Interactions between an endogenous oscillator and response to tap in C. elegans

Transcription

1 Psyhbilgy (4). 57/-58 Interatins between an endgenus sillatr and respnse t tap in C. elegans SUSAN SIKLOS, JENNIFER A JASPER, STEPHEN R. WICKS, and CATHARINE H. RANKIN University f British Clumbia, Vanuver, British Clumbia, Canada In this study, we investigated the interatin between the endgenus sillatr gverning defeatin in Caenrhabditis elegans and the wnn's respnse t a mehanial stimulus, a tap. The results shwed that the defeatin yle beame prgressively lnger ver the life span f the wnn. Taps hanged the phase f the defeatin yle, and taps delivered at different phases f the defeatin yle had signifiantly different effets n the phase hange bserved. In ntrast, the phase f the defeatin yle in whih the tap was delivered had very little effet n either the frequeny r the magnitude f the respnse t the tap. The phase hange by the taps habituated slwly ver multiple taps. There was n effet f tap delivery at a nsistent phase fthe defeatin yle; therefre, interatin between tap and defeatin annt aunt fr muh f the variane seen in a nnnal habituatin experiment. One f the simplest but least understd frms f learning is habituatin, whih is defined as a derease in respnding ver repeated stimulatin (Grves & Thmpsn, 197). In many rganisms, a striking feature f habituatin is that there are large individual differenes; thus, the mean urves are nt smth but shw a high level f variability. Fr examples, see the data frm Aplysia (e.g., Rankin & Carew, 1987), frm Caenrhabditis elegans (. elegans; e.g., Rankin & Brster, 1992), frm fish (e.g., Peeke & Ven, 1973), and frm rats (e.g., Davis, Parisi, Gendelman, Tishler, & Kehne, 1982). One pssible hypthesis fr the within-subjets variability bserved ver trials and the "nise" seen in the grup mean habituatin urves in respnse habituatin is that they reflet the effets f the stimulus' interating with different phases f an endgenus sillatr. C. elegans ffers a gd mdel system in whih t study rganismal fatrs affeting habituatin, beause its physilgy, genetis, and develpmental stages are well knwn. C. elegans has a small nervus system nsisting f nly 32 neurns and, therefre, is a prime andidate fr determining whih ells are invlved in learning. One stimulus, whih has been used in studies f habituatin in C. elegans, is the tap stimulus (Rankin, Bek, & Chiba, 199; Rankin & Brster, 1992; Wiks & Rankin, 1995). In respnse t a vibratry stimulus (a tap) t the Petri dish, adult C. elegans swim bakward. This respnse, whih has been named the tap-withdrawal respnse (Chiba & Rankin, 199), shws habituatin t repeated stimulatin. Althugh different grups f subjets trained with different interstimulus intervals (ISIs; the This wrk was funded by an NSERC perating grant t C.H.R. and an NSERC Predtral Fellwship t S.R.W. Crrespndene nerning this artile shuld be addressed t C. H. Rankin, Department f Psyhlgy, University f British Clumbia, Vanuver, BC V6T IZ4, Canada ( rankin@rtex.psyh.ub.a). times between stimulatins) shw systemati and signifiant differenes in their level f habituatin perfrmane, individual subjets within any given grup shw a high degree f variability arss trials. This individual variability annt be aunted fr by innate r experiential differenes amng subjets, beause the subjets are (I) genetially idential, (2) raised in similar nditins, (3) evaluated arding t a strit and preise prtl at the same develpmental stage, and (4) tested with the same equipment (Gallway, Wiks, & Rankin, 1997). One hypthesis t explain the within-subjets variability bserved ver trials in habituatin (nt nly in C. elegans, but in many rganisms studied) is that it reflets the effets f an interatin f respnse t tap with an endgenus sillatr. It is pssible that the magnitude fa given respnse might vary if the stimulus is delivered at different phases f sme endgenus sillatr. At sme phases f the sillatr, respnses may be larger, at thers smaller, than the average respnse. It is pssible that sme reprted ases f sensitizatin might simply be the result f a hane interatin between the time intervals f the experimental design and the peak f an internal sillatr. In studies f habituatin in whih stimuli are delivered at a set interval, it may be that this interval nflits/interats with an internal sillatr in suh a way that variatins in respnse amplitude are greater than they wuld be if all the stimuli urred at the same phase f the sillatr. C. elegans has several behavirs that are peridi (i.e., feeding, defeatin, and egg laying) and pssibly ntrlled by endgenus sillatrs (Liu & Thmas, 1994). The variability bserved in studies f habituatin t tap may be the result f an interatin between the animal's ability t respnd t tap and differing states f an internal sillatr. The interatin between the tap respnse and the endgenus defeatin yle was hsen fr these experiments beause the defeatin yle f wild-type 571 Cpyright 2 Psyhnmi Siety, In.

2 572 SIKLOS, JASPER, WICKS, AND RANKIN wrms is highly peridi, with deviatins f nly a few sends (Crll & Smith, 1978; Liu & Thmas, 1994; Thmas, 199), and is within the same time frame as the 1-6 se ISIs used in many habituatin studies (e.g., Rankin et ai., 199; Rankin & Brster, 1992; Wiks & Rankin, 1995). The defeatin yle f C. elegans lasts, n average, 45-7 se and ends with the expulsin f waste frm the bdy (defeatin). Defeatin itself nsists f three steps: the ntratin f the psterir bdy musle, fllwed by the ntratin f the anterir bdy musle, and ending with the ntratins f the intestinal musles and the anal depressr, whih results in the expulsin (Thmas, 199). Tgether, these musle ntratins signal that a defeatin has urred; thus, the defeatin yle f C. elegans is easy t identify and an be aurately sred. In additin, the behavir and genetis f the defeatin yle are well understd (Iwasaki & Thmas, 1997; Liu & Thmas, 1994; Reiner & Thmas, 1995; Reiner, Weinshenker, & Thmas, 1995; Thmas, 1994). Finally, a tuh stimulus, whih shares muh f the same neural pathway as the tap stimulus (Wiks & Rankin, 1995), resets the defeatin yle (Liu & Thmas, 1994; Thmas, 199). It is nt knwn whether a different mehansensry stimulus, the tap stimulus, als prdues a reset in the defeatin yle. The present experiments were designed t investigate the effets that tap has n the defeatin yle in C. elegans, the effet the defeatin yle has n the respnse t tap, and the interatin between the defeatin yle and the presses f habituatin. GENERAL METHOD Subjets A ttal f 458 C. elegans hermaphrdites frm the Bristl N2 wild-type labratry strain (Brenner, 1974) were used. The subjets were btained frm the Caenrhabditis Genetis Center and were grwn synhrnusly at 2 C n 5-m Petri plates that were filled with 1-ml Nematde Grwth Medium (NGM) agar and streaked with E. li strain OP5 (Brenner, 1974). Apparatus The animals were bserved n the NGM (Brenner, 1974) agar with a steremirspe (Wild Leitz, Canada, Ltd., Mdel M3Z). Fr data lletin and sring purpses, vide tape-rerding equipment (Panasni amera 5, Panasni 196 VCR, NEC lr mnitr) was attahed t the steremirspe. In rder t time the stimulus delivery and the defeatin yles, a time-date generatr (Panasni 81) superimpsed a stpwath nt the mnitr. During testing, the Petri plates that ntained individual wrms n NGM agar were held by a hlder designed ut f a Petri plate lid that had been munted nt a plasti rd. The rd was attahed t a Marzhauser mirmanipulatr (Mdel MM33) fr smth, nsistent mvement f the plate as it was kept within the field f the vide amera. Fr delivery f the tap stimuli, a mehanial tapper, whih nsisted f an eletrmagneti relay and a wire arm, was munted n the hlder. The eletrmagneti relay was nneted t a Grass stimulatr (Mdel S88), whih regulated the delivery f eah stimulus. The wire arm f the tapper was psitined s that the stimulus was delivered at a latin halfway up the side f the dish. The stimulatr was set t deliver a 25-mse, 6-V pulse t the relay. Predure Individual wrms were bserved n 5-m Petri plates filled with 1-ml NGM agar and streaked with E. li strain OP5 (Brenner, 1974). In the researh n endgenus sillatrs, an sillatr has traditinally been split int phase angles, instead f time intervals. Therefre, the defeatin intervals were determined fr eah wrm, and the perid split int 36 t determine the time f tap delivery. It was imprtant that the tap stimulus was delivered at a partiular plae in the yle. If the tap were administered at a set time interval, rather than at a phase angle, after the last defeatin f eah animal, it wuld ur at different phases f the sillatr, sine a 4- day-ld wrm's defeatin yle an range at least frm 3 t 6 se (Crll & Smith, 1978). Fr example, administering the tap stimulus at a time equivalent t 33 int the defeatin yle f ne wrm uld mean that the tap wuld be given at the beginning f the next yle fr a send wrm with a shrter defeatin yle. Test plates were washed with E. li (streaked) 48 h befre testing: The animals were preplated n the streaked plates 24 h later and left at rm temperature (2-22 C) vernight. Prir t bservatin, the animals were plaed n the steremirspe stage fr 1 min, with the light n the stage turned ff and the lid f the Petri dish remved. When rerding began, the light was turned n; measurements f defeatin yles were rerded frm the expulsin step f the defeatin yle until the expulsin f the next defeatin yle. Sring A wrm was nsidered t have made a reversal respnse t the tap stimulus if the videtape rerding shwed that the wrm mved bakward within I se f the tap stimulus. The magnitude f the reversal was sred by reviewing videtapes, using stpframe vide analysis t trae the path f the reversal nt an aetate sheet. Ifa wrm was spntaneusly reversing when the tap was administered, that wrm was nt used in the data fr respnse magnitude arss phase grups. The aetate traings were then digitally sanned and measured (DeskSanll, NIH Image sftware, Pwer Maintsh mputer). Fr analysis, the measurements were transferred int a statistial prgram (StatView 4.). EXPERIMENT 1 The first experiment was designed t investigate the effets f age n the defeatin yle. Thmas ( 199) reprted an inrease in the mean duratin f defeatin yles in wrms as a funtin f age: Cyles ranged frm 42 se at age 5 h t 59 se at 142 h f age. Blanwski, Russell, and Jabsn (1981) bserved a rapid deline in defeatin frequeny frm Day 3 t Day 6 f a C. elegans' life. Taken tgether, these data supprt the ntin that the defeatin yle inreases in length as wrms age. The studies f habituatin in C. elegans have primarily been dne using 4-day-ld wrms (e.g., Rankin et ai., 199; Rankin & Brster, 1992; Wiks & Rankin, 1995). The purpse f this experiment was t determine whether the length f the defeatin yle differed signifiantly between 3 and 5 days f age and, mre imprtant fr this study, whether it differed signifiantly ver the urse f the 4th day. Methd Subjets. A ttal f 4 hermaphrditi N2 C. elegans were used. Predure. The subjets were separated int fur grups f 1 wrms. The first grup nsisted f wrms that were 3 days ld

3 INTERACTION BETWEEN DEFECATION AND TAP RESPONSE 573 tested at 73 h f age. The send grup nsisted f 4-day-lds that were tested at 9 h f age. The third grup was made up f 4-dayld wrms that were tested at 99 h f age. The final grup nsisted f 5-day-ld wrms that were 126 h ld when they were tested. The wrms were left undisturbed n the mirspe stage fr 1 min, after whih three defeatin yles fr eah wrm were rerded. Fr eah wrm, a mean f the three interdefeatin intervals (!DIs) was alulated and used as the!di fr that wrm. Results and Disussin Figure 1 shws the mean IDI fr eah f the fur grups f subjets. An analysis f variane (ANOVA) shwed a signifiant differene in IDIs arss age grups [F(3,36) = 94.78, p <.5]. Fisher's prteted least signifiant differene tests indiated that the mean IDIs between eah grup, exept that between the 73-h and the 9-h grups, were signifiantly different. These results shw that age des in fat affet the duratin f the defeatin yle f C. elegans, nsistent with the researh f Blan w ski et ai. (1981) and Thmas (199). In additin, the results indiated that there was a signifiant differene in the IDIs ver the urse f Day 4 (grups 9 and 99 h f age). Sine the habituatin experiments in ur labratry use 4-day-ld wrms (e.g., Rankin et ai., 199; Rankin & Brster, 1992; Wiks & Rankin, 1995) and sine the defeatin yle hanges in duratin frm the beginning t end f the 4th day psthath, in all further experiments nly wrms h f age were used. EXPERIMENT 2 This experiment was designed t investigate whether a tap affeted the defeatin yle by resetting the yle 1 iii 8 = "E : 6 - (.) 4 "E : Age (in hurs) Figure t. Interdefeatin intervals (IDIs, in sends) fr fur different age grups (N = t/grup): 3-day-lds (73 hld), 4-daylds made late in the day (9 hld), 4-day-lds made early in the day (99 h ld), and 5-day-lds (126 h ld). With the exeptin f 73 hand 9 h, all the grups differed signifiantly in the duratin f the lois. in the same way as is dne by a tuh stimulus (Chalfie et ai., 1985; Thmas, 199) r whether it had any ther effets n defeatin. In additin, the predure als tested whether administering a tap stimulus at different pints in the defeatin yle f C. elegans affeted the frequeny r magnitude f reversals t tap. Methd Subjets. A ttal f 392 hermaphrditi N2 C. elegans ranging frm h f age were used. In additin t age, the defeatin yle an be influened by a number f envirnmental fatrs, suh as baterial ntaminatin, temperature, and humidity. In rder t ensure that nly healthy subjets were tested, it was imprtant t establish riteria fr the length f the!di used. The lwer limit riterin f 41 se was established using data frm Liu and Thmas (1994), wh reprted that the average defeatin yle was 45.3 se, with a standard deviatin f 4.3 sends. Data frm Experiment I shwed that the average defeatin yle f 4-day-ld wrms was 6 se, with a standard deviatin f S se; thus, the upper limit was set at 6S se. Any animals with defeatin yles falling utside f the se riterin were nt used. Predure. The experiment nsisted f 12 grups f animals, with wrms in eah grup. The 12 grups represented the defeatin yle divided int 3 phases (with indiating the expulsin), in rder t determine hw stimuli at eah phase f the yle might affet the defeatin yle and hw the tap-withdrawal respnse was affeted by taps at eah f these phases. Eah f the 12 grups rrespnded t a different phase in the defeatin yle:,3,6,9,12,15,18,21,24,27, 3, 33. Fr eah wrm tested, the first step was t measure the duratin f three defeatin intervals. A spreadsheet was used t alulate the average duratin f the individual wrm's defeatin yle. The average defeatin yle duratin was then used t alulate the time that wuld rrespnd t eah f the phases f the defeatin yle (by dividing the mean duratin int 36 ). This, in turn, was used t determine the time after the third defeatin at whih the tap stimulus shuld be administered. The tap stimulus was then delivered at the apprpriate time fr the apprpriate grup phase f the individual animal. The final step was t rerd five defeatin yles fllwing the tap. The five defeatin yles fllwing the tap were then used t alulate the phase hange fr eah wrm. The magnitude f reversals was alulated using the measurements taken frm the aetate traings. As a result f tehnial diffiulties in rerding and sring sme fthe data, the number f wrms prviding respnse magnitude data ranged frm 25 t 28 per grup. ANOVAs were perfrmed n all the data, using an alpha level fp <.5. Results The effets f a single tap n the defeatin yle. Tap des have a signifiant effet n the duratin f the defeatin yle, and that effet differs depending n the phase f the yle in whih the tap was delivered. The phase respnse urve fr the first defeatin interval after the tap is shwn in Figure 2A. If there were n hange in duratin f the yle fllwing the tap, the sre wuld be zer. Any sre greater than zer represents an inrease in the duratin f the yle and, thus, a phase hange. Tap stimuli delivered at all phases affeted the defeatin yle: Tap had a small effet n the phase f the defeatin yle immediately fllwing the defeatin, and the effet n the phase gradually inreased until 24 int the defeatin yle, at whih time there was a sharp drp in the effet n phase hange just prir t the next defeatin. An ANOVA mparing the phase

4 e 574 SIKLOS, JASPER, WICKS, AND RANKIN A e- Experimental Results Therelial Reset B 35 C 35 I :if 2 " i 15 5i 1 s:; () 5 CD UJ as s:; a.. -5 D E J, 15 5i 3 1 II 5 i... dp.... m... g, 15 as 3 1! Figure 2. Mean phase hanges in respnse t tap delivered at eah 3 phase f the defeatin yle (N = per grup) fr the first thrugh the fifth defeatins after a tap. In the first yle after tap, there were signifiant differenes in the phase hanges, depending n where the tap was delivered during the defeatin yle. There were n signifiant differenes in the effets f the tap at different phases f the defeatin yle fr the send thrugh the fifth yles after the tap. (A) Mean phase hange f the first defeatin yle after the tap (filled irles). Open bxes represent the theretial results predited if the tap aused reset f the defeatin yle at all phases. (8) Mean phase hange fthe send defeatin yle after the tap. (C) Mean phase hange f the third defeatin yle after the tap. (D) Mean phase hange f the furth defeatin yle after the tap. (E) Mean phase hange f the fifth defeatin yle after the tap.

5 INTERACTION BETWEEN DEFECATION AND TAP RESPONSE 575 hange arss grups shwed that there were signifiant differenes in the degree f phase hange, depending n when in the yle the tap was delivered [F(II,368) = 8.381,p <.5]. If the defeatin yle was entrained t sme "master sillatr," ne might predit that after being disrupted, the yle wuld gradually shift bak int phase ver the next few yles. The phase respnse urves fr the send thrugh the fifth defeatin intervals fllwing the tap are shwn in Figures 2B, 2C, 2, and 2E. Althugh the tap seems t have an effet n the length f the defeatin yle immediately fllwing the tap stimulus (Figure 2A), the tap des nt appear t have an effet n the length f the send, third, furth, r fifth defeatin yle fllwing the tap. These results suggest that the tap auses the endgenus sillatr regulating defeatin t permanently shift ut f phase. Thmas (199) reprted that the tuh stimulus aused a reset in the defeatin yle (i.e., aused the yle t begin again). In Figure 2A, the idealized data fr reset f the phase f the defeatin yle are pltted alng with the data frm this experiment, shwing the respnse f the defeatin yle t tap stimuli. The data shw that the tap nly resets the defeatin yle when administered at 9, 12, and pssibly 15. In rder fr the tap t reset the defeatin yle in eah phase grup, the pints fthe tw lines in Figure 2A wuld need t be inident with eah ther. The effets f phase f the defeatin yle n respnse t a single tap. An examinatin f the withdrawal respnses t taps delivered at different phases f the defeatin yle shwed that the phase f the defeatin yle at whih the tap was delivered did nt have a signifiant effet n the magnitude (Figure 3A) r the frequeny (Figure 3B) f reversals. Althugh the respnses appear t be smaller arund the time f the defeatin (, 3, and 33 ), an ANOVA n the magnitude f the reversals shwed n signifiant differenes between grups [F(II,29) =.81, p >.5]. Thus, variability in either the frequeny r the magnitude f the respnse t a tap is nt greatly influened by the plaement f the tap with regard t phases f the defeatin yle. EXPERIMENT 3 This experiment was designed t investigate whether multiple taps affet the defeatin yle by ausing a nstant phase hange in the defeatin yles fllwing eah tap r whether they have any ther effets n the defeatin yle. In additin, the data test whether administering multiple tap stimuli at a partiular phase in the defeatin yle f C. elegans affets the magnitude f the tap-withdrawal respnse and, nsequently, affets the shape f the habituatin urve by dereasing sme f the variability ften bserved when a tap is administered at a given lsi, irrespetive f the defeatin yle. Methd Subjets. A ttal f 66 hermaphrditi N2 C. elegans was used. The same riteria fr the length f the IDI as thse desribed in Experiment 2 were als used in this experiment. Predure. Fr all the subjets, three defeatin yles were initially rerded in rder t determine the mean defeatin yle fr eah individual subjet. The mean defeatin yle was then used t determine when, fllwing eah defeatin, the tap shuld be administered fr the experimental subjets, using a spreadsheet: The wrms were divided int tw grups, ne fr eah phase tested. The phases f the defeatin yle hsen fr tap delivery were 12 and 27. These phases were hsen t represent phases ne third and tw thirds f the way thrugh the defeatin yle. Experiment 2 shwed that a tap delivered at 12 aused a reset f the yle, whereas a tap delivered at 27 aused a large phase hange but did nt reset the yle. Sixteen experimental wrms frm the first grup ( 12 grup) reeived the tap stimuli 12 int the defeatin yle, and 17 experimental wrms frm the send grup (27 grup) reeived the tap stimuli 27 int the defeatin yle. Eah f the experimental wrms in eah grup (12 and 27 ) was paired with a yked ntrl wrm that was given taps at the same intertap intervals as its experimental unterpart, regardless f the length r phase f its wn defeatin yle. Eah subjet reeived 3 stimuli. The lengths ftw defeatin intervals fllwing the end f the 3 taps were als rerded. During Experiment 2, there were n defeatin intervals greater than 133 se rerded. Therefre, fr this experiment, if mre than 133 se went by withut a defeatin, it was assumed that either the experimenter had missed ne r the wrm had mved up the side f the Petri plate ut f the fd, interrupting the rhythmi defeatin. Fr the purpse f data analysis, these intervals were nsidered missing data pints (less than 4% f the ttal data pints). There were 16 experimental wrms fr the 12 grup and 17 experimental wrms fr the 27 grup. Results Effets f multiple taps n the defeatin yle. Fr bth grups tested, the first tap in the series aused an inrease in the length f the defeatin yle ver the baseline defeatin yle similar t that whih was reprted in Experiment 2. With repeated stimulatin, the phase shift f the!di habituated fr bth the 12 and the 27 grups (Figure 4A). T test fr habituatin, the mean f the first tw!dis was mpared with the mean f the last tw!dis, using a paired t test (Figure 4B). Bth lsi grups shwed signifiant derement frm firstt last IDIs [12 grup: t(15) = 3.12,p =.7; 27 grup: t(l6) = 2.8, p =.5]. Althugh bth grups shwed signifiant derement in the amunt f phase hange frm the first tap t the last tap, this derement was mdest, with nly a 16.53% drp in the 12 grup and nly a 14.13% drp in the 27 grup. Immediately after the end f stimulatin, the IDI in bth grups returned t its prehabituatin duratin (Figure 4A). Effets f phase f the defeatin yle n respnse t multiple taps. The habituatin urves fr respnse magnitude t tap fr the fur grups f wrms are shwn in Figures 5A-5. There were 16 wrms in eah grup (data frm ne fthe 27 ntrl wrms were lst, wing t mputer errr; therefre, its experimental partner's data were disarded). In rder t mpare the variability

6 576 SIKLOS, JASPER, WICKS, AND RANKIN A 6 5 "C :J -,4 as ::E 3 a. UJ &!. 2 : as ::E B UJ (ij.8.6 II: ' (;' :.4 :J C" U Figure 3. (A) Mean respnse magnitude f the tap-withdrawal respnse fr eah grup. There were n signifiant differenes in tap respnse magnitude, regardless fthe phase fthe defeatin yle when a tap was delivered. (8) The frequeny f reversals t tap fr taps delivered at the different phases f the defeatin yle. between the habituatin grups, an ANOVA was run n the mean abslute deviatin sres fr eah grup. The mean abslute deviatin sres were alulated by determining the abslute value f the differene between the mean sres fr eah stimulus and eah animal's sre n that stimulus. This mparisn f deviatins between respnses t taps delivered at a set phase f an individual wrm's defeatin yle with respnses t taps delivered withut referene t an individual's yle shws that administering taps at a set phase f the defeatin yle did nt affet the variability f the tap-withdrawal respnse f C. elegans [Figure 5E; F(3,116) =.614, p =.67]. GENERAL DISCUSSION The Effets f Tap n the Defeatin Cyle Tap des ause a phase hange in the defeatin yle. Studies f ther endgenus sillatrs have shwn that a disruptive stimulus an ause either a phase advane,

7 INTERACTION BETWEEN DEFECATION AND TAP RESPONSE 577 A 9 iii 85 -E Gi "E 65 "E ttl a be d e Interval B 8 iii 7 $ 6 5 :;::::: First 2 ttl Last "... 3 "E 2 ttl Figure 4. (A) The mean interdefeatin intervals (lois) fr the 12 (filled irles; N = 16) and 27 (pen bxes; N = 17) experimental grups. The first three pints fr eah grup represent 3 baseline lois prir t stimulatin; they are fllwed by 3 lois during habituatin training t 3 taps. The last tw pints n the urves represent the first 2 lois fllwing the end fthe tap stimulatin. (8) The mean fthe first 2 lois and the mean f the last 2 lois fr the 12 and the 27 grups. Within eah grup, there was a signifiant derement in the duratin f the 11 frm the beginning f tap habituatin training t the end. where the yle wuld beme shrter than the mean, r a phase delay, where the yle wuld beme lnger than the mean. The effets f the disruptive stimulus depend n the intensity f the stimulus and the initial phase f appliatin (Brda, Jhnsn, Taylr, & Hastings, 1989). In this study, the smallest phase hange urs lse t the time f defeatin, between 1 se befre and 1 se after the expulsin phase f the defeatin. The greatest phase hange urs in the send half (Figure 2A) f the defeatin yle, at 24. The data, therefre, shw that the tap stimulus has a different effet n the defeatin yle when it urs at different times during the yle. Thmas (199) reprted that administering a tuh stimulus t the head fthe wrm prdued a reset in the endgenus sillatr. If a tuh was administered 2 se int the defeatin yle, the fllwing defeatin yle was 2 se lnger, suggesting a reset t time zer at the time f the tuh stimulus. If that were the ase fr tap, there wuld have been an inrease in phase hange rrespnding t the higher phase hange grups. Hwever,

8 578 SIKLOS, JASPER, WICKS, AND RANKIN A ".2 " Cl ('(l en a. en a: ('(l :., B : '., f I,. I'.. ' E " :5 15 en. 1 < ('(l 5 C12 C27 E12 E27 Figure 5. (A) Habituatin fthe tap-withdrawal respnse fthe 12 experimental grup (N = 16) when the tap was delivered at 12 int the defeatin yle. (8) Habituatin fthe tap-withdrawal respnse f the 12 yked ntrl grup (N = 16). (C) Habituatin fthe tap-withdrawal respnse f the 27 experimental grup (N = 16). (D) Habituatin fthe tap-withdrawal respnse fthe 27 yked ntrl grup (N = 16). (E) Mean abslute deviatin sres (abslute value f mean sres - individual sres) fr eah f the fur grups. with tap there was an inrease in phase hange up t 24 in the defeatin yle, at whih time there was a sharp derease in the phase hange. The inreasing phase hange frm 3 t 24 was nt prprtinal t the phase angle at whih the tap was administered. If the tap stimulus prdued a reset in the sillatr, there shuld have been phase hanges equal t the phase grup we were testing. Fr example, if the tap was given 5 se after expulsin, the next defeatin yle shuld have been 5 se lnger. Hwever, the nly grups t display a phase hange equal

9 INTERACTION 8ETWEEN DEFECATION AND TAP RESPONSE 579 t its phase were the 9, 12, and 15 grups, and therefre these were the nly grups nsidered t displaya full resetting f the defeatin yle (Figure 2A). There are several differenes between the methd used by Thmas (199) and the present methd that might aunt fr the different results. These inlude the type f stimulus used, the timing f the stimulatin, and the number f stimuli tested. Thmas (199) used a headtuh stimulus, whereas the present study used a tap stimulus. The head-tuh stimulus nly ativates the head-tuh iruit (Chalfie et ai., 1985). The tap stimulus invlves a brad neural iruit ativating bth the head- and the tail-tuh iruits simultaneusly (Wiks & Rankin, 1995). Therefre, it is pssible that the tw different stimuli have different effets n the defeatin yle. In additin, the tuh stimulus used by Thmas was delivered by hand and, therefre, may have varied in intensity with eah delivery. Variatins in the intensity f the stimulus may have resulted in different effets n the defeatin yle. Thmas administered the tuh stimulus at speifi time intervals in the defeatin yle, rather than at different phases, at nly fur times in the defeatin yle (2,25,3, and 35 se, refleting phase angles f apprximately 15, 21, 24, and 27, respetively, if the yle was 45 se lng), all f whih were quite lse t eah ther, and nly during the send half f the defeatin yle, rather than spanning the entire defeatin yle. The data frm the present study indiate that tap aused the sillatr t shift ut f phase (Figure 2A) and stay ut f phase (Figure 28, 2C, 2, and 2E), ausing a lnglasting phase delay. The first defeatin yle after the tap was signifiantly lnger than the mean defeatin yle preeding the tap (Figure 2A). Hwever, the send thrugh the fifth defeatins after the tap were all apprximately the same length as the mean defeatin yles befre the tap (Figures 28, 2C, 2, and 2E). If the defeatin yle were t shift bak int phase, at least ne f the 2nd, 3rd, 4th, r 5th defeatin yles wuld be shrter r lnger than the mean defeatin yle preeding the tap, in rder t shift bak int phase. Hwever, sine this did nt ur in the data, it is lear that the tap stimulus aused a lng-lasting phase delay in the defeatin yle f C. elegans and that, within five yles, there was n revery t the riginal phase. The return t the riginal length fthe defeatin yle extends Thmas' (199) finding that the 2nd defeatin after the tuh stimulus was the same length as the mean defeatin yle prir t the tuh stimulus. Frm these data, it is pssible t nlude that the defeatin yle is nt strngly entrained t sme internal sillatr with whih it wuld have t retain a nsistent phase relatinship. Studies f iradian bilgial sillatrs have shwn that (I) mst iradian lks shift phase in respnse t light pulses and, therefre, rarely prdue a mplete reset and (2) the magnitude f the shift is dependent n when the stimulus is delivered (Ashff, 1965). Therefre, ur results, whih did nt shw resetting f the defeatin yle aused by the tap stimulus, rrespnd quite lsely t Ashff's desriptin f iradian lks: Our results shwed that the endgenus sillatr shifted phase and that the magnitude f the shift was dependent n when the stimulus was delivered and rarely prdued a mplete reset. The effets f multiple taps n the defeatin yle prvide evidene that the phase hange aused by a disruptive stimulus habituated with repeated stimulatins. The 11 gradually dereased tward the baseline IDI with repeated presentatins f the tap stimulus. Interestingly, 25 ut f the 3 lois fllwing tap were lnger in the 27 grup than in the 12 grup. This bservatin is nsistent with the results f Experiment 2, where we fund a phase hange that was apprximately 25 greater in the 27 grup than in the 12 grup. Experiment 3 prvided evidene that this inreased phase hange ntinued arss multiple taps and that even thugh the phase hange habituated fr bth grups, the phase hange still remained greater fr the 27 grup thrughut the experiment. The results f Experiment 3 shw that the disruptin f the defeatin yle fllwed the same mehanisms f habituatin as ther respnses, suh as the tap-withdrawal respnse and the tuh respnse, as fllws: (I) Similar t the dereased tap-withdrawal and tuh respnses with multiple stimulus presentatins, the phase hange f the defeatin yle was eliited less with eah tap given, and (2) similar t the habituatin urves f the tapwithdrawal respnse and the tuh respnse, the habituatin urve f the 11 als shwed sme variability (Figure 4A). Effets f phase fthe defeatin yle n respnse t tap. The phase f the defeatin yle in whih a tap was delivered had n signifiant effet n the tapwithdrawal respnse when nly a single tap was administered. There was a small trend apparent in the data (Figure 3A), hwever, that indiated that respnse magnitude slwly inreased frm the time f the expulsin until the middle f the defeatin yle at 18, at whih time the respnse magnitude dereased again. Figure 38 shws a small derease in the frequeny f reversals right befre the next defeatin wuld be abut t ur. A derease in respnding near the end f the defeatin yle wuld be expeted, beause tail musles needed fr reversing are nw invlved in defeatin (Thmas, 199). If the interatin between time f tap delivery and the phase f the defeatin yle played an imprtant rle in respnse magnitude during habituatin, we wuld expet less variatin in the habituatin urve in the experimental grup (Figures 5A-5C), where taps were administered arding t eah individual wrm's defeatin yle, than in the ntrl grups (Figures 58 and 5), where taps were given regardless f eah animal's defeatin yle. It is pssible that the attempt t design an experiment in whih the tap was delivered at a nsistent phase f the defeatin yle was nt suessful. One the first tap was administered, the defeatin yle was shifted ut f phase and, therefre, was lnger than the baseline defeatin yle. This was nt taken int aunt when

10 58 SIKLOS, JASPER, WICKS, AND RANKIN alulating the time fr administering the send, third, furth, and suessive taps. Fr example, if the mean IDI prir t the tap stimulus was 6 se, tap was administered 2 se after every defeatin fr the 12 grup. This did nt take int aunt the length fthe new defeatin yle. If the reset IDI was lser t 8 se and the tap was still administered 2 se after the expulsin, the tap wuld then rrespnd mre lsely t a phase f 9. Sine the phase length varied ver the urse f the habituatin series, the tap ranged ver a prtin f the defeatin yle, rather than being administered at a distint phase. Our riginal hypthesis abut the interatin between an internal sillatr and respnse magnitude t tap was prediated n a stable perid sillatr. In this experiment, we have demnstrated that the perid f the defeatin yle is altered by a single tap and is altered in a variable fashin by repeated taps. Thus, the perid bemes a "mving target" as it first resets and the reset then habituates slwly t repeated taps. This may ntribute t even greater variability f respnding than with a stable perid sillatr. It is pssible that if the timing f eah tap had been determined mre preisely, we wuld have btained habituatin urves with less variability than thse btained in Experiment 3. It may be pssible in future experiments t take this int aunt. Cnlusins Our findings suggest that tap stimuli have a large effet n the defeatin yle. When a single tap was administered, there was a phase hange that aused a lnglasting phase delay in the defeatin yle. The length f the phase delay depended n where in the defeatin yle the tap was administered. The defeatin yle was nly reset when the tap was delivered at 12 ± 3. When multiple taps were administered, the phase hange aused by the tap habituated slwly arss the urse f the experiment. With respet t the effet f the phase f the defeatin yle n the tap-withdrawal respnse, there was a weak relatinship. The results f Experiment 2 indiate that the phase f the defeatin yle at whih a tap is administered has n nsistent effet n the magnitude r frequeny fthe tap-withdrawal respnse. Repeated stimulatin at a nsistent phase f the defeatin yle did nt appear t have any effet n the variability bserved in the habituatin urves. It may be that the sillatr underlying defeatin des nt playa rle in the respnse t tap but that sme ther, as yet unidentified, sillatr des influene respnse t tap, r it may be that the sure f variability in the respnse t tap is independent f internal sillatrs. Further investigatin with a variety f rganisms is needed. REFERENCES ASCHOFF. J. (1965). Respnse urves in iradian peridiity. In 1. Ashff(Eds.), Ciradian lks (pp ). Amsterdam: Nrth Hlland. BOLANOWSKI. M. A., RUSSELL. R. L.. & JACOBSON. L. A. (1981 ). Quantitative measures f aging in the nematde C. elegans: I. 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