VOL. 53, 1965 ANATOMY: DOGGENWEILER AND FRENK 425

ginning to establish functional contact. In view of the fact that the only knownnerve growth factor is of protein nature and is highly specific, one may speculatethat selective establishment of synaptic contacts may be determined by specificproteins synthesized at the synaptic membrane of the receptive neuron. Followingthis line of reasoning, one can further visualize the possibility that the existence ofsubsynaptic ergastoplasm in the adult indicates a continued role of the adult neuronin adaptive adjustments of synapses, such as the formation of new contacts.Summary.-In electron micrographs of the anterior gray column of the spinal

cord of monkeys and of two juvenile chimpanzees, aggregates of ribosomes andendoplasmic reticulum, corresponding to ergastoplasm, were frequently noted to bein especially close apposition to the cell membrane in the region of proximal den-drites. This apposition did not appear to be random, but rather associated withoverlying synaptic boutons of definable characteristics, namely, large size (3-6 pi),and densely packed and segregated masses of mitochondria and of "synapticvesicles." Variation in amount of ribosomal material to be seen in subsynaptic re-lation to large boutons extended from large "Nissl bodies" to a subsynaptic cisternassociated with a small scatter of ribosomes. It is possible that the subsurfacecistern of Rosenblueth3 represents one extreme of a functional series of stages.Since ribosomal aggregates tend to avoid the cell and nuclear membranes in normalcircumstances, the possibility is suggested that the large boutons on soma or proxi-mal dendrites either possess special metabolic requirements associated with post-junctional protein synthesis, or perhaps a quantitatively greater need than otherjunctional regions on the adult motoneuron receptive surface. The possible role ofsubsynaptic ergastoplasm in the developing neuron is also discussed.

* Aided by a grant from The National Foundation.1 Bodian, D., Bull. Johns Hopkins Hosp., 114, 13-119 (1964).2 Bodian, D., and N. Taylor, Science, 139, 330-332 (1963).3 Rosenblueth, J., J. Cell Biol., 13, 405-421 (1962).4Cohen, S., these PROCEEDINGS, 46, 302-311 (1960).

STAINING PROPERTIES OF LANTHANUM ON CELL MEMBRANES*

BY C. F. DOGGENWEILERt AND S. FRENKIHARVARD MEDICAL SCHOOL, RESEARCH LABORATORY, MCLEAN HOSPITAL, AND

RESEARCH LABORATORY OF ELECTRONICS, MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Communicated by Jerome B. Wiesner, December 24, 1964

Baker' and Palay2 have suggested that Ca++ helps to stabilize the structure ofmembranes during fixation. Afzelius3 also has suggested that the replacement ofK+ by Ca++ in the permanganate salt of Luft's fixative improves membrane fixa-tion.

Recent experiments by Lettvin, Pickard, Moore, and Takata have shown thatLa+++ acts in the peripheral nervous system like a "super-Ca++."i Furthermore,La+++ has an electron scattering power high enough to produce contrast in electronmicroscope images.5The introduction of La+++ either prior to fixation (e.g., incubation in La(NO3)3)

426 ANAT'OMIY: DOGGENWEILER AND FRENK PROC. N. A. S.

Vn h;.Ag.a.4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

0.~~ ~ ~ ~ ~~

FIG. 1.-Frog retina through the optic fiber layer. The gap between the fibers is heavily stainedand in regions occluded (arrows). X 50,000.

or during fixation by using La(\1nO4)3 results in unusual tagging of several struc-tures, both intra- and extracellular. In particular, a surprising degree of density isproduced in the intercellular substance in the nervous systems of vertebrates andinvertebrates.

Methods. La(N03)3 (Fisher Scientific Co., Cat. #L-10, 1964) as a 1 per cent solu-tion made isotonic with NaCl was used as a prefixative agent and La(Mn\IO4)3 w-asused for fixation. The latter was synthesized in the following way. HydratedLa2(SO4)3 (Amend Chem. Corp., CP grade) was vacuum-dried at 300'C for 24 hir.This treatment is known to give an anhydrous product.' Twenty-five gm of theresulting salt were dissolved in a large volume of water to which was then added stoi-chiometric quantities of Ba(MnO1104)2 (Amiend Chem. Corp., CP grade) in aqueoussolution.The resulting solution was passed twice through Whatman #42 filter paper to

remove the BaSO4 precipitate, and then vacuum-evaporated at temperatures be-low 60'C, to give the 150 mnM solution of La(Mn104)3 used as a stock in this experi-meiit. This solution was kept in the cold in a dark brown bottle for periods notlonger than 1 month. The fixative was made isotonic by dilution with glass-distilled water. The pH of the lanthanum solution was adjusted to 5.8-6.2, withNaOH 0.1 N to avoid precipitation of La(OH)3.The specimens used were pieces of frog retina and sciatic nerve, as well as lobster

and crayfish walking leg nerves and crayfish nerve cord. In some cases, perfusion

VOL. 53, 1965 ANATOMY: DOGGENWEILER AND FRENK 427

brane.~~~XX12,0.Teista h peirgtcrehw ylnwt peidcty of 67

A_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Wh~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~p oW,>{

was ateped u os,^tfeunlfiaonwacrie otb mesio ncl

Some of theg spcimtcense wherseicubtedon1hwperhan cenlwthLa(eve riortomfixatidaons.Of thes,00 some ine ttelwereie in fcolgutralerhyderanenagimng ofro .toe6.25eo pnero chent;

Dehyrion waspearfored in acetone, and the sampnle waere themd inemrandite. Stions0. hese cut th a diamonne, in anL iBhUltrotomecat thick

.:#wX.;...M0 ..

nesssoatepdbout1ostforpaeqcontryfiastmiconwscpyrand othin scionesgiving inter-d

FeeIce co fromeiAaticaorv oher ectiow a icroa py Asemerns-Eliskop ib,withn obj the sperture owerf 50cuatandaccrnaingrvent of 8kws fiedoAtmehaeonssica l esheaieedpinted giutantadebydracnng. fren (15 Ptoerso625n Reoad

othrsane. 62,0.7 Thercenst atteupe ihcrenhosmei wt4eroiiy f6 t

Nasatic bmtat ion wasBradley's method7eodd bye Bauplfe wasempeoyedin

Resues ofandosucimens-nwre incasuted in LaMpy an) torincuateiod gvinLa(int

Ofolloede bym were thxed inteceld lulargpappearsdernsely stained,8thu obscupricng;

fiersypresetsiome scattperfredreionsaeofeocluindwheapeerebtmeraesmaedeincls

ference colors from gray to gold for electron wicrthnopy. A Siemens-Elmiskop lb,with ail objective aperture of 50 , at an accelerating voltage of 80 kv, was used>. Amechanically sharpened pointed filament made by C. W. French (15 Peterson Rload,Natick, M~ass.), after Bradley's method7modified by J. B. Caulfield, was employed.The double condenser aperture was 100-200 1A and the beam current kept below 10jRamp.

Results and Discussion.-In retinas fixed in La(Ai\,nO4)3 or incubated in La(NO3)3followed by KA\I1104, the intercellular gap appears densely stained,8 thus obscuringthe external dense strata of the unit membranes (Fig. 1). The gap between opticfibers presents some scattered regions of occlusion where both membranes are in closeapposition, producing a five-layered structure with an over-all width of 150 A.0

428 ANATOMY: DOGGEANWEILER AND FRENK PROC. N. A. S.

An:~~A

Y A. o o 4 'IE~~~~A

hevltie.4X 3300 The inset shows a magifictionof th are outined.X 10,000

ment mebane is no stind

The. spacAbetweena theraosvnatliecellsinlbtefncrayfish.h a ewedaetui periphraleinervesysained.in t300heryishcnrlnservhordalsmgicappnoftearsasutained(i. 3) whensub-

jectedat thiulstehiqe.otie ihfo cai erefbr hr h aeItwhseen suggesinted tatxon sand importantllionagin theatiytachment ofcel toi

ca++adbidtthe same site morerglan stronglyTheereffetoofitsi charg mayhe topifintroducegcerai shexrinageinte gapsuaebstanenwhchwnculdsexplainetheocclsionofentermembrane gas.no Incidentlyduha.efc a endecie o a

The splasma membrantesaofnerosanaelemets fiedin la(MnOr)ncafsho thericharac-

thivsfiativ is the difrafsentialsaningeofdtexeral dpenrsestratumd(in. cotrswithsub

Ithesinerna onge.tethetforme ismoehavilsmotainedn wiletheltattmert appcearstditictlltess9 denase (fig.4)WrvaenybLeve tihediferenpcteindenstydispldue tounadiffrentaldbindintofbtheaesie onestituntsmkinupethfeouteistrhatuema of the

unristimebrne,ime.,rthe lipidupolre grouscrand theasersocite noniptidnctmpvenfeturo

thifxatveishediferntaltanin o te etenaldese trtu incotrat7it

the.interaldionervTe ordmerayishToehegavil betweeneadjwhien unth latemrappeasiheaisiclystiessdens (Fig00.The inetshowsva hmag ificto ftearea otindenst sdue toa0,

twferentialbininnge aofLa+++ byhe nconstiunismagaing utheaouterstrained In this

menit membrane, ise.nthstaind.plrgop n h soitdnnii opnns

VOL. 53, 1965 ANATOMY: DOGGENWEILER AND FRENK 429

O )#f<+Ps~,~..

.4 ~ ~ Oh2 ~~~~~~~~~~~~~~~~~~~~~~~~~0.'1LFIG. 4.-Frog retina. The basal region of a rod outer segment is shown. The external coating

of the plasma membrane is stained. Note the different density of the two dense layers of theunit membrane. X 200,000. The inset is a microdensitometer tracing at the place and in thedirection shown by the arrow. The mark represents 100 A. All the material shown was fixedwith La(MnO4)3.

Clearly, either or both of these components could be involved. However, recentexperiments1121 have shown that La+++ displaces Ca++ from a monolayer of phos-phatidyl serine or phosphatidyl ethanolamine, whereas both ions are rejected froma monolayer of phosphatidyl choline. Since all three of these phospholipids arecomponents of plasma membranes, the differential staining of the outer surface ofthe membranes suggests to us that the inner cytoplasmic half of the bimolecularleaflet (making up the core of the unit membrane) might be mainly composed ofphosphatidyl choline, and the outer half might be relatively lower in its content ofthis phospholipid. If this is so, then the differential staining might be explained interms of the phospholipids alone.The radial repeat period of frog sciatic nerve myelin fixed in La(MnO4)3 is de-

creased to an average of 65 A (Fig. 2). This is due to an increase in the electronscattering of the intraperiod line, corresponding to the heavy staining of the ex-ternal dense stratum of the plasma membrane.The interlamellar matrix of retinal rod outer segments is also heavily stained by

lanthanum. Usually, the peripheral dense stratum of the unit membranes of thelamellae is obscured by the heavy staining of the matrix. Sometimes, however, itis visible because of fainter staining in this region. In addition, the space betweenthe membranes of mitochondria is frequently densely stained.

430 GENETICS: WEBBER AND DE SERRES PROC. N. A. S.

Other heavy cations fail to show the staining characteristics of La+++. For in-stance, we have used Ba(MnO4)2 as a fixative with no such effect, although it may bementioned that it reproduces with increased contrast the appearances of Ki\1nO0fixation.

The authors take pleasure in thanking Dr. J. Y. Lettvin for suggesting the use of lanthanum,Dr. William F. Pickard for synthesizing the lanthanum permanganate and for supplying us withthis reagent, Dr. J. D. Robertson for his numerous enlightening discussions with us, and Mr.Alfred Ley for the photographic work.

* This work was supported in part by the following research grants and contracts: NationalInstitutes of Health (grants B2665 and MH-04737-04); National Science Foundation (grantsGB-574 and GP-2495); a grant from the Bell Telephone Laboratories, Inc.; National Aeronauticsand Space Administration (grant NsG-496); U.S. Air Force (ASD contract AF33(615)-1747);the Joint Services Electronics Program [contract DA36-039-AMC-03200 (E)].

t Rockefeller Foundation postdoctoral fellow. On leave from Universidad Catolica de Chile.t Inter-American Guggenheim Fellow. On leave from Universidad de Chile.1 Baker, J. R., J. Histochem. Cytochem., 6, 303 (1958).2 Palay, S. L., S. M. McGee-Russell, S. Gordon, and M. Grillo, J. Cell Biol., 112, 385 (1962).3 Afzelius, B., "Chemical fixatives for electron microscopy," in The Interpretation of Ultra-

structure, Symposium of the International Society for Cell Biology (1962).4Lettvin, J. Y., W. F. Pickard, J. W. Moore, and M. Takata, Quarterly Progress Report,

Research Laboratory of Electronics, MIT, 75, 159 (1964).5 Kellemberger, E., and A. Ryter, in Modern Developments in Electron Microscopy (Academic

Press, 1964).6 Pitha, J. J., A. L. Smith, and R. Ward, J. Am. Chem. Soc., 69, 1870-1871 (1947).7 Bradley, D. E., Nature, 189, 298 (1961).8 Doggenweiler, C. F., S. Frenk, and W. F. Pickard, J. Cell Biol., 23, 25A (1964).9 Pethica, B. A., Exptl. Cell Res., Suppl. 8, 123-140 (1961).10 Robertson, J. D., in Cellular Membranes in Development (Academic Press, 1964)."Rojas, E., J. Y. Lettvin, and W. F. Pickard, manuscript in preparation.12 Rojas, E., Biochim. Biophys. Acta, in press.

INDUCTION KINETICS AND GENETIC ANALYSIS OFX-RAY-INDUCED MUTATIONS IN THE AD-3 REGION OF

NEUROSPORA CRASSA*

BY B. B. WEBBER AND F. J. DE SERRES

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY

Communicated by Alexander Hollaender, November 30, 1964

Forward-mutation experiments with a genetically marked balanced hetero-karyon of Neurospora have shown that two different classes of mutations are in-duced in the ad-3 region by X irradiation.' The firstclass consists of reparablemutants (ad-3R) that will grow as homokaryons on adenine-supplementedmedium, and the second consists of irreparable mutants (ad-3'5) that will notgrow as homokaryons either on adenine-supplemented or complete medium.2Genetic analyses by means of homology tests2' 3 indicate that the adj-3R mutantshave only the ad-3A or ad-3B locus inactivated, whereas in the ad-3IR mutants theinactivation covers other loci in the immediately adjacent regions. The most