Time-Resolved Imaging of Ca2+-Dependent Aequorin Luminescence of Microdomains and QEDs in Synaptic Preterminals

7 Pages·1994·2.9 MB·English

by  R B Silver

Reference: Biol. Bull 187: 293-299. (December. 1994)
Time-Resolved Imaging of Ca2+-Dependent Aequorin
Luminescence of Microdomains and QEDs
in Synaptic Preterminals
ROBERT B. SILVER1 3 MUTSUYUKI SUGIMORI2 \ ERIC J. LANG2-3
A,ND RODOLFO LLINAS23 ,
^Section andDepartment ofPhysiology, Cornell University, Ithaca, New York 14853-6401,
^Department ofPhysiology and Biophysics, New York UniversityMedical Center, 550 FirstAvenue,
New York, New York 10016, and^Marine Biological Laboratory, Woods Hole. Massachusetts 02543
Localizedelevationofintracellularfreecalcium[Ca2+J, domains, andIhat these[Ca2+],profiles are composedof
concentration serves as the trigger for a wide variety of groups ofshort-lived 0.5 fim diameter QEDs. In those
physiologicalprocesses, e.g., neurotransmitter release at records, obtainedwith 2:1 interlacingdevicesoperatingat
most chemical synapses (1-3). The details ofthe mecha- the RS-170 standard, QEDs appeared as striped dots or
nismsthatregulatetheseprocessesarestillunresolved(3- chevrons ratherthan as soliddots, indicatingthat a QED
6), but they must involve precise temporal sequences of lastedless than 16.6 ms (one videofield), andthus estab-
moleculareventsinitiatedbyatransientlocalizedelevation lishing the needfor higher sampling rates to better char-
oj'Ca2+ concentration (i.e., a Ca2+ microdomain [3, 7- acterizethe QED. In thepresent set ofstudies, wesought
15]). A microdomain is defined as an autonomous com- toextendourearlier work on calcium microdomains and
partment ofminimalspatio-temporal volume within which QEDs with the goal ofbetter understanding the lifetime
asignaledprocesscan occur(8, 10, 12). A quantum emis- ofthe QED and its relationship to the action potential.
sion domain (QED) is a quanta! signal element (3, 16, This is thefirst description ofthe methodology necessary
17). The concept ofa QED wasfirst appliedto Ca2+ sig- toimagetheCa2+ QED, andthetechniquesforobtaining,
naling at the synaptic preterminal (3. 4) and for large- processing, andanalyzing video information athighframe
diameter mitotic cells (16, 17). rates.
The concept ofCa2+ microdomains was tested by la-
belingpreterminals ofsquidgiant synapses with low-sen- Chevrons anddots in interpreting the duration ofQEDs
sitivityaequorin (aphotoproteinthat emitsaphoton upon QEDpatternsinaequorin-labeledsynapticpreterminals
bindingCa~*[18, 19]). That workconfirmedearliermod- were observed as discrete events following stimulation of
elingefforts(10, 16) andshowedthat, upondepolarization, single action potentials. These observationswere initially
the[Ca2*],profilereaches200-300^M within themicro- performed at a video frame rate of30 frames per second
(fps), using cameras conforming to the RS-170 standard.
Received 20September 1994;accepted 5 October 1994. Each video frame consisted oftwo interlaced video fields
SciPeonrtitfiiocnsMeofetthiinsgwsoartktwheereMaprriesneentBeidoliongipcraelliLmaibnoarryatfoorrym,aWtotohedGsenHeorlael. e(iv.ee.n, 2s:c1aninltienrels.aceA),neeawchfiecldomwpaossceadptoufretdheevseetryof16o.d7dmso,r
MA.on 16August 1994.
Abbreviations: CCD = chargecoupled device;dMCP = dual micro- and each scan line lasted for 63.5 /^s. Therefore, an event
channel plate image intensifier; N.A. = numerical aperture: NTSC lasting 16.7 to 33 ms would be seen as a solid dot, while
= NationalTelevisionStandardsCommittee;QED = quantumemission one lasting less than 16.7 ms would be observed as a set
domain; RS-170 = NTSC Resolution Standard 170: video frame reso- ofhorizontal stripes. Events ofduration close to 16.7 ms
lrauttei,on2:o1fi5n2te5rllaicneesooffvhiodreiozofinetladlswreistohliuntiaons,in6g0leHvzidveiodeforafmiee.ld3s3ammpslipnegr might also appear as dots, depending upon the timing of
frame (16.66 ms per video field): used primarily for black and white the initiation oftheevent relative tothetimingofthetwo
videodevices. video fields. Ifthe event began close to the end ofthe first
293
294 R. B. SILVER ET AL.
Figure 1. (A) Individual quantum emission domains(QEDs) activated by singleaction potentialsat a
preterminal appearas white chevrons when recorded at 30frames per second (fps). A few ofthese QEDs
show some fillingbetween the stripes, indicatingthat those eventsbegan closeto eitherthe startorend of
a single video field and overlapped to the preceding or following video field. The two QEDs seen in the
lowerright ofthis panel occurred in separateodd andeven fields. (B) Individual QEDsactivated bysingle
presynaptic spikesappearas white chevrons recorded at 200fps, indicating that the entire QED occurred
within the 2.5-ms sampling period ofthe video field. Note that in this case, unlike that in panel A. all of
the QEDsappearaschevrons, indicatingthat noobserved QEDs overlapped twoconsecutive videofields.
(C) The first 0.25-ms video frame recording ofa single evoked QED (red) superimposed onto its point of
origin inasynaptic preterminal (blue). The redcharacterssurroundingthe central imagearetypical ofthe
display from the Kodak high-speed videocamera. (D) The time course ofan evoked QED, and one for a
singleQEDemittedfromaequorinexpressedintocalciumbuffer,recordedatavideorateof4000 fps.Spots
oflight from single video framesare displayed asa row ofspotsat the same horizontal level as the terms
"Evoked" and "Aeq." The intensity profiles ofeach ofthe individual spots are seen directly above their
respectivespots. The spotsand intensity profileswere printed in pseudocolorto aid in visualization; black
andpurpleare tolow intensity, increasingthroughgreen, yellow,and orangetothe highest valuesofred
andwhite(8-bitgray-scalevalueof255). Thesevaluesarecorrelatedwith the luminance intensity incident
tothefaceplateofthedualmicrochannelplate(dMCP)intensifier.Smallwhitepointsmarkingeachindividual
video fieldappearbelowtheirrespectivespots. Thescale markerin panel C represents 10^m.
synSatpatnidcartdranmsemtihsosidosnw(e3r.e18u)s.edVitdoeodisismeacgtesstewlelarteecgaapntguliraedfrwiotmhsamadlilrecstquoipdti(cLaolliegxxtepnesailoenj}onatnodamodnMitCoPr
VideoIntensified Microscopycamerasystem (HamamatsuArgus 100; Hamamatsu Photonics, Inc.. Bridge-
water, NJ)operated in photon-countingmode. Threedifferent videocameraswereusedtodetectcloudsof
electrons emanating from the dMCP. For video at 30fps, a Hamamatsu saticon tube camera was used,
operatedundertheRS-170standard.ImageryfromthesaticoncamerawasrecordedonanS-VHSvideocassette
recorder.AnNACHSV-400(NAC,Inc.,Japan)high-speedcolorvideocamera,S-VHSvideocassetterecorder,
andPanasonicST-1000colorvideomonitor(MatsushitaElectric Industrial Co., Ltd., Fukuoka,Japan), all
operatedinB/Wmode,wereusedfor200-fpsimagingandrecording.TheNACHSV-400camerawasfitted
tothedMCPthroughadirectopticalcoupling.CarewastakentoensurethattheoutputimageofthedMCP
TIME-RESOLVED IMAGING OF CA2+ MICRODOMAINS AND QEDS 295
video field orended near to the start ofthe second video LuminanceIntensityforEvoked.SpontaneousandAequorinQEDs
field, the event would be recorded as a partially filled
chevron whose centroid coincided with the location of
the point of origin for the event. Events that were very
short compared with the video-field sampling rate would
nearly always appear as chevrons. This principle applies
toall 2:1 interlaced imagingdevices, irrespective ofvideo
frame rate.
QED images at 30 videoframespersecond
Ca2+j-dependent aequorin luminescence wasdetected 2345
as discrete QEDs at the preterminal side ofthe synapse
following presynaptic action potentials (Fig. 1A). Be-
cause QEDs were seen as chevrons, rather than solid Time (250microsecondvideoframes)
spots, they must havehad alifetime oflessthan 16.7 ms; IEvokedQED %^SpontaneousQED ElAequorinQED
nearly all ofthe presynaptic QEDs observed after such Figure 2. Luminance intensity for evoked, spontaneous, and ae-
action potentials appeared as chevrons. In several in- quorinQEDscorrectedforphosphorescentafterglow. Valuesaremeans
stances, chevron QEDs were seen to interlace. These ofthetotalluminanceintensityforeachoffiveQEDsforeachcategory,
were fortuitous observations, and would be expected and areexpressed asrelativetothebrightnessofthe initial frameofan
evokedQED. Evoked QEDs(solid black) lastedaslongasfive0.25-ms
for near-neighboring QEDs acting within microdo- videoframes; meanspontaneousQEDs(thindoublestnpes)lastedonly
mains. In several otherinstances, some ofthe QED sig- 0.5 ms;andaequorinQEDs(singlediagonalcrosshatches)wereseen for
nal extended to the following (or from the preceding) only one0.25-msvideo frame.
video field; that is, the blank space between the lines of Toensure that the imagery information was properly processed, the
the chevrons contained some record of a signal. We ifonlfloorwmiantgioanssoufmpthteioinnistiaanldvipdraecotifcreasmweerreecaodrodpitnegda.QThEeDphwoatsocnoninstiednesrietdy
interpret these images to represent a short-lived event torepresentlightfromthephotochemicalreactionofCa2+andaequorin.
that occurred near the end (or beginning, respectively) ThecontributionofafterglowinthefirstvideoframeofeachQEDrecord
ofa given video field that contained the majority ofthe wasconsideredtobenegligibleforimageryat4000fps.Imageinformation
chevron QED photon emission. for each subsequent frame was considered to consist ofa sum ofthe
Ca2+-dependent aequorin-based photon emission generated and inte-
grated onto the detector faceplate duringthat video frame, and the af-
QED images at 200 video framesper second terglow attributable to the preceding video frame. The contribution of
Because virtually all of the QEDs observed at 30 fps afofrtearegqluoowrwiansidnejetcetremdininetdowCiath2+e-mcpoinrtiacianlinvgalbuuefsfedreraisvefdolflorwosm.tThheeimlaugmeis-
appeared on the video monitor as chevrons, better tem- nance intensity values for QED photon emissions from each 0.25-ms
poralresolution wasclearly needed. Toresolveindividual videoframeweredeterminedfromdigitizedvideorecords.Photonemis-
QEDs in time, aequorin luminescence was videotaped at sionfromthereactionofCa2+andaequonnwasassumedtolast0.25ms,
200 fps (5-ms video frame, 2.5 ms per video field). Fig. coronpsoisdseirbeldytloesbs;esdoueadtdoitaifotnearlglloiwghtofdettheecdteMdCPby.tThheeCrCatDiocoafmtehreatwotaasl
1B shows examples ofQEDs recorded at a frame rate of intensityofthesecond framedividedbytheprecedingframewastaken
200 fps from a single evoked response. As at 30 fps, they asthe proportion ofthe total luminance intensity ofthe first frameas-
appeared as chevrons, so their lifetime must be less than signableasafterglow in thesecond frame.
2.5 ms. Moreover, fewer than 5% ofthe QEDs were seen
as partially filled chevrons, indicatingboth that the event
was less than the video frame rate and that no significant These results indicate that a further increase in video
degree of overlap occurred in which QEDs "began" in sampling rate would be needed to resolve the images of
one 1.25-ms video field and then extended into the next. individual QEDs.
wasfocusedtothe faceplateofthecamera. Higherspeed digital videoimagery wasobtained with a Kodak
Ektaprohigh-speedvideosystem(Hi-SpecProcessorandSIcamera)operatedatvariousframeratesbetween
30fpsand4000 fps. Highframeraterecordingswereperformedwithreducedframesize;e.g.. thecenter64
X256 pixel regionofthe fieldofviewat4000fps. The Kodak SI cameraand videoprocessorcombination
wasselectedasthedetectorofchoicebehind the Hamamatsu dMCP, foritsflexibility in videoframerates,
thelinearperformancecharacteristicsofthechargecoupleddevice(CCD)usedinthiscamera,andanadded
advantage that every image recorded by the CCD was a single, non-interlaced frame. Video imagery was
recorded in S-VHS formaton 3M ST 120 MasterBroadcast Videocassettes(3MCorp., Minneapolis, MN).
Hard-copy video prints were produced with a Kodak SV 6510 color video printer(Eastman Kodak, Inc.,
Rochester. NY).
23
296 R B. SILVER ET AL.
4
1
Figure3. Arecordingolthetimecourseofthecalcium-dependentphotonemissionbyaequorin.Columns
I through 4 show images from single sequential 0.25-ms video frames from the 4000-fps video record.
Column shows an image obtained before the injection ofaequorin. Row "d" shows the microinjection
pipette usingdifferentia] interference contrast (DIC) optics. Row "p" showsthe linearintensity profile for
each individual video framecorrespondingtothe DIC images in row "d"(above)and row"q"(below).
TIME-RESOLVED IMAGING OF CA2+ MICRODOMAINS AND QEDS 297
QED images at 4000 videoframespersecond When aequorin was injected into Ca2+-containing
buffer, time-resolved Ca2+-dependent aequorin lumines-
At4000 fps, individualQEDsfromwithinastimulated cencecould bedetectedwith thedMCP-SIcamerasystem
preterminal were observed and recorded as sets ofsolid operatedat4000 fps(Figs 1Dand 3). Priortotheinjection,
spots whose luminance intensity increased from back- the region around the tip of the micropipette appeared
ground to maximum in the first frame and decreased in black; 0.75 ms after injection, bright spots of light ap-
intensity with subsequent frames (Fig. 1C and D). Each peared. Following the initial and rapid rise in lumines-
QEDrecordwaspreceded byadark region (nolight). The cence, the signal diminished in intensity to background
first frame ofeach QED showed a dramatic increase in over a period offouror five 0.25-ms video frames. In six
luminance intensity, which resembled asymmetricdome trials, 0.75 ms elapsed between the initial mixing ofae-
of light distributed over an area less than 0.5 /urn in di- quorin andCa2+ bufferand theonsetofphoton emission.
ameter. Subsequent 0.25-ms video frames showed sym- Thus, the intensity ofCa2+-dependent aequorin lumines-
metrical domes with the same center as the first dome, cence was adequate to elicit a readily discernible signal
butwith lessoverall intensity. Each subsequent corrected from the dMCP-SI camera.
frame decreased in intensity by a factor that seemed to
follow an exponential decay curve (Fig. 2 and Table I). Shot noise
Shot noise was observed in recordings performed at all
Time-resolved imaging ofaequorin luminescence frame rates used in this study. In observations performed
in vitro at 30 fps to 200 fps, shot noise appeared asachevron. At
Anyanalysis ofthe QED imageswould require thatwe 4000 fps, shot noise appeared as a bright spot lasting for
determine the time course forthe aequorin reaction itself one video frame (Fig. ID). The intensity ofthese spots
underthe experimental conditions used. Published values was typically less than 5% of the initial frame for a
forthelagtimebetweentheaddition ofCa2+ toasolution real QED.
of aequorin in a test tube and the emission of photons
range between 2 and 10 ms(20, 21). That range isclearly Corrections of4000fps imagesforafterglow offaceplate
phosphors
too imprecise, so we sought a direct measurement using
4000-fps video, the imaging system being used for the We applied arithmetic correctionsto the rawQED im-
experiments. agestocompensate forthe phosphorescent afterglow(22,
Row"q"showsthespotsoflightrecordedasQEDs. Panel Idcorrespondstothesametimeperiodasshown
inpanels 1pand 1q;similarly2d,3d.and4dcorrespondto2pand2q,3pand3q,and4pand4q,respectively.
PanelOdshowsa 16-frameaverageoftheoil-andaequonn-filledmicropipettepriortoinjectionofaequorin
intothecalciumbuffer. PanelsOpandOq showthecorrespondingintensityprofileandQEDrecord;i.e.. an
absenceofaequorin-mediatedphotonemission.Thetipofthemicropipetteisnotedwithahorizontal, left-
pointingarrowheadinthispaneland panel4d.Column 1 showsthefirstvideoframefollowinginjectionof
aequorin intothecalcium buffer. The micropipette in Id isseen tobeempty oftheoil used toprotect the
aequorinfromthecalciumbufferpriortotheinjection.Theoildropletformedatthetipofthemicropipette
isseenasasphereatthemicropipettetip.Subsequentcolumnsshowthetimecourseoftheaequorinreaction,
totheemission ofthe first QED(column 4).
Separately, panels A through F show an image integration ofthe first millisecond ofQED emissions
detectedafterinjectionofaequorin intocalciumbufferforeachofsixseparateexperiments. TheQEDseen
inpanelCcorrespondstotheQEDseeninpanel4d. PanelGshowsanimageintegrationofalloftheQEDs
detected in panels A through F. Note the high degree ofspatial clusteringofthese initial QEDs recorded
duringone millisecond aboutthetipofthepipette.
To determine the lag time between the mixing ofaequorin and Ca2+, aequorin solutions were injected
into Ca2+ buffers as previously described (3), and the reaction sequence was observed at 4000fps. The
methodofinjectionofaequorinintotheCa2*bufferwasessentiallyidenticaltoquantitativemicroinjections
routinelyperformed(3, 19).Fortheseexperimentsamulti-spectral videomicroscopewasused(e.g.. 16. 17;
developedby R. B. S.).Thetimeofmixingwasconsideredtohavebegunwhenthetrailingmeniscusofthe
firstoildropletwasexpressedfromthetipofthepipette.Timingoftheinjectionwascoordinatedwithclock
timeofthevideosystem.ToassistinQEDfeatureextractionandquantification,videoimageswereprocessed,
filtered,andanalyzedwith an Imagel-AT(Universal ImagingCorp., WestChester, PA) runningon a Dell
325 AT-bus computer, with macro programs written by one of us (R. B. S.). Ca2+,-dependent aequonn
luminescenceimagesofQEDs,seenasdiscretespotsonadarkbackground, weredigitizedandanalyzedfor
each sequential video frame. The phosphorescent afterglow ofthe cameras and dMCPcombinations con-
tributes to the QED imagery (22, 23). The afterglow is due to the prolonged emission ofenergy from
phosphors at the detector faceplate and is common to all phosphor-based image detectors. The afterglow
and thecontribution ofshot noise tothe imagery dataareconsidered in thetextand in Figure 2.
298 R. B. SILVER ET AL
Table I
Meanluminancevaluesanddecayfunctionattributesforindividualevokedandspontaneous QEDsfromsynapticpreterminalsandforaequorin
QEDsproducedin vitro
TIME-RESOLVED IMAGING OF CA2+ MICRODOMA1NS AND QEDS 299
X 10 \ This number is very small, but it represents a 4. Llinas, R., M. Sugimori, and R. B. Silver. 1991. Imaging preter-
realistic estimate of the state of the art of optical com- minal calcium concentration microdomainsin thesquidgiantsyn-
ponents available forimaging single photon and photon- 5. aLplsien.asB,ioRl..,BMu.llSu1g8i1m:or3i1.6-a3n1d7.R. B. Silver. 1993. Presynapticcal-
limited events. ciumconcentrationmicrodomainsandtransmitterrelease./Physio/.
When extending observations to high-speed video, the (Paris)86: 135-138.
operational relationship between available light, detector 6. Llinas, R., M. Sugimori, and R. B. Silver. 1994. Localization of
esernesdi.tiWviittyh,inantdhefrspaemcetrraalteseonfsitthievictaymbearnadmoufstthebedectoencstiodr-, gcoiafalNncetiuusrmyontcaropansncese.mniPttprt.aetr1io3Rn3e-lm1eia3cs7re.oindLoM.moaSltiejncasurlnaeat,rtPha.enGdacrCteieevlnelguzalorandre,moeSf.cthGhraeinlsilqnsuemirsd,
observed image intensity is dependent upon the number J. Hbkfeltand D. Ottoson, eds. Raven Press, New York.
of photons interacting with the detector in a sampling 7. Llinas, R. R., 1. Z.Steinberg, and K. Walton. 1981. Relationship
period. This isaconsideration long known to those using between presynaptic calcium current and postsynaptic action po-
high-speed photographic emulsions at low light levels 8. tCehnatdia,lJi.nEs.,quainddgRi.anEtkesrytn.ap1s9e8.4.BiopChaylsc.iJu.m3d3o:m3a2i3n-s35a1ss.ociatedwith
for example, in photomicrography of fluorescent speci- individualchannelscanaccount foranomalousvoltagerelationsof
mens. In practice, the faster the frame rate, the less light Ca-dependent responses. Biophys. J 45:993-999.
available per image frame: as the frame rate doubles, the 9. Simon,S.M.,M.Sugimori,andR.R.Llinas. 1984. Modelingthe
light available foran image is halved. At 200 fps the light submembranouscalcium-concentration changesand their relation
torateofpresynaptictransmitterreleaseinthesquidgiantsynapse.
available per frame is roughly 7~' of that available at Biophys. J 45: p264a.
30 fps: at 4000 fps the light available per video frame is 10. Simon, S. M., and R. R. Llinas. 1985. Compartmentalization of
132~' ofthat available at 30 fps. At 4000 fps we are, at the submembrane calcium activity during calcium influx and its
best, abletodetect only 1.7 X 10~5 photonsemitted from significance in transmitter release. Biophys. J 48: 485-498.
theThsipsecpiampeenr irnepaorstisngtlieme0-.r2e5s-omlsvevdidiemoagfersamoef.Ca2+ entry 1121.. FZfourgcoekmlesrvoan,,riRAo..usSL.a,,rraaannydsdRo.Af.Ss.iLnZ.gulecFkoceghrea.lnns1eo9ln8s.5..1B9i8oP6prh.eyssy.nRaJep.ltia4ct8:icoan1ls0ch0ii3up-m1db0ie0f7tf.wuseieonn
to the synaptic preterminal for evoked and spontaneous transmitter release and presynaptic calcium influx when calcium
eventswithin microdomains. From the measurements of entersthrough discrete channels. Proc. Nail. Acad. Sci. U.S.A. 83:
3032-3036.
thereaction lagtimeandduration oftheaequorin reaction 13. Llinas,R.,L.Z.Steinberg,and K.Walton. 1981. Presynapticcal-
in vitro, italsoappearsthat high-speed video isan effective ciumcurrents in squidgiantsynapse. Biophys. J. 33: 289-321.
tool for the study ofmolecular reactions in solution. The 14. Pumplin, D. W., T. S. Reese, and R. Llinas. 1981. Are the pre-
implications ofthese observations for synaptic physiology synapticmembraneparticlesthecalciumchannels?Proc.Natl.Acad.
are discussed in the accompanying paper (18). 15. SPcuim.plL'i.nS,.AD..7W8.:.72a1n0d-7T.21S3..Reese. 1978. Membraneultrastructure
ofthegiantsynapseofthesquidLoligopealei.Neuroscience3:685-
696.
Acknowledgments
16. Silver,R.B. 1994. Videolightmicroscopicimagingofthecalcium
We thank Drs. O. Shimomura, S. Inouye. B. Musicki, signal that initiates nuclear envelope breakdown in sand dollar
(Echinaracniusparma)cells. Biol. Bull. 187: 235-237.
and Y. Kishi forthegenerousgiftsofthecustom aequorin 17. Silver, R. B., A. P. Reeves, M. Whitman, and B. Kelley.
preparations used in thisstudy. Thanks are alsoextended 1994. Analysisofspatial andtemporal patternsintheCa2+signal
to Mr. Paul W. DelGrego (Motion System Analysis Di- that signals nuclear envelope breakdown in sand dollar (Echinar-
vision, Eastman Kodak Co.) for his assistance with the acniusparma)cells. Biol. Bull. 187: 237-238.
digital high-speed video observations, and to Ms. Becky 18. rSeusgoilmuotriio,nmM.e,asEu.rJe.mLeanntg,ofRt.heB.tiSimlevecro,urasnedoRf.cLallicniausm.-c1o9n9c4e.ntrHaitgihon-
Hohman and Mr. Phil Presley (Carl Zeiss, Inc.) for in- microdomainsatsquid presynapticterminals. Biol. Bull. 187: 300-
formation regarding the characteristics of the objective 303.
lens used for observations of photon emissions in vivo. 19. Silver, R.B. 1989. Nuclearenvelopebreakdownandmitosisinsand
Thiswork supported bythe N. I. H. (NS 13742 to R. L.), cdoalllcairume-mrebvreyrossibliseifnahsihbiitoendabnydmbicyroainntjaegcotniiosntsofofcailnctiruacmelbluuflfaerrsCain2+a-
the U. S. Air Force (F49620-92-J-0363 to R. L.), and the channels. Develop. Biol 131: 11-26.
N. S. F. (DCB-9005343 and DCB-9308024 to R. B. S.). 20. Shimomura, O., B. Musicki. and V. Kishi. 1989. Semi-synthetic
aequorinswith improved sensitivity toCa2+ ions. Biochem. J. 261:
913-920.
Literature Cited 21. Shimomura, O., S. Inouye, B. Musicki, and Y. Kishi. 1990.
Recombmantaequonnandsemi-syntheticrecombinantaequorins.
1. Katz, B. 1969. TheReleaseofNeurotransmitterSubstances. The Biochem J 270: 309-312.
Sherrington LecturesX. Thomas. Springfield. IL. 22. Inoue,S. 1986. VideoMicroscopy. Plenum Press. New York. Pp.
2. I INI.is. R.R. 1977. Pp. 139-160inApproachesIKIlieCellBiology 149-262.
ofNeurons, Vol. 2. C. W. M. Ferendelli and J. A. Ferendelli. eds. 23. Wayne, R. P. 1988. PrinciplesandApplicationsofPhotochemistry.
Soc. forNeurosciences. Bethesda. MD. Oxford Science Publications, Oxford University Press, Oxford. Pp.
3. Llinas, R., M. Sugimori, and R. B. Silver. 1992. Microdomains 66-84.
ofhigh calcium concentration in a presynaptic terminal. Science 24. Hamamatsu PhotonicsK. K.,ElectronTubeCenter. 1991. Image
256: 677-679. Intensifies Shizuoka-ken, Japan.