Table Of ContentReference: Biol. Bull 180: 284-244. (April, 1941)
Photoresponsiveness of Aplysia Eye is Modulated by
the Ocular Circadian Pacemaker and Serotonin
JON W. JACKLET
Department ofBiologicalSciences, Neurobiology Research Center, State University
ofNew York atAlbany, Albanv. New York 12222
Abstract. The eye ofthe sea hare, Aplysia, contains a by the synchronous firing ofa population ofretinal pace-
circadian pacemakerthat controls rhythmic behaviors of maker neurons (Jacklet et ai, 1982), the axons ofwhich
theanimal. Thisreportshowsthatthepacemakercontrols enter the optic nerve and project to the central ganglia
the photoresponsiveness ofthe eye as well. The electro- (Olson and Jacklet, 1985). This rhythm ofCAP activity
retinogram (ERG) ofthe isolated eye-optic nerve prepa- is known to control rhythmic behaviors, such as loco-
ration, evokedby briefgreen light pulses in otherwisedark motor activity-rest, because eyeless animals lack the well
conditions, was recorded regularly, while the circadian defined circadian rhythm ofactivity-rest that normal an-
rhythm ofcompound action potential activity was con- imals display (Strumwasser et at., 1979; Lickey et ai,
tinuously recorded from the optic nerve. The waveform 1977). Feeding behavioralso exhibits a circadian rhythm
ofthe ERGchanged systematicallyandrhythmicallydur- (Kupfermann, 1974). but the contribution ofthe ocular
ingthecircadian cycle. Onewavecomponent ofthe ERG circadian pacemaker to that rhythm has not been tested.
was prominent during the subjective night phase ofthe The ocular circadian pacemaker probably controls
rhythm when the compound action potential frequency rhythmicbehaviorby neural connectionstocentral motor
was minimal; and it was inconspicuous during the sub- control centers, or by affecting physiological processes in
jective day phase ofthe rhythm when the compound ac- theeyeitself, although the mechanismsare notyet known.
tion potential frequency was maximal. Because eyes at- An eye contains the photoreceptors needed to entrain
tached to the central nervous system and isolated eyes the ocular circadian rhythm to the solar day light-dark
both exhibited the same rhythmic ERG changes, the cir- cycles,becausetherhythm isentrainedby light-darkcycles
cadian pacemaker in theeye isresponsible formodulation (Eskin. 1971), and thephaseoftheCAPfrequencyrhythm
of the ERG. Addition of serotonin, a putative efferent isshifted by light pulsesgivenduringthesubjective night,
transmitter, to the bathing saline induced the ERG wave yielding a phase response curve (Jacklet, 1974). The spe-
component characteristic ofthe subjective night phase of cific ocularphotoreceptor responsible forthe phase shifts
the rhythm. The threshold serotonin concentration was have not been identified. The eyes and other cephalic
10 7 AI. and serotonin had a long lasting effect. photoreceptors also mediate simple phototactic behaviors
(see Jacklet, 1980).
Introduction The electroretinogram (ERG) has been recorded from
the eye by an extracellular pipette placed in the retina
Each eye ofAplysia contains a circadian pacemaker (Jacklet, 1969b) and by a suction electrode placed on the
(Jacklet, 1969a) that produces a circadian rhythm in the cornea (Eskin, 1977; Eskin and Maresh, 1982). The ERG
frequency of optic nerve (ON) autonomous compound amplitude is increased by serotonin treatment or by ON
action potentials (CAPs). The CAP activity is produced stimulation, which presumably releases serotonin from
the terminal in theeyeoftheefferent neurons (Eskin and
Maresh, 1982). Eyescontain several types ofphotorecep-
Received 9 August 1990;accepted 28 December 1990.
Non-standard abbreviations: CAP, compound action potential: ON, tors, in addition to the pacemaker neurons involved in
optic nerve: CT, circadian time; ERG, electroretinogram; CM. culture the circadian rhythm. The largest and most numerous
medium. type is the R photoreceptor (Jacklet and Rolerson, 1982)
284
ORCADIAN CONTROL OF PHOTORESPONSIVENESS 285
that responds to light with a graded prolonged depolar- andCAPswere measured from computergenerated plots
ization. The light response ofthis receptor is largely re- ofthe recordings.
sponsible for the ERG (Jacklet, 1969b). Pairs ofeyes from eight animals were used in the cir-
Rhythmicchanges in the ERG ofAplysia. oranyother cadian rhythm study. Four eyes attached to the cerebral
gastropod, have not been reported, to my knowledge, even ganglion yielded 1 1 circadian cyclesofdata, and 7 isolated
though ERG circadian rhythms ofotheranimals arewell eyes yielded 12 circadian cycles ofdata. Eyes produced
known. For example, the ERG amplitude rhythm ofthe three to four cycles of CAP rhythm data routinely, but
compound eye of Limit/us has been intensively studied complete ERG datawere not collected from alleyes. Pairs
(Barlow. 1983). Photosensitivity increasesby 20-100 fold of eyes from seven animals were used in the serotonin
during the night, and the rhythm, driven by a central experiments.
nervous system circadian pacemaker, is mediated by ef- Lightpulseswere produced bydrivingan Archergreen
ferent innervation. The efferent transmitterappearstobe LEDwith 1-2 s(15, 30or 70V)pulsesfromaGrassmodel
octopamine (Battelle el ai, 1989; Kassand Barlow, 1984). S88 stimulator. Pulses were given at regular intervals of
I report here that the ERG waveform recorded from 10 min, 30 min, or 1 h in otherwise constant darkness.
the isolated Aplysia eye changes rhythmically, and the The LEDwas positioned 3 cm awayand directly overthe
rhythm maintainsastable phaserelationshipwiththecir- eye drawn into the PE tubing. The intensity incident on
cadian rhythm in CAP frequency. Thus, the ocular cir- the eye was measured by placing the sensor of a radi-
cadian pacemaker affects physiological processes within ometer/photometer(United DetectorCorp., Model 40X)
the eye itself. The waveform ofthe ERG that is charac- 3 cm from the LED. Light intensities for voltage pulses
teristic of subjective night is induced by the addition of usedtodrivethe LEDwere0.6 ^W/cm2at 15 V,0.8 ^W/
serotonin to the bathing saline during subjective day, cm2 at 30 V, and 3 ^W/cm2 at 70 V. TheAplysia eye has
mimicking the influence ofthecircadian pacemakerdur- high sensitivity to green light and the threshold intensity
ing subjective night. is about 0.06 ^W/cm2 at 500 nm (Jacklet, 1980).
Artificial seawater(ASW)wasmadeupofthefollowing
Materials and Methods salts in millimoles/liter: NaCl, 425; KC1, 10; CaCl:, 10;
MgCl:, 22; MgSO4, 26; NaHCO,, 2.5; adjusted to pH
Individuals ofAplysia californica were obtained from 7.8. Culture medium (CM) was composed ofASW, 20%
Marinus, Inc., Long Beach. California, and kept in Instant Aplysiablood, and 100 U/ml penicillin, 0.1 mg/ml strep-
Ocean tanks maintained at 16C under light-dark cycles tomycin. Serotonin (Sigma, creatinine sulfate)wasadded
of 12:12. Two isolated preparations were used: the eye- to the CM to final concentrations of 10 7, 10", or 10 -
ON, and the eye attached to the cerebral ganglion by the M. A 10-ml chamber fitted with polyethylene tubing for
ON. They were placed in a recording dish (100 ml) changing solutions inside the dark box was used for the
CM
equipped with tubing (polyethylene, PE) electrodes serotonin experiments. The wasremoved entirely by
embedded in the RTV silicone rubberbase. Preparations applying suction to the polyethylene tubing and was re-
were maintained in a dark box at 18C for several days placed within a few seconds with the serotonin solution.
of recording. The ON was drawn, by negative pressure The statistically significant differences between average
applied by a syringe, into one electrode (PE 10) that was latencies were determined using a two tailed / test. The
used to recordCAPs, and theeyewasdrawn intoanother level ofsignificance used was a = .05.
electrode (PE 50) for ERG recording. The negative pres-
sure was released, and theeye and ON remained in place Results
for recording. The eye was drawn completely into the Rhythmic changes in the ERG
electrode so that the activity ofall retinal cells could be
recorded. The basic ERG waveform recorded in these experi-
The eye is spherical and about 0.7 mm in diameter. It mentswastriphasic, even though theentireeyewaspulled
hasacentral lens, a poorlydeveloped cornea, and a com- into the tubing electrode. The waveform consisted of a
plex retina containing photoreceptors and neurons (see sharplyrisingwave, followedbyaslowerwaveofopposite
Jacklet el ai, 1982; Herman and Strumwasser, 1984). polarity and a weak slow third phase. There was no ob-
ERGelectroderecordingsroutinelypicked upsmallCAPs. vious "off" response. This triphasic ERG waveform is
Activitywasamplified with an A-M Systems model 1700 very similar to the ERG recorded by Eskin (1977) on a
(gain, XI000; bandpass 0.1-1000 Hz.) and displayed on Grass polygraph using a suction electrode applied on or
a Tektronix 5300 oscilloscope. ERGs and CAPswere re- near the cornea.
corded and stored with Asystant+ software in an IBM The latencyofthe ERG in the present studywasabout
AT computer, and the data were plotted with a Hewlett- 0.9 s in response to a 1 ^W/cm2 green light pulse. This
Packard model 7470A plotter. The latenciesofthe ERGs compares well with the latency ofabout 0.9 s obtained
286 J. W. JACKLET
3 sec. 3 sec.
1 -,
a
E
-
to
24 48
hr.
50O-,
hr.
ORCADIAN CONTROL OF PHOTORESPONS1VENESS 287
earlier in response to about 6 lux (<1 /jW/cm:) white used in Figure 1A. When the cycling ofthe relative am-
light (Jacklet, 1969b). This is a rather long latency, but plitude ofthe B-Cwaveiscompared totheCAPfrequency
similar to those of other gastropod eyes under similar rhythm plotted in Figure ID, the maximum B-C wave
conditions. Latencyis 1-3 sforOtala, aland snail (Ciliary amplitude seemstooccurat about CT 20and tocoincide
and Wolbarsht, 1967), and 0.2-0.5 s for the well-formed with minimal CAPfrequencyduringsubjective night. The
eye of Stronibu.s, another marine gastropod (Gillary. period ofboth rhythms is about 24 hours.
1974). The Aplysia eye latency is about 0.4 s in response A few eyes exhibited weak rhythmic changes in the A
to 600 lux white light (Jacklet, 1969b). wave ofthe ERG, but eitherthey did not persist overtwo
The ERG waveform was usually smooth during the cycles, ortheywerenotsufficiently robusttobeconsidered
subjectiveday,asshown in Figure 1AatCT2.5. CT refers true rhythms. The D wave was very stable and showed
to circadian time, which is measured from the actual pe- no rhythmicity.
riod ofthe free-running circadian rhythm ofinterest, in The ERG waveform recorded from eyes of different
thiscase theCAPfrequency rhythm shown in Figure ID. animals varied somewhat, but the basic waveform could
The period isdivided into 24 equal units. Circadian time always be observed. One ofthe most extreme waveforms
0-12 is subjective day, and CT 12-24 is subjective night. is shown in Figure 2A, B. This eye was attached to the
The phase point in the CAP rhythm corresponding to cerebral ganglion, but the paired isolated eye exhibited
subjective dawn (CT 0) is the CAP frequency at '/: max- thesame ERG waveform and changed rhythmically. The
imum marked in Figure ID. During subjective night, the A, B, C. and D waves are readily apparent. But the A
waveform developed a notch following the initial wave, wave is relatively small and the B-C wave is huge. The
as shown in Figure 1A at CT 20.5. For convenience, the relativeamplitudeofthe B-Cwavechangedrhythmically,
waveform hasbeen labeled in the figure. The initial wave but it never completely disappeared. It remained in the
is A, followed by the B-C wave or notch, followed by the appropriate phase relationship with the CAP frequency
slower D wave. The B-C wave has not previously been rhythm for many cycles (Fig. 2C, D). The period ofboth
reported. This waveform is typical ofthe ERGs recorded rhythms is about 23 hours.
during subjective night in these experiments. In most preparations, the latency (time from stimulus
The ERGsshown in Figure 1A were recorded from the onsetto '/: peakofAwave)oftheERG A wavewasshorter
same isolated eye at different phases (CT 20.5 and CT during the subjective night, when the B-C wave was
2.5) in the circadian cycle. Similar ERGs were recorded prominent, than during the subjective day. Forexample,
fromtheothereyeofthesameanimal atthesame phases, in Figure 1A and B the latency is about 100 ms shorter
eventhough theeyewasattachedtothecerebral ganglion duringsubjective night. The ERG latenciesforlightpulses
by the ON (Fig. IB). Being attached to the cerebral gan- given at CT 19-22, during subjective night, were com-
glion made noapparentdifference inthewaveform ofthe pared to latencies obtained at CT 1-4, during subjective
ERG or in the rhythmic changes in the waveform. In day, for 12circadiancyclesforboth isolatedeyesandeyes
general, eye pairsexhibited very similar ERG waveforms, attached to the cerebral ganglion. Mean latency and the
whether or not the eye was attached to the cerebral gan- standard error ofthe mean (SEM) were calculated, and /
glion. tests were performed to determine whether mean differ-
The ERG B-C wave changed rhythmically during the ences were statistically significant. The average latency
circadian cycle. It virtually disappeared duringsubjective for isolated eyes was 1000 ms (SEM, 20; N, 34) during
day and reappeared during subjective night. The relative CT 19-22, and 1050 ms (SEM, 10; N, 39) during CT 1-
amplitude ofthe B-C wave, measured from the peak of 4. The means were significantly different at the .05 level.
the B to the peak ofthe C wave, cycled continuously, as The average latency foreyesattached tothe cerebral gan-
shown in Figure 1C for the B-C wave ofthe isolated eye glion was920 ms(SEM, 20; N, 36)duringCT 19-22,and
Figure 1. Rhythmic changesinthe ERG. ERG waveformsrecorded from thesameisolatedeyeduring
subjective night(CT 20.5)and duringsubjective day (CT 2.5)areshown in A. The A. B. C. and Dwaves
are labeled. The 2-s light pulse (3 /jW/cm2) is indicated by the black bar. The other eye from the same
animal, but attached to the cerebral ganglion, exhibited the ERGwaveform shown in B taken at CT 20.5
and CT 2.5 asinA. Vertical scales in A and Bare 50/jV and 25 ^V perdivision. Thechangesin relative
amplitudeoftheB-CwaveoftheERGsrecorded fromtheisolatedeyeareplottedinCusingthesametime
scale asthe CAP frequency rhythm shown in D. Arrows identify the relative amplitude pointsofthe B-C
wavein C, and theCAP frequency in Dcorrespondingtothe ERGs in panel A taken at CT 20.5 andCT
2.5. Time reference forCT isthe thin labeled line in Dthat occursat 'A the maximum CAP frequency.
Theprojectedlight-darkcycleexperiencedbytheanimalbeforedissectionisshownbythewhite/crosshatched
bar.
288 J. W. JACK.LET
CT 15
sec.
400
ORCADIAN CONTROL OF PHOTORESPONSIVENESS 289
970ms(SEM, 20;N, 34)duringCT 1-4. The meanswere (CorrenU'M/.. 1978; Eskinand Maresh, 1982).Thenum-
significantly different at the .05 level. Although the mean ber ofCAPs evoked by the light pulse, especially those
A wave latencies are shorter for attached eyes than for evoked several seconds after the pulse, were also reduced
isolated eyes, both show similar shifts in ERG latency as shown in Figure 4B. However, coMmpared to the re-
during the circadian cycle. sponsejustbeforetheaddition of10~6 serotonin, there
To test for the involvement of chemical synapses in was no change in the latency ofthe initial CAP produced
the circadian pacemaker modulation ofthe ERG wave- by a light pulse. The mean latency for the initial CAP in
Mform, two eyes wereMsubjected to ASW containing 10 4 the light response was 1.40 s (SEM, 0.14; N, 7) before
Ca++ and 10 ' Mg++. This treatment drastically serotonintreatment, and 1.36 s(SEM, 0.05; N, 7) 15 min
reduced the ERG as expected (Eskin, 1977). Thus, a re- afterserotonin treatment. A /testshowedthatthese means
liable test for the involvement ofchemical synapses was were not significantly different. The initial CAPoccurs at
not possible. aboutthesametimeastheB-Cwave,andsmalldeflections
Removal ofthe ON from the isolated eye did not in- on the ERG waveform caused by CAPsare clearlyvisible
terrupt cycling ofthe ERG waveform, but it did reduce (Fig. 4A, B). During serotonin treatment, the CAP de-
the number ofcycles that an eye exhibited. The ON was flections on the ERG waveform, as well as the size and
cut away from four eyes at the bases oftheir retinas, and timing of the initial CAP during the light response, re-
ERGswere recorded as usual. Small CAPs recorded with mained the same. Thus, changes in the light-evoked ac-
the ERG electrode verified that theCAPcircadian rhythm tivity ofthe pacemaker neurons that produce the CAPs
continued. Twooftheeyesremained active fortwocycles, are not likely to account for the B-C wave induced by
and both exhibited cycling ofthe B-C wave, suggesting serotonin.
that the ON itselfis not necessary for circadian cycling Serotonin shortened the latency of the ERG and oc-
ofthe ERG. casionally increased the amplitude of the A wave. Both
Figures 3 and 4 show asubstantial decrease in the latency
Serotonin induces the ERG B-C wave and increase in the A wave. The average ERG latency
Serotonin induced the B-C wave in eyes at circadian before serotonin was 96M0 ms (SEM, 20; N, 9); 15 min
times when it was not normally expressed. The induced aftertheadditionof10 6 serotonin itwas900ms(SEM,
stBuh-beCjeBcw-taCivvweeanvcilegohswtea.lsyAwsreelsslehmdobewlvneedlionpthetedhewateaxvCaeTmpc2lh0ae,raoacsfteeFrxiipgsetucirtceedo3,,f wt30ea;nscNy8,6w0a9)sm,snaont(dSsEi4g5Mnimf4i0ic;nanNta,fatt7e)r.1a5Tnhmieand,ddiifbtfuietorneantoc4ef5sinemrimonteoaintniwnalais-t
jaaefnctdteirivttebhdeeacyaa.ddmAietisiohnoncrotonfstpi1im0~ceu6loMautsersleaatrteorCt,oTanti2nC.0Tt.oa0t.nh5d,ejdbuuasrttihni1g3ngsmusibon-- pAsiltginttiuhfdeiecatonhftrlteyhsehdoAilfdfwearcevonnetcieanntctrtrehaaetsie.od0n5o(nl1le0vyel71..1MT)thiemfeoasrveaEtrRa4g5GemaBim-n-.C
nluetairolnybiydenpteircfaulsitoon,thteheB-iCndwuacveed rBe-cCorwdaevdeat(FCigT. 32C0)(wFiags. wmasv(eNi,n4d)u,ctainodn,thteheAavwearvaegealmaptleintcuyddeecdriedansoetd cbhyaonngley.25
3A). Continued exposure to serotonin enhanced the B-C
wave (Fig. 3D) beyond the amplitude of the subjective Discussion
night B-C wave. Theeffectsofserotonin werelonglasting ERG waveform changes
and reached a maximum in about 1 h. Once induced, the
B-C wave required several hours ofwashout before it re- A major finding ofour study is that the ERG changes
turned to normal. Serotonin also enhanced the B-Cwave systematicallyand rhythmically duringthecircadian cycle
ofeyes tested during subjective night when the wave was ofCAP frequency. One component ofthe ERG, the B-C
already present. wave, is prominent during the subjective night phase of
During exposure to serotonin, the autogenous CAP the rhythm when the CAP rate is minimal, and incon-
frequency was reduced as expected from previous work spicuous during the subjective day phase when the CAP
Figure 2. Rhythmicchanges in the ERG ofaneyeattachedtothecerebralganglion. Waveformschar-
acteristic ofsubjective night (CT 15) and subjective day (CT 3) forthe same eye attached to the cerebral
ganglion by theON areshown respectively in Aand B. The 1-slight pulse(3 /jW/cm2) isindicated by the
blackbar. Vertical scalesin A and Bare50j/V perdivision. The B-Cwave became very prominent inthe
eyes ofthis animal after several days ofrecording. C shows the changes in relative amplitude ofthe B-C
waveplotted on thetimescaleoftheCAP frequency rhythmsin D. Arrows in Cand D markthetimesat
which the ERGsshown in A and B were taken. Time referenceforCT isthe thin labeled line in Dat '/:
the maximum CAPfrequency. The projected light-darkcycleexperienced by the animal beforedissection
isshown bythewhite/crosshatched bar. Light pulse isindicatedbyblack barin panelsA, B.
290 J. W. JACKLET
B
O
ec.
Figure 3. Changes in the ERG ofan isolated eye induced by serotonin. The ERG waveform recorded
adtayCTwav2e0fwoirtmh,alacchkariancgtetrhiestBi-cCBw-aCvew.avTheiristesehnowmnininafAt.erAttheCaTdd0i.t5iotnheofE1R0G~6hMadsecrhoatnogniend,ttohethEeRsGubjsehctoiwvne
in Cwith a prominent B-Cwavewasrecorded. The B-C wave progressively increased until the maximum
responseinDwasobservedat43 minaftertheadditionofserotonin. AtthattimethelatencyoftheAwave
had decreased about 100 ms, and the A wave amplitude had increased 140%. Vertical scale is 20 V per
division. Theblackbaron the time scalemarksthe 1-slight(.8nW/cnr) pulse.
rateismaximal. Thus, themaximal B-Cwaveoccursdur- mined. Isolated eyes, and eyes attached to the cerebral
ing minimal CAP rate in an antiphase relationship. The ganglion, exhibited similar ERG rhythms suggesting that
B-C wave decreases in size sharply during the increase in the B-C wave pacemaker resides within the eye and that
CAP rate at subjective dawn, suggesting that a high CAP it is likely to be the CAP circadian pacemaker. The in-
rate might suppress the B-C wave. However, the causal duction of the B-C wave by serotonin is an intriguing
relationship between these events has not been deter- observation that will beaddressed laterin thisdiscussion.
ORCADIAN CONTROL OF PHOTORESPONSIVENESS 291
>
3
o
sec.
sec.
Figure4. ChangesintheERGandCAPfrequencyinducedbyserotonin.Simultaneousrecordingswere
madeofCAPs(uppertraces)andtheERG(lowertrace)inresponseto 1-slightpulses(darkbar). Responses
atCT 1.5appearin A. CAPsappearontheONtraceandassmalldeflectionson the ERGtraceaswell. At
CT2.5,43 minafteraddition of10~6Mserotonin,the B-Cwaveseenin Bwasprominent, thelatencyhad
decreasedabout 100ms, and the numberofCAPsevokeddecreased. Black barson timescale markthe 1-
slight(.8MW/cnr) pulse. Vertical scalesare 50/W perdivision.
292 J. W. JACKLET
The ERG B-C wave characteristic ofsubjective night prolonged hyperpolarization following the initial depo-
has not been previously reported. Several years ago, I larization (see Fig. 3 in Jacklet and Rolerson. 1982).
looked for rhythmic changes in the ERG A wave ampli- However this hyperpolarization is also unlikely to cause
tude because ERG amplitude changes are well known in the B-C wave because it continues much longer than the
other animals (see Barlow, 1983), but did not find con- B-C wave. Responses from R photoreceptors have not
sistent rhythms. Inthepresentstudy, the useofcomputer been studied throughout the circadian cycle, especially
assisted recording to compare ERG waveforms soon re- notduringthesubjective night when the B-Cwave occurs,
vealedthatthe B-Cwaveoccursandchangesrhythmically. sochanges that might account forthe B-C wave have not
It also revealed that the A wave latency changes rhyth- been observed.
mically, butthatthe A waveamplitudedoesnot. Average Light responses ofR photoreceptors are not completely
latency differences of50 ms during the cycle were found blockedbylowCa++and high Mg* * ASW, but theresting
for both isolated and attached eyes. potential isdecreased, and the light-evoked depolarization
The ERG recordings were made from eyes completely is reduced and prolonged (Jacklet and Rolerson, 1982).
pulled into the tubing electrode, so signals could be re- This should account forthe reduction ofthe ERG in low
corded from any ofthe responding cell types in the eye, Ca+4 and high Mg++ ASW previously observed (Eskin,
including cells in the basal retina. Previous work had 1977) and confirmed in this study.
shown that the largest photoresponses, recorded with a The pacemaker neurons(orsecondary neurones. Jack-
glasspipette inthe retina, werecorneal negativeandwere let et a/., 1982) responsible for the CAPs also respond to
obtained near the distal segments of the large photore- light. Theydepolarizeand firesynchronousaction poten-
ceptors (Jacklet. 1969b). In the present study, light from tialsthat are correlated 1:1 with the ON CAPs. Asshown
the LED reached all surfaces ofthe isolated eye and was in this study. CAPs produce small but observable deflec-
notrestrictedtothepathway through thecorneaandlens. tionson the ERG waveform at all phases ofthecircadian
The photoreceptor organization ofthe Aplysia retina cycle, but they are tiny compared to the B-C wave, and
wasinvestigatedwith localized illumination by Blockand none are synchronized with the B-C wave. The light re-
McMahon (1983). They illuminated (100 lux, white light) sponses ofthe pacemaker neurons themselves seem un-
thedistal segmentsofthephotoreceptorssurroundingthe likely to contribute to the B-C wave. However, the B-C
lens and, as a result, unitary ON activity without CAPs waveismostconspicuousduringthephaseofthecircadian
was evoked in the ON. Illumination ofthe basal retina cycle when the autogenous CAP frequency is low or ab-
produced CAPs. Block and McMahon concluded that sent. Perhaps low autogenous CAP activity creates the
chemical synaptic inhibition, especiallythe inhibitoryac- conditions necessary for the B-C wave to occur. The re-
tion ofthe receptor layer onto the CAP generating neu- lationship between autogenous CAP activity and expres-
rons, shapesin part the light responsesoftheisolatedeye. sion ofthe B-C wave has not yet been tested directly.
Otherevidence ofsynaptic inhibition in the retina is pro- A retinal cell type that may contribute to the ERG B-
vided below. C wave is the H photoreceptor (Jacklet and Rolerson,
1982). Its typical light response is a volley ofaction po-
Retinalcells that may contribute to the ERG B-C wave tentials followed by brisk hyperpolarization, and then de-
polarization accompanied by action potentials. The hy-
The ERG consists ofa sharply rising A wave, a rhyth- perpolarization occursjust after the initial photoresponse,
mically changing B-C wave, and a stable D wave. The A at about the time that the B-C wave of the ERG is oc-
wave is likely to be caused by the R type photoreceptors curring. The H cell hyperpolarization appears to be syn-
ON
with microvillous distal segments adjacent to the lens apticallyevoked, becauseelectrical stimulation ofthe
(Jacklet. 1969b; Jacklet and Rolerson, 1982). Intracellular evokes a similar sharp hyperpolarization (Jacklet and
recordings show that dark adapted R photoreceptors re- Rolerson, 1982). Thiscelltypemaybeinvolvedinshaping
spond to white light pulses of600 lux after a latency of the light responses observed by Block and McMahon
400 ms, comparabletothe ERG latencyof400 msatthat (1983) duringselective illumination. The ERG B-Cwave
intensity (Jacklet, 1969b); the response isa prolonged de- might be produced by enhanced inhibitory synaptic in-
mV
polarization of60-70 (Jacklet and Rolerson, 1982). teractions within the retina controlled by the circadian
Light adapted, but not dark adapted, R photoreceptors pacemaker. Such enhanced interactions might improve
have a notch on the rising phase ofthe depolarization. the visual performance ofthe eye.
The notch is probably not responsible for the B-C wave Adetermination oftheretinalcell typesinAplysiathat
because it occurs early during the A wave, and because contribute to the rhythmic B-C wave must await a sys-
ERGs were recorded at interstimulus intervals ofupto 1 tematic intracellular study of cellular light responses
h when the R photoreceptor are dark adapted and not throughout the circadian cycle, preferably with simulta-
expectedtohavea notch. Some R photoreceptorsdisplay neous ERG recordings.
ORCADIAN CONTROL OF PHOTORESPONSIVENESS 293
The eye ofa marine gastropod. Stwmhux, may share neurons, because the initial CAP that most nearly coin-
some ofthe features ofthe Aplysia eye photoresponses, cides temporally with the B-C wave is unaffected by se-
including the B-C wave. The ERG exhibits two peaks of rotonin. Thus, serotonin does not allowexpression ofthe
negativity that are separable under certain conditions of B-C wave by suppressing the light-induced pacemaker
light and temperature (Gillary, 1974). The second peak neuron activity. However, the phase ofthe circadian cycle
resemblesthe B-C wave. Thiseye is 3 timesthe diameter, during low or zero CAP frequency is associated with
andcontainsabout 100timesasmanycells, astheAplysia expression ofthe B-C wave, and serotonin does suppress
eye. It appears to lack circadian pacemaker neurons, and autogenous CAP activity. Serotonin may create the nec-
changes in the photoresponse during the circadian cycle essary conditions forexpression ofthe B-C wave, in part,
have not been explored to my knowledge. This retina by suppressing autogenous CAP activity.
contains two types ofdepolarizing cells and one hyper- Because serotonin induces the ERG B-C wave and the
polarizingtype(QuandtandGillary, 1979), similartothe reduction oftheA wavelatency, one mayaskhowitmight
Aplysiaretina. Onedepolarizingcelltype(Type II)exhibits be involved in circadian control ofthe ERG. Serotonin
two peaks ofdepolarization that are similar, but of op- may just be mimicking a natural process, but because
posite polarity, to the ERG waveform (Quandt and Gil- thereareefferent synapticterminalscontainingserotonin
lary, 1980). in the eye, they may well be involved. Because isolated
eyesshowcircadian rhythmsin the B-Cwave, thecontrol
Role ofserotonin ofserotonin release from theefferentterminalsbycentral
Serotonin hasbeen shown byEskinand Maresh (1982) neurons is eliminated. But how then might serotonin be
to increase the first wave (A wave in this study) of the released by activity within the eye? Could processes con-
Aplysia ERG when it isrecorded with a suction electrode trolled by the circadian pacemaker in the eye release se-
applied to the cornea. They did not see a B-Cwave. That rotonin? Because the appearance ofthe B-C wave is as-
may be due to differences in recording methods, because sociated with minimal CAP frequency, release cannot be
they made polygraph recordingsanddid not report laten- adirecteffect ofCAPactivity. To producetheappropriate
cies. They found an average increaseMin the ERG ampli- response, autogenousCAPactivitywould havetosuppress
tude of 63% after 20 min in 10~6 serotonin, and a serotonin release, and inactivity would have to promote
threshold concentration near 10~7 M. Dopamine, acetyl- release. Otherwise another process controlled by the cir-
choline, and octopamine weretested but did not produce cadian pacemaker must be involved.
consistent ERG changes. They also reported a 20% in-
crease in the ERG in response to ON stimulation and Acknowledgments
proposed that the stimulation might cause the release of I thank Mark Goldberg for excellent technical assis-
serotonin from efferent terminals in the eye. Terminals tance. Research supported by NSF grant BNS 88-19773
have been identified by serotonin antisera (Goldstein el toJ.W.J.
al. 1984; Takahashi el ai, 1989).
Theeffect ofserotonin on the ERG suggeststhat cyclic Literature Cited
nucleotide second messengers may be involved. Cyclic
AMP mediates many of the serotonin effects on short- Barlow, R. B.,Jr. 1983. Circadian rhythmsintheLimulusvisualsys-
tem.J. Neumsci. 3: 856-870.
and long-term central synapses in Aplysia (Kandel and Battelle, B-A., J. A. Evans, and S. C. Chamberlain. 1982. Efferent
Schwartz, 1982), and serotonin phase shifts the CAP fibers to Limulus eyes synthesize and release octopamine. Science
rhythm (Corrent et al, 1978) by a mechanism involving 216: 1250-1252.
cAMP (Eskin et ai, 1982). In addition, cGMP mimics Block, G., and D. McMahon. 1983. Localized illumination of the
AplysiaandBullaeyerevealsnewrelationshipsbetweenretinallayers.
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CAP
phaseshiftsofthe rhythm(Eskinetai, 1984). During Corrent,G.,D.McAdoo,andA.Eskin. 1978. Serotoninshiftsthephase
thecGMPtreatment, the membrane potentialsofR pho- ofthecircadianrhythm fromtheAplysiaeye.Science202:977-979.
toreceptorswerenotaltered, butchangesinphotorespon- Eskin, A. 1971. Properties ofthe Aplysia visual system: in vitro en-
siinvcerneeasssedwetrheencotGeMxPplolreevdel. Abyte5n0%m..inCuytceliecxpGoMsuPremtaoyligbhet Eskietnyr,ea.iAnZ.me1nv9et7rg7ol.f.tPhhNeyescuiirorolcpoahgdyiiscaino7l4r:ohgy3it5c3ha-lm3a7M1ne.dchcaenntirsimfusgailnvroelgvuleadtiionnpohfottoh-e
elevated, either as a consequence of photoresponses, or entrainment ofthe circadian rhythm from theAplysiaeye. J. Neu-
because it is involved in the phototransduction process robtoi 8: 273-299.
(Eskin el ai, 1984). Ifany rhythmic changes in the levels Eskin, A., and R. Maresh. 1982. Serotonin or electrical optic nerve
ofcyclic nucleotides occur, they might be involved in the stimulationincreasesthephotosensitivityoftheAplysiaeye. Comp.
B-C wave and the A wave latency changes. EskiBni,ocAh.,emJ..PSh.ysTiaokla.ha7s3hCi:,27M-.31Z.atz, and G. D. Block. 1984. Cyclic
Serotonin does not appear to induce the B-C wave by guanosine 3':5'-monophosphate mimics theeffectsoflight on a cir-
a direct effect on the initial light response ofpacemaker cadian pacemakerin theeyeofAplysia. J Neurosci. 4:2466-2471.
