Physiological and Morphological Identification of Photosensitive Neurons in the Opisthosomal Ganglia of Limulus polyphemus

2004·3.5 MB·

by  Kazuo Mori

Reference: Bint. Bull. 207: 209-216. (December 2004)
> 2004 Marine Biological Laboratory
Physiological and Morphological Identification of
Photosensitive Neurons in the Opisthosomal Ganglia of
Limulus
polyphemus
KAZUO MORI*. TAKEHIKO SAITO, AND TAKETERU KURAMOTO
Shimoda Marine Research Center, University ofTsukuba, Shimoda, Slu'~.uokii, 415-0025, Jupun
Abstract. The motor outputs ofthe isolated opisthosomal studied extensively because they are accessible for intracel-
ventral nerve cord in Limuhtspolyphemus are modulated hy lular recording (Wilkens and Larimer, 1972, 1976: Krus-
light. We have identified the photosensitive neurons and zewskaand Larimar, 1993) andbecause theiractivity can be
examined their physiological and morphological properties correlated with the animal's behavior (Welsh, 1934; Ed-
using intracellular recording and staining techniques. We wards, 1984; Simon and Edwards, 1990).
found that photosensitive neurons are present in each gan- The horseshoe crab, often referred to as a "living fossil,"
glion ofthe opisthosomal ventral nerve cord. These neurons has proved useful for studying the phylogenetic develop-
often discharged action potentials spontaneously in the ment of arthropods. Because of its accessible eyes, the
dark, and they increased the frequency of this discharge in animal is also an admirable model for vision research:
the light. The mean latency (SD) of the light-induced extensive studies have been performed on its lateral eyes
action potential was 2.2 l.ls. Cells responded in agraded (Hartline and Ratliff, 1957; Ratliff. 1974; Barlow et al..
fashion overa2-log unitoflight intensity. The peak spectral 2001) and ventral photoreceptor (forreview, see Dorlochter
sensitivity was 425 nm or lower. The Lucifer-yellow-la- and Stieve, 1997). Studies on extra-retinal photoreceptors in
beled photosensitive neurons had oval somata with mean the central nervous system have, however, been limited
(SD) diameters of 102 3 ju,m (long axis) and 75 5 /am (Snodderly, 1971).
(short axis), and extended their axons to the contralateral Using extracellular recording techniques, we recently ob-
region ofthe ventral nerve cord. The soma had nodendrites, served that motor outputs ofthe isolated opisthosomal ven-
and the axon had thin branches. tral nerve cord of Limuhts are modulated by direct light
stimulation (Mori and Kuramoto, 2004). Encouraged by this
Introduction finding, we used intracellularrecording techniques to search
for photosensitive neurons in isolated ganglia ofopisthoso-
Extra-retinal photoreceptors have been detected in the mal ventral nerve cord and examined their physiological
c1e9n8t2r)a.l nFeorrvoeuxsasmypsltee,m pohfoatrostehnrsoiptoidvse(fnoreurreovnisew,hasveee Pbaegeen, and morphological properties. The results indicate that pho-
tosensitive neurons are present in each ganglion ofopistho-
f1o9u3n4;d Kienntnheedy6,th1a95b8d,om1i9n6a3l: BgarnugnloioanndofKecnrnayefdiys,h 1(P9r6o2s)s,eri,n rseosmpaolnsveenitsr4a2l5nnermveorclorodw,er.anTdhethfeunpcetiaoknaolfsitghneiirficsapneccteraolf
the abdominal ganglion and the brain ofinsects (Arikawael the photosensitive neurons in the Limulus central nervous
a!.. 1991; Ichikawa, 1991; Hariyama, 2000). in the brain of
system is compared with those found in other arthropods.
spiders (Yamashita and Tateda, 1981, 1983). and in the
telson (Zwicky, 1968) and the metasomata of scorpions
Materials and Methods
(Geethabali and Rao. 1973). Among these extra-retinal pho-
toreceptors, the caudal photoreceptors in crayfish have been Preparation
The horseshoe crabs (Limulus polyphemus (Linnaeus.
R*eTcoeiwvehdom10cMoarrrceshpo2n0d0e4n;ceaccsehpotueldd 2b4eAaudgduresstse2d00a4t.Center for Vision 1758). both sexes. 17-21 cm in carapace width) were pur-
Research. SUNY Upstate Medical University, 750 East Adams Street. chased from the Marine Resource Center (Marine Biologi-
Syracuse, NY 13210. E-mail: [email protected] cal Laboratory, Woods Hole. MA). They were reared in the
209
210 K. MORI ET AL
aquaria of Shimoda Marine Research Center (Shizuoka, Intracellular staining
Japan) under a natural light/dark cycle. The temperature in Lucifer yellow was iontophoretically injected into phys-
the aquaria ranged between 1 1 and 25 "C throughout the iologically examined cells with negative electrical currents
year. of 5-15 nA at 2.5 Hz for 30-90 min. Ganglia containing
For these experiments, an animal was inverted, and the stained cells were kept overnight at 4 C and then fixed for
gwiellrepldaitsessetcotgeedthferreewiftrhomthethoepiansitmhaols.omUanldveerntarasltneererovmeiccorrod- s2alhinine4(%PBpSa)raatfoprHma7l.d4.ehAyfdteerinw0a.s1himnolg/1foprho1.s5phhatinePbuBfSfearnedd
scope, a chain of 9th to 16th opisthosomal ganglia with several minutes in distilled water, they were dehydrated in
intact dorsal and ventral nerves were freed from surround- a graded ethanol series and cleared in methylsalicylate. The
ipnhgysitoilsosugei.calThseaylinweesroelutpiuotn ainntdokeaptpeattri4 dCishunttiillluesdedw.itIhn wstiatihneadfnleuuorroesncsenincewhmoilcer-osmcooupnet(pOrpetpiaprhaotti,onNsiwkeorne)oabnsderpvheo-d
ecvhaeirny oefxpoepriismtehnots,omaalsignagnlgeligaaangnldiomnouwnatsedisvoelnattreadl sfirdoemupa tographed with a digital camera (Coolpix 990, Nikon).
on a silpot platform in an acrylic chamber (3 X 2 X 0.7 Results
cm3). The sheath of the ganglion and the connective tissue
surrounding cell bodies were carefully removed. The prep- Intniccllnlcir and t:\tracelliiliir recordingsfrom isolated
aration was perfused with saline solution saturated with air opisthosomal gang/id
bNyaClb,ubb1l0.i7ngK.C1T,heISs.aOliCnaeCUs.olu2t4i.o6nMcgonCtUain2e.d3 N(ianHCmOM,),5a3n0d Each opisthosomal ganglion contains a pair ofsegmental
nerves (dorsal and ventral nerves) with the dorsal nerves
3 glucose; adjusted to pH 7.4. The temperature ofthe saline lying anterior to the ventral nerves. The dorsal nerves in-
was maintained between 17 and 19 "C during the experi- nervate visceral organs such as the intestine and the heart,
ment.
and the ventral nerves innervate the muscles of the gill
plates (Patten and Redenbaugh, 1900: Carlson, 1905). A
Electrodes clusterofcell bodies is located at thebase ofeach segmental
nerve.
For intracellular recordings from photosensitive neurons, Using intracellular electrodes, we located photosensitive
micropipettes were made from glass capillary tubing neurons in single isolated opisthosomal ganglia. We also
(BF100-86-IO. Sutter) using a Brown-Flaming micropipette recorded extracellularly from dorsal or ventral nerves to
M
puller (P-97, Sutter) and filled with 3 KC1 solution. The correlate the activity of motor neurons with the activity of
resistance of the electrodes was 20-30 Mil. The electrode photosensitive neurons. Figure 1 illustrates the extracellular
was connected to a DC amplifier (MEZ-8301. Nihon discharge pattern of the right ventral nerves of the 9th
Kohden) through a high-impedance negative-capacitance ganglion (Fig. 1A) and the intracellular response of a pho-
preamplifier (JZ-101J, Nihon Kohden). For extracellular tosensitive neuron in the cluster of cell bodies of the same
recording of segmental nerves, glass suction pipettes filled ganglion (Fig. IB). In darkness, a periodic burst discharge,
with saline were connected to an AC amplifier (AB621G, corresponding to gill plate activity, was recorded extracel-
Nihon Kohden). Forintracellular staining, the micropipettes lularly from ventral nerves, and spontaneous action poten-
werefilled with 37r Luciferyellow (Polyscience Inc.) in 100
mM
LiCl (15-80 Mil). All nerve signals were digitized
(Digidata 1200B. Axon Inst.), saved on a computer, and
analyzed with Axoscope software (version 9. Axon Instru-
50pV
ments).
Light stimulus
The preparation was illuminated with a halogen lamp
(300 W) through a heat-absorbing filter, lens, mechanical
shutter, and mirror system. The intensity oflight of0.0 log
units roughly corresponds to 1.5 X 1017 quanta/cnr-s at the
Light
surface of the preparation and is attenuated with neutral-
density filters between 0.0 and 3.0 log units. Monochro- Figure 1. Simultaneousrecordingsofextracellularresponsesfrom the
matic light (425. 450. 475, 500. 550. and 600 nm) was rfirgohmtvaenpthroatlosneenrsvietiivneisnoeluatreodn9wtihtghainnglaicolnus(tAe)r,oafndceilnltrbaocdeilelsulairnrtehseposnasmees
obtained by narrow-band interference filters (Optical Coat- ganglion(B).In(A),illuminationproducedtheinhibitionofrhythmicburst
ings Japan) in the light path. Neutral-density filters adjusted activity andthe appearance ofsustained bursting. In (B), the frequencyof
the quanta! flux for each filter to 1.6 X 1014 quanta/cnr-s. action potentials increased by illumination.
PHOTOSENSITIVE NEURONS IN L1MULVS GANdl I \ 211
tials with an irregular interval were recorded intrucellulurly Physiological pnipertic.'i ofthe photosensitive neurons
from a single neuron. Illumination ofthe ganglion increased
the frequency ofaction potentials and inhibited the periodic- Photosensitive neurons are not abundant in isolated opis-
burst discharge of ventral nerves, producing a sustained thosomal ganglia. Thus far. less than 2'7r ofthe neurons that
discharge. we examined (20 out of 1050) exhibited photosensitivity.
Todetermine whetherthe photosensitive neurons respond All 20 photosensitive neurons exhibited spontaneous action
to light directly or through synapses, the preparation was potentials in the dark and responded to light with an in-
treated with Co2"1", a blocker of chemical transmission. crease in the frequency of action potentials. The level of
Figure 2 shows simultaneous recordings from the left ven- spontaneous activity and the response to light varied from
tral nerves ofthe 13th ganglion and a photosensitive neuron cell to cell and, occasionally, during the course of experi-
in the same ganglion before (A) and after (B) application of ment on the same cell. Three examples of photoresponses
Co2+. In the presence of 20 mA/ Co2+, the periodic burst are shown in Figure 3. Responses just before and at the
discharge was inhibited within 3 min, while the frequency beginning of illumination (dotted line under each left trace)
of action potentials without illumination was not signifi- are shown on the right with an expanded time scale. Re-
cantly changed. Illumination increased the frequency of sponses shown in Figure 3A and B were obtained from the
aticvteionneuprotoenntiisaalspr(Fiimga.ry2Bp)h,otsourgegceespttionrg.tThhatetehfifsecpthsotoofsCenosi- psoatmeenticaelllofinabotuhet -132t0h mgVangalnidone.xhTihbeitecdelflewhasdpoantarensetoiunsg
partially recovered after washout (not shown). action potentials in the dark within several minutes after
penetration (Fig. 3A). In the course of the experiment,
however, the frequency of action potentials gradually in-
creased to about 1 impulse/s (ips) without a significant
change in the membrane potential (Fig. 3B). After illumi-
nation, the membrane potential depolarized about 2 mV.
10 and the frequency of action potentials increased to 2 ips.
The latency ofthe response was 0.6 s in Figure 3A and 2 s
in Figure 3B. The response in Figure 3C wasrecorded in the
15th ganglion. In the dark, the cell discharged an irregular
spike frequency ofabout 0.1 ips. Light stimulation caused a
slight depolarization of the membrane potential and in-
creased the spike frequency to about 0.4 ips.
Figure 4 shows the effect of duration of light on a
10 mV photosensitive neuron in the 12th ganglion. Light stimula-
tion increased the frequency ofaction potentials from a rate
Light 10s ofabout 0.3 ips to 0.8 ips. The light-evoked response of0.8
ips was independent of the stimulus duration up to 60 s,
indicating minimal adaptation to the light stimulus. Photo-
B 20m/WCo2+ sensitive neuronsdidnotexhibit substantial adaptation tolight.
Photosensitive neurons have a graded response to light
intensity. Figure 5 (A-F) shows responses recorded from a
10 photosensitive neuron in the 9th ganglion to light ofdiffer-
ent intensities. The frequency ofaction potentials increased
at light intensities greater than 2.0 log units, reaching a
maximum of0.9 ips at 0.0 log units. The number of action
potentials to different light intensities was normalized to
mV
10 that evoked by the maximal light intensity (0.0 log units)
and plotted against the log intensity of light for the three
10s cells in Figure 5G. The data from the cell in Figure 5 (A-F)
Light are illustrated by filled circles. The intensity-response curve
Figure 2. Effect of Co2+ on extracellular responses from the left indicates that the photosensitive neurons can convey infor-
ventral nerve in isolated 13th ganglion, and intracellularresponses from a mation about light intensity over a 2-log unit.
photosensitive neuron within aclusterofcell bodies in the same ganglion Photosensitive neurons respond maximally to shortwave-
(mfAAr/)e)q.bueefMnocoryteoorafppaalcctiticivaoitntiyopnointoeftnhtCeioalv2se*n;t(rl(aoBlw)enr1e5rtvrmeacie(n)upawfpiteterrhotaurptapcleii)lclawutamiisonnartoeifdounCcoead2n.*dT(th2he0e wleengetxhamililnuemdinatthieons.pecUtsrianlgredisfpfoenrseentprwoapveerlteinesgtohfsfoivfelnigehut-,
increase in the frequency ofaction potentials with illumination were not rons. Figure 6 (A-D) shows a representative spectral re-
significantly changed in the presence ofCo2+. sponse pattern of a photosensitive neuron in the 9th gan-
K. MORI ET AL.
longitudinally in the cord. These morphological properties
are based on observations of the Lucifer yellow (LY) that
was iontophoretically injected into 12 photosensitive neu-
rons after recording their electrical responses. Of 12 cells
injected with LY, 6 were successfully stained. Figure 7
shows a photomicrograph of the cell in Figure 3A and B.
10s The cell has an oval soma with a diameter of 103 /u,m (long
Light axis) and no dendrites. A single axon extends toward the
contralateral region without any branches, then turns, as-
cending in the connective toward the anterior ganglion
(Figure 7, inset). Several thin branches are sparsely distrib-
uted along the axon within the 12th ganglion. Diagrams in
Figure 8 show the 9th-16th opisthosomal ventral nerve cord
with the recording sites of the 20 recorded photosensitive
5mV neurons marked by filled circles (Fig 8A) and 6 LY-stained
photosensitive neurons (Fig. 8B). The diameter of the cell
h-H =
10s bodies (n 6) was 102 3 jum (long axis) and 75 5 /mi
Light Light (short axis). Each cell extended its axon to the contralateral
region. In five of the six cells, the axons ran along the
longitudinal ventral nerve cord toeitheranteriororposterior
connectives (Fig. 8B, circled A-D and F); the axon of the
sixth cell was restricted in the ganglion (Fig. 8B, circled E).
Discussion
10mV
r-H Extra-retinal photoreceptors that have been found in
Light 20s Light 5s Liiuulnx include ventral photoreceptors (Millecchia et al..
Figure 3. Three examples of the dark-adapted activity patterns and
light responses ofphotosensitive neurons. (A and Bl Responses from the
same neuron in the cluster of cell bodies at the right dorsal nerve in an
isolated 12th ganglion. In (A), the record was obtained 5 min after pene-
tration. The cell was essentially quiescent in the dark. In (B), the record
was obtained 80 min after the recording in (A). The frequency ofaction
potentials in the dark increased without significant change in membrane
potential. (C) Response from a neuron in the I5th ganglion. This neuron
had a relatively low frequency of spontaneous action potentials with
irregularspikeintervals.Allcellsrespondedtolightwithan increaseinthe 10s
frequencyofaction potentialsandacorresponding increase in theslopeof
the pacemaker potential, as seen in responses just before and at the B
beginningofillumination(dottedlineundereachlefttrace)andontheright
with an expanded time scale.
glion. Monochromatic light of equal quuntul flux hut
different wavelength was applied successively to the gan-
30s
glion from 425 nm to 600 nm. The response increased
slightly at 500 nm and became maximal at 425 nm. The
number of action potentials at each wavelength was nor-
mali/ed with respect to the number of action potentials at
425 nm and plotted as a function ofwavelength for five cells
(Fig. 6E). The spectral-response curve suggests that photo- /' / ! |f lfpft\ft 10 mV
sensitive neurons are maximally sensitive at 425 nm or
shorter wavelengths.
60s 10s
Morphological properties ofthe photosensitive neurons Figure 4. Effect of light of different duration on a photosensitive
neuron within aclusterofcell bodies at the base ofthe right dorsal nerve
All photosensitive neurons extend axons to the contralat- ofthe 12thganglion. An increaseinthefrequencyofactionpotentialswith
eral region ofthe ventral nerve cord, and mostextend axons illumination was maintained during illumination.
PHOTOSENSITIVE NEURONS IN L1MULVS GANGLIA 213
transient peak depolarization followed by a steady-state
depolarization (Hartline etal, 1952; Millecchiaelai. 1966;
Nolle and Brown, 1972). On the otherhand, the response of
photosensitive neurons in the Limulus ventral nerve cord
exhibits tonic discharges to light with little adaptation,
which is similar to the response of the caudal crayfish
-3.0 -1.5
photoreceptor (CPR) ofthe crayfish (Kennedy, 1958, 1963;
Wilkens and Larimer, 1972). In the crayfish ventral nerve
cord, primary photoreceptors are identified by at least two
criteria: the production of a slow depolarization, with su-
perimposed action potentials, upon illumination, and the
5mV
10s
1.0 -
0.9
0)
ta
O 0.8
Qin. 425nm 450 nm
0) 0.7
tr
0.6
0.5
0.4
0.3
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0
log/ 500nm 600 nm
Figure 5. (A-F) Effect oflight ofdifferent intensitieson a photosen-
sitive neuron within a cluster ofcell bodies at the base ofthe left dorsal
nerve ofthe 9th ganglion. Light stimuli between 3.0 and 0.0 log units
were successively appliedat intervalsofseveral minutes. (G)The number
of action potentials at each intensity of light was normalized with the 1.0
numberofaction potentials at the maximum intensity oflight and plotted o>
against different intensities. <cn 0.9
o
a
VI 0.8
as
ca
1966), photoreceptors adjacent to the optic ganglia (Snod-
a> 0.7
derly, 1971), photoreceptors in the median optic nerves
(Sarnie et /., 1995), and photoreceptors in the telson JS 0.6
(Hanna et nl.. 1988). Here, we found new photosensitive rr
neurons in the opisthosomal ventral nerve cord. These often 0.5
exhibited spontaneous action potentials in the dark and an
0.4
increase in the number of action potentials in light. The 450 500 550 600
latency ofthe response to light was on the orderofseconds. Wave Length (nm)
The frequency of action potentials was maintained during
illumination and modulated by light intensity over a 2-log Figure 6. (A-D) Effect oflight ofdifferent wavelengths on a photo-
unit. The peak spectral response was at 425 nm or lower. sensitive neuron within a cluster ofcell bodies at the base of left dorsal
nerve in the 9th ganglion. Monochromatic light with aduration of30s is
shownbeloweachrecord.Lightstimuli wereappliedatintervalsofseveral
Are photosensitive neurons photoreceptors? minutes. The maximum spectral response was seen at 425 nm. (E) The
number of action potentials at each wavelength was normalized to the
In Limitlus. ventral photoreceptors and photoreceptor numberofaction potentials at425 nm and plotted againstdifferent wave-
cells ofthe lateral and median eyes respond to light with a lengths. Values are mean (SD).
214 K MORI ET AL.
Figure 7. Ventral view ofa Lucifer-yellow-injected photosensitive neuron in the 12th ganglion. The cell
body (103 jam in diameter, long axis) extends an axon toward the contralateral region, where the axon turns
sharplyandascendstowardthe 1 Ithganglionwithsomedendriticbranches(arrows). Inset: aschematicdrawing
ofthe neuron in the ganglion.
resetting of the endogenous rhythm by interpolated anti- quency. Although we did not examine whether an anti-
dromic impulses (Kennedy 1963). In the present study. dromic pulse stimulation resets the spontaneous activity
many photosensitive neurons also responded to light with a rhythm, the fact that, when chemical transmission was sup-
slow depolarization and an increase in action potential fre- pressed by Co:+. photosensitive neurons did not lose spon-
10th 11th 12th 13-16th
B
Midline
200pm
Figure 8. Ventral view of the opisthosomal ventral nerve cord (A), and the morphology of 6 Lucifer-
yellow-labeledphotosensitive neurons (B). In (A), filledcircles indicate the location ofsomataof20photosen-
sitive neurons that are examined physiologically. In (B). morphology ofthe labeled neurons is schematically
illustrated. Circled letters in (B)correspondtothose in (A). The midlmeoftheopisthosomal ventral nervecord
is shown.
PHOTOSENSITIVE NEURONS IN LIMULUS GANGLIA 215
taneous action potentials in the dark and photosensitivity to directions. In crayfish, a pair of CPR interneurons also
light suggests that they are primary photoreceptors. consists of a contralateral soma and ip.silateral axonal
branches (Wilkens and Larimer, 1972). The axon of the
Comparison ofextra-retinalphotoreceptors in Limulus CPR projects the length of the ventral nerve cord from the
and other arthropods 6th ganglion to the brain (Simon and Edwards, 1990). In
contrast, Limulus has photosensitive neurons in each gan-
From aphylogenetic pointofview, it is important topoint glion of the ventral nerve cord. In one of six LY-stained
out similarities and differences in the extra-retinal photore- cells, the axon was restricted to a single ganglion (Fig. 8B.
ceptors of Liinnliis and other arthropods. Like Limulus. the circled E), whereas the other five cells extended their axons
scorpion is a chelicerate, and it shares a number offeatures to anterior or posterior connectives (Fig. 8B, circled A-D
in its structure and development with the horseshoe crab. It and F). Therefore, it appears that they extend axons to more
has been reported, using extracellular recording (Zwicky. than one ganglion. Another morphological difference be-
1968; Geethabali and Rao, 1973) and behavioral techniques tween the photoreceptor ofthe two species is the paucity of
(Zwicky, 1968, 1970). that the scorpion has extra-retinal dendrites in Limulus relative to those in crayfish. Further-
photoreceptors in some abdominal ganglia, and that the more, in crayfish, these dendrites and not the cell body have
peak spectral response is 568 nm with a secondary peak at been assumed to be photosensitive. The site of the light
440 nm. A more detailed comparison between the extra- sensitivity ofthe photosensitive neurons in the opisthosomal
retinal photoreceptors of Limulus and the scorpion must ganglion of Limulus is not known.
await further analysis using intracellular recordings in scor-
pions. In the brain of spiders, extra-retinal photoreceptors Functional significance ofextra-retinal photosensitive
with a peak spectral response at 420-440 nm have been neurons
detected using extracellular recording techniques (Ya-
Extra-retinal photoreceptors in arthropods are often func-
mashita and Tateda, 1981. 1983).
The photosensitive properties ofLimulus neurons can be rtihoyntahllmys a(sYsaomcaisahtiedta waintdh Ttahteedae,ntr1a9i8n1m;enPtageo,f 1c9i8r2c)a.diaInn
bwehtitcerhchoamspabreeedntosttuhdeiceadudmaolrpehoetxotreencseipvteolyr.ofTthhee cCraPyRfishi,n cernatyrfaiisnhe,dfbory eixlalmupmilnea,ticoinrcoafditahne lborcaionmo(tPoargeactainvdityLacrainmebre,
clroacyaftiesdhiinstchoemp6tohseadbdoofmtiwnaolbiglaantgerlailolny.sLyimkmeettrhiocsaelinneLuirmoun-s m1o97v2e)m.enFutrsth(eWremlosrhe,, 1i9l3l4u;miEndatwiaorndso.f 1t9h8e4;CPSiRmoenvoakneds Elde-g
lus, they exhibit spontaneous action potentials in the dark wards. 1990) and disrupts circadian locomotion (Fuentes-
and a tonic increase in the frequency ofaction potentials in Pardo and Inclan-Rubio. 1987; Inclan-Rubio and Fuentes-
rliegshpto(nPsreosisseart,5109034n;mKe(nBnreudnyo,a1n9d63K)e;nbnuetdyth,ei1r9p6e2a;kLsapreicmterra.l Pardo, 1987). In scorpions, Zwicky (1968. 1970) has
1966) rather than at 425 nm or lower. It is worth noting that speculated that the negative phototaxis might be mediated
by extra-retinal photoreceptors.
the peak spectral response ofextra-retinal photoreceptors in In the present and previous studies (Mori and Kuramoto,
Limulus is similar to that in scorpion (the secondary peak) 2004), we have observed that illumination of isolated opis-
and spider, which are phylogenetically closer to the horse- thosomal ganglia of the Limulus ventral nerve cord either
shoe crab.
ofrtoofarrnT2egh.cCe2eePpotlRfoartlsalent.nerelcuansyrcn.oiognefeSssdutcmhf(hear0y.ora3meb-sle01po.2on5pngasstr,elotal5ityKneesdn,nLcuinywemeiudhttalyhosu,masbeme1eaex9est5anur8nra)a-e.lrmseeoTtiSnhrnteDaelpevolrpararhltroouegr-edes hbpvuielenreaanhettrtilwetrbieaimletbitenehnndaeaettrrohyvraeiecnetgsaxii.clsvpclieWeotprlmyelieeamrtoaesetfanusetngdsigov,mefi(ssaoabctlrlesrurtebaehso(lattdtlrhaiao)gltihirantggtehhanftdenortssaoatncmisitamhimanuvoandlilwtaLnytag)iEic.oolcDflneTldemhpeolmiarrasabsyttaeeelrsdmde.dosatuednhIld-dent
caused by a fluctuation in the intervals between spontaneous
action potentials. Nevertheless, latencies on the order of snuegugreosntsstthhraotugehxttehrenaslemliitgrhatnscpaanrernetacshkinthoefpthhoetoisnteancstitainvie-
seconds are significantly longer than those reported for mal and perhaps have a role in behaviors such as an animal
glmaeitlseltrieasldecoforonrvdesn.tthrAeallspoehncogotnordreecmsepepostnsoseresn,gelwrahtieccnachsycaardieneohcnarsatyhfebiesoehrndeCsruPgoR-f bgrrieegcahattuiesnregrtoihtlseeeylfi.anrPethhometoorbseeehnastvriatiniosvrpeaornfeenujturvotenhnsainlmeaaduhylotrshs.aevsHehooawenceernvavbei-sr
(mKercuhsazneiwssmkareamnadinsLaurinmreers,olv1e9d93f)o.r Texhtera-prheottinoatlrapnhsodtuocsteino-n ronmental light modulates organ activities and animal be-
havior are subjects for future research.
sitive neurons in Limulus ventral nerve cord.
Dye-injection experiments demonstrate that all somata of
Acknowledgments
photosensitive neuronsexamined in Limulus extend an axon
to the contralateral region, where it runs longitudinally We thank Dr. Robert B. Barlow (SUNY Upstate Medical
along the opisthosomal ventral nerve cord in either or both University) for his critical reading of the manuscript and
216 K. MORI ET AL.
comments. This research is a study of the Shimoda Marine Krus/ewska, B.,andJ. L. Larimer. 1993. Specific second messengers
Research Center (Contribution No. 700). activate the caudal photoreceptorofcrayfish. Brain Res. 618: 32 K).
Larimer,J. L. 1966. A functional caudal photoreceptor in blind caver-
mcolous crayhsh. Nature 210: 204-205.
Literature Cited Millniliia. R.,J. Bradbury,and A. Mauro. 1966. Simple photorecep-
tors in Liniiilu.\ polvpliemus. Science 154: 1199-1201.
Arikawa, K., K. Uchiumi, and E. Eguchi. 1991. Extraocular photore- Mori, K.. and T. Kuramolo. 2004. Photosensitivity ofthe central ner-
ceptors in the last abdominal ganglion of a swallowtail butterfly. vous system ofLimiting po/yphemns. Zool. Sci. 21: 731-737.
Papilioxuthus. Naturwissenschaften 78: 82-84 Nolle, J., and J. E. Brown. 1972. Electrophysiological properties of
Barlow, R.B.,J.M. Hitt,and F.A.Dodge.20(11. l.imiiliisvision in the cells in the median ocellus ofLinmlns. J. Gen. Phvsiol. 59: 167-185.
marine environment. Binl. Hull. 200: 169-176. Page, T. L. 1982. Extraretinal photoreception in entrainment and pho-
Bruno, M. S., and D. Kennedy. 1962. Speetral sensitivity ofphotore- toperiodism in invertebrates. Expericntia 38: 1007-1013.
ceptor neurons in the sixth ganglion of the crayfish. Camp. Biochem. Page, T. L., and J. L. Larimer. 1972. Entrainment of the circadian
Pliysiol. 6: 41-46. locomotoractivity rhythm in crayfish.J. Comp. Pliysiol. 78: 107-120.
Carlson,A.J. 1905. Comparativephysiologyoftheinvertebrateheart. I. Patten. W.,and \V. A. Redenbaugh. 1900. Studieson Limiilus. II The
The innervation ofthe heart. Rial. Hull. 8: 123-109. nervous system of Limiilus polvphemns, with observation upon the
Dorliichter, M., and H. Stieve. 1997. The Limiilus ventral photorecep- general anatomy. J. Morphol. 16: 91-200.
tor: light response and the role of calcium in a classic preparation. Prosser. C. L. 1934. Action potentials in the nervous system of the
Prog. Neurobiol. 53: 451-515. crayfish. II. Responses to illumination oftheeye andcaudal ganglion.
Edwards, I). H., Jr. 1984. Crayfish extraretinal photoreception. I. Be- ./. Cell Comp. Pliysiol. 4: 363-377.
havioural and motoneuronal responses to abdominal illumination. J. RatlilT, F.,ed. 1974. Studies on E.\cilutinn ctntlInhibition in the Rerinu.
Ev/>. Binl. 109: 291-306. A Collection ofPaper* front the laboratories of H. Kcfter Hurtlinc.
Fuentes-Pardo, B.,and V. Inclan-Rubio. 1987. Caudal photoreceptors Chapman and Hall, London.
synchronize the circadian rhythms of crayfish. I. Synchronization of Sarnie,F.H.,R.N.Jinks,W.W.Weiner,andS.C.Chamberlain. 1995.
ERGandlocomotorcircadianrhythms. Comp. Biochem. Pliysiol. A 86: The morphology and physiology of a "mini-ommatidmm" in the me-
523-527. dian optic nerve ofLiiiiulu.\ po/vp/ieimis. Vis. Neurosci. 12: 69-76.
Geethabali, and K. P. Rao. 1973. A metasomatic neural photoreceptor Simon. T. W., and D. H. Edwards. 1990. Light-evoked walking in
in the scorpion. ./. EV/J. Biol. 58: 189-196. crayfish: behavioral and ncuronal responses triggered by the caudal
Hanna, \V. J. B.,J. A. Home, and G. H. Renninger. 1988. Circadian photoreceptor. ./. Comp. P/nsiol. A 166: 745-755.
photoreceptor organs in Limiilnx. II. The telson. ./ Comp. Pliysiol. A Snodderly. M., Jr. 1971. Processing ofvisual inputs by brain ofLimii-
162: 133-1411 Ins. ./. NcurapliYMol. 34: 588-611.
Hariyama.T. 2000. Thebrain asaphotoreceptor: intracerebral ocelli in Welsh, J. H. 1934. The caudal photoreceptor and responses of the
the firefly. Naturwissenschqften 87: 327-330. craUish to light. / Cell Comp. Physiol. 4: 379-388.
Hartline, H. K.,and F. RatlitT. 1957. Inhibitory interaction ofreceptor Wilkens, L. A., and J. L. Larimer. 1972. The CNS photoreceptor of
units in the eye ofLimn/us. J. Gen. Pliysiol. 40: 357-376. crayfish: morphology and synaptic activity. ./. Comp. Pliyxiol. 80:
Hartline, H. K.. H. G. Wagner, and E. F. MacNichol,Jr. 1952. The 389-407.
CM
peripheral origin ofnervous activity in the visual system. Spring Wilkens, L. A., and J. L. Larimer. 1976. Photosensitivity in the sixth
HmhorSymp. (Jiumt Binl. 17: 125-141 abdominal ganglion of decapod crustaceans: a comparative study.
Ichikawa,T. 1991. Brainphotoreceptorsinthepupalandadull butterfly: J. Comp. Pliysiol. A 106: 69-75.
fate of the larval ocelli. Znnl. Sci. 8: 471-476. Yamashita.S.,and H.Tateda. 1981. Efferentneuralcontrol intheeyes
Inclan-Rubio, V.,and B. Fuentes-Pardo. 1987. Caudal photoreceptors ot orb weaving spiders. ./. Comp. Phvsiol. A 143: 477483.
synchronize the circadian rhythms ofcrayfish. II. Functional relation- Yamashita,S.,andH.Tateda. 1983. Cerebralphotosensitiveneuronsin
ships between caudal and visual photoreceptors. Com/'. Biochem. theorbweaving spiders.Arf>iopehnicnnichiiand,4. amoenu.7. Comp.
Physinl. A 86: 529-536. P/;y.W/. A 150: 467-472.
Kennedy, I). 1958. Responses from the crayfish caudal photoreceptor. Zwieky,K.T. 1968. AlightresponseinthetailofUrmlncus. ascorpion.
Am. J. Oplitlwlmol. 46: 19-26. Life Sci. 7: 257-262.
Kennedy, D. 1963. Physiology ofphotoreceptor neurons in the abdom- Zwieky,K.T. 1970. Behaviouralaspectsoftheextraocularlightsensenl
inal nerve cord ofthe crayfish. J. Gen. Phvsiol. 46: 551-572. Vrodacus, a scorpion. E\perit'ntin 26: 747-748.