for Ba2+, demonstrating its critical role in modulating ion selectivity of the BSC1 channel (Zhou et al., 2004). It is likely that such a substitution will have a similar effect on the ion selectivity of the DSC1 channel. In sodium channels, the selectivity-filter motif “DEKA” and the outer-ring motif “EEMD” (mammals) or “EEID” (insects) in the pore region of domain I-IV are critical for TTX binding (Catterall, 2000). In DSC1/BSC1 channels the amino acid sequences “DEEA” and “EEIN,” respectively, are found in the corresponding positions. Because the DSC1 channel is insensitive to TTX (Fig. 2-3 C), l speculate that the K to E substitution in the DEEA motif of the DSC1/BSC1 channels might be involved in TTX resistance since a charge-reversal substitution of the K residue in the DEKA motif renders rNav1.2 channels extremely resistant to ‘l'l'X (Catterall, 2000). On the other hand, the N residue in the EEIN motif may not be involved in TTX resistance because an S to N substitution made varroa mite sodium channels more sensitive to TTX (Du et al., 2009a). Besides these two motifs, a non-aromatic residue, cysteine or serine, immediately after the first 52 E of the EEMD motif has been shown to be responsible for TTX resistance of rNav1.5, rNav1.8 and rNav1.9 channels (Satin et al., 1992; Sivilotti et al., 1997). Interestingly, both DSC1 and BSC1 channels have a non-aromatic residue (N359 in DSC1, N366 in BSC1) in this position. Therefore, N359/N366 may also contribute to TTX-insensitivity of DSC1/BSC1 channels. In conclusion, DSC1 gene encodes a voltage-gated cation channel that is permeable to Ba2+, Ca2+, and Na+. DSC1 transcripts undergo alternative splicing just like transcripts of the sodium channel gene para. Different exon usage might be a mechanism in regulating DSC1 expression level or in yielding channels with distinct gating properties to regulate neuronal excitability. 53 Table 2-1 DSC1 cDNA sequencing primers Primer Name Primer Sequence T7 5’- TAATACGACTCACTATAGGG -3’ DSFO 5’- GGCGGAGTACATCTITCTGGCC -3’ DSF1 5’- CCATGCTGACGACATTCCAGC -3’ DSF2 5’- GCCATAGCGCAACTGAACGCC -3’ DSF 3 5’- GGTGGTGATCGATGACCTACCCG -3’ DSF3A 5’- CTCGACGATCCGCGCTCTTGG -3’ DSF4 5’- CAAGAGCGCAAGGATCGCAAGG -3’ DSF5 5’- CTCTI'CGGGAGTGAGTACCC -3’ DSF6 5’- CTTGCAGGAGGAAGAGGAACTGC -3’ DSF7 5’- GCAAGATCGATGAGGACTI'TAGC -3’ DSF8 5’- GCTGGCTGCTCTTACCGAGC -3’ DSF9 5’- CCTCAGAAGTGCTACGACC -3’ DSF10 5’- GGCAAGGAATGCGGATTGTAG -3’ DSF11 5’- GAAGAAGTATGAAGGAGGAG -3' DSF12 5’- CGTTACTTCGGGTGGTCCGC -3’ DSF13 5’- CTTCAATCAGGCGCACCAGG -3’ Zhang77a 5’-TTATCGA'ITGTTI'GGTCGAA -3’ Zhang77b 5’-CGGATCGTTCTTCACACTGA -3’ Zhang77c 5’-AATGGTGCTCCTCAAAATGC -3’ Zhang77d 5’-GGCCTCTATCTTCGGGATTC -3’ 54 Figure 2-1. Molecular characterization of 20 DSC1 cDNA clones. A. The genomic organization of the coding region of the DSC1 gene deduced from the sequence of DSC1-1.1 (Genbank No. DQ466888.1). Solid boxes represent exons. Positions of the alternative exons are labeled with arrows. B. Schematic drawing of the topology of the DSC1 protein indicating locations of alternative exons. All alternative exons are optional. C. Exon usage of 20 DSC1 cDNA clones. Splice variants are named according to the splice types. Variants of each splice type contain sequence differences due to scattered amino acid differences. For example, the two variants in splice type 3 are designated as DSC1-3.1 and DSC1-3.2. A 11A, B, C 17A. B. C 47‘ 9A I "SAW I 1%A l r I W 1 2 3 456 7891011 12 13 1415161 1819 20 & B 6A 4 11A11811C 16A 17A17B 17C 19A * * * * --l-----I----=_-----I----_------ * * rllxllrlkfll'l TIT" D r\ A AA’ {IUD +H3 * * 000' 55 Figure 2-1 (continued) Alternative Exons Splicing 6A 4A 11 11 11 16 17 17 17 19A #of type A B C A A B C clones 1 I - 6 2 I * I - 5 3 I * I - 2 4 --—- 2 * 5 *IZ:II I 1 El * * 6 Im-_- 1 8 . 1 * 9 :ll- I 1 56 Figure 2-2. Alignment of amino acid sequences of DSC1-1.1, BSC1 and Para proteins. Dots represent amino acid residues in B801 and Para that are identical to those in DSC1-1.1. Dashes indicate gaps introduced to maximize sequence alignment. The four homologous domains (I to IV) and six transmembrane segments (S1 to $6) in each domain are marked above the sequences. Exons 17B and 19A are highlighted. Locations of other optional exons (which are not included in DSC1-1.1) are marked with arrowheads. The optional region C in BSC1 is highlighted. Locations of optional regionsA and B (which are not present in BSC1) are marked with arrows. Amino acid residues in DSC1-1.1 and BSC1 corresponding to the MFM motif in Para (i.e., IFM motif in mammalian sodium channels) that is critical for fast inactivation are boxed. The amino acid residues which are important for ion selectivity and TTX binding are indicated with asterisks. The GenBank accession numbers are DQ466888.1, AF312365.1 and M32078.1 for DSC1-1.1, BSC1 and Para, respectively. 57 DSCH-11 1 BSC1 1 para 1 39 48 51 72 81 101 120 129 147 168 177 197 218 227 247 268 277 297 315 322 347 364 371 397 414 421 447 MGDDQAT ----------- FNDEKAVAKHQVVAYTQRSQVKHENRHIQ-LV .ATPASSNPGPSSTM--PTAQKP.PG.A..KPF.KE.LERM..KTV.-.. .TE.SDSISEEERSLFRP.TR.SL.QIE.RI.AEHEK.KEL.RKRAEGE. REYG --------- FH ------- PRTKASVEDGDVLPRKFE-PFPEHMYGK K... --------- .Q ------- . .R.L ..... s. ..G... .SRL. .R PR. .RKKKQKEIRYDDEDEDEG. QPDPTL. Q. VPI. VRLQGS. .PELAST region A—Q, exon 6A v PLEEIDTFIYEE--TFCVVSKRFQKNYIHRFTGTKSLFLFYPWSPARRVC ...... N...D.--.........R........A.N.F.I.S..NAI..T. .D..PY-.SNVL..V....--G.D-.F..SAS.AMWMLD.FN.I...A IS1 ISZ VYIATNQFFDYCVMATILFNCIFLAM-—TETVEEAEYIFLAIYSIEMVIK IFLS...Y...V..I...L..V....--.D ............. TA..I.. I..LVHPL.SLFIIT...V...LMI.PT.P...ST.V..TG..TF.SAV. IS3 IS4 IIAKGFLLNKYTYLRNPWNWLDFVVITSGYATIGMEVGNLAGLRTFRVLR S...}.I ........................................... VM. R. .1. CPF. .DA ......... ALA.V.M.IDL....A ........ IS5 ALKTVSIMPGLKTIINALLHSFRQLAEVMTLTIFCLMVFALFALQVYMGE ...................... K........................... ..... A.V......VG. VIE. VKN. RD. II. .M. S.S....MG..I...V ERNKCVRQVPTD—-WTNVSHTDWQIWVNDTDNWLYDEDELPV-LCGNLTG ....... KI--.--SS.A.DIE.TE....EE...FT.EDE..-I...V.. .TE..IKKF.L.GS.G.LTDEN.DYHNRNSS..YSEDEGISFP. .IS. exon 4A ARHCPFEYVCLCV- -GENPNHGYTNFDNFMWSMLTTFQLITLDYWENVYNM' ...... G. P. .Q. -.........s. .L..................... .GQ.DDD....QGF.P...Y. .s. .s. G. AF. SA. R. M. Q. P. .DL. QL IS6 VLATCGPMSVSFFTVVVFFGSFYLINLMLAVVALSYEEEAEITNEERKKD .PS...I..V ................................ Q...R.. LLDHRDDSTFSFDPSVLNVKKLNKNNKKKIDS ------- RKGVLLASY-- .T ............. .0. D. K. RRRV. ................ TR EEAI. EAE----EAAAAKAA. .EERANAQAQAAADAAAAEEAA. HPEMAK 58 Figure 2-2 (continued) DSC1-1.1 455 BSC1 464 para 493 504 514 543' 533 563 593 566 603 643 597 634 693 623 668 743 657 703 793 707 753 837 756 802 887 806 852 937 SKKKTRRKKTKGGKEGGTNGNGNSSNGDDNKSDSATPSPGPSPRHSATE- .R ..... R.RPRAGGS.SG.EN.NN.NNGQSEHGGSR.AT ...... GSGP .PTYSCISYELFVGGEKG.DDN.KEKMSIRSVEVESE.VSVIQ.QP.PTT ---------- RPSALTM-----------QAQKQYQQMEQQHKLAKSGSGG CPPSAPSPES..HS..LS-PPSVVKGLSNPEQEEEEEQ..EP.QQEEQPP AHQATKVRKVSTTS.SLPGSPFNIRRGSRSSHK.TIRNGRGRFGIP..DR -------- SNTP-----MAPTPKGRISFQDSGMGVKNP--NML--YPSDY -------- QQP.QLQRFLQ.PIAAPAPAEHASDTLHPV--.T.GKLGPPH KPLVLSTYQDAQQHLPYADDSNAVTPMSEEN.AIIVPVYYGN.GSRH.S. ----------- KGQLIANSG-----QPSSNSSGVNRESSQDDSGVVD-~- ----------- R...L.SRH--—--PS.N..DSN.....L.......--- TSHQSRISYTSH.D.LGGMAVMGVSTMTKE.KLR..NTRNQSV.ATNGGT --------- DHEERDTTND--------MGHVSTVELALSPREV--—---- --------- ....G.V.SEDVAQIPNVVQR.EP.TV...SK.I------- TCLDTNHKL..RDYEIGLECTDEAGKIKH.DNPFIEPVQTQT.VDMKDVM ---RLIKCNG-NIARIKNH--NVYALHQE -------- FSSEV--VVIDDL ---.V ..... VSPGHGTQK--HL.T.PSD -------- YL.HI--..LN.. VLNDI.EQAAGRHS.ASDRGVS..YFPT.DDDEDGPT.KDKALE.ILKGI I|S1 PDRNCDRCVHWCTDYESWLQFQNCLYKVVRDPLFELAITLCIVLNTAFLA ...... K. TQC. V. .DG. .R. .SG..........D.L.....I. ..M.. . - VF. VW--DC. W---V. K. .EWVSLI. F. .FV. .F ...... V. .M. M. _ IISZ MEHHGMSESFRNALDVGNKVFTSIFTFECIVKLMALS-KDFFLCGWNIFD ......... V.Q...I..........L..FL.IL...-.EY.A....... .D..D.NKEMERV.KS..YF..AT.AI.ATM....M.P.YY.QE ...... II83 |IS4 LLIVTASLLDIIFELVDGLSVLRGLRLLRVLKLAQSWTTMKVLLSIIIST .I..S ..... LS...M.S.V...C .......... R ............... FI..AL...ELGL.G.Q ...... SF ..... F...K..P.LNL.I..MGR. ||S5 IRALGNLTLILVIVIYIFAVIGMQLFSKDYTP--EKFDPDPVPRWNFNDF .G ...... FV ..................... A--D..Y...I ........ MG ...... FV.C.I.F....M ..... G.N.HDHKDR.PDGDL ..... T.. 59 Figure 2-2 (continued) DSC1-1.1 854 BSC1 900 para 987 904 950 1033 954 999 1070 1004 1012 1070 1054 1041 1106 1104 1057 1118 1154 1060 1131 1204 1078 1131 1254 1088 1148 1304 1097 1148 IISG * * FHSFMMIFRILCGEWIEPLWDCMRAEEEQGASTCFAIFLPTLVMGNFMVL ...... V........T..........KLT..E........A......... M....IV..V ....... SM.. .----YV.DVS.IPF..A.V.I..LV.. exon11A,11B,11Cv W— region B NLFLALLLNSFNSEELKSKKEEVGEESKLARSIERVRDLIRKKRQERKDR D ............................... F..L.SIV....HA-.EK ........ SN.G.SS.SAPTAD-NDTN.I.EAFN,IGR—----------- KERKFAEKFQQIVLDAQQAHAQTLSHQAAVGLERGDKPGVLAETKFHRLS T.NEQNMRLEEM. ------------------------------------- YQESMNRPVSGSDFGFQIPLHDGLHTIVDGLEYDDTGDLPEQIQLQAHAL -H.V.S.HAAEKCYT ----- AS.V.ET.I.RTI.H --------------- FKSWVK.NIAD-C.KL---IRNK.TNQISD---QPS.ERTN.. ------- PPTSDSMPPTYESAMMATTGGSFSSVNGNGTCQNLTPFVQAERRLQHQIS -------------------------------- KRHAAL..ETMS..RA-- --------------------- .WIWSE.K.v.R----------------- SGVSTQQYDSCEEATYTESIELRLLGQYNSTDTDPYANDQRSGCGSFNRG ----------------------------------------------- Q.Q ---------- .IS.EHGDN-..E.----------—--------------- DSLQDNSSRRYGSEEHDEAFLKYQKSLLTRSPSYRKSLDRLSQSSGQSQR QQE.Q.DGS..NPDAENN -------------------------------- SLLKSEEAEMRRHSSGQSLNSMSIEQDELLSQQGNLREELLNCDQKELFQ ------------------------ .DEDRNG.HS—--------—------ ------------------------------ GHDEI.ADG.IKKGI..-—- FLQEEEELQKGTKLRRISNVMRSRRPSSQMGQPENETMVEHSEFDNIIQS ----------------------------------------- PT.PRVVN. 60 Figure 2-2 (continued) DSC1-1.1 1354 33011104 para 1160 1404 1105 1160 1454 1122 1185 1504 1128 1201 1554 1137 1201 1604 1150 1219 1654 1195 1238 1703 1245 1262 1750 1288 1298 1800 1338 1348 AADEVVLPLNPYDSYDLSSVPRRSQSVSAAAQRQSVKLKRRSLEKQRKID EDFSISNEIRKICDQIHAPFVAMEAMAVAATSASQAQPNQSPFLRRKVDP RKY.V. ------ .HEN --------------------------- .NKHMED -E.T.HGDMKN ------------------------ NK.KK.KY.NNAT.D FTVQFDRFKRLSLIERVEEVPEEEKPISTLRIESEKMPRKFLHGPDQLRL -------- ..Y..V---------—---------------—---------- D.ASINSY ------------------------- GSHKN.P. --------- DSLSLKSTNSYENLLIQKQKLGMATPPAVPATPPTSLKSSIEPPTLAQIS ---------------------------- L.KSAQSA.------------- SLKTTPPLAALTEHQQHFHATSIQAAPTPAHTHAHSQAHAHSMAGQRRRM ------------------------------ .DEDNK.SEDSVL------— ------------------------------- KDESHKGS.ET.E.EEK.- EHPQSTLDKAASFQSARTESHSSGAADASSALALAMAQKTEQSQSTAPDA ---HPLV..RT.LTTQQ...KDESNPEE.--IE.EVLD.APGETE.MLPP --------------------------- DA.KED.GLDEELDEEGEC---- region C TQKPSAFTRLTEKPWHCLVSCVDDLTVGGRRNSQGAYNDPM-TFPSYGAT AET.KKTRSI.K...NA.APY..E.......D..-H.V.G.GS..GEARN ---------------------- EEGPLD.DIIIHAHDE.ILDEY.A---- KAAKVPDDCFPQKCYDHFYFRCPWFMSC---MDTQSAKHWTRVRTAVLTV .TV...Q.....H..Q----..---LC.DKYLE.PCGQR..HI..Q..S. ------- ..C.DSY.KK.----.-ILAGDD--.SPFWQG.GNL.LKTFRL IHS1 IHSZ VDTPAFEWFVLVLIFASSITLCFEDINLDKNKALKRVLYWINFSFCLIFV ....V....I ................ Y..Q.LV..N....T.LG..AL.S IEDKY..TA.ITM.LM..LA.AL..VH.PQRPI.QDI..YMDRI.TV..F |I|S3 VEMILKWLALGFSKYFTSFWTILDFIIVFVSVFSLL--IEENENLKVLRS I..M ........ WR ................. I....--M ........... L..LI ....... KV.L.NA.CW...V..M..LINFVASLVGAGGIQAFKT 61 Figure 2-2 (continued) IHS4 IHSS DSCH-1J 1848 LRTLRALRPLRAISRWQGMRIVVNALMYAIPSIFNVLLVCLVFWLIFSIM 33011386 .................................................. para 13 98 M ........... M . M V ..... VQ ............. I ..... A exon 16Av 1898 GVQFFGGKFFKCVNEMGELLPITEVNDKWDCIEQNYTWINSKITFDHVGM 1436 ............. DDE.N....SV.DGMLE.EDK..S.V ....... N..N 1448 .L.A..Y...EDMN.TK.SHEIIPNRNA.ESE....V..AMN ..... N * . IHS6 1948 GYLALLQVATFEGWMEVMADAVDARGVDLQPQREANLYAYIYFVIFIVCG 1486 A....F ................. E...NM ........... L...V ..... 1498 A..C.F ..... K..IQI.N..I.S.E..K..I..T.I.M.L...F..IF. exon 17Ay ‘ exon 17B 1998 SFFTLNLFIGVIIDNFNMLKKKYEGG FLT SQKHYYTAMKKLGRKK 1536 ............................................. 1548 ................. EQ...-A s M M D. K s M s exon 17C y 2048 FQKVIKRPINHFLAMFYDLSNSRRFEIAIFVLIFLNMLTMGIEHYDQPHA 1586 ........ M.QV..T .............................. Q.... 1597 .L.A.P..RWRPQ.IVFEIVTDKK.D.I.MLF.G...F..TLDR..ASDT IVSZ IVS3 2098 VFFILEVSNAFFTTVFGLEAIVKIVGLRYHYFTVPWNVFDFLLVLASIFG 1636 ........................ I ............ L ...... v...L. 1647 YNAV.DYL..I.VVI.SS.CLL..FA ...... IE...L..VVV.IL L IVS4 2148 ILMEDIMIDLPISPTLLRVVRVFRIGRILRLIKAAKGIRKLLFALVVSLP 1686 ......... F.V ...................................... 1697 LVLS..IEKYFV .......... AKV..V...V.G ..... T ..... AM... IVSS 2198 ALFNIGALLGLITFIYAILGMSLFGNVKLQGALDDMVNFQTFGRSMQLLF 1736 ......... A ........ I...V..H..Q .......... E .......... 1747 ..... CL..F.VM..F..F...F.MH..EKSGIN.VY..K...Q..I... 2248 RLMTSAGWMDVLESLMIQPPDCDPFIHGHT-NGNCGHPLLAITYFTSFII 1786 ........... VG ....... N.N.TYNNQP-..D..S ............. 1797 QMs ..... DG..DAI-.NEEA...PDNDKGYP....SATVG..FLL.YLV IVS6 exon 19A 2297 ISYMIVINMYIAIILENFNQAHQEEEIGIVEDDLEMFYIRWSKY ------ 1835 ............................................ DPHATQ 1846 .FL ........ V....YS..TEDVQE.LTD..YD.Y.EI.QQFDPEGTQ 62 Figure 2-2 (continued) DSC1-1.1 2341 ._;_ ------------------------------------------------ BSC” 1885 FIRFSQLSDFIASLDPPLGIPKPNTVALVSFNLPIARGNKIHCLDILHSL para 1 8 9 6 YIRYDQLSEFLDVLBPPLQIHKPNKYKI I SMDI PICRGDLMYCVDILDAL 2341 1935 1946 2345 1981 1987 2366 2031 2027 2381 2081 2063 2400 2130 2107 2400 2180 2148 2403 2230 2181 2406 2280 2181 .----11---______-; --------- 4‘-------4--4---+-—-vrss VKYVLGH----VEETDDFKKLQDQMDIKFKKQFPTRKELEIVSSTRIWKR TKDFFARKGNPIEETGEIGBIAAR --------- PDTEGYEPVSSTLWRQR DSRPNRRI----4--;¥-----4------+ ------- SPKAARQTIQRTL QDKAA.T.QGAFREYIRLKREREREPLDLEDEMTQTS..GGGW.SRLSAF EEYCA.L.QHAWRK--HKARGEGGGSFEPDTDHGDGGD.D.G -------- LTIP ------------------- SDLLADTIHMP ---------------- .HVHRGSRASSRKSSRASDASEL.E.GGAWLNL.LLFLSGAHQGQTEDLL ---DPAPDEATDGDAPAGGDGSVNGTAE ----------- GAADADESNVN ------------------------- PNLY--TSRNCPILLPLSIHH-—-- DPGHGSNGNSNVCITVSEPSPDT-G.P.NEE.KPSTSMV..V.VKDELRE SPGEDAAAAAAAAAAAAAAGTTTAGSPGAGSAG.QTAV.V ------ ESDG PLTVGDVSILVTQPSPEGAGVPVDRGTEDRRPSNSSGESFHQIDSSESRT FVTKNGHKVVIHSRSPSITSRTADV-.ARPRPPLQDARE-Y.H ------- ----------------------------------------------- WAP MPGVETTLSSSGTTTAALDRHPLGLLRPVGTVLSLFPMQLGNGGDLGST. EYVLDVACCISDAAAT-VS.QQ ------- QQQGE-QQA.--FSA ------ RSP ——————————————————————————————————————————————— .K.ASEAVTGPVDMHPLRVRPGTAFSLPPSEIVKPLAKSSPERRRARSRR _________ IM—————--—-----SL. RPSDGTLVRVLVHRESEESNDDKG.-.W --------- ...KNTIQTSKQQHKKNNKQTRIN 63 Figure 2-3. DSC1 currents recorded from Xenopus oocytes expressing DSC1-1.1 channels. A. Recording protocol consisted of a 15 ms depolarization from -100 mV to 80 mV followed by repolarization back to -100 mV. B. Superimposed Na+, Ca2+ and Ba2+ currents recorded from an oocyte four days after injection with 3 ng DSC1-1.1 cRNA using the protocol illustrated in A. C. Superimposed . + . . . . DSC1-1.1 currents recorded usmg a Ba containing recording solution from an oocyte two days after injection with 3 ng DSC1-1.1 cRNA before and after exposure to 10 nM TTX using the protocol illustrated in A. A 15ms 80mV -100mV _ B 10- 332+ . LI Ca2+ 5- Na+ ’m‘ . 5 4-0 0" E c 92 . S 0 -5- -10 W.j 5 1015 20 25 30 35 Time (ms) 64 Figure 2-3 (continued) w/oTTX 10‘ F —10p.MTTX :a. 5‘ 75' 0‘ W + Na: -5- m - O ............ 51015 20 25 30 35 Time (ms) 65 CHAPTER 3 BEHAVIORAL AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF DSC1 KNOCKOUT MUTANTS 66 3.1 Abstract The Drosophila Sodium Channel 1 (DSC1) gene encodes an ion channel protein that is homologous to voltage-gated sodium channel 0 subunits. However, functional study in Xenopus oocytes shows that DSC1 is a voltage-gated cation channel that is permeable to both sodium and calcium ions. The physiological role played by the DSC1 channel in insects is not clear. In this study, DSC1 mutant D. melanogaster lines were generated and tested for phenotypic changes at both behavioral and electrophysiological levels. Behaviorally, DSC1 knockout flies jumped more frequently during heat shock compared with wild-type flies although they have comparable kinetics of temperature-induced paralysis. Moreover, the locomotor activity of DSC1 knockout flies recovered significantly more slowly than wild-type flies and failed to recover completely following a 40-min recovery period after heat shock. DSC1 knockout flies also jumped more frequently and had a shorter life span than wild-type flies when they were subjected to starvation. Electrophysiological study of the giant fiber (GF) pathway showed that the long latency refractory period of DSC1 knockout flies was similar to that of wild-type flies at room temperature but was shortened more dramatically in response to heat shock and starvation. These results provide strong evidence for a critical role of the DSC1 channel in regulating neuronal activities and associated behavior in response to environmental stresses. 67 3.2 Introduction Voltage-gated ion channels are a class of transmembrane proteins located on the surface of every excitable cell. Modulation of the membrane potential causes voltage-gated ion channels to open, permitting current, in the form of ions, to pass across the membrane. The ion component is determined by the ion selectivity of the channel. The current mediated by a channel protein in turn depolarizes or hyperpolarizes the membrane potential and facilitates cellular functions, such as action potential (AP) initiation and propagation and neurotransmitter release. Voltage-gated ion channels can be classified into several groups based on their ion selectivity and play important roles in modulating specific cellular functions. For example, voltage-gated sodium channels are responsible for the rising, or depolarizing, phase of an AP, whereas voltage-gated potassium channels mediate the falling phase of an AP (Hodgkin and Huxley, 1952). There are nine voltage-gated sodium channel genes in mammals (Yu and Catterall, 2003). In contrast, only one confirmed sodium channel encoding gene, para, has been reported in D. melanogaster(Loughney et al., 1989). The Drosophila Sodium Channel 1 (DSC1) gene was identified over two decades ago (Salkoff et al., 1987). The deduced amino acid sequence of DSC1 was highly similar to those of sodium channels, including the Para sodium channel. RNA in situ hybridization studies indicated that para is expressed in the central nervous system (CNS) and peripheral nervous system (PNS) at all developmental stages, whereas DSC1 is expressed only in a few neurons in embryonic and larval stages. The expression of DSC1 almost overlaps with that 68 of para in the CNS in pupae and adult stages (Hong and Ganetzky, 1994). An immunohistochemical study showed that the DSC1 protein is found only in synapse regions and axonal processes, but not in the cell bodies of the cortex, where Para is found. DSC1 is also widely distributed in the PNS, such as motor neurons in thorax muscles, neuromuscular junctions, and sensory neurons in compound eyes, antenna, proboscis, and tibia (Castella et al., 2001). Electrophysiological analysis of BSC1, a DSC1 orthologue from cockroaches, revealed that BSC1 is not a sodium channel, but the founding member of a novel family of voltage-gated cation channel with ion selectivity toward Ca2+ (Zhou et al., 2004). My work described in Chapter 2 showed that the DSC1 channel mediates currents characteristic of those conducted by the BSC1 channel. Despite these advances, the physiological role of BSC1/DSC1 channels in insects remains elusive. In this study, I performed behavioral and in vivo electrophysiological characterization of two 0801 null mutants. My data reveals a critical role of the DSC1 channel in regulating neuronal activities and associated behavior in response to environmental stresses. 3.3 Materials and Methods 3.3.1 Generation of DSC1 knockout lines As illustrated in Fig. 3-1, total DNA was isolated from Drosophila and ~4.5 kb genomic DNA fragments (from primers (5’-AGCAGCGGCCGCAACAGA 69 ATATCTCCGGTTGCC-3’) and (5’-TACCGGTACCAGATTI'GACGGACTCAC OTC-3’) and primers (5’- AGCAGGCGCGCCTAATGGCTCTGCAGGAACG-3’) and (5’- TACCCGTACGTCACCTCTCTCCAGACCAAC-3’) containing DSC1 gene were PCR-amplified using platinum Taq DNA polymerase High Fidelity (lnvitrogen, lnc., Carlsbad, California). The DNA fragments were cloned into PCR2.1 vector (lnvitrogen, Inc., Carlsbad, California), respectively. One stop codon was introduced into both the upstream and downstream fragments by PCR from primers (5’-CGTCATGGCCACCATTCTGTTC-B’) and (5’-CGTTAGTCGAAG AACTGGTTGG-3’) and primers (5’- TTCTGTTAAACACAGCCTTTTTGGCC-3’) and (5’- TTCAAAGTGTGATGGCCAGCTC-3’) with Pfu DNA polymerase (Agilent Technologies, Inc., Santa Clara, CA), and a Mlu l or an EcoR I cleavage site was introduced together with each nonsense mutation, respectively. The upstream stop codon terminates translation after the first 129 amino acids that only cover the N-tenninal of the DSC1 protein. The downstream stop codon results in a DSC1 protein truncated after the 687th amino acid that only contains the first two domains and part of the second intracellular linker. The upstream fragment and the downstream fragment were then cloned into the pW25 vector (generously provided by Kent Golic) through Notl and A0065! sites, and BerI and Ascl sites, respectively. Two DSC1 knockout lines, DSC1-3 and DSC1-6, were generated using an ends-out targeting technique (Gong and Golic, 2003). The mini-white (w+) marker gene in the donor plasmid provides additional enzyme sites for identifying correct gene targeting events. DSC1 knockout was confirmed by Southern blot analysis 70 and sequencing of the region spanning the downstream premature stop codon (Fig. 3-1). The two initial DSC1 knockout founder lines, DSC1-3 and DSC1-6, were back- crossed to w1118 for five generations to generate 08018 and DSClb, respectively. W1118 was used as the wild-type control in all experiments. Flies were fed on regular cornmeal-molasses—agar medium and raised at room temperature with 70% humidity and under a 12-hr light/dark cycle. 3.3.2 Climbing assay An adult fly climbing assay was adopted from a previous study (Godenschwege et al., 2009). Briefly, five- to seven-day-old flies were collected and slightly immobilized by 002. Ten flies (five males and five females) were transferred into a 95XZ5 mm plastic vial (Genesee Scientific, Inc., San Diego, CA). After the flies had acclimated for 30 minutes at room temperature, another empty vial was placed upside-down on top of the first vial. The rims of the two vials were aligned and sealed so that the flies could climb into the top vial freely. The bottom vials were tapped gently and files were given 30 seconds to climb. The number of flies that climbed into the top vial was recorded, and a climbing score was given. For example, if there were eight flies that climbed into the top vial, the climbing score would be eight. This procedure was repeated five times for each group of flies and mean values were calculated for statistical analysis. 71 3.3.3 Heat shock assay Flies were collected and transferred as described above (3.3.2). To avoid using damaged flies, a climbing assay was performed and only flies that could climb into the top vial were collected for the heat shock assay. Flies (10 flies in each vial) were incubated in a 40°C humidified hybridization oven (Hybaid, Thermo Scientific, Inc. Waltham, MA) with a glass front door which allowed direct observation of fly behavior during heat shock. The number of paralyzed flies was counted every minute during the heat shock period (15 minutes). Paralysis is defined as loss of the ability to walk. Percentage of paralysis was calculated. Additionally, the number of jumps in each vial (1O flies) was recorded from the 5th to 10th minutes during the heat shock period. A jump was defined as taking off from the wall followed by landing on the bottom of the vial. 3.3.4 Recovery assay Flies were returned back to room temperature after heat shock. A modified climbing assay was performed at various time points to determine the recovery of flies. The flies were tapped down to the bottom and given 30 seconds to stand up and climb. The number of flies that could climb on the wall of the vial was recorded. The assay was performed every 2 minutes during the first 20 minutes and every 5 minutes during the second 20 minutes of the recovery period. At the end of the recovery period, the locomotor activity was recorded after a brief vortex (Vortex- Genie 2, VWR Scientific Products, West Chester, PA) at the highest speed for 1 minute. 72 3.3.5 Starvation assay The method for this assay is similar to that described in the study done by Walker et al. (Walker et al., 2006). Thirty, two- to three-day-old, male flies were evenly divided into six groups and raised on regular fly food for another two days before being transferred to vials containing 0.5% agar (five ml) instead of food. Flies were kept in the agar-containing vials to determine lifespan. The number of live flies was counted every 3-8 hours for six days. Percentage survival was calculated. A jumpy phenotype similar to that induced during the heat shock was elicited by a single bang of the vial on the bench and videotaped. The number of jumps per vial (five flies) was counted during the second to tenth seconds after tapping. The jump phenotype was examined every day for three days. 3.3.6 Giant fiber system (GFS) recording Giant fibers (GF) are a group of neurons which are associated with the animal escape response in many invertebrates and some lower vertebrates (T rimarchi and Schneiderman, 1995b; Trimarchi and Schneiderman, 1995a). The large axons of the giant fibers extend from the brain to the thoracic ganglion. In the thoracic ganglion, the terminal of giant fiber axons forms synaptic connections with two different neurons: a large motorneuron that innervates the tergotrochanteral muscle (TT M) and a peripherally synapsing intemeuron (PSI). The PSI axon cross the midline and synapses with another motor neuron which innervates a group of muscles called dorsal longitudinal muscles (DLM). Thus, the 73 entire circuit, including the giant fiber neurons, intemeurons, motor neurons, and muscle cells, is termed as the GF system and is illustrated in Fig. 3-2 (Engel et al., 2000). Methods for recording GF-driven muscle potentials with electrical stimulation were adopted from the study of Engel and Wu (Engel et al., 2000). Briefly, adult flies were exposed to ether for 10 to 20 seconds before being affixed on the top of a wire mount that was glued behind the neck of the fly. The fly was placed in a position that does not interfere with wing or limb mobility. All stimulating and recording electrodes were uninsulated tungsten wires sharpened by electric etching. Stimulating voltage pulses (0.1-ms duration) generated by a Grass S88 stimulator (Grass Technologies, Inc., West Warwick, RI) were delivered with two stimulating electrodes that were inserted just beneath the cuticle of left and right compound eyes, respectively. The muscle potential of the number 3 dorsal longitudinal muscle (DLM) fiber was recorded with tungsten electrodes inserted beneath the cuticle at the middle point between the midline and the root of the anterior dorso-centrals. A ground electrode was inserted into the abdomen. Muscle responses were amplified by a DAM50 DC amplifier (World Precision Instrument, lnc., Sarasota, FL) and converted to digital signal by a Digidata 1440A (Axon Instrument, lnc., Foster City, CA) coupled with Clampex 10.2 and Clamfit 10.2 software (Axon Instrument, Inc., Foster City, CA) . The GF pathway can be triggered from different neurons by different stimulation intensities (Fig. 3-2). The time interval between the stimulus and the first muscle potential is termed the response latency. It reflects the time required 74 for the stimulation signal to conduct along neural circuits from the activated neuron to the innervated muscle, DLM. Based on the length of latency, the responses can be grouped into two classes, long latency (LL) and short latency (SL) responses. When a relatively low-strength stimulus (i.e., low voltage) is applied, a muscle potential with longer latency is usually elicited. As the strength of the stimulus becomes higher, the short latency muscle response is triggered. The lowest voltage by which the long latency response is elicited is termed the long latency threshold and the lowest voltage to elicit the short latency response is called the short latency threshold. If the fly was given two pulses with the same voltage amplitude and duration time but varying intervals, the longest interval at which muscle potential is elicited by the first pulse but not by the second one is termed the refractory period, which is an indication of the stability of the GF circuit (Tanouye and Wyman, 1980). The refractory period of the long latency response was tested using the mean value of the long latency threshold and the short latency threshold. The short latency refractory period was tested using stimuli at 30 volts, which is high enough to elicit the short latency in all flies examined. 3.3.7 Statistics Student’s t-test was used to analyze the jump phenotype during heat shock and the climing assay resuts. Two—way analysis of variance (ANOVA) with Tukey’s test employed as the post hoc test were used to analyze the data of GF recording. P < 0.05 was set as the criterion for statistical significance. 75 3.4 Results 3.4.1 DSC1 knockout flies exhibit an abnormal jumping response during heat shock DSC1 knockout flies exhibited normal locomotor activity at room temperature (Fig. 3-3A). However, in this study, I examined a possible role of DSC1 in insect stress responses. First, I evaluated the sensitivity of DSC1 knockout flies to heat shock. Flies were incubated at 40°C for 15 minutes and their responses recorded at different time points. All flies, wild-type and mutant, were paralyzed at the end of the 15-min heat shock period (Fig. 3-3B). The paralysis curves of W1118, D8018 and DSC1b overlapped, indicating that knockout of DSC1 did not affect heat-induced paralysis kinetics. However, a distinct ‘jumpy’ phenotype was observed during heat shock, particularly from the fifth to tenth minutes. DSC1 knockout flies were significantly more jumpy than W1118 flies (Fig. 3-3C). 3.4.2 DSC1 knockout flies showed a defect on recovery from heat shock To test if DSC1 knockout mutants are altered in recovery from heat shock, the vials containing paralyzed flies were returned back to room temperature at the end of the 15-min heat shock period. As shown in Fig. 3-4, wild-type flies recovered significantly faster than the DSC1 knockout flies of both DSC18 and DSC1b lines. At 10-min time point, 80% of the wild-type flies, but only about 40% of DSC1 knockout flies had resumed climbing the wall of the assay vial following heat-induced paralysis. It took another 30 min for DSC1 knockout flies to 76 achieve 80% - 85% recovery (Fig 3-4). 3.4.3 Abnormal jump response and shortened life span of DSC1 knockout flies during starvation To test if DSC1 knockout mutants are defective in other stress responses, we examined the response of DSC1 knockout mutants to starvation. Flies from both 08018 and W1118 lived on 0.5% agar for three days. After 24, 48, and 72 h, the number of jumps by 08018 flies was significantly higher than those of wmaflies (Fig. 3-5A). Beginning on day 4, both 08013 and W1118 flies began showing mortality. At day 5, 53% of the DSC1a flies had died, but only 33% of the W1118 flies. Within 12 h (day 5.6), all the 0301a flies had died, yet 13% of W1118 flies were still alive, indicating a shorter life span of D8018 flies during starvation (Fig. 3-5B). To determine whether this difference was caused by differences in body 1118 0 mass between DSC1a and W1118 flies, we weighed 40 individuals of w r DSC1a (20 male flies and 20 female files). No significant difference was found in body weight between W1118 and DSC1a flies (Fig. 3-50). 3.4.4 Reduced long latency refractory period (LLRP) in response to heat shock in DSC1 knockout flies The abnormal jumpy phenotype of DSC1 knockout flies during heat shock 77 and starvation suggests that the DSC1 channel may be involved in regulating stress-associated neuronal activities. To test this hypothesis, we examined the effects of the DSC1 knockout on electrical signaling in the GFS. The GFS was chosen for two reasons. First, the GFS is a well-characterized and major neural circuit (Fig. 3-2) that mediates jump and flight responses in Drosophila (T rimarchi and Schneiderman, 1993). Second, DSC1 proteins are densely expressed in GF neurons (Castella et al., 2001). In this study, I measured the long latency and short latency responses of DLMs evoked by brain stimulation of different intensities (see the Materials and Methods for the details). No differences were detected in the latencies or the refractory period of long or short latency pathways between W1118 and DSC1 knockout flies at room temperature (Fig. 3-6, Table 3-1, 3-2, 3-3, 3-4.). To examine whether the abnormal jumpy phenotype in the DSC1 knockout flies is caused by altered electrical activities of the giant fiber circuit, I compared the activities of the GFS of 08018, and wm8 flies. Specifically, I measured the long latency and short latency responses of DMLs at 5 min, 10 min and 15 min of the 15-min heat shock and determined the response latency and refractory period of both short and long latency pathways. The SL was significantly shortened after 5 min of the heat shock, but gradually increased over the rest of the heat shock period for both mutant and wild-type flies (Fig. 3-6A, Table 3-1.). The short latency refractory period (SLRP) was gradually increased for the duration of heat shock. The SLRP was significantly greater after 15 min of heat shock than it was at RT for both W1118 and DSC1a flies. However, 78 no differences in the response latency or refractory period of the short latency pathway were detected between W1118 flies and D8018 flies (Fig. 3-63, Table 3-2.). The LL was also shortened after 15 min of heat shock for both DSC1a and W1118 files; but no difference was found between Wm8 flies and DSC1a flies (Fig. 3—60, Table 3-3.). The long latency refractory period (LLRP) of DSC1a and w1118 were reduced by heat shock. Interestingly, the LLRP of DSC1a flies was significantly shorter than that of w1118 flies, measured at the fifth and tenth minutes of the heat shock treatment (Fig. 3-6D, Table 3-4.). 3.4.5 The heat-induced reduction of LLRP was rapidly reversed for W1118 flies, but not for DSC1a flies The heat stress-induced changes in the latency and refractory periods of the short latency pathway (Fig. 3-7A and B) and the latency of the long latency pathway (Fig. 3-7C) were readily reversed when DSC1a and W1118 flies were returned to room temperature (Fig. 3—7A, B, C, Table 3-5, 3-6, 3-7). Similarly, the heat stress-induced LLRP reduction of wma flies was almost completely reversed during the first 10 min of recovery at room temperature (Fig. 3-7D, Table 3-8). However, the recovery of LLRPs of DSC1a flies following heat shock 79 lagged significantly behind that of W1118 flies. Furthermore, LLRPs for 08018 flies did not recover fully from heat stress by the end of the 40 min recovery period at room temperature (Fig. 3-7D, Table 3—8). Based on my behavioral assay (Fig. 3-4), at the 40-min time point, the locomotor activities of DSC1a flies seem to be fully recovered from the heat shock. To determine whether the remaining LLRP reduction at the end of the 40 min recovery from heat shock could alter the locomotor activity of DSC1a flies, we vortexed flies in vials for 1 min at the highest speed and then immediately examined the locomotor activity. We reasoned that the stimulation introduced by a brief tapping of the vial in the locomotor activity assay (See 3.3.4) may not have been sufficient to reveal subtle behavioral defects. Indeed, the climbing scores of DSC1a flies were significantly lower than that of w1118 flies (Fig. 3-7E) indicating that stress-induced deficits in locomotor activity of DSC1a flies had not fully recovered to the level of W1118 flies even after 40 min of recovery at room temperature. 3.4.6 The long latency refractory period (LLRP) of DSC1 knockout flies was reduced by starvation Both wma and 0301 knockout flies did not exhibit mortality in the first three days of starvation. From the 4th day, both genotypes started showing some mortality. Therefore, for my experiments to examine the GFS, I used flies that 80 had been starved for 3 days. No difference in the short latency pathway was found between w’"8 and DSC1a flies following starvation (Fig. 3-8A and 3-8B). 1118 a A slight, but significant reduction in the long latency was detected for w nd DSC1a flies as a result of starvation, however, there was no difference between the two lines (Fig. 3-80). Interestingly, the LLRP was significantly shortened by starvation for 08018 flies, but not for w’"8 flies (Fig. 3—8D, Table 3-9.). 3.5 Discussion DSC1 was identified as a putative sodium channel gene more than two decades ago based on sequence similarity to mammalian and insect voltage sensitive sodium channels (Salkoff et al., 1987). Functional characterization, however, revealed that the DSC1 channel is a novel voltage—gated cation channel (Chapter 2). Despite this exciting finding, the physiological role of the DSC1 channel in insects remains a mystery. The successful generation of two DSC1 knockout lines, using gene targeting via homologous recombination, made it possible for me to address the physiological role of DSC1 in Drosophila. In this chapter, I described several behavioral and electrophysiological characteristics of the DSC1 knockout lines. Under standard laboratory rearing conditions DSC1 knockout flies are viable and exhibit no obvious behavioral defects, indicating that the DSC1 channel does not play an indispensible role in electrical signaling under these conditions. To determine whether DSC1 knockout flies can tolerate adverse environmental 81 conditions, I examined their responses to heat shock and starvation. Interestingly, I observed abnormal behavior of the DSC1 knockout flies in response to both heat shock and starvation. The DSC1 knockout flies jumped more frequently than wm8 flies under these conditions. To understand the mechanism underlying this behavioral defect, I examined the GFS that expresses the DSC1 channel and mediates jump response, specifically the long and short latency pathways of DLMs. The neuronal activities of the GFS were examined at room temperature, after heat shock for 5 min, 10 min, and at the end of the 15—min heat shock. Consistent with the behavioral results, no difference in the neuronal activities of the GFS was detected between w’"8 and DSC1 knockout flies at room temperature. The LLRP was reduced in wild-type flies following heat shock at 40°C, in agreement with a previously published report (Elkins and Ganetzky, 1990). Interestingly, the LLRP of the DSC1 knockout files is significantly shorter than that of W1118 flies at 5 min and 10 min during heat shock. The reduction in LLRPs in DSC1 knockout flies during the heat shock correlates well with the occurrence of the abnormal jumpy phenotype. Refractory period controls the frequency of action potentials; therefore the reduction of LLRP could increase the frequency of firing in the long latency pathway. Therefore, I predict that the reduction in the LLRP could enhance thoracic jump muscle activities, which could lead to the jumpy behavior of DSC1 knockout flies. The reduction in LLRP was also observed in starved DSC1 knockout files that exhibit the jumpy phenotype, further supporting my prediction. 82 1118 a Although at the end of the 15-min heat shock, the LLRPs of both w nd DSC1 knockout flies were reduced to the same level, the LLRP of W1118 flies returned to the initial level within a few minutes after the flies were transferred back to room temperature. In contrast, the recovery of LLRP of DSC1 knockout 1 files lagged significantly behind that of w" 8 flies. In fact, after a 40-min recovery from heat shock, although the locomotor activity of the DSC1 knockout flies was recovered, the LLRP of DSC1 knockout flies was restored to only 75% of the original level before the heat shock. These results indicate that recovery from heat shock is mediated by neural circuits other than the GFS, suggesting the involvement of the DSC1 channel in other neural circuits as well. The requirement of the DSC1 channel for maintaining the neuronal excitability under heat shock highlights an important role of DSC1-regulated neuronal circuits (and possibly other cellular processes) in insect stress responses. In this study, I also observed jump behavior and reduced tolerance of DSC1 knockout flies when they were challenged with another stressful condition, starvation, although there is no difference in the biomass between W1118 flies and DSC1 knockout flies. In the past several decades, extensive studies have been carried out to understand the starvation responses, mainly focusing on metabolic pathways (Piper et al., 2005; Rion and Kawecki, 2007). Little information is available regarding the involvement of ion channels in regulating tolerance to starvation. Shahidullah et. al. (2009) showed that a Drosophila potassium channel binding protein, Slob, modulates fly tolerance to starvation, probably through regulating 83 neuronal excitability (Shahidullah et al., 2009). I found in this study that the LLRP of DSC1 knockout flies was shorter than that of W1118 flies after 3 days of starvation, indicating a relationship between enhanced neuronal excitability and reduced starvation resistance in DSC1 knockout flies. However, how starvation shortens the LLRP and whether there is a direct connection between the shortened LLRP and altered starvation tolerance requires further investigation. In summary, heat shock and starvation both enhance the neuronal excitability of the GFS in D. melanogaster. These stress conditions affect DSC1 knockout files more than to W1118 flies, highlighting the important role of the DSC1 channel in dampening neuronal excitability under these extreme conditions. How the DSC1 channel modulate neuronal excitability is not clear. It has been shown that expression of para and DSC1 transcripts completely overlap in the CNS of Drosophila adults (Hong and Ganetzky, 1994), suggesting that the DSC1 channel may function along with the Para channel in regulating neuronal activities. Sodium channels are responsible for the rising phase of the action potential. Because the DSC1 channel requires high depolarization voltage to be activated (Chapter 2), it is possible that this channel could be activated only during the rising phase of an action potential. If so, the D801 channel may prolong or enhance depolarization of the membrane potential during the rising phase of the action potential. It is possible that the modification of the action potential by the DSCI channel is too subtle to be detected at room temperature. However, in response to stress conditions when the conductance of the sodium channel and the potassium channel are altered (Huxley, 1959), the modulating role of the DSC1 channel 84 becomes evident. Future experiments recording the action potential of the GF neuron from both W1118 and DSC1 knockout flies will be able to test this hypothesis. Interestingly, a Cay-activated plateau action potential has been detected in dorsal paired median neurons from the terminal abdominal ganglion of the cockroach Periplaneta amen'cana, but the ion channel responsible for the action potential has remained elusive (Amat et al., 1998). It is possible that the DSC1 channel is involved in the generation of the Ca2+-activated plateau action potential. Direct measurement of the action potential in dorsal paired median neurons in the DSC1 mutant using voltage clamp could test this prediction. Therefore, I predict that the DSC1 channel dampens the neuronal excitability by modulating the initiation and propagation of the action potential mediated by the sodium channel. 85 Table 3-1. Response latencies (ms) of the short latency pathway of w 1118 and DSC1a flies measured at different time points of heat shock process (mean :I: SD) Heat Shock Tlme RT]I 5 min 10 min 15 min 1fl8 12:02 10131 10:01 151%? W (n=17) (n=5) (n=5) (n=10) a 12:02 10i02 12:00 IAiOJ DSC1 ("=10 (n=5)3 (n=5) (n=10)5 Note: SL - short latency 1 0 min (Response latency measured at room temperature before heat shock). 2 p<0.05, compared with the SL value of w recorded before heat shock, Two-way ANOVA. a 3 p<0.05, compared with the SL value of DSC1 recorded before heat shock, Two-way ANOVA. 4 1118 p<0.05, compared with the SL value of w process, Two-way ANOVA. a 5 p<0.05, compared with the SL value of DSC1 recorded at five-minute time point of heat shock process, Two-way ANOVA. recorded at five-minute time point of heat shock Table 3-2. Refractory periods (ms) of the short latency pathway of W1118 and 08018 files measured at different time points of heat shock process (mean :I: SD) Heat Shock Tlme RT1 5 min 10 min 15 min 1118 5.9 :l:1.2 7.6 :I: 0.9 _ 8.5 :I: 1.9 w (n=20) (n=5) 8.0 :I: 1.6 (n-5) ("=11)2 a 6.5 i 1.0 6.2 :t 1.6 __ 9.8 :I: 2.3 DSC1 “1:23) (n=5) 8.8 :I: 1.6 (n-5) (”=11)3 Note: SLRP - short latency refractory period 1 0 min (i.e. Refractory period measured at room temperature before heat shock). 2 p<0.05, compared with the value of w recorded before heat shock, Two-way ANOVA. a 3 p<0.05, compared with the value of DSC1 recorded before heat shock, Two-way ANOVA. 86 Table 3-3. Response latencies (ms) of the long latency pathway of W1118 and 08018 files measured at different time points of heat shock process (mean :I: SD) Heat Shock Tlme RT1 5 min 10 min 15 min 1118 3.8 :I: 0.4 3.8 a: 0.3 3.9 :I: 0.4 3.5 120.1 W (n=22) (n=7) (n=9) (F7) a 3.8 s 0.3 3.5 s 0.2 3.9 s 0.3 3.4 :t 0.1 DSC1 (n=5) (n=11)3 Note: LL - long latency 1 0 min (i.e. response latency measured at room temperature before heat shock). 8 2 p<0.05, compared with the value of w recorded before heat shock, Two-way ANOVA. a 3 p<0.05, compared with the value of DSC1 recorded before heat shock, Two-way ANOVA. Table 3-4. Refractory periods (ms) of the long latency pathway of W1118 and DSC1a flies measured at different time points of heat shock process (mean :I: SD) Heat Shock Tlme RT1 5 min 10 min 15 min 1118 46.0 :I: 9.3 _ 44.3 :I: 8.0 26.9 :I: 7.0 w (mm) 39.1 :I: 5.2 (n-9) 01:9) (n=7)2 8 46.8 :I: 153 31.0 :I: 4.8 23.4 :I: 2.8 23.6 :t 3.1 DSC1 (n=z5) (n=8)4 (n=8)5 (n: 1 1)3 Note: LLRP - long latency refractory period 1 0 min (i.e. Refractory period measured at room temperature before heat shock). 1118 2 p<0.05, compared with the LLRP of w recorded before the heat shock, Two-way ANOVA. a 3 p<0.05, compared with the LLRP of DSC1 recorded before the heat shock, Two-way ANOVA. 1118 4 p<0.05, compared with the LLRP of w w recorded at the same time point (five-minute), Two-way ANOVA 1118 5 p<0.05, compared with the LLRP of w recorded at the same time point (10-minute), Two-way ANOVA 87 Table 3-5. Response latencies (ms) of the short latency pathway of W1118 and 08018 files measured at different time points of recovery process (mean :I: SD) Recovery Tlme 10 min 20 min 30 min 40 min Wma 1.21:0.1 1.1:I:0.1 1.11:0.1 1-2i10-1 (n=12) (n=5) (n=5) (n=9) a 12:01 1.1101 1.11:0.1 121:0.1 DSC1 (n=6) (n=11)2 Note: SL - short latency 1118 1 p<0.05, compared with the SL of w recorded at the end of the heat shock, Two-way ANOVA. a 2 p<0.05, compared with the SL of DSC1 recorded at the end of the heat shock, Two-way ANOVA. Table 3-6. Refractory periods (ms) of the short latency pathway of Wm8 and DSC1a flies measured at different time points of recovery process (mean 1- SD) Recovery Time 10 min 20 min 30 min 40 min 1113 7.5 i 2.1 _ _ 6.5 $1.3 w (n=12) 6.2 d: 1.3 (n 5) 6.6 d: 1.5 (n 5) (n=10)1 a 16il9 _ _ 69109 DSC1 (n=10) 8.2 :I: 1.2 (n 6) 8.7 :I: 1.2 (n 6) (n=11)2 Note: SLRP - short latency refractory period 1 p<0.05, compared with the SLRP of w recorded at the end of the heat shock, Two-way ANOVA. a 2 p<0.05, compared with the SLRP of DSC1 recorded at the end of the heat shock, Two-way ANOVA. 88 Table 3-7. Response latencies (ms) of the long latency pathway of wmaand DSC1a flies measured at different time points of recovery process (mean 1 SD) Recovery Tlme 10 min 20 min 30 min 40 min 1118 4.0 1 0.1 4.1 1 0.2 3.9 1 0.2 3.9 1:102 W (n=12) (n=11) (n=6) (hey) a 3.8 1 0.2 3.9 1 0.1 3.6 1 0.2 3-7 i 0-2 030’ (n=9) (n=8) (n=8) (n=9)2 Note: LL - long latency 1118 1 p<0.05, compared with the LL of w recorded at the end of the heat shock, Two-way ANOVA. a 2 p<0.05, compared with the LL of DSC1 recorded at the end of the heat shock, Two-way ANOVA. 1118 a Table 3-8. Refractory periods (ms) of the long latency pathway of w nd DSC1a flies measured at different time points of recovery process (mean 1 SD) Recovery Time 10 min 20 min 30 min 40 min W1118 44.6 1 7.8 48.7 1 8.6 44.8 1 6.0 49711 10.0 (n=12) (n=11) (n=6) (n=7) 26.3 1 2.1 32.6 1 5.6 30.1 1 4.3 31.4 1 4.8 a DSC1 (n=9)3 (n=8)4 (n=8)5 (n=9)2’6 Note: LLRP - long latency refractory period 1118 1 p<0.05, compared with the LLRP of w recorded at the end of the heat shock, Two-way ANOVA. a 2 p<0.05, compared with the LLRP of DSC1 recorded at the end of the heat shock, Two-way ANOVA. 1118 3 p<0.05, compared with the LLRP of w recorded at the same time point (10-minute), Two-way ANOVA 1118 4 p<0.05, compared with the LLRP of w recorded at the same time point (20-minute), Two-way ANOVA 1118 5 p<0.05, compared with the LLRP of w recorded at the same time point (30-minute), Two-way ANOVA 1118 6 p<0.05, compared with the LLRP of w recorded at the same time point (40-minute), Two-way ANOVA 89 Table 3-9. Response latencies and refractory periods of W1118, and DSC1a flies after 72 hours starvation (mean 1 SD) SL (ms) SLRP (ms) LL (ms) LLRP (ms) 1118 1.1 1 0.1 5.5 1 0.6 3.5 1 01-2 49.2 1 13.8 (n=13) (n=13) (n=13) (n=13) a 1.1 1 0.1 6.0 11.6 3.3 t 0.2 35.3 1: 5.8 DSC1 (n=12) (n=12) (n=12)2 (n-."-12)3 Note: SL- short latency SLRP- short latency refractory period LL- long latency LLRP- long latency refractory period 18 1 p<0.05, compared with the LLRP of w recorded before starvation, Two-way ANOVA. a 2 p<0.05, compared with the LLRP of 0801 recorded before starvation, Two-way ANOVA. 1118 3 p<0.05, compared with the LLRP of w recorded after stewed for 72 hours, Two-way ANOVA. 90 Figure 3-1. Generation of DSC1 knockout D. melanogaster lines. A. Schematic illustration of the targeting strategy. Stop codons (ST) were introduced in upstream and downstream homologue regions. A wild type genomic DNA fragment containing part of the DSC1 gene was replaced through ends—out homologous recombination. The mini w+ gene was used as a marker to indicate the successful replacement. B. Southern blot analysis of the genomic DNA of DSC1 knockout flies. Kpn l was used to digest genomic DNA for Southern blot analysis. The positions of Kpn I sites are labeled and sizes of DNA fragments are shown in A. Sizes of DNA fragments are demonstrated. DSC1 KO: DSC1 knockoutflne. A w* ST1 1 ’ 8T2 l t * I Donor construct >< >< ll/ Jl I } I ,l/ Wild type Kpn l 16'9 kb Kpn l genomic DNA ST1 W+ ST; II/ I * , * L I// | 33kb I 61kb Kpn l Kpn I Kpn I 91 Figure 3-2. Schematic illustration of the giant fiber pathway (one side shown). High-voltage stimulation (high) activates the cervical giant fiber (CGF) to induce a short-latency response, whereas low voltage stimulation (low) excites brain afferent neurons to trigger a long-latency response. ('ITM) Tergotrochanteral muscle; (TT Mn) TTM motoneuron; (DLM) dorsal longitudinal muscle; (DLMn) DLM motoneuron; (PSI) peripherally synapsing intemeuron. (modified from Engel etal.2000) stim. v: low high TrMn 5. electrochemical -4 chemical head 92 Figure 3-3. DSC1 knockout flies have normal locomotor activity but are more jumpy than Wm8 flies. A. Climbing assay carried out on W1118 flies (n=5), DSC1a (n=5), and DSC1b (n=5) flies at room temperature. 3. Time course of paralysis in response to heat shock (40°C) of w’"8 (n=5), DSC1a (n=5) and 0301” (n=5) flies. C. The number of jumps of W1118 (n=5), DSC1a(n=5) or DSC1b (n=5) flies from the fifth to tenth minutes during heat shock (mean 1 SD, * p<0.05, Student’s t-test). The values are the averages of a total number of jump of 10 flies per vial. A Climbing Scores W1118 03013 0301b _._W1118 12; -'- 08013 —4— 0301” Knockdown Ratio 0 A 02166101271416 Heat Shock Time (min) 93 Figure 3—3 (continued) C Number of Jumps l 10 files <75 W1116 DSC18 DSC1” 94 Fwifiure 34. DSC1 knockout flies recovered more slowly from heat shock than 18flleS The recovery from heat shock was determined using a climbing) assay to measure the locomotor actlvrty ofw118(n= 5), DSC1a (n= 5), and DSC1 (n= 5) files. (mean 1 SD) 121 V i 3 Climbing Scores _\ .5 t- 6 0° 0 th. l———I= I b l—-I O l . I I l r T 1 0 4 8 12 1W620242832 364044 Recovery Time (min) 95 Figure 3-5. DSC1 knockout flies jumped more and died earlier during starvation. A. Number of jumps of w1118 (n=10) and 08018 (n=10) flies counted during the first three days of starvation. A jumpy phenotype similar to that induced during the heat shock was observed by a brief tapping of vials and videotaping. The number of jump per vial (five flies) was counted during the second to tenth seconds after tapping. (mean :t SD, * p<0.05, Two-way ANOVA). B. Survival curves of w1118 (n=6) and 08018 (n=6) flies. (mean i SD) C. Weights of individual fly of w1118(n=40) and DSC1a (n=40) line. A 4.5- 4.02 3.5-3 3.01 2.5 2.03 1.53 1.03 0.55 0.05 Number of Jumps I Vial 0 hr 24 hr 48 hr 72 hr 100- - Survival (%) 3fo ' 3:5 '4fofi 4T5 ' 5fo ' 55 so ' 6:5 Starvation Time (days) 96 Figure 3-5 (continued) C Weight (mg) 1.0- 0.9! 0.81 0.71 0.6; 0.52 0.42 0.31 0.21 0.11 0.0j Male Female 97 -W1118 -DSC1a Figure 3-6. Giant fiber recording of the short latency pathway (A. B.) and the long latency pathway (C. D.) carried out on w1118 and 08018 flies at different time points during heat shock. The latencies and refractory periods measured at room temperature before heat shock were included for comparison (RT). SL: short latency; SLRP: short latency refractory period; LL: long latency; LLRP: long latency refractory period; RT: room temperature. (mean i SD, * p<0.05, Two-way ANOVA) A * 1.8- ' * 1.5: [—11—] 1.4 r—*—I 1.2 131.05 E 0.8- <71) 06‘ 0.49 0.2{ 0'0‘ RT 5 1o 15 Heat Shock Time (min) 98 Figure 3-6 (continued) _3 _L 0) O N l I l l l l SLRP (ms) 4.5- 4.0-‘ 3.5% 3.01 2.55 2.01 1.51 1.0- 0.51 SLRP (ms) 0.05 - W1118 * I:JDSC18 W l * l l RT 5 1o 15' _ Heat Shock Time (ms) I * I - w1118 * I l:lDSC1a RT 5 10 15 Heat Shock Time (min) 99 Figure 3-6 (continued) LLRP (ms) N (:0 O l O 1 l _x O l A O L . l * ' mosc1a RT 5" 1o 15" Heat Shock Time (min) 100 Figure 3-7. Giant fiber recording of the short latency pathway (A. B.) and the long latency pathway (C. D.) carried out on W11 and 08018 flies at different time points during a recovery process after heat shock. The latencies and refractory periods measured at room temperature before heat shock were included for comparison (RT). SL: short latency; SLRP: short latency refractory period; LL: long latency; LLRP: long latency refractory period; RT: room tem1p1e1r8ature. (mean :t: SD, * p<0.05, two-way ANOVA) E. Locomotor activity of w (n=60) and DSC1a (n=60) flies tested at the end of recovery process with a vortex. (mean 1 SD, # p<0.05, Student’s t-test) - W1118 I I [208013 RT 0 1o" 20 3o 40“ Recovery Time (min) 101 Figure 3-7 (continued) B * -W1118 . r ... ' :Joscra 12_ If I 103 SLRP (ms) 0 1o 20 36 40 Recovery Time (min) 4.5- * 1 EDSCTa 4.05 3.5% I 3.05 37 2.5l . . .5 E 2.01 ' r. 3 1.5L 1.01 - 5% 05' . l 0.03 RT 0 1o 20 30 40 Recovery Time (min) 102 Figure 3-7 (continued) D * -W1118 50: F7 A 4o- 30; 201 LLRP (ms 10« RT 0 1o 20 3o 40’ Recovery Time (min) E l 10. a) 8‘ 9 8 . U) 6‘ 2’ . '5 4‘ E ‘ Z3 2- 0.. W1118 08018 103 Figure 3-8. Giant fiber recordlng of the short latency pathway (A. B.) and the long latency pathway (C. D.) in w and DSC1a flies after 72 hours of starvation. SL: short latency; SLRP: short latency refractory period; LL: long latency; LLRP: long latency refractory period; CT: values recorded before starvation. (mean i SD, * p<0.05, Two-way ANOVA) A -w1118 EEDSC1a 72h$ [:IDSC13 SLRP (ms) A l 72 hrs 104 Figure 3-8 (continued) C * -W1118 4.0L 353 3.0L A 2.53 :13, 2.0L j 1.53 1.0-3 0.53 0.05 - w1118 E08013 72h$ 105 CHAPTER 4 DSC1 KNOCKOUT MUTANTS ARE MORE SUSCEPTIBLE TO PYRETHROID INSECTICIDES 106 4.1 Abstract Voltage- or ligand-gated ion channels are transmembrane proteins that mediate critical neuronal functions, such as action potential (AP) and neurotransmitter release. They are often the molecular targets of natural or synthetic neurotoxins, including insecticides. Voltage—gated sodium channels are the primary targets of two important classes of insecticides: sodium channel-gating modifying pyrethroid insecticides and sodium channel blocker insecticides (SCBls). The DSC1 channel shares up to 50% identity in the transmembrane domains with Para sodium channels. However, whether the DSC1 channel is involved in the toxicology of pyrethroids and/or SCBls remains unclear. In this study, using both contact and topical bioassays, I examined the susceptibility of adult DSC1 knockout flies to four pyrethroids (perrnethrin, bioresmethrin, deltamethrin and fenvalerate), N—decarbomethoxyllated JW062 (DCJW), a potent metabolite of a SCBl, indoxacarb, and fipronil, a y-aminobutyric acid (GABA)-gated chloride channel blocker insecticide. I found that DSC1 knockout flies were more susceptible to all pyrethroids tested, but remain as susceptible as wild-type flies to DCJW and fipronil. To determine whether the increased susceptibility to pyrethroids is due to enhanced sensitivity of the nervous system, I examined the response of a well-defined neural circuit, the giant fiber system, to deltamethrin. Indeed, compared with that of wild-type flies, the giant fiber system of DSC1 knockout flies was more sensitive to pyrethroids. These results indicate that the DSC1 channel is involved in dampening the neuronal excitability induced by pyrethroids, which is reminiscent of the role of the 107 DSC1 channel in modulating neuronal excitability in response to heat shock and starvation reported in the Chapter 3. 4.2 Introduction Voltage-gated ion channels (VGICs) are transmembrane proteins located on the surface of every excitable cell. Opening and closure of these channels are regulated by the membrane potential. Opening of VGle leads to either outward or inward currents, which in turn may either depolarize or hyperpolarize the membrane potential to facilitate critical cellular functions such as initiation and propagation of APs and neurotransmitter release. VGle can be classified into several groups based on their ion selectivity and play different roles in modulating specific cellular functions (Yu et al., 2005; Catterall et al., 2007). For example, voltage-gated sodium channels are responsible for the rising phase of an AP, whereas voltage-gated potassium channels mediate the falling phase of an AP. There are more than nine voltage-gated sodium channel genes identified in mammals by far, but only one confirmed sodium channel gene in insects, such as para in Drosophila melanogaster. The Drosophila sodium channel 1 (DSC1) gene was discovered in 1987, and was initially predicted to encode a sodium channel based on its high sequence similarity with sodium channels. However, functional characterization in Xenopus oocytes showed that DSC1 encodes a novel voltage—gated cation channel, instead of a conventional voltage-gated sodium channel (Chapter 2). VGICs are some of the most important targets of insecticides (Soderlund and 108 Bloomquist, 1989; Soderlund, 1990; Narahashi, 1996; Narahashi, 2000; Ray and Fry, 2006). In particular, sodium channels are a well-known target of two widely used classes of insecticides, pyrethroids (Narahashi, 1996; Narahashi, 2000; Narahashi et al., 2007) and SCBIs (Silver and Soderlund, 2005). The modes of action of pyrethroids and SCBIs are different. Pyrethroids inhibit channel deactivation and inactivation, causing persistent activation of sodium channels (Narahashi, 1996; Narahashi, 2000), whereas SCBls block sodium channels in a state-dependent manner (Zhao et al., 2003). Pyrethriods are classified into type I and type II pyrethroids based on the absence (type I) or presence (type II) of a a—cyano-3-phenoxybenzyl alcohol (See Fig. 1-3) (Soderlund and Bloomquist, 1989). 'Although both type I and type II pyrethroids are sodium channel gating modifiers, type II pyrethroids alter gating kinetics more drastically than type I pyrethroids, causing a much slower decaying tail current upon repolarization. At the cellular level, type I pyrethroids produce repetitive discharges, whereas type II compounds do not (Soderlund and Bloomquist, 1989). Instead, type II pyrethroids cause stimulus-dependent membrane depolarization and block of nerve conductance (Narahashi, 1988; Soderlund and Bloomquist, 1989; Narahashi, 1992) The predicted topology of the DSC1 protein is highly similar to that of the sodium channel, and DSCI shares the highest amino acid sequence similarity to the Para sodium channel, with up to 50% identity in the four transmembrane domains. However, it is not known whether DSCI is involved in the toxicology of pyrethroids and/or SCBIs. In this study, I evaluated the susceptibility of adult wma 109 and DSC1 knockout flies using both contact and topical bioassays to four pyrethroids, as well as DCJW, a potent metabolite of indoxacarb, and a GABA-gated chloride channel blocker, fipronil. 4.3 Materials and Methods 4.3.1 Fly stocks Two initial DSC1 knockout D. melanogaster founder lines, DSC1-3 and DSC1-6, were generated by Dr. Ningguang Luo, a former post-doc associate in our lab, using homologous recombination-mediated gene disruption (Gong and Golic, 2003). DSC1-6 was backcrossed to W1118 for five generations to generate DSC1a, while DSC1-3 was backcrossed to wma for five generations to b b 1118 . generate DSC1 . DSC1 was further back crossed to w for four generations to generate DSC1C. DSC1a and DSC1c were used in insecticide bioassays. 1118 . . . . w was used as a wnld-type control In all expenments. All these lines were raised on regular cornmeal-molasses-agar medium at room temperature. 4.3.2 Contact bioassay: A 0.5-ml insecticide solution dissolved in acetone was delivered into a 25-ml glass scintillation vial. To coat the inner surface of the vial with insecticide, the vial was rolled on the side in a chemical fume hood for 3 to 5 min. \fials were kept in the hood for another 30 min to evaporate residual acetone. Twenty flies at the age 110 of two to three days old were slightly immobilized by 002 and put into the insecticide-coated scintillation vials. Vials were plugged with a cotton ball and 3 ml of 20% sugar water was then added on the cotton ball. The vials were then kept at room temperature and the number of dead flies was counted 24 hours later. Three to five replicates were carried out for each concentration. The bioassay was repeated at least three times. The median lethal concentration (LC50) and 95% confidential interval were calculated using the POLOplus software (LeOra Software Company, Petaluma, CA). The L050 ratio is defined as the LC50 of w1118 flies divided by the LC5o of DSC1 knockout flies. 4.3.3 Topical bioassay: For topical bioassay, 2- to 3-day-old male flies were slightly immobilized by 002 and 0.2 ul insecticide acetone solution was delivered onto the dorsal side of thorax of individual flies. The treated flies were held in disposable plastic Petri dishes (6-cm diameter x 1-cm height) lined with a moistened filter paper (Whatman, Clifton, NJ) at room temperature for 15 minutes. Two toxicity-associated phenotypes, abdomen elongation and knockdown, were recorded. The abdomen elongation phenotype was defined as rigid, elongated abdomen (See Fig. 4-28) in comparison with shorter, flexible abdomen (See Fig. 4-2A) before insecticide exposure. The knockdown phenotype was defined as losing the ability to walk. The median effective dose (EDso) and 95% confidential 111 interval for both phenotypes were calculated using the POLOplus software (LeOra Software Company, Petaluma, CA). 4.3.4 Giant fiber recording . . 1118 . A dose of 0.4 ng deltamethrin (i.e., E095 of knockdown of w fires) or 4 ng bioresmethrin was topically delivered to individual 2- or 3-day old male flies. The dose of bioresmethrin was chosen based on contact bioassay results which show that the LC50 of bioresmethrin was about 10 times higher than that of deltamethrin. The activities in the giant fiber system (GFS) were recorded 15 min after pyrethroid exposure. The methods for recording the response latency and refractory period are the same as those described in Chapter 3. To examine the stability of the GFS, I used a protocol modified from those described in (Tanouye and Wyman, 1980; Fayyazuddin et al., 2006). Atrain of 50 pulses of 30 V was delivered at the frequency of 100 Hz. The response (i.e., muscle potentials) of dorsal longitudinal muscles (DLMs) to this stimulation was recorded. The 50 pulses were divided into 5 groups with 10 pulses in each group; and the number of muscle potentials in each group was counted. 4.3.5 Statistics Student’s t-test was used to analyze the data of muscle potentials elicited by high frequency stimulus. Log-rank test was used to analyze the data of the percentage of knockdown that are obtained after being exposed to deltamethrin 112 for various periods. Two-way analysis of variance (ANOVA) with Tukey’s test employed as the post hoc test were used to analyze the other GF recording data. P < 0.05 was set as the criterion for statistical significance. 4.4 Results 4.4.1 DSC1 knockout flies are more sensitive to pyrethroids, but not to DCJW and fipronil To determine whether the DSC1 channel could be involved in the toxicity of pyrethroids, a contact bioassay was performed using W1118, 08018, and DSC1c flies. DSC1a flies and DSC1c were about 2-fold more susceptible, measured by lethality, than W1118 to all four pyrethroids tested: two type I pyrethroids, perrnethrin and bioresmethrin, and two type II pyrethroids, deltamethrin and fenvalerate (Table 4-1, 4-2, 4-3, 4-4). In contrast, w1118and DSC1 knockout flies exhibited similar susceptibility to DCJW and fipronil (Table 4-5, 4-6). 4.4.2 0861 knockout flies are more sensitive to knockdown by deltamethrin Being extremely lipophilic, pyrethroids can be taken up by insects easily and affect sodium channels yielding the acute knockdown symptom, which is an important characteristics of pyrethroid intoxication (Narahashi, 1996). To study if DSC1 knockout flies are also more easily knocked down by pyrethroids, I performed a topical bioassay using deltamethrin on W1118 and 08018 flies. 113 Results showed that DSC1a flies were more susceptible to deltamethrin than Wm8 flies (Fig. 4-1, Table 4-7). During these experiments, I noticed another phenotype. After being exposed to deltamethrin for 15 minutes, the shape of the abdomen of some files changed from a smooth and soft state (Fig. 4-2A) to an elongated and rigid state (Fig. 4-28). 08018 flies more frequently displayed this deltamethrin-induced abdomen elongation phenotype (Fig. 4-20, Table 4-8). To compare the kinetics of intoxication of w1118 and DSC1a lines, I topically applied 0.04 ng deltamethrin, which is close to the E050 of DSC1a flies, to individual fly and observed the knockdown phenotype for two hours. The number of flies that were knocked down was recorded every 15 minutes during the first hour and every 30 minutes during the second hour. A time-response curve was generated (Fig. 4-3). wma flies began to exhibit the knockdown phenotype at about 15 minutes after pyrethroid exposure, and the percentage of knockdown peaked at 90 minutes and then decreased. The kinetics of knockdown of DSC18 flies was parallel to that of wma flies. However, at 30, 45, and 60 minutes after pyrethroid exposure, the percentage of knockdown of DSC1a flies was significantly higher than that of wma flies (p<0.05, Log-rank test). 114 " ...__i_.fl__;‘ __.._-_— _ _ 4.4.3 Pyrethroids destabilized the GFS of DSC1 knockout flies to a greater extent To investigate whether the enhanced pyrethroid susceptibility of DSC1a flies is correlated with an enhanced sensitivity of the nervous system to pyrethroids, we examined the response of a well-defined neuronal circuit, the GFS, to bioresmethrin (a type I pyrethroid) and deltamethrin (a type II pyrethroid). I first examined the activities of short and long latency pathways of 08018 and W1118 flies 15-min after topical application of bioresmethrin (type I) and deltamethrin (type II). Unexpectedly, bioresmethrin had no effect on the long or short latency pathways of W1118 flies (Fig. 4-4). However, bioresmethrin increased the short latency (Table 4-9, Fig. 4-4 A), but reduced the short latency refractory period (SLRP) of DSC1a flies (Fig. 4-4 B). Bioresmethrin also did not affect the latency or the refractory period of the long latency pathway of DSC1a flies (Fig. 4-4 C, D). In contrast to bioresmethrin, deltamethrin did not alter the latency or the refractory period of the short latency pathway of either line (Table 4-10, Fig 4—5 A, B). However, deltamethrin shortened both the latency and the refractory period of the long latency pathway of both w1118 and 08018 files, but there was no difference between the two lines (Fig. 4-5 C, D). In wild-type Drosophila flies, the short latency pathway to the DLM can follow stimuli at frequencies as high as 100 Hz (T anouye and Wyman, 1980). To further characterize the function (i.e., stability) of the GFS, we examined the response of 115 both w1118 and DSC1a flies to high frequency stimulation in the absence and presence of pyrethroids. In response to a train of 50 pulses delivered at 100 Hz at the amplitude of 30 V, each stimulus evoked a muscle potential from the DLM and a total of 50 muscle potentials were recorded. A recording trace of 10 muscle potentials in response to the first 10 pulses is shown in Fig. 4-6 A. No difference was observed between W1118 and 08018 flies after topically applying 0.2 pl acetone (Fig. 4-6 B). In the presence of deltamethrin, the number of muscle potentials was reduced to about seven in the fifth 10 stimuli in w1118 flies, indicating that deltamethrin destabilizes the function of the GFS. Interestingly, in 08018 files the reduction in muscle potentials was already evident in the second 10 stimuli and this reduction progressed in the remaining three groups of 10 stimuli (Fig. 4-6 C). Bioresmethrin affected the function of the GFS more drastically than deltamethrin. In wma flies, the number of muscle potentials during the third 10 stimuli was reduced to about 3; and no muscle potentials were elicited in the remaining two groups of 10 stimuli (Fig. 4—6 D). Again, the function of the GFS in 08018 flies was more sensitive to bioresmethrin; a reduction in muscle potential number was observed in the second 10 stimuli (Fig. 4—6 D). Thus, both type I and type II pyrethroids affect the GFS, with bioresmethrin being more potent than deltamethrin. The DSC1a flies are hypersensitive to 1 pyrethroids, compared with W1 18 flies. 116 4.5 Discussion Ion channels are transmembrane proteins that are essential for neuronal functions. Many natural or synthetic neurotoxins and insecticides target ion channels. For example, voltage-gated sodium channels are the primary targets of pyrethroid insecticides and sodium-channel-blocker insecticides, such as DCJW. Synthetic pyrethroids are a group of environmentally friendly, highly effective, and very selective insecticides used globally for pest control. Unfortunately, with intensive application, insecticide resistance has emerged in numerous insect populations, resulting in reduced efficacy of insecticides. As a result, pest control often requires higher insecticide dosages, which poses greater risks to humans, wildlife, and environment (Jeyaratnam, 1990; Konradsen, 2007). There is a great need to enhance the effectiveness of available insecticides and also to discover new insecticides that are highly selective against insects. In this study, I found that DSC1 knockout flies were more susceptible to both type I and type II pyrethroids, compared with wmaflies. The effects of the DSC1 channel on the toxicity of pyrethroids are evident in both short— and long—duration exposures since DSC1 knockout flies are sensitive to both pyrethroid-induced rapid knockdown and eventual lethality. I predict that an inhibitor of the DSC1 channel may enhance the efficacy of pyrethroids against pests. Therefore, this channel may be a potential target for the development of a new generation of insecticide synergists. To gain an understanding of the involvement of the DSC1 channel in insect 1118 a susceptibility to pyrethroids, I compared the activities of the GFS in w nd 117 DSC1 knockout files before and after pyrethroid application. The three main findings and the implications of these findings are summarized below. First, the type II pyrethroid deltamethrin, but not type I bioresmethrin, reduced the response latency and the refractory period of the long latency pathway in both w1118 and DSC1 knockout flies. The effects of deltamethrin, but not bioresmethrin, on the long latency pathway may be caused by differences in the modification of sodium channel gating by type I vs. type II pyrethroids. It is known that type II pyrethroids modify the gating of sodium channels to a greater extent than type I pyrethroids, resulting in more drastic alterations in neuronal excitability (Narahashi, 1992; Bloomquist, 1993). At the cellular level, type I pyrethroids induce repetitive firing, whereas type II pyrethroids cause membrane depolarization (Narahashi, 1988; Narahashi, 1992). Splice variants with differential sensitivity to type I and type II pyrethroids have been isolated from Blattella gennanica and D. melanogaster (Tan et al., 2002; Du et al., 2009b; Hu et al., unpublished data). Perhaps neurons that are important for the long latency pathway but not for the short latency pathway (e.g., those in the brain that are presynaptic to the giant fiber) express sodium channel variants that are more sensitive to deltamethrin or more resistant to bioresmethrin. Secondly, the refractory period of the short latency pathway was reduced by bioresmethrin in DSC1 knockout flies although neither deltamethrin nor bioresmethrin altered the latency or refractory period of the short latency pathway in wma flies. Thus, knockout of the DSC1 gene possibly potentiates the repetitive firing induced by type I pyrethroids. This finding supports my 118 hypothesis in Chapter 3 that the DSC1 channel dampens neuronal excitability, based on enhanced excitability of DSC1 knockout flies in response to heat shock and starvation. The lack of an effect of deltamethrin on the refractory period of the short latency pathway in DSC1 knockout flies may be explained by the unique mode of action of type II pyrethroid, which does not induce repetitive firing, but mainly causes depolarization of the membrane potential. Thirdly, both pyrethroids reduced the ability of the short latency pathway to follow stimulus at high frequency; and the effect was more pronounced on DSC1 knockout flies than on w1118 flies. These results indicate that the nervous system of DSC1 knockout flies is more sensitive to pyrethroids than that of Wm8 flies. The DSC1 mutation does not alter the action of DCJW, a sodium channel blocker. I found this result intriguing because the effect of the DSC1 channel is evident only when sodium channels are activated (by pyrethroids), but not when sodium channels are blocked (by DCJW). DSC1 knockout flies are also not more sensitive or resistant to fipronil compared with W1118. Fipronil blocks GABA-gated chloride channels, thereby enhancing the overall excitability of the nervous system, similar to that of pyrethroids. The fact that DSC1 knockout files are not altered in fipronil sensitivity indicates that enhanced nerve excitability per se may not be the basis for the effect of the DSC1 knockout. Instead, my results support the hypothesis the DSC1 channel functions specifically along with sodium channels to regulate the initiation and propagation of action potential in the 119 nervous system. In conclusion, the results reported in this chapter show that the knockout of the DSC1 gene increases the toxicity of the sodium-channeI-activators insecticides pyrethroids. This effect is accompanied by an enhancement of the sensitivity of the nervous system to pyrethroids in DSC1 knockout flies. The lack of an effect of DSC1 knockout on the action of blockers of sodium channels or GABA-gated chloride channels indicates that the DSC1 channel dampens neuronal excitability likely by modulating the initiation and propagation of the action potential, in which activation of sodium channels is crucial. 120 Table 4-1 Susceptibility of W1118 and DSC1 knockout flies to perrnethrin (contact bioassay) LC5o (95%01) (pg/vial) n Slope (SE) LC50 ratio "/1"? 14.00 (11 56-1657) 1680 3.47 (0.23) - 03616 5.01 (4.37-5.75) 700 3.06 (0.25) 2.8 03016 5.37 (4.17-6.68) 600 3.95 (0.34) 2.6 Note: LC50, median lethal concentration LC50 ratio = (LC50 ofw1118) /(Lc50 of os01 knockout line) 8 Table 4-2 Susceptibility of W111 and DSC1 knockout flies to bioresmethrin (contact bioassay) LC50 (95%c1) (pg/vial) " Slope (SE) L050 ratio W11“ 3.80 (3.34431) 640 2.68 (0.21) - 03018 2.18 (1.95-2.43) 640 3.32 (0.27) 1.7 03010 2.04 (1 .83—2.27) 640 3.41 (0.27) 1.9 Note: L050, median lethal concentration LC50 ratio = (LC50 of w1118) / (L050 of oscr knockout line) 121 ..-.—I Table 4-3 Susceptibility ofw1118 and DSC1 knockout flies to deltamethrin (contact bioassay) LC50 (95%Cl) (pg/vial) " Slope (SE) LC5o ratio (”1118 0.46 (0.38-0.55) 2220 2.79 (0.14) - 03013 0.25 (0210.30) 640 1.96 (0.15) 1.9 03010 0.20 (0.16-0.25) 240 3.43 (0.62) 2.3 Note: LC50. median lethal concentration L050 ratio = (LC5o of Wm”) / (L050 of DSC1 knockout line) Table 4-4 Susceptibility of W1118 and DSC1 knockout flies to fenvalerate (contact bioassay) L050 (95%01) (pg/vial) ’7 Slope (SE) LC50 ratio W11"? 3.09 (2.73-3.37) 860 3.28 (0.32) - DSC1a 1.4 (0.85-1.92) 1160 2.5 (0.23) 2.2 03010 1.65 (0.99-2.41) 860 2.15 (0.21) 1.9 Note: L050, median lethal concentration L050 ratio = (L050 of W1118) / (LC5o of DSC1 knockout line) 122 Table 4-5 Susceptibility ofw1118 and DSC1a flies to 00.1w (contact bioassay) LC50 (95%01) (pg/vial) " Slope (SE) L050 ratio W11“? 2.10 (1.69-2.53) 720 1.48 (0.15) - 03018 1.93 (1532.34) 720 1.43 (0.15) 1.1 Note: L050, median lethal concentration L050 ratio = (LC50 of W1118) / (L050 of DSC1 knockout line) Table 4-6 Susceptibility of w"18 and DSC1” flies to fipronil (contact bioassay) LC50 (95%Cl) (pg/vial) n Slope (SE) LC50 ratio “/1"? 1.58 (0715.49) 420 1.58 (0.15) - 08018 1.13 (0.77-1.91) 480 1.74 (0.16) 1.4 Note: L050, median lethal concentration L050 ratio = (L050 ofw1118) / (L050 of DSC1 knockout line) 123 Table 4-7 5050 ofw1118 and DSC1a flies to knockdown phenotype ED5o (95% CI) ED95 (95% CI) n Slope (SE) ("Q/fill) (nglfly) w1118 0.067 (0055-0083) 0.376 (0262-0638) 240 2.20 (0.24) 03013 0.034 (0026-0044) 0.298 (0195-0568) 240 1.75 (0.21) ED5o, median effective dose Table 4-8 E050 of W1118 and D8018 flies to abdomen elongation phenotype 5050 (95% CI) 15095 (95% Cl) n Slope (SE) (rig/fly) (nglfly) W1118 0.029 (0027-0031) 0.055 (0049-0065) 300 5.89 (0.61) 03018 0.024 (0022-0025) 0.044 (0040-0051) 300 6.12(0.59) ED50, median effective dose 124 Table 4-9. Response latencies and refractory periods of male wma and DSC1a flies before and after exposure to 4ng/fly bioresmethrin for 15 minutes (mean :I: SD) Before After W1118 0801a W1118 D8016 SL (ms) 1.2 e 0.2 (n=9) 2313)? 1.3 :I: 0.1 (n=6) 116:6?” SLRP (ms) 5.9 :I: 1.4 (n=7) 6123150 5.1 :I: 0.6 (n=6) 48:5? LL (ms) 4.1 s 0.3 (n=8) 23:1 352 3.9 s 0.2 (n=6) 4'(1n:6(;°3 45.4 s 10.1 43.5 :I: 8.7 42.7 a 6.2 41.3 :t 4.4 LLRP (ms) (n=12) (n=12) (n=6) (n=6) Note: SL - short latency SLRP - short latency refractory period LL - long latency LLRP - long latency refractory period 1 p<0.05, in comparison with the SL of 08018 before bioresmethrin application, Two-way ANOVA. 2 p<0.05, in comparison with the SLRP of DSC1a before bioresmethrin application, Two-way ANOVA. 125 Table 4-10. Response latencies and refractory periods of male w1118 and DSC1a flies recorded before and after exposure to 0.4ng/fly deltamethrin for 15 minutes (meantSD) Before After 1118 1118 W W osma DSC1a SL (ms) 1.2 a 0.2 (n=9) 1.2 :l: 0.2 (n=12) 1.2 :I: 0.1 (n=9) 1.2 1 0.2 (n=9) SLRP (ms) 5.911.4(n=7) 6.011.0(n=12) 6.31:1.0(n=8) 6.1 i0.9(n=9) 36:02 35:02 LL (ms) 4.1 1: 0.3 (n—8) 3.9 :I: 0.2 (n-12) (n=8)1 (n=6)2 45.4 :I: 10.1 43.5 s 8.7 38-6 t 3-4 33-2 i 6-1 LLRP (m8) (n=12) (n=12) (n=8)3 (n=6)4 Note: SL - short latency SLRP - short latency refractory period LL - long latency LLRP - long latency refractory period 1118 1 p<0.05, in comparison with the LL of w before deltamethrin application, Two-way ANOVA. 2 p<0.05, in comparison with the LL of 08018 before deltamethrin application, Two-way ANOVA. 1118 3 p<0.05, in comparison with the LLRP of w before deltamethrin application, Two-way ANOVA. 4 p<0.05, in comparison with the LLRP of 08018 before deltamethrin application, Two-way ANOVA. 126 Figure 4-1. Deltamethrin dose-response relation to knockdown phenotype of w1118 and DSC1a flies. Probit values were calculated using software SAS and plotted using software Originlab 8.0. ED5, ED5o, and ED95 were marked by dash lines. Percent of Knockdown 127 Figure 4-2. Abdomen elongation phenotype of w1118 and DSC1a files. A. Abdomen of a DSC1b fly before deltamethrin exposure. B. Abdomen of the same DSC1b fly after deltamethrin exposure. The abdomen is elongated and rigid. C. Deltamethrin does-response relation to abdomen elongation phenotype w1118 and DSC1a flies. Probit values were calculated using software SAS and plotted using software Originlab 8.0. ED5, ED50, and ED95 were marked by dash lines. A 128 Figure 4-2 (continued) a a. .o n S w D V . $305 7 6 5 4 o m T . 2 my 5 . mumunwmnwnlunw Munw 2 9 9 8 76543 2 1 5:855 5:522. .o 39:85.”. 129 Figure 4-3. Tlme-response curves of knockdown phenotype of W1 11 a 8 and DSC1 flies. The dose of deltamethrin applied was 0.04ng/fly. (mean :I: SD, p<0.05, Log-rank test). Percentage of Knockdown (%) _D_W1118 —-— DSC1a Exposure Time (min) 130 0 '1'5'3'0'4'5'6'0'7'5'90105120 Figure 4-4. The GFSs of wma and DSC1a flies response differently to bioresmethrin. Response latencies and refractory periods were measured in W1118 and 08018 male flies before and after exposed to 4 ng/fly bioresmethrin for 15 minutes. SL, short latency; SLRP, short latency refractory period; LL, long latency; LLRP, long latency refractory period. (* p<0.05, Two-way ANOVA) Before 131 Figure 44 (continued) l SLRP (ms) .3 N (12.) A 1 l O I Before I: DSC1a 4.5- 4.0- 3.53 3.05 1;; 2.55 L5, 2.02 :1 1.51 1.03 0.5- 0.01 Before After 132 Figure 44 (continued) D -W1118 [:3 D8018 01 O) O O L 1 l N (JO A O O O l . 1 . L . LLRP (ms) _x O I 1 0 Before 133 Figure 4-5. The GFSs of w1118 and 08018 flies response differently to . . . . 1118 deltamethlrn. Response Iatencres and refractory periods were measured In w and DSC1a male flies before and after exposed to 0.4ng/fly deltamethrin for 15 minutes. SL, short latency; SLRP, short latency refractory period; LL, long latency; LLRP, long latency refractory period. (* p<0.05, Two-way ANOVA) - W1118 L:I D8018 Before 134 Figure 4-5 (continued) 7: [:1 DSC1a U1 1 A l l l l SLRP (ms) —k N (1») A O 2; Before I C - W1118 ' l. ' :Ibsma 4.5- I 4.03 3.55 3.05 7,; 2.51 E, 201 j 15- 1.01 05-1 0.0I Before After 135 Figure 4-5 (continued) D LLRP (ms) —‘ N (JO 0 O O l . l t l O I . Before After 136 Figure 4-6. Pyrethroids destabilized the GFS of DSC1 knockout flies to a greater extent. A. Representative traces of DLM muscle potentials elicited by a train of stimuli before pyrethroid application. The frequency of the train pulse is 100 Hz. The amplitude and duration of each pulse are 30 volts and 0.1 ms respectively. B. Number of DLM muscle potentials triggered by each of groups of stimuli after exposed to 0.2 pl acetone for 15 minutes. C. Number of DLM muscle potentials triggered by each of groups of stimuli after exposed to 0.4 ng deltamethrin for 15 minutes. D. Number of DLM muscle potentials triggered by each of groups of stimuli after exposed to 4 ng bioresmethrin for 15 minutes. (* p<0.05, Student’s t-test) A 0.1 ms II ll H H II II II II II ||100Hz,30volts -, (\DLM muscle potential [:3 08013 Number of Muscle Potentials 137 Figure 4-6 (continued) Sosaa 3rd * l—I 2nd 1st . — a — a — a q a q a — a _ 2086420 11 mas—86¢ 2822 Co 59:32 D - W1118 [:1 08013 _ a u a u a q a - 2 0 8 6 4.2.0. 11 23:90.“. 0.82). so 528:2 2nd 3rd 1st 138 CHAPTER 5 SUMMARY AND CONCLUSIONS 139 5.1 Summary and conclusions The goal of this project was to investigate the role of the DSC1 channel in neurophysiology in Drosophila melanogaster. Two DSC1 knockout D. melanogaster lines were studied for behavioral defects under both normal and stressful conditions (heat shock and starvation). The DSC1 knockout flies were also evaluated for their susceptibilities to several classes of insecticides. The activities of a well-define neuronal circuit, the GFS, of DSC1 knockout flies were examined. My main findings are: 1) the DSC1 channel is a voltage-gated cation channel that is permeable to Na+, Ca2+, and Ba”; 2) compared with wild-type flies, the DSC1 knockout flies are more sensitive to heat shock and less tolerant to starvation; 3) the DSC1 knockout flies are more susceptible to pyrethroids than wild-type flies; and 4) modifications in the activities of the GFS in DSC1 knockout flies correlated with the behavioral alterations. Based on my findings and the information previously obtained from the study of the DSC1 ortholog, BSC1, Below, I proposed a working model on the role of the DSC1 channel in insect neurophysiology. The DSCI channel functions along with sodium channels to dampen neuronal excitability by modulating the initiation and propagation of the action potential mediated by the sodium channel. The DSC1 channel could be activated during the rising phase of an action potential in which could enhance the depolarization and/or prolong the duration of the action potential. This modulation by the DSC1 channel may be too subtle to be detected 140 at room temperature. In response to heat shock which alters the conductance of sodium and potassium channels (Huxley, 1959), or when sodium channels are activated by pyrethroid insecticides, the modulating role of the DSC1 channel becomes evident. A practical implication of the findings reported in Chapter 3 and 4 is that the DSC1 channel may be used as a novel target for the development of new insecticides. Exposure of insects to chemicals that block the DSC1 channel could make insects hypersensitive to pyrethroids and possibly more vulnerable to environmental stresses in nature. Moreover, the DSC1 family of ion channels is unique in invertebrate animals. Thus, DSC1 channel-targeted chemicals may have the advantage of being highly selective and safe to mammals. However, the interaction between pyrethroids and the DSC1 channel is not fully understood and needs further investigation. Additional studies focusing on identification of DSC1 orthologs in beneficial insects and investigations of the interactions between these orthologs and pyrethroids could further our understanding of the selectivity of DSC1channeI-targeted insecticides to insect pests. 5.2 Future studies The discoveries I made in my dissertation study are the first step toward comprehensive understanding of the role of the DSC1 channel in modulating 141 neuronal excitability. Below I highlight the major research areas for future studies. Molecular analyses indicate that DSC1 transcripts are alternatively spliced. However, the tissue and/or developmental distributions of these DSC1 variants are not clear. It is possible that alternative splicing of DSC1 transcripts is regulated in a tissue- and/or developmental stage-dependent manner. If so, study of the underlying regulatory mechanisms would be important. Furthermore, some of the alternative exons contain in-frame stop codons that would result in truncated DSC1 proteins. Why the truncation occurs and whether the truncated protein forms functional channels are worth of further research. More extensive in vitro electrophysiological recording in dissociated neurons or in vivo recording of specific neuronal circuits should be used, for example, to measure the resting membrane potential, action potential, and neurotransmitter release. Such studies could be combined with a more detailed examination of the distribution of DSC1 channels in axons and synapses or the distribution of specific DSC1 variants in distinct neurons. Together, these experiments could provide us with more clues to understand how the DSC1 channel regulates neuronal excitability. Genetically, our DSC1 mutant alleles could be crossed with other ion channel mutants to study the interactions between ion channels. It will also be interesting to knockout the DSC1 gene in a specific group of neurons, such as odor receptor neurons, to characterize the roles played by the DSC1 channel in 142 these particular neurons. Voltage-gated sodium channel has been showed to regulate the development of the nervous system (Brackenbury et al., 2008; Brackenbury et al., 2010). Although the DSC1 knockout flies exhibit normal appearance and behavior under standard laboratory conditions it is not known whether the DSC1 channel contributes to neuronal development. The giant fiber system and neuromuscular junction are good models in adults and larvae for studying the development of the nervous system, such as the axon guidance and the generation of synapses. Therefore, detailed morphological study of the giant fiber system and the neuromuscular junction of DSC1 knockout files may be another direction to take in the future. Of course, the morphological study could always be coupled with genetic and electrophysiological approaches to obtain a more comprehensive understanding. Sodium channels and calcium channels consist of several subunits. For example, the Para channel requires the accessory subunit TlpE for maximal function (Feng et al., 1995). Although expression of DSC1 alone in Xenopus oocytes results in detectable currents, it is not clear whether the DSC1 channel requires another subunit for maximal function. Because DSC1 is highly homologous to the Para channel, it might need TlpE or a TlpE-like protein for optimal function. In Drosophila, there are four TipE homologous genes (TEH1-4). Possible interactions between DSC1 channels and these proteins could be 143 investigated using multiple methods, such as protein-protein interaction assays, two-electrode voltage clamp recordings in Xenopus oocytes, gene mutation analyses, and co-localization assays. 144 BIBLIOGRAPHY Agnew WS, Moore AC, Levinson SR, Raftery MA (1980) Identification of a large molecular weight peptide associated with a tetrodotoxin binding protein from the electroplax of Electrophorus electricus. Biochem Biophys Res Commun 92:860-866. Allen M, Godenschwege, TA, Tanouye, MA, Phelan, P. (2006) Making an escape: development and function of the Drosophila giant fibre system. Semin Cell Dev Biol 17:31-41. Aman TK, Grieco-Calub TM, Chen C, Rusconi R, Slat EA, lsom LL, Raman IM (2009) Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels. J Neurosci 29:2027-2042. Amat C, Lapied B, French AS, Hue B (1998) Na” -dependent neuritic spikes initiate Ca2*-dependent somatic plateau action potentials in insect dorsal paired median neurons. J Neurophysiol 80:2718-2726. Beneski DA, Catterall WA (1980) Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin. Proc Natl Acad Sci U S A 77:639-643. Bloomquist JR (1993) Neuroreceptor mechanisms in pyrethroid mode of action and resistance. Rev Pestic Toxicol 22185-230. Borjesson SI, Elinder F (2008) Structure, function, and modification of the voltage sensor in voltage-gated ion channels. Cell Biochem Biophys 52:149-174. Brackenbury WJ, Djamgoz MB, lsom LL (2008) An emerging role for voltage-gated Na+ channels in cellular migration: regulation of central nervous system development and potentiation of invasive cancers. Neuroscientist 14:571-583. Brackenbury WJ, Calhoun JD, Chen C, Miyazaki H, Nukina N, Oyama F, Ranscht B, L. IL (2010) Functional reciprocity between Na+ channel Nav1.6 and beta1 subunits in the coordinated regulation of excitability and neurite outgrowth. Proc Natl Acad Sci U S A 107:2283-2288. 145 Burr SA, Ray DE (2004) Structure-activity and interaction effects of 14 different pyrethroids on voltage-gated chloride ion channels. Toxicol Sci 77:341-346. Casida JE (1980) Pyrethrum flowers and pyrethroid insecticides. Environ Health Perspect 34:1 89-202. Castella C, Amichot M, Bergé JB, Pauron D (2001) DSC1 channels are expressed in both the central and the peripheral nervous system of adult Drosophila melanogaster. Invert Neurosci 4:85-94. Catterall W (1986) Voltage—dependent gating of sodium channels: correlating structure and function. Trends in Neurosciences 927-10. Catterall WA (1992) Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 72:315-48. Catterall WA (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26:13-25. Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J (2005) International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharrnacol Rev 57:411-425. Catterall WA, Cestele S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon 49:124-141. Clements AN, May TE (1977) The actions of pyrethroids upon the peripheral nervous system and associated organs in the locust. Pestic Sci 8:661-680. Defaix A, Lapied B (2005) Role of a novel maintained low-voltage-activated inward current permeable to sodium and calcium in pacemaking of insect neurosecretory neurons. Invert Neurosci 5:135-146. Derst C, Walther C, Veh RW, Wicher D, Heinemann SH (2006) Four novel sequences in Drosophila melanogaster homologous to the auxiliary Para sodium channel subunit TlpE. Biochem Biophys Res Commun 339:939-948. 146 Dong K (2007) Insect sodium channels and insecticide resistance. Invert Neurosci 7:17-30. Du Y, Nomura Y, Liu Z, Huang ZY, Dong K (2009a) Functional expression of an arachnid sodium channel reveals residues responsible for tetrodotoxin resistance in invertebrate sodium channels. J Biol Chem 284:33869-33875. Du Y, Lee JE, Nomura Y, Zhang T, Zhorov BS, Dong K (2009b) Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides. Biochem J 419:377-385. Elkins T, Ganetzky B (1990) Conduction in the giant nerve fiber pathway in temperature-sensitive paralytic mutants of Drosophila. . J Neurogenet 6:207-219. Elliott M (1980) Established pyrethroid insecticides. Pestic Sci 11:119-128. Elliott M, Janes NF (1978) Synthetic pyrethroids - a new class of insecticide. Chem Soc Rev 7. Elliott M, Famham AW, Janes NF, Needharn PH, Pulman DA, Stevenson JH (1973) A photostable pyrethroid. Nature 246:169-170. Engel JE, Xie XJ, Sokolowski MB, Wu CF (2000) A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila. Learn Mem 72341-352. Fayyazuddin A, Zaheer MA, Hiesinger PR, Bellen HJ (2006) The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS Biol 41420-431. Feng G, Deak P, Chopra M, Hall LM (1995) Cloning and functional analysis of TlpE, a novel membrane protein that enhances Drosophila para sodium channel function. Cell 82:1001-1011. Genneraad S, O'Dowd D, Aldrich RW (1992) Functional assay of a putative Drosophila sodium channel gene in homozygous deficiency neurons. J Neurogenet 8:1-16. 147 Godenschwege T, Forde R, Davis CP, Paul A, Beckwith K, Duttaroy A (2009) Mitochondrial superoxide radicals differentially affect muscle activity and neural function. Genetics 183: 1 75-1 84. Goldin AL (1992) Maintenance of Xenopus Iaevis and oocyte injection. Methods Enzymol 207. Goldin AL (2001) Resurgence of sodium channel research. Annu Rev Physiol 63:871-894. Goldin AL (2003) Mechanisms of sodium channel inactivation. Curr Opin Neurobiol 13:284-290. Gong WJ, Golic KG (2003) Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad Sci U S A10012556-2561. Gorczyca M, Hall JC (1984) Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis. J Neurogenet 12289-313. Gray AJ, Soderlund DM (1985) Mammalian toxicology of pyrethroids. New York: Wiley. Guy HR, Seetharamulu P (1986) Molecular model of the action potential sodium channel. Proc Natl Acad Sci U S A 83:508-512. Hall CA (1978) The efficiency of cyperrnethrin (NRDC 149) for the treatment and eradication of the sheep louse Damalinia ovis. Aust Vet J 54:471-472. Heinemann SH, Terlau H, StiJhmer W, Imoto K, Numa S (1992 ) Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356:441-443. Hille B (1992) Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates, Inc.. Hodgkin AL, Huxley AF (1952) The components of membrane conductance in the giant axon of Loligo. J Physiol 116. Hong CS, Ganetzky B (1994) Spatial and temporal expression patterns of two sodium channel genes in Drosophila. J Neurosci 14:5160-5169. 148 Hoopengardner B, Bhalla T, Staber C, Reenan RA (2003) Nervous system targets of RNA editing identified by comparative genomics. Science 301 2832-836. Huxley AF (1959) Ion movements during nerve activity. Ann N Y Acad Sci 81:221-246. lsom LL, Ragsdale DS, De Jongh KS, Westenbroek RE, Reber BF, Scheuer T, Catterall WA (1995) Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif. Cell 83:433-442. lsom LL, De Jongh KS, Patton DE, Reber BF, Offord J, Charbonneau H, Walsh K, Goldin AL, Catterall WA (1992) Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science 256:839-842. Jeyaratnam J (1990) Acute pesticide poisoning: a major global health problem. World Health Stat Q 43:139-144. Kerr NC, Holmes FE, Wynick D (2008) Novel mRNA isoforms of the sodium channels Na(v)1.2, Na(v)1.3 and Na(v)1.7 encode predicted two-domain, truncated proteins. Neuroscience 155:797-808. Koenig JH, lkeda K (2005) Relationship of the reserve vesicle population to synaptic depression in the tergotrochanteral and dorsal longitudinal muscles of Drosophila. J Neurophysiol 94:2111-2119. Konradsen F (2007) Acute pesticide poisoning—a global public health problem. Dan Med Bull 54:58-59. Kontis KJ, Rounaghi A, Goldin AL (1997) Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains. J Gen Physiol 110:391-401. Kosaka T, Ikeda K (1983) Possible temperature—dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol 14:207-225. Kulkami NH, Yamamoto AH, Robinson KO, Mackay TF, Anholt RR (2002) The DSC1 channel, encoded by the smi60E locus, contributes to odor-guided 149 behavior in Drosophila melanogaster. Genetics 161 :1507-1516. Lin WH, Wright DE, Muraro NI, Baines RA (2009) Alternative splicing in the voltage-gated sodium channel DmNav gegulates activation, inactivation, and persistent current. J Neurophysiol 102:1994-2006. Littleton JT, Ganetzky B (2000) Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26:35-43. Liu Z, Chung I, Dong K (2001) Alternative splicing of the BSC1 gene generates tissue-specific isoforms in the German cockroach. Insect Biochem Mol Biol 31:703-713. Liu Z, Song W, Dong K (2004) Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. Proc Natl Acad Sci USA 101:11862-11867. Loughney K, Kreber R, Ganetzky B (1989) Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58:1143-1154. Lowenstein O (1942) A method of physiological assay of pyrethrum extracts. . Nature 150:760-762. Lund AE, Narahashi T (1983) Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs. Pestic Biochem Physiol 20:203-216. Mannikkb R, Elinder F, Larsson HP (2002) Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. Nature 419:837-841. McLaughlin GA (1973) History of pyrethrum: New York: Academic. Morgan K, Stevens EB, Shah B, Cox PJ, Dixon AK, Lee K, Pinnock RD, Hughes J, Richardson PJ, Mizuguchi K, Jackson AP (2000) beta 3: an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics. Proc Natl Acad Sci U S A 97:2308-2313. Motomura H, Narahashi T (2000) Temperature dependence of pyrethroid modification of single sodium channels in rat hippocampal neurons. J 150 Membr Biol 177223-39. Narahashi T (1962a) Nature of the negative after-potential increased by the insecticide allethrin in cockroach giant axons. J Cell Comp Physiol 59:67-76. Narahashi T (1962b) Effect of the insecticide allethrin on membrane potentials of cockroach giant axons. J Cell Comp Physiol 59:61-65. Narahashi T (1988) Molecular and cellular approaches to neurotoxicology: past, present and future. In: Neurotox '88: molecular basis of drug and pesticide action: New York: Elsevier. Narahashi T (1992) Nerve membrane Na+ channels as targetes of insecticides. Trends Pharrnacol Sci 13:236-241. Narahashi T (1996) Neuronal ion channels as the target sites of insecticides. Pharrnacol Toxicol 79:1-14. Narahashi T (2000) Neuroreceptors and ion channels as the basis for drug action: past, present, and future. J Pharrnacol Exp Ther 29421-26. Narahashi T, Zhao X, lkeda T, Nagata K, Yeh JZ (2007) Differential actions of insecticides on target sites: basis for selective toxicity. Hum Exp Toxicol 26:361-366. Noda M, Suzuki H, Numa S, Sttihmer W (1989) A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel ll. FEBS Lett 259:213-216. Noda M, lkeda T, Suzuki H, Takeshima H, Takahashi T, Kuno M, Numa S (1986) Expression of functional sodium channels from cloned cDNA. Nature 322:826-828. Noda M, Shimizu S, Tanabe T, Takai T, Kayano T, Ikeda T, Takahashi H, Nakayama H, Kanaoka Y, Minamino N (1984) Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312:121-127. O'Dowd DK, Gee JR, Smith MA (1995) Sodium current density correlates with expression of specific alternatively spliced sodium channel mRNAs in 151 single neurons. J Neurosci 15:4005-4012. Olson R, Liu Z, Nomura Y, Zhang T, Song W, Dong K (2008) Molecular and functional characterization of Para sodium channels in Drosophila melanogaster. Insect Biochem Mol Biol 38:604-610. Park Y, Taylor MF, Feyereisen R (1999) Voltage—gated sodium channel genes hscp and hDSC1 of Heliothis virescens F. genomic organization. Insect Mol Biol 82161-170. Piper MD, Skorupa D, Partridge L (2005) Diet, metabolism and lifespan in Drosophila. Exp Gerontol 40:857-862. Plummer NW, McBumey MW, Meisler MH (1997) Alternative splicing of the sodium channel SCN8A predicts a truncated two-domain protein in fetal brain and non-neuronal cells. J Biol Chem 272:24008-24015. Ramaswami M, Tanouye MA (1989) Two sodium-channel genes in Drosophila: implications for channel diversity. Proc Natl Acad Sci U S A 86:2079-2082. Ratcliffe CF, Westenbroek RE, Curtis R, Catterall WA (2001) Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain. J Cell Biol 154:427-434. Ray DE, Fry JR (2006) A reassessment of the neurotoxicity of pyrethroid insecticides. Phannacol Ther 111 :174-193. Rion S, Kawecki TJ (2007) Evolutionary biology of starvation resistance: what we have Ieamed from Drosophila. J Evol Biol 20:1655-1664. Rohl CA, Boeckman FA, Baker C, Scheuer T, Catterall WA, Klevit RE (1999) Solution structure of the sodium channel inactivation gate. Biochemistry 38:855-861. Salkoff L, Butler A, Wei A, Scavarda N, Giffen K, lfune C, Goodman R, Mandel G (1987) Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosophila. Science 237:744-749. Satin J, Kyle JW, Chen M, Bell P, Cribbs LL, Fozzard HA, Rogart RB (1992) A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 256: 1 202-1 205. 152 Shafer TJ, Meyer DA (2004) Effects of pyrethroids on voltage-sensitive calcium channels: a critical evaluation of strengths, weaknesses, data needs, and relationship to assessment of cumulative neurotoxicity. Toxicol Appl Pharrnacol 196:303-318. Shahidullah M, Reddy S, Fei H, Levitan IB (2009) In Vlvo Role of a Potassium Channel-Binding Protein in Regulating Neuronal Excitability and Behavior. J Neurosci 29: 1 3328-1 3337. Siddiqi O, Benzer S (1976) Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A 73:3253-3257. Silver K, Soderlund DM (2005) Action of pyrezoline-type insecticides at neuronal target sites. Pestic Biochem Physiol 81 :136—143. Sivilotti L, Okuse K, Akopian AN, Moss S, Wood JN (1997) A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS. FEBS Lett 409:49-52. Soderlund D, Bloomquist, JR (1990) Molecular mechanisms of insecticide resistance. In: Pesticide resistance in arthropods: Chapman and Hall, New York,. Soderlund DM, Bloomquist JR (1989) Neurotoxic actions of pyrethroid insecticides. .Annu Rev Entomol 34:77-96. Song JH, Narahashi T (1996) Modulation of sodium channels of rat cerebellar Purkinje neurons by the pyrethroid tetramethrin. J Pharrnacol Exp Ther 277:445-453. Song W, Liu Z, Tan J, Nomura Y, Dong K (2004) RNA editing generates tissue-specific sodium channels with distinct gating properties. J Biol Chem 279:32554-32561 . Stafstrom CE (2007) Persistent sodium current and its role in epilepsy. Epilepsy Curr 7:15-22. Sttihmer W, Conti F, Suzuki H, Wang XD, Noda M, Yahagi N, Kubo H, Numa S (1989) Structural parts involved in activation and inactivation of the sodium channel. Nature 339:597-603. 153 Suzuki DT, Grigliatti T, Williamson R (1971) Temperature-Sensitive Mutations in Drosophila melanogaster, Vll. A Mutation (parats) Causing Reversible Adult Paralysis. In, pp 890-893. Tan J, Liu Z, Nomura Y, Goldin AL, Dong K (2002) Alternative splicing of an insect sodium channel gene generates pharrnacologically distinct sodium channels. J Neurosci2215300-5309. Tanouye MA, Wyman RJ (1980) Motor outputs of giant nerve fiber in Drosophila. J Neurophysiol 44:405-421. Tatebayashi H, Narahashi T (1994) Differential mechanism of action of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J Pharrnacol Exp Ther 270:595-603. Terlau H, Heinemann SH, Stt'lhmer W, Pusch M, Conti F, Imoto K, Numa S (1991) Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett 293293-96. Thackeray JR, Ganetzky B (1994) Developmentally regulated alternative splicing generates a complex array of Drosophila para sodium channel isoforms. J Neurosci 14:2569-2578. Thackeray JR, Ganetzky B (1995) Conserved alternative splicing patterns and splicing signals in the Drosophila sodium channel gene para. Genetics 141 2203-214. Trimarchi JR, Schneiderrnan AM (1993) Giant fiber activation of an intrinsic muscle in the mesothoracic leg of Drosophila melanogaster. J Exp Biol 177:149-167. Trimarchi JR, Schneiderrnan AM (1995a) Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns. J Comp Physiol A 176:355-364. Trimarchi JR, Schneiderrnan AM (1995b) Different neural pathways coordinate Drosophila flight initiations evoked by visual and olfactory stimuli. J Exp Biol 198:1099-1104. Trimarchi JR, Schneiderrnan AM (1995c) Initiation of flight in the unrestrained fly, Drosophila melanogaster. J Zool 235:211-223. 154 ‘11 Verschoyle RD, Aldridge WN (1980) Structure-activity relationships of some pyrethroids in rats. Arch Toxicol 45:325-329. \fijverberg-HPM, van der Bercken J (1990) Neurotoxicological eggects and the mode of action of pyrethroid insecticides. CRC Crit Rev Toxicol 21:106-126. Vljverberg HPM, van der Zalm JM, van der Bercken J (1982) Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves. Nature 295:601-603. Walker DW, Muffat J, Rundel C, Benzer S (2006) Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan. Curr Biol 16:674-679. Wannke JW, Reenan RA, Wang P, Qian S, Arena JP, Wang J, Wunderler D, Liu K, Kaczorowski GJ, Van der Ploeg LH, Ganetzky B, Cohen CJ (1997) Functional expression of Drosophila para sodium channels. Modulation by the membrane protein TlpE and toxin pharmacology. J Gen Physiol 110:119-133. Welsh JH, Gordon HT (1947) The mode of action of certain insecticides on the arthropod nerve axon. J Cell Physiol 30:147-171. West JW, Patton DE, Scheuer T, Wang Y, Goldin AL, Catterall WA (1992) A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation. Proc Natl Acad Sci U S A 89:10910-10914. VWcher D, Walther C, Wicher C (2001) Non-synaptic ion channels in insects-basic properties of currents and their modulation in neurons and skeletal muscles. Prog Neurobiol 64:431-525. Wood JN, Boorrnan JP, Okuse K, Baker MD (2004) Voltage-gated sodium channels and pain pathways. J Neurobiol61255-71. Yu FH, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome Biol 4:207-213. Yu FH, Yarov-Yarovoy V, Gutman GA, Catterall WA (2005) Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharrnacol Rev 57:387-395. ' 155 Yu FH, Westenbroek RE, Silos-Santiago l, McCormick KA, Lawson D, Ge P, Ferriera H, Lilly J, DiStefano PS,’Catterall WA, Scheuer T, Curtis R (2003) Sodium channel beta4, a new disulfide—linked auxiliary subunit with similarity to beta2. J Neurosci 23:7577-7585. Zhao X, lkeda T, Yeh JZ, Narahashi T (2003) Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW. Neurotoxicology 24:83-96. Zhao Y, Yarov-Yarovoy V, Scheuer T, Catterall WA (2004) A gating hinge in Na+ channels; a molecular switch for electrical signaling. Neuron 41 :859-865. Zhou W, Chung I, Liu Z, Goldin AL, Dong K (2004) A voltage-gated calcium-selective channel encoded by a sodium channel-like gene. Neuron 42:101-112. 156 MICHIGAN STATE UNIVERSITY LIBIRAR'IES III III II' III III I II II ......