From the Department of Applied Physics and Section of Neurobiology and Behavior, Division ofBiology, Cornell University, Ithaca, New York
ABSTRACT
The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuro-muscular junction was determined quantitatively by electron microscope radioautographyafter incubation of muscle fragments in DFP'H . Most of the sensitive sites were located inthe subneural apparatus at a concentration of 90,000 sites per a' of cleft tissue or 12,000sites per µ2 of postjunctional membrane surface area . A considerable concentration is alsopresent in the teloglial cap . It has previously been demonstrated (Rogers et al ., 1966) thatone-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)-a compound which selectively reactivates phosphorylatedacetylcholinesterase . In the present study, it was found that this ratio of 1 :2 holds also on afine-structural level . Muscle mast cells were found to have a heavy concentration of boundDFP.
The use of radioactive diisopropylfluorophosphate(DFP) to label tissue acetylcholinesterase (AChE)was first suggested by Ostrowsky and Barnard(1961) . Rogers et al . (1966, 1969) improved andstandardized these procedures (see also review byBarnard and Rogers, 1967) . DFP, one of theorganophosphate nerve gases, is a potent in-hibitor of cholinesterases as well as of othercarboxylic acid esterases, and acts by phosphorylat-ing the enzyme sites (for review, see Dixon andWebb, 1964) . When muscle fragments are incu-bated in radioactive DFP, the distribution of tissueesterases is reflected by the distribution of tissueradioactivity and can be studied quantitatively .
A similar procedure was used by Waser and Relier(1965) .
Rogers et al . (1966, 1969) manipulated thesystem in order to label acetylcholinesterase(AChE) separately from the other DFP-reactivesites. They accomplished this by combining the useof radioactive DFP with that of nonradioactiveDFP and with the oxime, pyridine-2-aldoximemethiodide (2-PAM), which selectively reactivatesphosphorylated AChE under their experimentalconditions (Wilson, Ginsburg, and Quan, 1958 ;Wilson, 1966) . Reversible enzyme inhibitors (suchas eserine) were also used to protect different sitesfrom radioactive DFP . They concluded that ap-
proximately one-third of the DFP-sensitive sitesat the endplate was AChE and that the remainingtwo-thirds were 2-PAM-resistant sites . Less than10% of the total DFP-phosphorylated sites repre-sented serum cholinesterase (ChE) .
In an earlier paper, I analyzed the distributionin the endplate of the 2-PAM-reactivated siteson a fine-structural level (Salpeter, 1967) . In thepresent study, a similar analysis is performed forthe DFP-phosphorylated sites which are noteasily reactivated by 2-PAM, and the distributionsof the two are compared .
MATERIALS AND METHODS
LABELING OF TISSUE : Mouse sternomastoidmuscle was used. The tissue was labeled by the pro-cedures standardized by Rogers and Barnards (Rog-ers et al ., 1966, 1969 ; Barnard and Rogers, 1967) .Two different labeling regimes were employed .Regime 1 was designed to phosphorylate only AChEwith DFP- 3H. The procedure was as described in aprevious publication (Salpeter, 1967) . Small piecesof muscle were first fixed in glutaraldehyde (1 .5% in0.06 M phosphate buffer at pH 7 .4) for 2 hr and thenthoroughly rinsed in buffer . Glutaraldehyde fixationdecreases the uptake of DFP at motor endplates by, atmost, about 10% (Rogers et al ., 1966). The musclewas then incubated in nonradioactive DFP (10 -3 M
in phosphate buffer pH 7 .4) at room temperature for20 min to phosphorylate all DFP-sensitive sites, andthen washed several times in buffer for a total of 20min. It was then incubated in 2-PAM (10 -3 M) atroom temperature for 40 min to reactivate the AChE,washed in cold buffer, and left in buffer overnight .Radioactive DFP was then introduced into the reac-tivated sites by incubating the muscle in DFP- 3H(10-4 M at 2 .56 or 4.32 c/mmole) for 30 min at roomtemperature. The tissue was finally rinsed first inbuffer and then in nonradioactive DFP (10 min in10-3 M followed by 1 hr in 10-4 M), and then over-night in buffer . This sequence (nonradioactive DFP-2-PAM-DFP-3H) introduces radioactivity only intoDFP-sensitive sites that had been reactivated by2-PAM. The final washes in nonradioactive DFP andbuffer minimize nonspecific radioactivity looselybound to the tissue. The muscle was then postfixed inOSO4, stained in uranyl nitrate, and embedded inEpon 812 .
Regime 2 was designed to phosphorylate only 2-
i The labeled muscle material used was kindly pre-pared by Dr. A. W. Rogers at the Molecular Enzy-mology Unit, Department of Biochemical Pharma-cology, State University of New York at Buffalo in aprogram of A. W. Rogers and E. A . Barnard sup-ported by NIH GM 11754.
PAM-insensitive sites with DFP- 3H. The tissue wasfixed in glutaraldehyde as in regime 1 and then incu-bated in DFP- 3H (10-4 M at 4.32 c/mmole) for 30min so as to introduce radioactivity into all the reac-tive sites . A washing sequence in nonradioactiveDFP and buffer followed as in the last step of regime1 . The muscle was then incubated in 2-PAM (10 -3 M)
at room temperature for 40 min to reactivate theAChE and washed in buffer overnight . This se-quence (DFP 3H-2-PAM) thus leaves radioactivityonly in those DFP-sensitive sites not reactivated by2-PAM. The specificity and reliability of these pro-cedures have been demonstrated by Rogers et al .(1966, 1969) and Barnard and Rogers (1967) . SinceDFP phosphorylates enzyme sites stoichiometricallyin a 1 to 1 ratio, one can equate the number of boundDFP molecules with active enzyme sites. DFP com-bines also with nonenzymatic sites, but at a con-siderably slower rate .
RadioautographySPECIMEN PREPARATION : Specimens
wereprepared for EM radioautography by the procedureof Salpeter and Bachmann (1964, 1965 ; see alsoSalpeter, 1966) . Conditions for quantitative measure-ments were strictly applied (Bachmann and Salpeter,1965, 1967). Sections having silver and pale gold in-terference colors were placed on collodion-coatedslides, and their thickness was then measured with aninterferometer (Reichert Instruments, W . Caldwell,N. J .) . We found that in each case pale gold sectionsmeasure on the average 1,000 A and silver sectionsabout 800 A, with all measurements deviating fromthe mean by less than 10%. This confirms the findingsof Peachey (1958) and Bachmann and Sitte (1958) .The sections were then stained with 1 0% uranyl ace-tate for 3 hr and vacuum coated with carbon . Thegold sections were coated with a monolayer of IlfordL4 (judged by purple interference color) while thesilver sections were coated with a monolayer of KodakNTE (judged by pale gold interference color) . Thecorrelation between emulsion thickness and inter-ference color is given in previous publications(Salpeter and Bachmann, 1964, 1966). After 15-27wk of exposure, the Ilford L4-coated slides weredeveloped with Microdol X (Eastman Kodak Co.,Rochester, N . Y.) for 3 min at 24 °C and the KodakNTE slides were developed with the gold latensifica-tion-Elon ascorbic acid method, also at 24 °C. Thespecimens were stripped off the slides, picked up oncopper electron microscope grids, and examined withan RCA EMU3 electron microscope .ANALYSIS OF EM RADIOAUTOGRAPHS : Ran-
dom pictures of muscle were first taken for obtain-ing a value for the extent of muscle background(i .e . developed grains per unit area of muscle) . Graindensities associated with the different regions of the
M. M. SALPETER Radioautography as Quantitative Tool . II
endplate were then obtained . Three regions were con-sidered : the nerve endbulb, the junctional fold zone,and the teloglial cap-a region of connective tissueand Schwann cell cytoplasm overlying the nerve bulb(Couteaux, 1958) . Grids were scanned completely,and all endplates were photographed, whether or notthere were developed grains associated with them . Allradioautographs were magnified to 30,000 . The loca-tion of each developed grain was defined by its centerwhich was determined as follows : a small plastic maskon which were drawn differently sized circles con-centric around a common perforated center was laidover the grain, and the grain was fitted into thesmallest circle which fully circumscribed it . Thecenter was then punched with a dissecting needle ontothe print .
So as to provide an easy means for tabulating dataas grain density (i .e ., grains per unit area), the areaoccupied by the different regions of the endplate wasdetermined as follows : a board containing a grid ofequally spaced nails was fitted exactly over eachradioautograph, and the nails were punched throughthe print . The resultant lattice of points, which arecompletely unbiased (random) with respect to anytissue components, were then tabulated in the samemanner as were the grains . Grain density was ex-pressed either as grains per point or, since the averagenumber of points per square micron of radioauto-graph was easily calculated and the thickness of thesections known, as grains per µ 3. Under the conditionsof this study, the number of developed grains wasfound to increase linearly with exposure time . Thenumber of developed grains was, therefore, converti-ble to the number of radioactive decays in the tissueper unit time of exposure (using sensitivity valuespreviously established-Bachmann and Salpeter,1967) . Finally, since the specific activity of the DFP-3 H was known (giving the number of decays per moleof DFP per unit of time), and since one DFP moleculephosphorylates one active enzyme site, an easy calcu-lation further converted grain density to molecules ofDFP or phosphorylated enzyme per cubic micron oftissue.
One cannot, however, convert grain density toradioactive decays in the tissue without consideringthe scattered grains . Because of the spread of radiationfrom a radioactive source, developed grains do notlie immediately above a source in the tissue, butdistribute themselves in some statistical manneraround the source . Therefore, the developed grainsimmediately over a structure represent only a fractionof the developed grains which are due to radioactivedecays within that structure . The extent to which thiswould effect the accuracy of any conversion fromgrain density to radioactive decays depends on,among other things, the size of the radioactive area .The relative number of total grains that lie outside aradioactive structure decreases as the size of the struc-
124 THE JOURNAL OF CELL BIOLOGY • VOLUME -f2, 1969
ture increases . (Salpeter et al ., 1969 have shown that,under the average conditions of resolution used in thisstudy, 2 less than 30% of the developed grains wouldlie outside a radioactive structure that is approxi-mately 1 s in radius, whereas more than 75°J% wouldlie outside this structure were it 0 .1 .s in radius .) Thusfor small radioactive structures, the radiation spreadmust be considered before a conversion from graindensity to radioactive decays is valid .
It was, therefore, first essential to define the radio-active structures as accurately as possible . This wasdone by plotting grain density distributions arounddifferent components of the endplate and determiningthe peaks and general shapes of these distributions.In the postjunctional region, grain density distribu-tions were obtained in relation to both axonal andpostjunctional membranes . This was done as follows :the perpendicular distance from every developedgrain center and from every lattice point was meas-ured first to the axonal membrane and then to thepostjunctional membrane . A grain distribution histo-gram (total grains per unit distance from the mem-brane) was plotted separately for these twomembranes. The distance had a negative sign if thegrain or point lay on the axonal side of the particularmembrane and a positive sign if it lay on the muscleside of the membrane . Similar histograms were alsoplotted for the lattice points . When the number ofgrains in each histogram column was divided by thenumber of points in the same histogram column, anaccurate correction for area was made and the graindistributions were converted to a grain density dis-tribution.
All distances were measured in units of "HD"-ameasure of resolution . 2 When plotted in distance unitsof HD, grain density distributions have a universalshape . This allows data from different specimens to becombined (Salpeter et al., 1969) .
RESULTS
A typical section through an endplate is illustratedin Fig. 1, and typical radioautographs in Figs . 2and 3. The nerve endbulb sits in a trough at the
2 In a recent study, Salpeter et al . (1969) have meas-ured resolution experimentally for a variety of EMradioautographic specimens differing in section thick-ness and emulsion and developer combinations . Theyfound that for each specimen there is a value for"half-distance" (HD) which is characteristic of itsresolution . HD is the distance from a line source insuch a specimen within which 50/0 of the developedgrains would fall. The HD values for the two specimenconditions used in this study are 1200 A (for the silversection, Kodak NTE emulsion, Elon ascorbic aciddevelopment) and 1600 A (for the pale gold section,Ilford L4 emulsion, and Microdol X development) .
FIGURE 1 Typical electron micrograph of a motor endplate in this material . Nerve terminal (N) sits intrough formed by the invagination and infolding of postjunctional muscle membrane (jf) . The teloglialcap consists of tongues of Schwann cytoplasm (Sch) and connective tissue (CT) . The Schwann cell doesnot make a close seal over the nerve (see also Fig . 2) . The clefts (c) separate axon from junctional folds .Dense granules (arrows) are characteristically seen on the muscle side of the endplate . In radioautographsof this material, such granules were never selectively associated with developed grains . X 80,000 .
surface of the muscle fiber . The plasma membrane AChE) between postjunctional region and teloglialof the muscle is thrown into characteristic junc- cap was - 85 : 10. A similar distribution was seentional folds . Dense-core vesicles are seen amongthese folds . Overlying the nerve bulb is the telo-glial cap consisting of loosely interwoven Schwanncytoplasm and bands of connective tissue . TheSchwann cytoplasm does not seal the nerve from theextracellular space-separations of several thousandAngstroms between nerve endbulb and Schwanncell are common, as are large gaps in the Schwanncell cover . In this study the teloglial cap wasarbitrarily defined as the connective tissue andSchwann cells within a zone 4HD (6400 A) wideoverlying the nerve endbulb .
Location of Radioactivity
In the first paper of this series (Salpeter, 1967),the relative radioactivity (representing labeled
in the present study with both labeling regimes .Preliminary counts indicate that in the teloglialcap there was a peak grain density on the Schwannplasma membrane, with less over the connectivetissue, and possibly only scattered grains overSchwann cytoplasm . The total number of grainsin the cap was too low, however, (66 grains) tomake a statistically valid comparison of radio-activity in connective tissue versus Schwanncytoplasm . The cap was, therefore, treated as aunit.
In the postjunctional region, however, thegrain yield was much higher (848 grains) and amore detailed analysis was possible . Figs . 4 and 5are grain density distributions with respect to theaxonal plasma membrane, obtained from radio-
M. M . SALPETER Radioautography as Quantitative Tool . II 125
FIGURE 3 Radioautograph of tissue treated as in Fig. 2 . Silver section coated with Kodak NTE, goldlatensification-Elon ascorbic acid developed . Note developed grains (arrows) mainly over junctionalfolds . X 45,000.
autographs after the two DFP-labeling regimes .It is clear from these histograms that the bulk of theradioactivity after both labeling regimes lies tothe muscle side of the axonal membrane in aband corresponding to the junctional fold zone .
When tabulated in relation to the postjunctionalmembrane, the location of the radioactivitybecomes much better defined (illustrated inFigs . 6-8) . There is a distinct peak of graindensity over the postjunctional membrane afterboth labeling regimes . The theoretical distributionssuperimposed on the experimental histograms arefrom Salpeter et al . (1969) . They represent theexpected grain density distribution around a linesource with a slight curvature equal to the typicalcurvature of the nerve endbulb. Since the func-tional folds have a somewhat more complicatedgeometry than would be given by a straight linesource, the theoretical curve is only an approxi-mation. It fits the experimental distributionsremarkably well, however. Owing to the limits set
by the resolution of the EM radioautographictechnique, the data are compatible with eitherof two hypotheses : (1) that the radioactivity isassociated with the junctional membranes, or (2)that it is associated with the postjunctional clefts(a band of -700 A) .
Enzyme Concentrations
Grain density was converted to density of boundDFP 3H molecules (i .e ., active sites) in the tissue(Table I) . Because of the greater stability ofIlford L4 with prolonged exposures (Bachmannand Salpeter, 1967), only radioautographs fromtissue coated with Ilford L4 and developed withMicrodol X were used for this conversion. Underthe conditions of this experiment, one developedgrain represented, on the average, 10 radioactivedecays (sensitivity value from Bachmann andSalpeter, 1967) . The specific activity of the DFPgave the relationship between radioactive decaysand molecules of DFP and, thus, active sites .
M. M . SALPETER Radioautography as Quantitative Tool . II 127
FIGURE 4 Grain density (i .e ., grains/points) with distance either side of axonal membrane (axonal mem-brane at 0) . Labeling sequence was DFP-2-PAM-DFP- 3 H, which introduces radioactivity selectively into2-PAM-reactivated sites, i .e ., AChE. On the left, the histogram goes into the nerve ; on the right, into themuscle. Distance was measured in microns and then divided by the HD value characteristic for the givenspecimen . (Tabulating distance in units of HD allowed data from the two specimen conditions, i .e . IlfordL4 coated and Kodak NTE coated, to be combined [Salpeter et al . 1969] .) Density was normalized to 1 .0at the origin . The grain density peaks over a broad zone which is coincident with the junctional folds .(Based on 272 grains .)
1 .4 -
L
DFP-2 PA M-DFP3H
axonal membrane
IIIIIIIIIIIIII6.5
5.2
3.9 2 .6
1 .3
0
1 .3 2.6
3.9
5.2
6.5
7.8 9.1
10.4 11 .7
Distance (in units of HD)
FIGURE 5 Histogram obtained as in Fig . 4, except for the labeling sequence DFP- 3H-2-PAM, whichintroduces radioactivity selectively into 2-PAM-nonreactivated sites . The general distributions in Figs .4 and 5 are similar. (Based on 437 grains .)
In the postjunctional region, the grain densitydistributions (Figs. 6-8) indicated that the radio-activity was confined to a relatively narrow bandand, as discussed above, there was, therefore, aneed to make a correction for the spread of radia-tion. From the density histograms, the radioac-tivity in the postjunctional region could be related
FIGURE 6 Histogram of grain density(grains/points) with distance eitherside of postjunctional membrane forthe labeling sequence DFP-2-PAM-DFP- 3H (i.e ., 2-PAM-reactivatedsites) . Left side of histogram goes to-wards the nerve, right side towardsmuscle. Distance was measured inunits of HD as in Fig. 4, and the datafrom Ilford IA and Kodak NTE spec-imen were combined . Grain densitywas normalized to 1 .0 at origin . Thesmooth curve superimposed on thehistogram represents the expected dis-tribution, if the radioactivity wasconfined to a line (or thin band) fol-lowing the postjunctional membrane .(A correction for the slight curvaturegiven by the shape of the muscletrough produces the left-to-rightasymmetry.) The theoretical distri-bution is from Salpeter et al . (1969) .(Histogram is based on 307 grains) .
DFP3H-2PAMpostjunctionalmembrane
7.8 6 .5 5.2 3 .9 2 .6
1 .3
0
1 .3 2.6 3 .9 5 .2 6 .5Distance (in units of HD)
FIGURE 7 Histogram and superimposed curve obtained as for Fig . 6 except for 2-PAM-nonreactivatedsites . (Based on 541 grains .)
either to the junctional membranes or to the
-700A cleft adjacent to them . Enzyme concen-trations were calculated for both of these possi-bilities, in each case correcting for scattereddeveloped grains. This correction was done byconsidering all the grains which fitted the expecteddensity distribution (Figs. 6 and 7) as belonging
M. M . SALPETER Radioautography as Quantitative Tool . II 129
FIGURE 8 Grain density around postjunctional membrane for total DFP-sensitive sites obtained byadding data (grains and points) from both labeling sequences (DFP- 3H-2-PAM and DFP-2-PAM-DFP-3H). (Based on 848 grains .)
to the radioactive region . The surface area of
junctional membrane was obtained by measuringits length in all the radioautographs with a mapmeasurer and multiplying this length by thethickness of the section . The volume of the cleftwas obtained from the number of lattice pointslying over cleft tissue in the radioautographs .Values for grains per µ2 of surface membrane or
µ3 of cleft were then converted to enzyme concen-trations .
Since no precise localization of radioactivity
was accomplished for the teloglial cap, the entirevolume of the teloglial cap was used for the densitydetermination . The value given in Table I for theteloglial cap thus represents a lower limit.
Rogers et al . (1966, 1969) have shown that inthe endplate, as a whole, the two sites (2-PAMnonreactivated, and 2-PAM reactivated) appearquantitatively in the ratio of about 2 to 1 . FromTable I, it can be seen that the two sites appear inthe same ratio also on a fine-structural levelwithin the limit of resolution attained in thisstudy.
Labeling Other than at Endplate
MAST CELL : An immediate and strikingobservation when looking at radioautographs ofmuscle fragments which had been incubated in
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THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969
DFP-3 HpostlunCtional
membrane
I
i
I
I
I
I
I5.2 3.9 2 .6
1 .3
0
1.3 2.6Distance (in units of HD)
3 .9 5 .2 6 .5
II
DFP 3H is the massive labeling over the cytoplasmof mast cells lying between the muscle fibers .(The sensitivity of mast cells to DFP has earlierbeen reported by Langunoff and Benditt, 1963 .)Rogers et al. (1966) have suggested that, withunstained tissue and light microscope radioautog-raphy, images of labeled muscle mast cells couldhave been mistaken for endplates in the study by
Ostrowski and Barnard (1961 ; see also Barnard
and Ostrowski, 1964) . Fig . 9 shows a fragment ofa mast cell labeled by the regime DFP3H-2-PAM . Darzynkiewicz and Barnard (1967) haveshown that the DFP-sensitive sites in the mast cellare not reactivated by 2-PAM. This thus repre-sents all the DFP-sensitive sites . The radioauto-graphic exposure time is equal to that of the end-plate in Fig . 2. With this exposure, the emulsionover the mast cell is clearly saturated and no
quantitation was possible . Yet with similar mate-rial exposed for only 2 wk, a rough tabulation gavea value of 1 .9 X 10' DFP molecules bound percubic micron of mast cell cytoplasm . The grainsappear concentrated in the granules rather thandistributed uniformly throughout the cytoplasm(see also Budd et al ., 1967) . Thus, the value percubic micron of granule would be even higher(almost twice that value) since the granulesoccupy just over half the volume of the cytoplasm .
In a recent publication, Darzynkiewicz andBarnard (1967) give a value of 6 X 10 1 moleculesof DFP bound per peritoneal mast cell which hasa diameter 16 .1 /. A rough tabulation indicates
TABLE I
Concentration of DFP Molecules Bound at Endplate
Density X 103
Concentration of DFP molecules bound at theendplate was determined from grain density valuesafter appropriate corrections were made for scat-tered radiation . (A correction was also made for apossible 10% loss of active sites due to glutaralde-hyde fixation .) Total DFP bound per tissue com-ponent is given by the sum of columns 1 and 2 .* In the subneural zone, enzyme densities werecalculated both per µs of cleft tissue and per /2 ofpostjunctional membrane surface .t Grain density distributions (Figs . 4-8) suggestthat any developed grains over the nerve terminalare due to radiation spread from its radioactivesurroundings .§ The volume of the teloglial cap was determinedby the total number of grid points which fell overSchwann cytoplasm and collagen fibrils within4 HD (6,400 A) from the nerve endbulb . Since apreliminary analysis suggests that the radioactivityis concentrated within a smaller volume, the densityvalue given here represents a lower limit, but isprobably correct to within a factor of two .
that in these cells approximately 16 of the cellvolume is nucleus . The cells that Darzynkiewiczand Barnard studied thus had a cytoplasmicvolume of 1 .8 X 103 µ3 and a density of boundDFP molecules equal to 3.3 X 10 ;5 per /3 ofcytoplasm . This value agrees, within a factor oftwo, with the value calculated here per /3 ofmuscle mast cell cytoplasm (i .e., 1 .9 X 10 1 ) .MUSCLE : There is a certain amount of grain
density over the muscle, which we have calledmuscle background. It is about two to three timesabove off-section, or true, background, and isequivalent to about 500 DFP molecules bound perµ 3 of muscle tissue (Table I) . This quantity isapproximately the same after both labeling re-gimes (DFP 3H-2-PAM and DFP-2-PAM-DFP-3H) . (See also Salpeter, 1967 .)
There is some evidence for the localization ofesterases in muscle (Barrnett and Palade, 1959 ;and Miledi, 1964) . Preliminary histograms ofgrain density distributions within the muscleshow no concentration over either the M or the Zbands. However, there is a tendency to have aslightly higher concentration near the musclemembrane. Rogers et al . (1966, 1969) suggest thatmuch of the muscle activity represents low levelsof residual adsorption of DFP rather than phos-phorylated enzyme sites, although the latterpossibility cannot be excluded . It appears, how-ever, that whatever muscle esterases are present,their concentration is too low to be detected abovethe general background of adsorbed radioactivity,and must, therefore, be considerably less than500 sites// 3 .
DISCUSSION
DFP-sensitive sites at the endplate were deter-mined quantitatively by electron microscoperadioautography. Most of these sites (-80 0 0 )
FIGURE 9 Mast cell between muscle fibers after labeling sequence DFP-3H-2-PAM. Radioautographicconditions as for endplate in Fig . 2 . X 7,700 .
M. M . SALPETER Radioautography as Quantitative Tool. II
are within the subneural apparatus at a concen-tration of 90,000 sites per µ3 of cleft tissue or 12,000sites per µ2 of postjunctional membrane . A signifi-cant concentration is also present in the teloglialcap (at least 7,900 sites per µ3) .
Rogers et al . (1966, 1969), using DFP32P andlight microscope radioautography, demonstratedthat only about 30-40% of the DFP-sensitive sitesat the endplate can be reactivated by 2-PAM (orare sensitive to eserine) . They, therefore, concludethat only this one-third represents true AChE .The present study has shown that the 2 to 1 ratiofor the 2-PAM-nonreactive and 2-PAM-reactivesites exists in the subneural apparatus also on thefine-structural level (Table I) and thus that thegeneral distribution of these two DFP-sensitivesites is indistinguishable within the resolutionlimits set by the EM radioautographic technique(see also Fig . 8) .It is not yet clear what the 2-PAM-nonreac-
tivated sites represent . Waser and Reller (1965)claim that all the DFP-reactive sites at the end-plate are AChE . Yet Barnard and Rogers (1967)argue that the conditions of reactivation in theirprocedures exclude the possibility that ageingprevented the full reactivation of the phosphoryl-ated AChE. Furthermore, Wiescowski and Bar-nard (1967) have shown that under conditionssimilar to those of this experiment only the DFP-phosphorylated enzyme sites which are reactivatedby 2-PAM are essential for neuromuscular trans-mission . This is consistent with other data whichindicate that 2-PAM can produce completerecovery of neuromuscular transmission afterDFP poisoning (Wills et al ., 1957; Namba andHeraki, 1968) . The evidence thus suggests thatthe 2-PAM-insensitive sites are not directlynecessary for neuromuscular transmission .
The 2-PAM-nonreactive sites may representnonspecific or ali-esterases which are known to besensitive to phosphorylation by DFP (see Dixonand Webb, 1964, for review) . Yet this wouldcontradict the available histochemical studieswhich find little evidence for the presence ofeserine-insensitive esterases at the endplate(Denz, 1953 ; Zacks, 1964; Koelle and Gromadzki,1966 ; Davis and Koelle, 1967) . The histochemicalliterature is, however, not yet definitive, since thereis still some disagreement on the relative concen-trations of the different esterases at the motor end-plate . Eranko and Teravainen (1967 a, b) andTeravainen (1967), for instance, do find some
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THE JOURNAL OF CELL BIOLOGY • VOLUME 42, 1969
eserine resistant or ali-esterases at normal, as wellas at regenerating motor endplates . However,even in these studies, the nonspecific esterasesappear to represent a smaller component thando the cholinesterases . The various histochemicalresults and the DFP data still have to be reconciledin determining the nature of the 2-PAM-nonreac-tive sites . The similarity in distribution of the twotypes of DFP-sensitive sites suggests the possibil-ity that they are anatomically and (or) functionallylinked . This suggestion is strengthened by thefact that in a completely different system, the ratmegakaryocyte, there exists a similar two to oneratio between DFP-sensitive sites which are2-PAM-resistant and those which are 2-PAM-sensitive (Darzynkiewicz et al ., 1966) .
The exact localization of esterases at the end-plate is also still an open question . Light micro-scope histochemical studies had early designatedthe subneural apparatus as the primary site ofesterase activity at the endplate (see review byCouteaux 1955, 1958) . This is confirmed by thedistribution of radioactivity in the present study .One hopes that finer localization will come fromEM histochemistry which potentially has a muchhigher resolution than does EM radioautography.Unfortunately, however, problems of diffusion ofreaction product have prevented, in several studieswith EM histochemistry, the utilization of thishigher resolution and the reaching of an agree-ment on whether the enzymes are located only onthe membranes (Davis and Koelle, 1967 ; Bergmanet al., 1967) or in the clefts as well (Barrnett,1962 ; Zacks and Bloomberg, 1961 ; Lehrer andOrnstein, 1959 ; Csillik, 1965) . Recent studies haveeven suggested a postjunctional cytoplasmiclocalization for AChE (Teravainen, 1967) . Untilagreement is reached, it is not clear which ofthe values for enzyme concentration given in TableI provides the most meaningful biological in-formation .
The number of developed grains found over thenerve was small . The data are compatible with ahypothesis that all the developed grains over thenerve could be due to the spread of radiation fromits radioactive surroundings (see histograms inFigs . 4-8) . Hoskin et al . (1966) have claimed thatthere is a DFPase present in squid nerve . If aDFP-hydrolyzing enzyme is also present in mousenerve and if it remains active after glutaraldehydefixation, it could explain the relative absence ofbound radioactivity in the nerve . Yet the absence
histochemical studies which similarly do not get areaction product deposited inside the nerve bulb .
A question frequently raised is whether or notthere are any esterases on the axonal membrane .Many histochemical studies suggest that there are
(e .g . Davis and Koelle, 1967 ; Barrnett, 1962) .Although EM radioautography cannot resolveradioactivity on the axonal membrane from thaton the adjacent postjunctional membrane, thedata in Figs . 4 and 5 do provide some quantitativeinformation . The grain scatter seen in these figuresis roughly what one would expect if the radioac-tivity was distributed in a band over the junctional
fold region with an edge on or near the axonalmembrane. There is no significant increase ingrain density in the histogram column whichcontains both postjunctional membrane andaxonal membrane (i .e ., at the origin Figs . 4 and 5) .
BIBLIOGRAPHY
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Therefore, at most, the radioactivity on the axonal
membrane cannot exceed in density (sites per unitarea of membrane surface) that on the postjunc-tional membrane . Owing to the small surfacearea of the axonal membrane relative to the post-junctional membrane (1 :6), even in the extreme
case the total activity at the axonal membranecannot represent more than about 1J of theactivity in the subneural apparatus .
I wish to thank Drs. A. W. Rogers and E. A . Barnardfor giving me the DFP-labeled muscle and Mrs .Frances McHenry for technical assistance .This study was supported by United States PublicHealth Service Research grants GM 10422 from theInstitute of General Medical Sciences and a CareerDevelopment Award K3-NB-3738 from the Instituteof Neurological Diseases and Blindness .
Received for publication 2 December 1968, and in revisedform 27 January 1969 .
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