Contraction, Serotonin-Elicited Modulation, and Membrane Currents of Dissociated Fibers of Aplysia Buccal Muscle

8 Pages·1991·3.4 MB·English

by  J L Ram

Reference: Biol. Bull. 180: 276-283. (April, 1991)
Contraction, Serotonin-Elicited Modulation, and
Membrane Currents of Dissociated Fibers
of Aplysia Buccal Muscle
JEFFREY L. RAM, FENG ZHANG, AND LI-XIN LIU
Department ofPhysiology, WayneState University, Detroit, Michigan 48201
Abstract. Feeding muscles ofthe buccal mass ofAplysia immediate effect, but rather, it modifies the influence of
are innervated by cholinergic and serotonergic neurons. other effectors. These modulatory influences appear to
Buccal muscle 15 contracts in response to acetylcholine fine-tune the nervous system and the muscles it controls
(ACh). During feeding arousal, ACh-elicited contraction forswitchingbetween differentbehavioraltasks. The same
ofmuscle 15 ispotentiatedbyserotonin(5-HT). Thispaper circuits can subserve several different behaviors; the be-
demonstrates a dissociated cell preparation ofmuscle 15 havioral output depends on the relative weighting ofdif-
in which cellular mechanisms regulating contraction can ferent synapses and the excitability of circuit elements.
be investigated. Modulation ofmuscle is part ofthis integrated scheme.
Dissociated muscle fiberscontracted in responsetoboth Although the relative strength ofcontractions appropriate
KC1 and ACh. Serotonin (10~6 M) significantly poten- for different behaviors could be manipulated by discrete
tiated the shortening caused by both KC1 and ACh. Po- relative changes in motoneuron activity, an additional,
tentiation lasted at least 4 min, similarto potentiation in possibly more efficient method may be to give different
intact muscles. "global commands" that change the relative strengths of
Fourtypes ofcurrents recorded by patch clamp meth- contractile responses in ways appropriate for particular
ods are illustrated. With 540 m/U KG in the patch elec- behaviors. An example ofa modulatory effect ofa neu-
trode, stretch-activated channels having a chord conduc- rotransmitter mediating a change in behavior is the po-
tance of 150 pS are observed in on-cell patches. In whole tentiatingeffect ofserotonin (5-HT) on buccal massmus-
cellconfiguration, AChelicitsinward currentataholding clesofAplysia, which isbelievedtomediate, in part, feed-
potential of -60 mV. With high potassium in the elec- ing arousal.
trode, depolarization elicitsanoutwardcurrent. Thevolt- Thebuccal massmusclesofAplysiaaresmooth muscles
age-dependent outwardcurrent isblocked withcesium in used in voluntary feeding movements. These musclesare
the electrode and 4-aminopyridine and tetraethylam- innervated by cholinergic, peptidergic, and serotonergic
monium outside the cells. The remaining voltage-depen- neurons. Acetylcholine(ACh)isthedirecteffectorofthese
dent inward current is calcium dependent. The voltage- muscles,asexemplified bythecontractile responsetoACh
dependent inward and outward currents are activated ofbuccal muscle 15. Muscle 15, also known as the acces-
within the range ofdepolarization produced by ACh and sory radular closer muscle (Cohen et al., 1978), is the
maythereforeplayrolesin regulatingcontractileresponses most intensively studiedAplysia muscle. 15 isinnervated
elicited by ACh. by buccal ganglion neurons B15 and B16 (Cohen et al.,
1978; Ram, 1983), both ofwhich synthesize ACh(Cohen
Introduction c/ al., 1978) as well as several peptides (Cropper et at.,
1987, 1988). In addition, 15 is innervated by the seroto-
Neurotransmitters can cause both direct and modula- nergic metacerebral giant cell (MCG). Activity of MCG
tory effects on target tissues. A "modulatory effect" ofa causes no direct response ofthe muscle; however, it po-
neurotransmitter is one in which the transmitter has no tentiatessubsequent contractile responsesto B15 and B16.
This modulation isachieved largely through post-synaptic
Received 9 August 1990: accepted6 November 1990. actions on the muscle (Weiss el a/., 1978). In isolated 15
276
DISSOCIATED MUSCLE FIBERS OF APLYSIA 111
buccal muscles were dissected from the animal, teased
intothin strips, and incubated for2-4 hat28Cin Instant
Ocean containing 10 mAI HEPES (pH 7.0, adjusted with
NaOH), 0.15% collagenase (SigmaType I). 0.1% soybean
trypsin inhibitor(SigmaType I-S), and 1 //g/ml leupeptin
Figure 1. Typical dissociated buccal muscle fiber. At the right is a
patchelectrodepointingtothefiber.Scale:calibrationmarksare7.8
apart.
muscles 5-HT produces no contractile response itselfbut
does potentiate ACh-elicited contractions (Ram el a/..
MCGs
1981). areactiveduringfeeding(Weisseta/.. 1978).
Lesion of serotonergic neurons changes (although does
not completely block) feeding motor activity (Rosen et
a/.. 1983, 1989). Thus, the modulatory effect of the se-
MCG
rotonergic neurons on feeding muscles appears to
have an important role in feeding arousal.
Although previous experiments on mechanisms me-
diating the modulatory effect of5-HT on buccal muscles
AMP
havesuggested roles forcyclic (Mandelbaum, 1980:
Rametal.. 1983, 1984a)andcalcium(Ram etal.. 1984b;
Ram and Parti. 1985), the cellular targets of these me-
diators have not been determined. For example, it is un-
knownwhethercyclicAMPorcalcium modify membrane
mechanismssuch asion channelsorchange thesensitivity
oractivity ofcontractile proteins. For studyingeffects on
contractile proteins, this laboratory developed a skinned
muscle preparation, which is described elsewhere (Ram
and Patel, 1989). To study membrane mechanisms, we
developed a dissociated muscle fiber preparation. This
paper describes the contractile properties of this disso-
ciated muscle preparation, including its modulatory re-
sponseto 5-HT, anddemonstratesitssuitability forpatch
clampanalysisofsinglechannel andwholecell ioniccur-
rents. Preliminarydescriptionsofsomeofthesedatahave
appeared previously (Ram and Liu, 1990; Zhang and
Ram. 1990). traFcitigounr.eD2i.ssoSceiraotteodnimnus(c5l-eHTfi)beprosteinntiaastmesalhligchhapomtbaesrsi(u0m.-4elmilcivteodlucmoen)-
wereconstantlysuperfusedwithartificialseawater(ASW)at5.5ml/min.
Materials and Methods (A) Fibersat rest. (B) Contractile response to a 3-s pulseofASW con-
taining 100 mAI KG. All but the fiber in the upper right contracted.
taiInneddivfirdoumalMsaorfiAnpulsys(iLaocnagliBfeoarcnhi,caC(al2i0f0o-r4n0ia0)ga)nwdermeaionb-- aFtibtehresernetduronfed1 tmomresstulpeenrgftuhsiaotnthweitehndASofWthceopnutlasienionfgK1G0.~6(MC) F5i-bHeTr.s
tcyacilnee.dTaot o1bt8aCinidnisIsnosctiaanttedOcmeuasnclweiftihberas,1a2:m1o2diLf:iDcaltiigohnt oF1ifmb1eir0ns0srmuepAmeIarifKunseGidonAatSwiWrte.shti1ld0ee~nn"gttiMhc.al5(-tDo)HTtChoaAntStrgWai.cvteAinllleincro(enBst)p,roanicsmteminetgdoifaaibtee3r-lssysphauoflrtstee-r
ofthe methods of Ishii et al. (1986), previously used to ened more after 5-HT treatment. Scale: calibration marks are 12 pm
dissociate muscle fibers in Mytilus, was used. Both 15 apart.
RAM
278 J. L. /:/ .11.
KCI contractions Electrical stimuli and digital recording of currents were
mean resting length=248 28 fj.m. n=9 controlled by pCLAMP software (Axon Instruments,
150T *, p<0.05, paired t compared to pro 5HT ( 2 min) Burlingame, California).
Results
100 .Morphology andcontraction
The typical appearance ofadissociated fiberfrom buc-
50 cal muscle 15 isillustratedin Figure 1. Dissociated muscle
fibers were spindle-shaped and ranged from 5 to 25 nm
mm
-2024 indiameteranduptoa inlength. Thewidestdiameter
usually occurred near the middle ofthe fiber, adjacent to
the nucleus, and averaged 13.6 0.9 ^m (mean S.E.,
Time post 5HT (min) n = 19). The average diameter of the fibers, measured
every 20 ^m alongthe length ofthe fiber, was 10.8 0.7
Figure3. Magnitudeandtimecourseof5-HTpotentiationofK.C1- urn. The average length offibersat rest was 270 10 ^m
elicited contractions. Fibers were measured in images similar to those =
gililvuesntreatveedryintFwiogumrien2.. FHiibgehrspowtearsesieuxmpo(s1e0d0tmo.U5-KHCTI(A1S0~W6)MpulisnesAwSeWr)e (nDis3s7o)c.iated musclefiberscontractedinresponseto KG.
ASW
for 1 min immediately priortothe time point. ImnMresponse to a 2- or 3-s pulse of containing 100
KCI, 22 fibersthat hadaveragerestinglengthsof262
13 iim shortened to 218 12 ^m. An illustration ofa
(Sigma). Whencellsbeganappearinginthemedium, fibers subset ofthesefibersisshown in Figure 2. in which Figure
in remaining muscle pieces were dispersed by gentle trit- 2A shows the fibersat rest and Figure 2B shows the max-
uration. The resultant dissociated cells were washed by
centrifuging and resuspending them in wash medium
containingall ingredients ofthe dissociating medium ex-
ceptcollagenase. Washedcellswereplated ontoglasscov-
4C
erslips in 30-mm plastic petri dishes and stored at in
a humidified chamber. Cells were usually used within 1-
4days, although viable, contractilecellshavesurvived for
as long as 10 days under these conditions. Experiments
weredoneat room temperature(20-24C) afterallowing
the cells to warm gradually for at least 30 min.
A plexiglassinsert havingacentral hole approximately B
1 cm in diameterwasclamped intothe 30-mm petridish.
The insert formed a small chamber, approximately 0.4
ml in volume. Medium was constantly pumped into the
chamber (5.5 ml/min) and removed by suction from a
surface wick opposite the inflow. The dish was mounted
ona movable stage ofan inverted microscope. The shape
and movement ofmuscle fibers were recorded by a VHS
camcorder (RCA CC310). A videotape demonstrating
many ofthecontractile andelectrophysiological responses
reported in this paper ("Dissociated Muscle Fibers of
Aplysia,"byJ. L. Ram)isavailable from theauthorsupon
request. Morphometric analysis was done by measuring
still-images on the tape playback. Photographswere made
TV
by oscilloscope camera directly offthe monitor.
A Dagan 8900 Patch Clamp-Whole Cell Clamp was
used for single channel and whole cell recording. Data
were filtered by the 1 kHz low-pass filter in the Dagan Figure 4. Serotonin (5-HT) potentiates ACh-elicited contractions.
ahmepplairfiineirz.edElheecmtartoodcersiwtegrleassf,apbroilciastheedd tforobmubbFlisehnerumnboenr- TwreahssepeolpnirscoiecteetdodubAryCeahi.3s-s(iCdpe)unltCsioecnoatlfra1tc0ot"i4tlhMeatrAeoCsfhpo.Fni(sgAeu)rteFoi2biederexsntcaietcpartelsttp.hua(ltBs)ecCooonfnttrAraaCcchttiiolanes
3-4 (Corey and Stevens, 1985), and coated with Sylgard. in (B) immediately after 1 min superfusion with 10~6 A/ 5-HT ASW.
Pipet solutions are described in relevant figure captions. Scale: calibration marksare20^m apart.
DISSOCIATED MUSCLE FIBERS OF APLYS1A 279
ACh contractions
mean resting length=264 20 ^zm, n=6
1SU- *, p<0.05. paired t compared to pre-5HT (-2 mln)
If
280 J. L. RAM ET AL
10~5 M ACh preparation forstudying mechanisms regulatingcontrac-
tion and its modulation. First, isolated fibers have appro-
priate contractile responses: They contract in response to
both high potassium and ACh. and the contractions to
both are potentiated by 5-HT. Second, the dissociated
0.5 nA fibersare suitable forpatch clampanalysisofsinglechan-
nel and whole cell currents.
-Vm=-60 mV Previous studies have used indirect methods for inves-
Vm=-80 mV tigating the roles of specific ion channels in regulating
contraction of molluscan muscles. One set ofquestions
that arise concernsthesourcesofactivatorcalcium in the
10 15 20 25 30 35
physiological responses to neurotransmitters. Is contrac-
TIME
(s) tion dependent upon the influx ofextracellular calcium?
Figure 8. Response to ACh in whole-cell recording configuration. If so, are the channels receptor operated or voltage de-
Pipet solution contained (in mA/) 20 NaCl. 406 K.C1, 2 MgCU, 100 pendent, and are they specific for calcium? Many mol-
HEPES. 5 EGTA, 5 MgATP, 0.1 NaGTP. 10glutathione, pH 7.0 (ad- luscan muscles are highly dependent upon extracellular
jw3uasst.teAerd.1wH5io-tslhdpiuKlnOsgeHp)oof.te1nF0ti~ib5aelMrswAawesCrh-e8ec0loincmisttVeadnatilnnydwaswruadpsecrumfrourvseneetddawttiobtoh-t6ahr5tmimfeimVcibalrevaseenraey- ccoanltcriaucmtitoontsriogfgebruccocnatlramcutsicolne. FEolr(eaxnaomtphleer,mAuCshc-leeliocfittehde
Aplvsui buccal musculature whose contraction is poten-
potentials.
tiated by 5-HT) fail within two minutes of removal of
extracellular calcium (Ram et ai, 1984b). Similarly, cal-
suction-activatedchannel,determined byelicitingchannel cium-dependence ofACh-elicited contractions have also
activity at several different holding potentials, averaged been demonstrated in a non-spiking muscle ofAplysia
=
140 20pS(n 3),asexemplifiedbythepatch illustrated gill (Reilly and Peretz, 1987) and in four different pro-
in Figure 7.
Aftergigaseal formation, wholecell configuration could
be achieved by applyinggreater suction than is necessary
to activate suction-activated channels. With a pipet so-
lution containing high potassium and other ingredients
meant to mimic the normal intracellular milieu of the
fibers (complete composition is given in the caption to
Fig. 8), AChelicited an iMnward current (Fig. 8). The peak
current elicited by 10 5 ACh at a holding potential of
-80 mV was -2.4 0.5 nA (n = 20); at a holding po-
- ms
tential of-60 mV. the peak current averaged 1.4 0.4
nA (n = 20). Under the same ionic conditions, depolar- 15 nA
ization activated outward current (Fig. 9). In observations
of more than 20 cells, a net voltage dependent inward
current was never seen under conditions ofhaving high
potassium in the pipet and normal sea wateroutside. Oc-
casionally, therewasaslightdelayinactivation ofoutward
5
current (not seen in Fig. 9), possibly indicating an initial
mV
counterbalancing inward current. -Me1m0b0rane Potenti-a5l,0 50
Voltage-dependent inward current can, however, be
seen under conditions that block potassium channels. i
With cesium in the electrode and 4-aminopyridine and
tetraethylammonium in the extracellular solution, de-
L -5 nA
polarization elicited an inward current. The peak inward
currentaveraged2.4 .4 nA(n = 11 fibers). Asillustrated Current
in Figure 10, the voltage dependent inward current was Figure9. Voltage-dependent outward current in whole-cell config-
dependent upon calcium in the extracellular medium. uration. Pipetsolutionandexternalmediumwerethesameasin Figure
8. Holdingpotentialwas-80mV.Currentswereelicitedby60-mspulses
Discussion to various potentials, from -100 mV to +20 mV. given at intervalsof
Thispaperdemonstratesthatsmooth musclefibersdis- p1e.r2)s.TyLpiinceaalrrleeaskpoannsdectaopac3i0tatmiVv.et(rLaonwsieern)tsCuhrarveenbteveonltsaugbetrraecltaetdi.on(sUhpi-p
sociated from buccal muscles of Aplysia are a suitable forpeak currentduringthepulse.
DISSOCIATED MUSCLE FIBERS OF APLYSIA 281
V =-80 mV ergicactivation, about 30% ofthe increasein intracellular
H
free calcium may be attributed to the release of stored
calcium (Ishii tf ai. 1988).
Studieson the influx ofcalcium-45 in responseto ACh
nA havebeen usedtodeterminewhethercalciumdependence
1 of contraction involves physical movement of calcium
10 ms normal into the cell. ACh stimulates influx of calcium-45 into
calcium
Aplysia buccal muscles El and 15 (Ram and Parti, 1985;
Gole el ai. 1987); however, ACh does not significantly
increase calcium-45 influx into ABRM (Tameyasu and
Sugi. 1976). The lack ofsignificant ACh-stimulated cal-
ABRM
cium-45 influx in not only contrasts with the ob-
servations in Aplysia muscles but also stands in apparent
contradiction with FURA-2 measurements showing a
Membrane potential (mV) -T 1 nA significant extracellular dependenceAofBRthMe ACh-stimu-
lated rise in intracellular calcium in (see above).
Calcium-45 influx measurements are inherently more
variable than FURA-2 measurements. Therefore, the lack
ofsignificant effectofAChon calcium-45 influxinABRM
probably reflectsa relatively lowerimportanceofcalcium
ABRM
influx in compared to Aplysia buccal muscles
ratherthan acompleteabsence ofACh-stimulated influx.
A possible route for calcium entry into muscle cells
during the response to ACh is via voltage-dependent cal-
cium channels. As discussed below, ACh causes depolar-
ization ofmolluscan muscles. Previous evidence that de-
polarization could activate voltage dependent calcium
channels included demonstrating that another depolar-
izing stimulus, a high potassium medium, causes con-
Figure 10. Voltage-dependentcalciumcurrent inwhole-cellconfig- traction. High potassium induces contractions ofBusycon
uration. Pipetsolutioncontained(inmA/)20NaCl,406CsCI.2 MgCl:. proboscis retractor muscles (e.g.. Huddart el ai. 1990a.
100 HEPES, 5 EGTA, 5 MgATP. 0.1 NaGTP. 10glutathione. pH 7.0 b),ABRM (e.g., TwarogandMuneoka, 1972),and.Aplysia
(adjustedwithCsOH).Externalmediumwasartificialseawatercontaining buccal muscles (Ram, unpub. data). Furthermore, high
w(MiignthSmON.a4U,O)2H55)0.MgtFCeoltrr:a,ec1at0lhcyKilCua1m,mmmo1e0ndiCiuaumCm,I,,,5tha4en-dCaam1Cm0loH:pEwyanPsdEiSlen,fet.pouH4t87a5.n8dN(aaaldCljlou.tsht2ee5dr ipnotAAapBslsyRisuMimaebluiccictaslcomnutrsacclteio(nFiogfs.is2olaatnedd f3i,betrhsi,sapsadpeesrc)riabnedd
ingredients increased in concentration by 1%. Holding potential was in (Ishii elai. 1986), unambiguouslyprovingthat
-80 mV. Currentswere elicited by 60 ms pulsesto various potentials, contraction elicited by high potassium is a direct effect
from -100mVto +10mV atintervalsof1.2 s. Linearleakandcapac- on single fibers and is not dependent upon either release
diteaptoilvaeritzraantsiioennttsoh-a3ve0bmeVenisnubtthreactperde.se(nUcpepearn)dTaybpsiecnacleroefspcoanlsceisumt.o of neurotransmitters from nerve endings in the muscle
(Lower) Current-voltage relationships for peak current in the presence or mechanical or electrical coupling between fibers. Ishii
andabsence ofcalcium. el ai (1988) has also used FURA-2 to show that high
potassium causesan increasein intracellularcalciumthat
is completely dependent upon extracellular calcium.
boscis musclesofthe marine snail Busycon (Huddart and Blockersofvoltage-dependent calcium channels reduce
Hill, 1988; Huddart el ai. 1990a, b; Hill and McDonald- thecontractile responsesproduced byboth AChand high
Ordzie. 1979; Hill el ai, 1970). Another molluscan mus- potassium. Thus, Huddart el ai (1990b) found thatdilti-
cle, the anterior byssus retractor muscle (ABRM) seems azam, verapamil, and nifedipine all decrease ACh-elicited
less dependent upon extracellular calcium because ACh- contractions ofBusycon proboscis muscles. Similarly, ni-
elicited contractions ofABRM are not abolished by the fedipinereducesACh-elicitedcontraction ofAplysiabuc-
removal ofextracellular calcium for up to 10 min (Sugi cal muscle (Ram and Liu, 1990).
and Yamaguchi, 1976). Recent investigations of intra- Theaboveindirectevidencefortheexistenceofvoltage-
cellular calcium levels in ABRM using the calcium-sen- dependent calcium channels is now supported in the
sitive fluorescent indicator FURA-2 have shown that al- present paper by voltage clamp recordings ofa voltage-
thoughextracellularcalcium mayaccount forthe majority dependent inward current that is dependent on extracel-
ofthe rise in intracellular calcium in response to cholin- lular calcium (Fig. 10). As described in a preliminary re-
282 J. L. RAM ET AL.
port (Ram and Liu, 1990). we have also demonstrated This paperalso demonstrates that buccal muscle fibers
that this current is partially inhibited by nifedipine and contain stretch-activated channels. Becausethesechannels
completely blocked by lanthanum. were observed with on-cell patch electrodes containing
For voltage-dependent calcium channels to play a role only KC1, the inward currents illustrated are almost cer-
in mediating ACh responses, it must also be shown that tainly due to potassium current. Similarly, stretch-acti-
the range ofmembrane potentials at which a voltage-de- vatedchannelsconductingprimarilypotassium ionshave
pendent calcium current can be activated is within the been reported previously in molluscan neurons (Morris
range of membrane potentials caused by ACh. As illus- and Sigurdson, 1989; Sigurdson and Morris, 1989) and
trated in Figure 10, voltage-dependent calcium current cardiac muscle (Brezden and Gardner, 1986; Sigurdson
begins to activate with depolarizations to 40 mV. In ctai. 1987). Stretch-activated channelsthat aresomewhat
other cells (data not shown), voltage-dependent calcium less selective forpotassium have been reported in various
current has been activated with depolarization to as little mammalian tissues, including skeletal muscle (Guharay
as -50 mV. Asdiscussed below, ACh candepolarizecells and Sachs, 1984)andsmooth muscle (Kirberrt ai. 1988).
toapproximately -35 mV. Thus, the membrane potential In futureexperimentsit shouldbepossibletodetermine
required to activate voltage-dependent calcium channels whether 5-HT potentiates contraction of dissociated
is clearly within the range ofdepolarization produced by muscle fibers by modifying the ionic currents illustrated
ACh in Aplysia buccal muscles. This paper provides the here. One indication in a molluscan muscle that 5-HT
strongest evidence yet that activator calcium enters mol- may change membrane currents ofmolluscan muscles is
luscan muscle fibers during cholinergic stimulation by that, in ABRM. 5-HT potentiates the rise in intracellular
voltage-dependent calcium channels. calcium caused by 100 mA/KCl (Ishii et ai. 1989). The
This paper also initiates the analysis ofreceptor-oper- rise in intracellular calcium in response to KC1 is com-
ated channels activated by ACh. ACh causes depolariza- pletely dependent upon extracellular calcium (Ishii etai,
tion of molluscan muscle fibers in clam heart (Wilkens 1988) and is presumed to be due to voltage-dependent
ABRM
and Greenberg, 1973), (Twarog, 1954), Bmycon calcium current, similar to the current described in this
proboscis muscles (Huddart ct ai, 1990b; Hill and Licis, paperin Figure 10. Inaddition, 5-HT might alsobe mod-
1985; Hill and McDonald-Ordzie, 1979), Aplysia gill ifying potassium channels, receptor-operated channels,
muscle(Reillyand Peretz, 1987)andAplysiabuccal mus- and stretch-activated channels.
cles (Ram ct ai. 1990). Detailed quantitative studies of
Aplysia buccal muscle revealed (a) an average resting po- Acknowledgments
mV
tential of-65 (Gole et ai. 1987), (b) no contraction
elicited with depolarizations less than approximately 10 This work was supported by the Muscular Dystrophy
mVaboverest, (c)a non-linearrelationshipbetween con- Association and NIH grant RR-08167. I am indebted to
traction and depolarization in which increasing ACh be- R. B. Hill for his critical comments on the manuscript.
yond a certain concentration was accompanied by in-
creasing contraction with little or no further increase in Literature Cited
depolarization, and(d) maximal ACh-eliciteddepolariza-
tion of approximately 30 mV above rest (Ram et ai, Brezden, B.L.,and D. R.Gardner. 1986. Apotassiumselectivechannel
in isolated Lymnaca stagnalisheart musclecells. J. Exp. Bio/. 123:
1990).
175-189.
TmheVlimit on depolarization produced by ACh to only Cohen, J. L., K. R. Weiss, and I. Kupfermann. 1978. Motor control
30 above rest may result from several mechanisms. ofbuccal musclesinAplysia. J. Neitmphys. 41: 157-180.
One possibility is that the reversal potential for ACh-ac- Corey, D.,andC.S. Stevens. 1985. Scienceandtechnologyofpatch-
tivatedchannels isonly 30 mV above rest. An alternative rmeacnorndianngdelEe.ctNreohdeers,.Pepd.s.5P3-l6e8nu'mmSPirnegsls,eCNheawnnYeolrRke.cording, B.Sak-
explanation is that depolarization activates voltage-de- Cropper, E.C, M. \V. Miller, R.Tenenbaum, M. A.G. Kolks, I. Kup-
pendent potassium channelsthatactasaneffective brake fermann,and K. R. Weiss. 1988. Structureandactionofbuccalin:
on further depolarization even in the face ofactivation amodulator, neuropeptidelocalizedtoanidentifiedsmallcardioac-
ofmore ACh-activated channels. Data in this papershow tivepeptide-containingcholinergicmotorneuronofAplysiacalifor-
that analysis ofthis question is feasible. As expected for nicii. Proc. Nal.Acad. Sci. 85: 6177-6181.
a depolarizing stimulus, ACh activates inward current CropWpeeirs,s.E.19C8.7,.R.MTyeonmeondbuaulmi,n:Ma.biAo.acGt.ivKeolnkesu,roIp.eKputpidfeerpmraensenn,tKi.naRn.
(Fig. 8). The reversal potential of the ACh response is identified cholinergic buccal motor neuron ofAplysia. Proc. Nal.
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current is activated within the voltage range elicited by effectsonbuccal musclesofAplysia. Camp. Biochciu. Physial. 88C:
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4-AP outside the cell and Cs in the electrode (Fig. 10). 352:685-701.
DISSOCIATED MUSCLE FIBERS OF APLYSIA 283
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1970. Electromechanicalcouplinginamolluscanmuscle,theradula actuated adenylate cyclase and the possible role ofcyclic AMP in
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