the relationship of pinocytosis and synaptic vesicles at the frog neuromuscular junction

Bram Research, 290 (1984) 1-18 1 Elsevier

Research Reports

The Relationship of Pinocytosis and Synaptic Vesicles at the Frog Neuromuscular Junction

CHARLES K. MESHUL* and GEORGE D. PAPPAS**

University of lllinois at Chicago, Health Sciences Center, P. O. Box 6998, Chicago, 1L 60680 (U.S.A.)

(Accepted May 3rd, 1983)

Key words: synaptic vesicles - - pinocytosis - - horseradish peroxidase - - coated vesicles - - neuromuscular junction - - electron microscopy

The fate of the extracellular marker horseradish peroxidase (HRP), following intense transmitter release was studied using identified muscle fibers from the frog sartorius nerve-muscle preparation. The muscle was stimulated indirectly via its nerve at 10 Hz or K +- depolarized for 15 min. Other preparations were also stimulated or K+-depolarized for 15 rain and then rested for an additional 15 min. Endings from only identified muscle fibers were photographed with the electron microscope. It was found that in the par- adigms studied above, less than 10% of the mean number of synaptic vesicle profiles per section contained the marker. Following electrical stimulation, there was a statistically significant decrease in the mean number of synaptic vesicle profiles per section. After a 15 min rest period, the vesicle profile number had returned to the control value. At this time point, the endplate potential was but 25% of the control. K+-depolarization caused no significant change in the mean number of synaptic vesicle profiles per section. Experi- ments were also performed to rule out any direct effect of the label on the number of coated and synaptic vesicle profiles. The mean number of labeled coated vesicle profiles increased during either electrical stimulation or K+-depolarization, and then fell during the subsequent rest period. Their numbers accounted for less than 2% of the total number vesicles/section. A surprisingly high number of coated vesicle profiles (as high as 41%) contained no label. This finding is inconsistent with the exclusive role of coated vesicles associated with synaptic vesicle membrane recycling. The low level of HRP labeling of synaptic vesicles is also inconsistent with synaptic vesicles undergoing exo- and endocytosis along the presynaptic plasma membrane.

INTRODUCTION

The most c o m m o n l y accep ted hypothes i s of the

morpho log ica l basis o f quan ta l re lease of t r ansmi t t e r

at the n e u r o m u s c u l a r junc t ion involves the ves icular

re lease o f t r ansmi t t e r via exocytos is , with the subse-

quen t r e fo rma t ion of vesicles by re t r ieva l of p lasma

m e m b r a n e ( e n d o c y t o s i s ) at or nea r the p resynap t ic

ending. H e u s e r and R e e s e 13 r e p o r t e d that fo l lowing

electr ical s t imula t ion , the to ta l m e m b r a n e area , in-

c luding the synapt ic vesicle , c is ternal , coa t ed vesicle

and p lasma m e m b r a n e s tayed constant , but that

there was a shift f rom one m e m b r a n e c o m p a r t m e n t

to another . Whi le the synapt ic vesicle m e m b r a n e was

drast ically r educed by m o r e than 5 0 % , the re was a

compensa to ry increase in the o t h e r m e m b r a n e com-

pa r tmen ts , i .e. c is ternal , c o a t e d vesicle and p lasma

m e m b r a n e . A f t e r s t imula t ion and a 1 h rest per iod ,

these inves t iga tors r e p o r t e d that 50% of the synapt ic

vesicles were fil led with an ex t race l lu la r m a r k e r , this

being the a p p r o x i m a t e n u m b e r of newly f o r m e d vesi-

cles.

Ut i l iz ing the edge musc le f ibers of the frog sar tor i -

us muscle , R o s e et al. 24 w e r e able to visual ize wi th

the light and the e l ec t ron mic roscope , the ne rve ter-

minal which synapsed on iden t i f ied musc le f ibers , af-

ter r ecord ing end pla te po ten t ia l s (EPPs) . The i r find-

ings indica ted e x t r e m e var iabi l i ty in the popu la t i on

of synapt ic vesicles in iden t i f ied con t ro l endings.

They r e p o r t e d that the d e c r e m e n t of the ampl i tude of

* Current address: Department of Pharmacology and Experimental U.S.A.

** To whom correspondence should be addressed.

Therapeutics, University of Maryland, Baltimore, MD 21201,

0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

the end plate potential following tetanic stimulation cannot be correlated with changes in the population of synaptic vesicles. The number of vesicles was independent of the decrement of the amplitude of the EPP.

The purpose of this study is to clarify the relationship of pinocytotic events (exo- and endocytosis) with the release of transmitter brought about by indirect electrical stimulation or K+-depolariza - tion of the frog sartorius muscle. Horseradish peroxidase (HRP) was used as the extracellular tracer. Muscle fibers were monitored electrophysiological- ly, and their respective nerve endings were later visu- ally identified with the electron miscroscope.

We wanted to ascertain whether a substantial amount of HRP becomes internalized into membrane-bound presynaptic organelles (i.e. synaptic vesicles, coated vesicles, cisternae), following transmitter release and subsequent rest periods. We find that less than 10% of the mean number of total synaptic vesicle profiles per section contain label. The

number of coated vesicles does increase following transmitter release, but their numbers accounted for less than 2% of the mean number of total synaptic vesicle profiles per section. It was also found that a

surprisingly high number of coated vesicles did not contain label. Experiments were also carried out to rule out a direct effect of HRP on the vesicle population. It is concluded that our findings are inconsistent with the notion that transmitter release exclusively involves synaptic vesicles undergoing exocytosis and

subsequently new vesicles reforming by endocytosis of the presynaptic plasma membrane. A preliminary report of this work has appeared elsewhere21.

MATERIALS AND METHODS

Animals The experiments were conducted on the isolated

sartorius muscle from Rana pipiens. The animals were maintained in running tap water, and all experiments were carried out at room temperature. The flogs were fed crickets every other day.

Electrophysiology The frogs were pithed and the sartorius muscle ex-

posed. Care was taken to avoid damaging the edge

muscle fibers. The edge fibers could be separated by

pulling the fascia away from the muscle. This assisted in identifying the muscle fibers from which recordings were made and later identification under the electron microscope. It was shown by Rose et al. 24

that junctions are located laterally on either side of the nerve, and this helped to make recordings near the junctional area.

The nerve was drawn into a suction electrode, which was connected to a stimulator (Grass SD 9). End plate potentials or miniature end plate potentials (MEPPs) were monitored intracellularly throughout the stimulation period with a glass micropipette filled with 3 M potassium citrate. The micropipettes had resistances ranging between 10 and 40 MQ. The ref- erence electrode was a silver-silver chloride wire. The micropipette was mounted on a Leitz microma- nipulator, and the signals from the micropipette fed into a conventional preamplifier (WP-Instruments M4-A). The output of the preamplifier was delivered into a dual beam oscilloscope (Tektronix 5A20N) which recorded both resting potentials and either EPPs or MEPPs. The preparation was stimulated with square wave pulses, 0.5 ms in duration and amplitude 2.5 times threshold.

In experiments using horseradish peroxidase (HRP Type VI, Sigma Chemicals), the muscle was rested in a frog saline solution containing 10 mg/ml HRP. The frog saline contained: 115 mM NaC1, 2.7 mM KC1, 1.8 mM CaC12, 1 mg/ml dextrose, and 15 mM sodium bicarbonate. The saline solution was made flesh before each experiment from stock solu- tions (all kept at room temperature). The solution was bubbled with 95% air, 5% CO2, until the pH reached 7.4. To prevent twitching of the muscle, a flesh solution of tubocurarine (Sigma Chemicals)

was made and an aliquot added to the frog saline -HRP solution, to bring the final concentration to 3 x 10 -6 g/ml. The final volume of the solution was always 1 ml. It generally took 30-40 min to reach a stable EPP. The resting potential ranged from --85 to

--95 mV. The EPP ranged between 4 and 6 mV before the start of the stimulation. If the resting potential was not at least - -85 mV, the preparation was not used. Also, if at any time during the 15 min recording period, an EPP could not be recorded, the preparation was discarded. For the control preparation, in order to be certain that the preparation was physiologically sound, it also was stimulated once or

twice and the EPP observed. The experimental and control preparati0ns'were paired, in that one muscle from the frog was used as the experimental, the other

side as the control. During the stimulation period, the EPPs from three edge muscle fibers were monitored. After penetration of one fiber, the potentials were generally monitored for 1-2 min. The poten-

tials from the 3 fibers were monitored twice during the course of the experiments, and generally the last fibers were monitored up to 15-20 s prior to the end of the experiment. At this time the muscle was im-

mersed in fixative and 1 min later the stimulation was stopped.

Electron microscopy Following 10-20 min in fixative, the tissue con-

taining the studied junctions was cut and placed into fresh fixative. The fixative was made fresh prior to each experiment and consisted of the following: 2.5% glutaraldehyde (Tousimis Research Corp.), 3% formaldehyde (depolymerized from paraformalde- hyde), in 0.1 M sodium cacodylate buffer, pH 7.2, containing 10 mM CaC12. The tissue was kept at 4 °C for 3-5 h, and washed overnight in 0.1 M sodium cacodylate buffer, pH 7.2, at 4 °C (with several changes). The tissue was processed for histochemical localization of HRP, according to the method of Gra- ham and Karnovsky12, as modified by Adams 1. For the cobalt enhancement procedure, the tissue was incubated in a shaker at room temperature with 0.5%

COC12, in 0.1 M sodium cacodylate buffer, pH 7.2, for 10 min. The tissue was then washed for 3 × 5 min in buffer, and preincubated in 3,3'-diaminobenzidine (DAB) (1 mg/ml of buffer) for 35 min. The solution was changed to one containing DAB plus 0.03% H202, and incubated twice for 20 min (fresh DAB + H202 was added for the second change). The tissue was washed for 3 × 30 rain in buffer (4 °C), placed in cold 1% osmium tetroxide (in buffer) for 3 h, washed in several changes of distilled water, and then en bloc stained for 12 h (4 °C) with 0.5% aqueous uranyl ac-

etate. The tissue was then washed in distilled water and quickly dehydrated through a graded series of cold (4 °C) ethanols, cleared in propylene oxide, and embedded in Epon 812. The blocks were oriented in the molds for cross-sections.

Thick sections (0.25/~m) were cut and stained with toluidine blue until the identified junctions could be

located. Then thin sections were cut and picked up with a single holed(slot) grid. This was then placed on a formvar-coated aluminum holder and dried at room temperature. The sections were then stained with lead citrate for 1 min. Generally 4-6 sections were placed on a single grid.

Under the electron microscope, edge fibers could be easily identified since there was less connective

tissue along the edge where the recordings were made (due to the stretching of the fascia). Ten series of adjacent sections were cut (usually 0.25/~m be-

tween adjacent sections) and only one section per grid was photographed. The identified neuromuscular junction was photographed only at or near the region of the active zone.

Experimental procedure in the presence of HRP Two experimental protocols were used. The nerve-

muscle preparation was electrically stimulated at 10 Hz for 15 min and then fixative was added. In other experiments, the preparation was electrically stimulated at 10 Hz for 15 min, rested for 15 min, then the fixative added to the bath. The electrophysiological response of the preparation was tested at the end of the rest period. The nerve was stimulated once or twice, and the EPP monitored from an identified muscle fiber. If no EPP could be recorded, the preparation was discarded. At this point, the EPP ranged between 0.5 and 1.0 mV in amplitude. At the end of the stimulation period, the EPP ranged between 0.25 and 0.50 mV.

The preparation was also K+-depolarized, with the addition of 20 mM excess KC1 to the saline-HRP solution (no curare was added). Miniature end plate potential frequency was monitored before and during the K+-depolarization. The resting frequency ranged between less than 1/s to 3/s. During the depolarization, frequencies ranged from 200-300/s and the resting potential ranged between - -50 to - -55 mV. Fol- lowing 15 min of depolarization, the muscle was fixed and processed as described above. Also after 15 min of depolarization, some preparations were rested for an additional 15 min. In this procedure, the excess KCl-sal ine-HRP solution was discarded and quickly replaced with a fresh HRP-saline solution. Once the identified muscle fibers were pen-

etrated during the rest period, the MEPP frequency had returned to its resting frequency. The time taken

to record again was about 1.5-2.0 min after discard-

ing the high potassium solution. If the frequency did

not return close to its resting frequency, the prepara-

tion was discarded.

Experimental procedure in the absence of HRP Some preparations were electrically stimulated or

K÷-depolarized (with the usual rest periods as de-

scribed above) without HRP in the bath. This was

used to control for the possible influence of HRP on

membrane recycling as suggested by Gennaro et al. n

Statistical analysis Synaptic vesicles and coated vesicle profiles were

counted from electron micrographs after placing a

mark in each vesicle. Vesicle profiles were counted

only with a defined membrane around their perime-

ter. The mean and standard error of the mean was

determined for each experimental and control group,

and significant differences between the groups were

determined using the t-test.

RESULTS

The nerve terminal cytoplasm contains an accumu-

lation of round membrane-bound synaptic vesicles

clustered close to the prejunct ional membrane.

These vesicles range in diameter from 400 to 600 A.

Coated vesicles can occasionally be seen among the

synaptic vesicles. The mitochondria are located be-

hind the synaptic vesicles, along with microtubules,

neurofilaments and smooth endoplasmic reticulum

(SER). Occasionally SER can be seen interspersed

among the synaptic vesicles. Clear areas within the

nerve terminal cytoplasm are frequently seen in

Epon sections. The active zone can be seen as an in-

crease in cytoplasmic density along the prejunct ional

membrane. This density is located opposite a post-

junctional muscle fold, a region known to contain the

acetylcholine receptors9. Synaptic vesicles can be

seen clustering very close to the region of the active

zone.

Fig. 1 shows a series of identified junctions, which

were incubated (with curare) in the absence (a and b)

or presence (c and d) of HRP-sa l ine solution. The

amplitude of the recorded EPP ranged between 4 and

6 mV. It is apparent from the micrographs that there

is a wide variation in the number of synaptic vesicle

profiles per section. All micrographs of nerve end-

ings used in this study were taken at or near the active

zone. There was very little uptake of H RP into syn-

aptic vesicles in these control preparations. Tables I,

II, IV and V show that for the controls, there was a

mean of less than one vesicle profile labeled with the

tracer per section. The number of coated vesicles

also accounted for a very low percentage compared

to the total number of vesicle profiles per section. A

comparison of the mean number of vesicle profiles/

section was made between the control preparations

from the various experimental groups. It was found

that there were statistically fewer vesicle profiles/sec-

tion in those control preparations soaked in H R P -

saline solution in contrast to those control prepara-

tions soaked in just frog saline solution (P < 0.05).

Also, statistically fewer vesicle profiles/section were

found in those control preparations used in the K +-

depolarization experiments in contrast to those con-

trol preparations not used in K+-depolarization ex-

periments (P < 0.05). Therefore, pooling of all the

control values could not be done, and all comparisons

for the experimental t reatments must be specifically

made against their respective controls. The same ap-

Fig. 1. Electron micrographs of sections from frog sartorius neuromuscular junction. The preparation was not tetanically stimulated and served as a control. In a and b the preparation was incubated in frog saline solution, in the presence of curare, and the nerve stimulated once or twice and the end plate potential recorded. The end plate in a showed an end plate potential of 5 mV. In a the nerve terminal contains a number of round synaptic vesicles (V), smooth endoplasmic reticulum (SER), mitochondria (M), microtubules (MT), and neurofilaments (NF). The active zone (*) appears as an increase in cytoplasmic density along the prejunctional membrane, and is located opposite a post-junctional muscle fold. Synaptic vesicles are lying along the prejunctional membrane. These structural details can also be seen in b. In b there is an increase in packing of the vesicles. In b there is a coated pit near a Schwann cell finger(at arrow). Note in a and b, area apparently devoid of structure (X). These areas may have had glycogen that was lost during tissue proc- essing. Note that many of the subsequent sections in this study show such clear areas. In c and d, the preparation was incubated in frog saline solution containing 10 mg/ml HRP. After 45 min of rest, the tissue was fixed in a buffered glutaraldehyde/formaldehyde solution. In c, there is a labeled vesicle (arrowhead) and an unlabeled coated vesicle(arrow). In general, the control preparation contained very few HRP-labeled vesicles. Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-reaction product. Magnification: a = 37,800 x ; b--d = 27,000 x.

Fig. 2. Electrical stimulation in the presence of HRP. The preparation was electrically stimulated for 15 min at 10 Hz in a frog saline solution containing 10 mg/ml HRP, and then fixed in buffered glutaraldehyde/formaldehyde solution. In a and b a number of vesicles are labeled with HRP. These micrographs also show large vacuolar-like structures containing the label (arrow). In c this section shows very little uptake of HRP. Only one synaptic vesicle contains the tracer(arrow). Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-reaction product. Magnification: a, b and c = 27,000 x.

plied when considering the mean number of coated

vesicle profiles/section.

Electrically st imulated junct ions

Six frogs were used for this s tudy and recordings

were made from 3 identif ied muscle fibers from each

animal. Within the first 3-5 min following electrical

st imulation at 10 Hz, the EPP decreased from a con-

trol value of 4-6 mV to about 1 mV. This ampli tude

was mainta ined for several minutes, until near the

end of the 15 min st imulat ion period. A t this point ,

the final ampl i tude of the EPP ranged between 0.25

to 0.50 mV. Only those prepara t ions were used in

which a final EPP could be recorded. It is apparent

from Fig. 2 that even though all the junctions behav-

ed physiologically, more-or- less in an identical man-

ner, the number of HRP- l abe l ed vesicles var ied

greatly. Also the extent of decrease in vesicle popula-

tion was not constant . It should be noted that in gen-

eral, the HRP- l abe l ed synaptic vesicles were ran-

domly dis t r ibuted throughout the entire terminal

area. They were not g rouped together in any partic-

ular region of the ending. The degree of mitochondri-

al swelling was variable from ending to ending. There

were many instances where the mi tochondr ia were

not swollen, and the terminal had a marked decrease

in synaptic vesicles. There was also no correlat ion be-

tween the degree of swelling and the number of

HRP- labe led synaptic vesicles. It is apparent that the

lack of mi tochondr ia l swelling cannot be used as a cri-

terion to establish whether or not that nerve terminal

was actually s t imulated.

Table I shows that following electrical s t imulat ion

there was a 37% decrease in the mean number of syn-

aptic vesicle profiles per section. This decrease was

statistically significant compared to the control val-

ue. St imulat ion also caused an increase in the mean

number of HRP- l abe l ed synaptic vesicles. This in-

crease was not statistically significant, even though

there was a statistically significant increase in the

percentage of labeled vesicle profiles. The increase

in the HRP- labe led synaptic vesicle profiles ac-

counted for less than 10% of the total number of syn-

aptic vesicles. There was also a statistically signifi-

cant increase in the mean number and percentage of

coated vesicle profiles following st imulation. The

percentage of coated vesicle profiles accounted for

just over 1.3% of the total number of vesicles. The

TABLE I

Effects of electrical stimulation~

Vesicle groups Experimental b Control b

Number of synaptic vesicles/section 44.5 + 5.1 c 70.0 + 4.9

Number of HRP-labeled vesicles/section 2.6 + 0.950 0.92 + 0.24

% of HRP-labeled vesicles 5.4 + 1.6 c 1.3 + 0.4 Number of coated

vesicles/section 0.52 + 0.08e 0.16 + 0.05 % of coated vesicles 1.31 + 0.03 e 0.25 + 0.08

a Preparation was electrically stimulated at 10 Hz for 15 rain. b Mean + S.E.M. (n = 6). c Significantly different vs controls at P < 0.01. a Not significantly different vs controls. e Significantly different vs controls at P < 0.05.

increase was similar to that repor ted by Heuser and

Reese 13. It is impor tant to point out that even though

many junctions contained a number of HRP- l abe l ed

synaptic vesicle profiles, the vast major i ty of junc-

tions contained very little HRP. It is apparent not

only from the micrographs but also from Table I that

on the average most endings, al though electrically

st imulated, took up very little of the extracel lular

tracer. This was despite the fact that the t racer filled

the extracellular space surrounding the nerve termi-

nal.

Af te r 15 min of s t imulat ion at 10 Hz, the prepara-

tion was rested for 15 min. Toward the end of the rest

per iod, the nerve was st imulated once or twice and

the EPP recorded from the identif ied muscle fibers

previously moni tored during the s t imulat ion period.

At the end of the rest period, the ampl i tude of the

EPP ranged between 0.5-1.0 mV. The controls were

also moni tored in exactly the same manner as the ex-

per imenta l prepara t ion . If the fixative (due to its

abili ty to depolar ize membrane15. 24) or the one or

two impulses were responsible for the uptake of H R P

into synaptic vesicles, coated vesicles, or vacuoles,

then the controls would also have demons t ra ted this.

As will be shown, this was not the case.

Fig. 3 shows a series of micrographs from prepara-

tions electrically s t imulated and then rested. Table II

shows the results from this exper iment . The mean

number of synaptic vesicle profiles per section recov-

ered to the control level following the 15 min rest pe-

riod. The mean number of HRP- l abe l ed vesicles also

increased compared to the electrically s t imulated

junctions. Again , the percentage of labeled synaptic

Fig. 3. Electrical stimulation plus rest in the presence of HRP. The preparation was electrically stimulated for 15 min at 10 Hz in a frog saline solution containing 10 mg/ml HRP, then rested 15 min, and then fixed in a buffered glutaraldehyde/formaldehyde solution. A wide range of labeled vesicles are evident. The HRP-labeled vesicles appear to be scattered randomly throughout the nerve terminal. In a the ending contains a number of labeled vesicles. In b and HRP-filled coated pit(arrowhead) and non-coated pit(arrow) along the prejunctional membrane, is seen. Except for a few vesicles, the ending is relatively free of the tracer. Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-reaction product. Magnification: a = 27,000x; b = 41,400x.

vesicles accounted for about 10% of the total number

of synaptic vesicles. The percentage of coated vesi-

cles decreased during the rest period and was similar

to the control value. An interesting example of pino-

cytotic pits filled with HRP can be seen in Fig. 3b.

One pit is coated and the other lacks a coat. It should

be noted that the number of coated vesicles make up

a very small percent compared not only to the total

number of synaptic vesicle profiles per section, but

also to the mean number of HRP-labeled vesicle profiles per section.

Table III shows the percentage of coated vesicles

that were not labeled with the tracer following elec-

trical stimulation and/or rest. A surprisingly high per-

centage of unlabeled coated vesicles was found. What

was of interest was that the percentage also increased

following stimulation and then decreased again after

the 15 min rest period. Ceccarelli et al. 4 also report-

ed finding unlabeled coated vesicles. Examples are

shown (see Fig. 5) in the present study where unla-

beled and labeled coated vesicle profiles are located

in close proximity to each other. The proximity of the

TABLE II

Effects o f electrical stimulation plus rest ~

Vesicle groups Experimental b Control b


Number of HRP-labeled vesicles/section 4.53 + 0.42 d 0.72 + 0.08

% of HRP-labeled vesicles 7.9 + 0.6 e 1.3 + 0.20 Number of coated

vesicles/section 0.33 + 0.11 c 0.19 + 0.02 % of coated vesicles 0.61 _+ 0.20 c 0.35 + 0.02

a Preparation was electrically stimulated at 10 Hz for 15 min, rested for 15 rain, then fixed.

b Mean + S.E.M. (n = 6). c Not significantly different vs controls. d Significantly different vs controls at P < 0.01. e Significantly different vs controls at P < 0.001.

TABLE III

Percentage o f coated vesicles not filled with H R P ~

Treatment

Electrical stimulation Control (not stimulated) 8 Electrical stimulation (10 Hz, 15 rain) 22 Electrical stimulation (10 Hz, 15 min)/ Rest (15 rain) 15

Potassium depolarization Control (not depolarized) 33 Potassium depolarization (15 rain) 41 Potassium depolarization (15 rain)/ Rest (15 min) 37

a The total number of coated vesicles was first calculated. Of these coated vesicles, a certain number were not labeled with HRP. The number of unlabeled coated vesicles/total number of coated vesicles × 100 is the percentage presented in this table.

TABLE IV

Effects o f potassium depolarization a

Vesicle group Experimental ~ Control b


Number of HRP-labeled vesicles/section 4.7 + 0.56 d 0.53 + 0.13

% of HRPqabeled vesicles 9.3 _+ 1.6 e 1.2 + 0.3 Number of coated

vesicles/section 0.44 __+ 0.09 d 0.06 _+ 0.01 % of coated vesicles 0.89 + 0.22 d 0.12 _+ 0.03

a Preparation was potassium depolarized (20 mM excess KC1) for 15 min then fixed.

b Mean + S.E.M. (n = 6). c Not significantly different vs controls. d Significantly different vs controls at P < 0.01. e Significantly different vs controls at P < 0.001.

labeled and un l abe l ed coa ted vesicle prof i les wou ld

exclude the possibi l i ty of fa i lure to de tec t the histo-

chemica l reac t ion p roduc t .

P o t a s s i u m d e p o l a r i z a t i o n

Six animals were used for this s tudy, and the pre-

pa ra t ion was depo l a r i zed for 15 min with 20 m M ex-

cess po tass ium chlor ide . T h e M E P P f r e q u e n c y was

m o n i t o r e d dur ing this t ime per iod , and in genera l ,

the f r equency inc reased f rom less than 1-3/s to as

high as 300/s. T h e f r equency was fairly wel l main-

ta ined t h roughou t the 15 min t ime per iod , wi th little,

if any, d e c r e m e n t in the ampl i tude . This d e c r e m e n t

could have b e e n due to the dec rease in the rest ing po-

tential . For the contro ls , the rest ing f r equency was

m o n i t o r e d for a few minu tes p r io r to f ixat ion.

Fo l lowing po ta s s ium depo la r i za t ion , synapt ic vesi-

cles, coa ted vesicles , and vacuo la r or tubular - l ike

s t ructures l abe led with H R P cou ld be obse rved . Ex-

amples of this are shown in Fig. 4. T h e var ia t ion in

the n u m b e r of H R P - l a b e l e d vesicle prof i les is appar-

ent. Tab le IV shows that fo l lowing depo la r i za t ion ,

there was actual ly a slight iiacrease in the m e a n num-

ber of synapt ic vesicle prof i les pe r sect ion. This resul t

must be v iewed with cau t ion since the m e a n n u m b e r

of synapt ic vesicle prof i les pe r sect ion for the cont ro ls

was surpris ingly low ( c o m p a r e d to cont ro l va lue in

Tab le I). T h e r e was a stat ist ically signif icant increase

in the m e a n n u m b e r of H R P - l a b e l e d ves ic le prof i les

per sect ion fo l lowing depo la r i za t ion as c o m p a r e d to

the controls . The H R P - l a b e l e d vesicles accoun ted

for slightly less than 10% of the to ta l n u m b e r of syn-

aptic vesicles. T h e p e r c e n t a g e of coa ted vesicles also

increased fo l lowing K+-depo la r i za t ion . E x a m p l e s of

n o n - H R P coa ted vesicle prof i les are shown in Fig. 5.

Tab le II shows that of the to ta l n u m b e r of coa t ed ves-

icles for the cont ro l p repa ra t ion , 33% of those were

not l abe led with the t racer . Fo l lowing depo la r i za t ion

the pe rcen t age of un l abe l ed c o a t e d vesicles in-

c reased to 41%. This increase was no t as d rama t i c as

af ter e lectr ical s t imula t ion . H o w e v e r , the p e r c e n t a g e

of un labe led coa t ed vesicles for the cont ro ls was

h igher in the K + - d e p o l a r i z e d p r e p a r a t i o n c o m p a r e d

to the e lectr ical ly s t imula ted p repa ra t ion .

Fo l lowing the 15 min K+-depo la r i za t i on , the solu-

t ion was r ep laced with fresh s a l i n e - H R P and the pre-

pa ra t ion res ted for 15 rain. As soon as the high K +-

solut ion was changed , the M E P P f r equency of the

10

Fig. 4. Potassium depolarization in the presence of HRP. The preparation was potassium depolarized (20 mM excess KC1) for 15 min in a frog saline solution containing 10 mg/ml HRP, and then fixed in a buffered glutaraldehyde/formaldehyde solution. In a, HRP- filled coated pit is in the region of the Schwann cell finger(arrow). A couple of HRP-labeled vesicles are also evident. In a there is a tubular-like structure filled with the tracer(arrowhead). In b a number of vesicles are labeled with HRP, none of which are coated. Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-reaction product. Magnifica- tion: a and b = 30,600x.

identified muscle fibers was once again monitored. In

most instances, the frequency returned to the control

value. In those cases where the frequency remained

well above the control level (greater than 10/s), the

preparation was discarded. In two instances, the fre-

quency was about 5/s, and these preparations were

included in the analysis. However, there was no cor-

relation between the value of the MEPP frequency

during the rest period and the number of HRP-la-

beled vesicles. Examples of the variation in the num-

ber of vesicles containing the tracer are illustrated in

Fig. 6. The populat ion density of synaptic vesicles

appeared normal. This observation was substan-

tiated when the mean number of vesicle profiles per

section was calculated (Table V). Since the number

of vesicles for the controls was smaller than for the

electrically stimulated preparat ion (see Table I),

there was actually a small increase in the average

number of vesicles following the rest period.

There was no significant difference in the mean

number of synaptic vesicle profiles/section (experi-

mental minus control values) between the K+-depo -

larized or electrically stimulated preparations. This

was independent of whether the preparations were

rested or not.

11

Fig. 5. Potassium depolarization in the presence of HRP. As in Fig. 4, the preparation was potassium depolarized (20 mM excess KCI) for 15 rain in a frog saline solution containing 10 mg/ml HRP, and then fixed in a buffered glutaraldehyde/formaldehyde solution. In a an unlabeled coated vesicle(arrow) is adjacent to a labeled coated pit(arrowhead). A pinocytotic structure containing label is seen in b(arrowhead), while the terminal also contains an unlabeled(double arrow) and labeled(arrow) coated vesicle. Large vacuolar-like structures are also evident. Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-reaction product. Magnification: a and b = 30,600 x.

There was also an increase in the mean number of

HRP-labeled vesicle profiles per section at the end of

the rest period, but this value was not statistically sig-

nificant compared to the control value. There was a

statistically significant difference between the mean

number of HRP-labeled vesicle profiles in the K+-de -

polarization experiments compared to the electrical-

ly stimulated experiments (P < 0.05) (compare Ta-

ble I and Table IV). This could point out a difference

in the neurotransmit ter release mechanism between

K+-depolarization and electrical stimulation. Fol-

lowing a 15 min rest period, there was no statistical

difference between K+-depolarization and electrical

stimulation in terms of the mean number of HRP-la-

beled vesicles.

Table IV shows that the percentage of coated vesi-

cles increased after depolarization, and then de-

creased by more than 50% following the rest period.

The percentage of unlabeled coated vesicles fol-

lowed a similar pattern (see Table III), but the de-

crease at the end of the rest period was not as dramat-

ic. There was no statistical difference in the mean

number of coated vesicle profiles for the K+-depola -

rization experiments compared to the electrically

12

Fig. 6. Potassium depolarization plus rest in the presence of HRP. The preparation was potassium depolarized (20 m M excess KCI) for 15 min in a frog saline solution containing 10 mg/ml HRP, the high K+-solution washed out, and fresh HRP-sal ine added. The preparation was then rested for 15 min and then fixed in a buffered glutaraldehyde/formaldehyde solution. In a and b, there are a variable number of HRP-labeled synaptic vesicles, while in c, there is a single labeled vesicle(arrowhead). In c there is an unlabeled coated vesicle(arrow). Note that the increased opacity of the extracellular space surrounding the nerve terminal contains the HRP-react ion product. Magnification: a, b and c = 30,600x.

TABLE V

Effects of potassium depolarization plus rests, b

Vesicle group Experimental c Control c

Number of synaptic vesicles/section 53.8 + 5.3 48.1 + 6.0

Number of HRP-labeled vesicles/section 3.51 + 1.07 0.84 + 0.21

% of HRP-labeled vesicles 6.6 + 2.2 1.8 + 0.4 Number of coated

vesicles/section 0.19 + 0.08 0.10 + 0.02 % of coated vesicles 0.37 + 0.17 0.22 + 0.06

a Preparation was potassium depolarized (20 mM excess KCI) for 15 min, the excess KC1 washed out, and then rested for 15 min.

b All experimental values were not significantly different vs controls. Mean _+ S.E.M. (n = 6).

TABLE VI

Effects of electrical stimulation in the absence of HRP ~

Vesicle group Experimental b Control b


Number of coated vesicles/section 0.35 + 0.01 d 0.07 + 0.01

% of coated vesicles 0.75 + 0.14 c 0.11 + 0.03

a Preparation was electrically stimulated at 10 Hz for 15 min in the absence of HRP, then fixed.

b Mean + S.E.M. (n = 2). c Not significantly different vs controls. d Significantly different vs controls at P < 0.01.

TABLE VII

Effects of electrical stimulation plus rest in the absence of HRP ~,b

Vesicle groups Experimental c Control c

Number of synaptic vesicles/section 69.2 + 6.0 85.6 + 16.6

Number of coated vesicles/section 0.69 + 0.53 0.28 + 0.06

% of coated vesicles 0.94 + 0.68 0.33 + 0.01

a Preparation was electrically stimulated, 10 Hz, 15 min, in the absence of HRP, rested 15 min, then fixed.

b All experimental values not significantly different vs controls.

c Mean + S.E.M. (n = 2).

s t i m u l a t e d e x p e r i m e n t s , i n d e p e n d e n t of w h e t h e r t he

p r e p a r a t i o n s w e r e r e s t e d o r no t .

Exper iments in the absence o f H R P

T o be c e r t a i n t h a t H R P was n o t i n f l u e n c i n g t he

n u m b e r of c o a t e d a n d synap t i c ves ic les , t h e e lec t r ica l

TABLE VIII

Effects of potassium depolarization in the absence of HRP a,b

13

Vesicle groups Experimental c Control c

Number of synaptic vesicles/section 46.4 _+ 2.2 56.0 + 1.6

Number of coated vesicles/section 0.29 + 0.14 0.13 + 0.14

% of coated vesicles 0.63 + 0.06 0.23 + 0.04

a Preparation was potassium depolarized (20 mM excess KCI) for 15 rain, in the absence of HRP, then fixed.

b All experimental values not significantly different vs controls.

c Mean + S.E.M. (n = 2).

TABLE IX

Effects of potassium depolarization plus rest in the absence of HRP ~

Vesicle groups , Experimental b Control b


Number of coated vesicles/section 1.15 + 0.01 d 0.14 _+ 0.0

% of coated vesicles 2.07 + 0.22 c 0.23 + 0.5

a Preparation was potassium depolarized (20 mM excess KC1) for 15 rain, in the absence of HRP, the excess KCI washed out, rested for 15 min, then fixed.

b Mean + S.E.M. (n = 2). c Not significantly different vs controls. d Significantly different vs controls at P < 0.01.

s t i m u l a t i o n a n d K + - d e p o l a r i z a t i o n e x p e r i m e n t s w e r e

r e p e a t e d in t h e a b s e n c e o f H R P . T w o a n i m a l s w e r e

u sed for e a c h e x p e r i m e n t a n d 4 m u s c l e f ibe rs we re

m o n i t o r e d d u r i n g t h e s t i m u l a t i o n o r d e p o l a r i z a t i o n

pe r iod . T h e s a m e c r i t e r i a u s e d in t he p r e p a r a t i o n s

c o n t a i n i n g H R P w e r e u sed in t h e s e e x p e r i m e n t s .

A n y p r e p a r a t i o n w h i c h fa i l ed to r e l e a se t r a n s m i t t e r

a t any p o i n t d u r i n g t he e x p e r i m e n t was d i s ca rded .

T a b l e s VI , V I I , V I I I a n d I X s u m m a r i z e t h e r e su l t s

f r o m t h e a b o v e e x p e r i m e n t s . F o l l o w i n g e lec t r ica l

s t i m u l a t i o n t h e r e was a 2 5 % d e c r e a s e in t he m e a n

n u m b e r of synap t i c ves ic le p ro f i l e s p e r sec t ion . Th i s

d e c r e a s e was no t s ta t i s t ica l ly s ign i f ican t c o m p a r e d to

t he con t ro l s . Th i s c o m p a r e s to a 3 7 % d e c r e a s e in t he

m e a n n u m b e r of synap t i c ves ic le p ro f i l e s p e r sec t ion

in t he p r e s e n c e o f H R P . F o l l o w i n g K + - d e p o l a r i z a -

t ion , t h e r e was a 17% d e c r e a s e in t he m e a n n u m b e r

of ves ic le p ro f i l e s p e r sec t ion . I t is di f f icul t to com-

p a r e t h e s e r e su l t s wi th t h o s e in T a b l e IV , s ince t he

con t ro l v a l u e in T a b l e I V was su rp r i s ing ly low. I f t he

14

numerical values for the mean number of vesicle pro-

files per section are compared in the two experi-

ments, the numbers are very comparable (52.9 in the presence of HRP versus 46.4 in the absence of HRP).

It was also observed that very little difference in the

number of vacuoles (or cisternae-like structures) ex-

ists between the two experiments. The mean per-

centage of coated vesicle profiles per section was also

very small (less than 2%) regardless of the presence or absence of HRP. The major difference was that in

the absence of HRP, the percentage of coated vesi-

cles appeared to increase during the rest period (fol-

lowing either electrical stimulation or K+-depolariza -

tion). The opposite took place in the presence of

HRP, in that there was a decrease in the mean number of coated vesicles following the rest period. It is

unknown why this occurred. It should be emphasized

again that the number of coated vesicles was so small,

the significance of this increase must be viewed with caution.

A statistical comparison of the experimental and

control groups can be made between those preparations bathed in the presence of HRP and those

bathed in the absence of HRP. In terms of the mean

number of synaptic vesicle profiles/section or the

mean number of coated vesicle profiles/section,

there was no statistically significant difference be-

tween HRP and non-HRP soaked preparations. It

was also found that the variability between the vari-

ous preparations was mainly due to the variability between the sections examined.

It was reported by Heuser and Reese 13 that HRP

caused bursting of MEPPs and following prolonged

incubation (greater than 1 hour) in HRP, membrane

whorls appeared within the nerve ending. Neither of

the above effects were observed in the present study.

The tissue was also incubated in the absence of HRP

and processed for histochemical localization of peroxidase according to the method of Graham and Kar-

novsky 12. Endogenous peroxidase activity was not

detected within the synaptic or coated vesicles in this preparation.

DISCUSSON

The findings in the present study point out the im-

portance of recording from identified muscle fibers. The sartorius muscle contains edge fibers which can

be visualized while making electrophysiological re-

cordings. Later, their respective nerve endings can

be located and photographed in the electron micro-

scope. Since muscle fibers were monitored during the stimulation period, any sudden change in the ampli-

tude of the EPP would have been detected and the preparation subsequently discarded. The amplitude

of the EPP at the end of the stimulation period

ranged between 0.25 and 0.50 mV. This small range of amplitude is important since the wide variation in

the number of vesicles remaining or in the number of

HRP-labeled vesicles at the end of the stimulation

period could indicate a wide variation in the electro-

physiological response of the preparation. Since the electrophysiological response was not that variable,

the ultrastructural findings become even more signif-

icant.

Rose et al. 24 reported that after 9-28 min of elec-

trical stimulation, the mean number of vesicles had

decreased by 24%. The percentage was significant at

the 0.005 level. The present study showed about a

25% decrease in the mean number of vesicles after

stimulation for 15 min in the absence of HRP; a 37% decrease, in the presence of HRP, was found not to

be statistically different compared to 25%. In the

present study, K+-depolarization, in the absence of HRP, caused the average number of vesicles to de-

crease by 17%. In the presence of HRP, there was a

slight increase in the mean number of vesicles, biat this was not statistically different compared to the non-HRP study. Earlier, Heuser and Reese 13 report-

ed greater than 50% depletion of vesicles after

15 min of stimulation. Since they did not use identi-

fied endings, their findings were probably based on

selected micrographs. Following a rest period after stimulation or depola-

rization, the synaptic vesicle numbers returned to their control value. This is of interest since after

15 min of electrical stimulation and 15 min of rest,

the amplitude of the EPP increased to about 1 mV

(compared to 4-6 mV for the controls). There was no

obvious change in the resting membrane potential.

The mean number of vesicle profiles per section was similar to the controls but the EPP was well below the

control value. This could mean that the source of

ACh for evoked release was not the synaptic vesicles,

or that many vesicles were not filled with ACh, so that quantal content had been reduced. Another pos-

sibility is that release had been reduced since calcium

was no longer able to interact with its binding site 22.

The most surprising result, from the present study was the relative lack of uptake of HRP into cytoplas-

mic membrane-bound compartments after 15 min of

electrical stimulation or K+-depolarization followed

by 15 min of rest. Following electrical stimulation, a mean of 2.6 synaptic vesicle profiles per section were

labeled with HRP. This number was not significantly

different compared to the control. Following 15 min

of rest, there was less than a two-fold increase in the

mean number of HRP-labeled vesicles. The fact that

in both instances (electrical stimulation or K+-depo -

larization) the percent labeling of synaptic vesicles was less than 10%, was unexpectedly low.

The relative lack of labeling of vesicles with HRP

(around 10%) was similar to the report of Birks 2. Us-

ing thorium dioxide as the extracellular tracer, he

found that potassium isethionate depolarization

caused no increase in the number of vesicles con-

taining the marker. There was also no change in the population density of synaptic vesicles.

Simple depolarization of the presynaptic mem-

brane alone cannot explain the effects of high potas-

sium (20 mM) in increasing the frequency of MEPPs 7,10. The total output of acetylcholine may be

about the same following K+-depolarization com-

pared to electrical stimulation as measured biochem- icallyW. However, Ceccarelli et al.4 did find a 1.5-

fold increase in the density of vesicle fusion sites ad-

jacent to the active zone following K+-depolarization

in contrast to electrical stimulation. This indicated

that perhaps K+-depolarization might increase the number of vesicles labeled with HRP. If Tables I and

IV are compared, it can be seen that K+-depolariza -

tion did increase the average number of HRP-labeled

vesicles by less than 2-fold. Very little difference was

found in the mean number of HRP labeled vesicles following electrical stimulation or K+-depolarization plus 15 min of rest.

Heuser and Reese 13, using HRP, reported that 1 h

of rest following 10 Hz stimulation for 15 min labeled

about 50% of the vesicles. Ceccarelli et al. 6 and Cec-

carelli and Hurlbut 5 used low frequency stimulation for 2 h (with or without hemicholinium-3) or high fre-

quency stimulation in the presence of dextran, and

reported labeling of half the number of vesicles. The number of endings examined or the mean number of

15

labeled vesicles was not given. In the present study,

there were a few examples where the percent uptake

of HRP into synaptic vesicles was greater than 10%.

This was not the usual situation since the sections

show a wide range of HRP uptake (see Figs. 2, 3, 4 and 6).

It could be argued that the increase in HRP uptake

was due to the depolarizing action of the fixative it- self. It has been shown that following the addition

of glutaraldehyde, an increase in the frequency of

MEPPs occurs during the first minute15. 24. In the pres-

ent study, electrical stimulation was stopped 1 min

after the muscle was immersed in fixative. This might

suggest that the fixative could be causing a low per-

centage of vesicles to become labeled. However, it

would seem that the unstimulated preparation would

also have been expected to have the same percentage of vesicles labeled. This was clearly not the case.

While the high K+-solution was kept in the petri dish

during the application of the fixative, it can be seen

that the same percentage of vesicles were labeled as

compared to the electrically stimulated preparation.

The low percentage of labeling of synaptic vesicles

following electrical stimulation could suggest that

blockage of the action potential took place at an axo-

nal branch point. Even though only those prepara-

tions showing EPPs at the end of the stimulation peri-

od were used for the final analysis, branch blockage

could still have taken place without being detected. However, with K+-depolarization, all membranes

should be depolarized and therefore, the extent of

transmitter release from all nerve terminals would

be, more or less, the same. Even in this situation, less

than 10% of the total number of synaptic vesicles

were labeled with HRP. With no branch blocking

taking place during K+-depolarization, far fewer syn-

aptic vesicles were labeled than would be expected.

Labeled synaptic vesicles were scattered throughout the presynaptic cytoplasm and not localized close to the areas of release. Since electrical stimulation or

K+-depolarization was continued during the time of

fixation, and if vesicle exo- and endocytosis occurs

predominantly in the region of the active zone, then

more examples of HRP-labeled vesicles near the presynaptic membrane should have been seen. It could

be argued that the depolarizing action of the fixative

caused the release of HRP-labeled vesicles near the

active zone. Unlabeled synaptic vesicles would then

16

have moved forward to take their place. However, it

would appear that the intense electrical stimulation

or K+-depolarization should have overshadowed the relatively weak depolarizing action of the fixative.

To properly answer this question, future experiments

will focus on fast-freezing of the neuromuscular junc-

tion following K÷-depolarization in the presence of

an extracellular tracer. Only in the absence of alde-

hyde fixation will we be able to determine whether or not the scattering of labeled synaptic vesicles and the

lack of labeled vesicles near the presynaptic mem-

brane was a fixation artifact. Recent work by Gennaro et al. 11 has shown that a

very high K+-propionate solution can cause depletion of vesicles and infoldings of the axolemmal mem-

brane. They found numerous examples of coated

vesicles forming from these infoldings and located in

regions not extending beyond the extent of the axo-

lemmal infolding. Their criticisms of the use of HRP

in studying membrane recyling mainly rested on the

evidence that HRP can decay to form superoxide an- ions 30 and promote lipid peroxidation 23. It had also

been shown by previous work in their laboratory 25.26

that HRP can cause an additional 20--40% decrease

in the number of synaptic vesicles following depolarization compared to the controls. To answer the crit-

icism of whether HRP influences synaptic transmis-

sion, experiments were conducted with electrical

stimulation or K+-depolarization in the absence of

HRP. Tables VI, VII, VIII and IX should be com-

pared to Tables I, II, IV and V. Following electrical

stimulation, a 25% decrease in the mean number of synaptic vesicle profiles was observed, vs 37% in the

presence of HRP. Following K+-depolarization in

the absence of HRP, the mean number of vesicle pro-

files per section decreased 17%. Potassium depolari-

zation in the presence of HRP caused a modest in-

crease in the number of synaptic vesicles vs the controls. If the mean number of vesicles following K ÷-

depolarization in the presence or absence of HRP are

compared (52.9 vs 46.4, respectively), they are very

similar. There was no statistical difference in the

number of synaptic vesicle or coated vesicle profiles per section in preparations soaked in HRP versus

preparations not soaked in HRP.

It was also of interest that Gennaro et al. H could

K+-depolarize their preparation in a magnesium-

substituted EDTA Ringer solution and still obtain

exactly the same results (i.e. membrane infoldings,

vesicle depletion). This was accomplished in the absence of extracellular calcium. Liley 17 found that

MEPP frequency decreased in the presence of Mg 2÷

while Ceccarelli et al. 4 found that 20 mM KCI, in the

absence of calcium, caused freeze-fractured replicas

of the neuromuscular junction to appear identical to the controls. Gennaro et al. 11 used a very high potas-

sium solution (115 mM) for a long period of time

(1 h). In the present study, a 20 mM potassium chlo-

ride solution was used for only 15 min. Also propionate was used as the counterion by Gennaro et al. 11 in

contrast to chloride in the present study. The results of Gennaro et al. 11 should be viewed with caution

since the propionate could have effects of its own.

Another interesting finding in our study was the

percentage of coated vesicles that were not labeled with HRP. Heuser and Reese 13 reported that after

1 min of stimulation nearly all the HRP-labeled vesi-

cles were coated. Unlabeled coated vesicles were quantitated in the present study. Table III shows that

of the low number of coated vesicles found in the

nerve terminal, 8% were unlabeled in the control.

This percentage increased to 22% with electrical

stimulation and fell to 14% following a rest period.

These percentages were higher following K+-depola - rization, but so was the control value. The percent-

age did increase during depolarization and fell during

the rest period.

That coated vesicles may also be associated with other functions besides the possible role in vesicle

membrane recycling 13 is suggested by the work of

Waxman et al. 29. They reported that coated pits were

also located postsynaptically, such that transfer of material from pre- to postsynaptic structures could

conceivably take place. The increase in number fol-

lowing stimulation could mean that pinocytosis may not be directly related to transmitter release.

It was recently suggested by Lentz and Chester16

that there may be two separate populations of coated

vesicles. They used alpha-bungarotoxin, linked to HRP, to label not only postsynaptic acetylcholine re-

ceptors, but also presynaptic acetylcholine receptors.

The synaptic vesicle membrane does not contain any

acetytcholine receptors, and therefore, is not labeled

with the toxin-HRP. After labeling, the preparation

was stimulated. If stimulation caused vesicle mem-

brane to fuse with the prejunctional membrane, and

if the recycled vesicle membrane contained any part

of the prejunctional membrane, the new vesicle

membrane should be labelled with toxin-HRP. The

synaptic vesicles were free of the label, strongly sug-

gesting that if there was recycling according to the Heuser and Reese 13 model, then the prejunctional

and synaptic vesicle membranes did not intermix to

any significant degree. If there was mixing, there

must be some system which specifically recognizes

the vesicle membrane and incorporates that into a regular synaptic vesicle 20. There was evidence for

coated vesicle membrane not stained with the toxin-HRP, although Lentz and Chester 16 did show

some coated vesicle membrane that was stained. This

suggested that coated vesicles can pick up prejunction-

al membrane, even though none of the regular synaptic vesicles contained the label. It is apparent that

these labeled coated vesicles are not involved in syn-

aptic vesicle membrane recycling. Coated vesicles

need not be involved in the formation of new synaptic

vesicles. It was shown that many non-coated pinocytotic vesicles, located near or away from the active

zone, were also not labeled. The fate of the labeled

coated vesicles is unknown, although they could have

been transported retrogradely in the axon.

In the present study, since most of the pits sugges-

tive of pinocytosis were coated and filled with the ex-

tracellular tracer HRP, it is curious that unlabeled

coated vesicles were found within the nerve terminal

and their numbers increased during stimulation. It could first be argued that those unlabeled coated ves-

icles underwent pinocytosis in a region of the prejunctional membrane where the tracer had not pen- etrated the extracellular space. However, in all in-

stances there was good penetration of the tracer in

the space between the nerve terminal and the

Schwann cell. Also, in no instance was a pit along the

prejunctional membrane not seen containing HRP. Coated vesicles could be forming locally within the

nerve ending, perhaps from the smooth endoplasmic

reticulum (SER). There is also no evidence to suggest that coated pits are exclusively undergoing en-

docytosis. Material within the coated vesicle may be released by exocytosis at the prejunctional mem-

brane. This material may consist of metabolites not

directly related to acetylcholine. The increase in the number of coated vesicles following stimulation may

be in response to a metabolically active ending. The

17

source of these coated vesicles could again be the

SER found within the ending or be transported to the

ending by way of the axon from the cell body.

It could be argued that 15 min was a very short rest

period since the time used by others was 60 min 6,13.

After a longer rest period, when the electrophysiolog-

ical response was back to the control level, it had

been reported by Ceccarelli et al. 6 and Heuser and

Reese 13 that 50-60% of the vesicles were labeled

with HRP. We found that after a 15 min rest period,

there was no statistical difference between the mean

number of synaptic vesicle profiles per section in the control and the rested preparations. This is of inter-

est since the EPP after 15 min of rest (0.5-1.0 mV)

was still well below the value of the EPP for the con-

trols (4-6 mV). It was rather curious that the rest period brought

the vesicle numbers back to the control level, but the

number of labeled vesicles was still surprisingly

small. This might suggest that new vesicle membrane

is not formed from the presynaptic membrane or that the tracer is preferentially excluded from the newly

formed vesicles. The total presynaptic membrane area was not

measured, so it is not known whether the 37% loss of

vesicles following electrical stimulation was compen-

sated for by an increase in the axolemmal membrane. Tremblay and Phileppe 28 reported a net loss of mem-

brane following transmitter release, and Birks 3 ob-

served no noticeable expansion of the bouton area in

sympathetic nerve endings despite a 56% depletion

of vesicles following stimulation. Whether the 'lost'

vesicles were actually transported retrogradely in the axon away from the terminal is not known. Litchey is

had demonstrated retrograde flow of HRP at the frog

neuromuscular junction. If the vesicle membrane

was incorporated into the axolemmal membrane,

then it is puzzling as to why, after the 15 minute rest

period, the new synaptic vesicles found in the ending

did not contain HRP. The source of these new synaptic vesicles could be either the cell body 14 or the agranular endoplasmic reticulum s,27.

We can conclude from our findings that because of

the relative lack of HRP labeling of synaptic vesicles

formed after stimulation and/or rest periods, that the

new vesicle membrane does not originate directly

from the internalization of the presynaptic plasma

membrane.

18

ACKNOWLEDGEMENTS

Par t of this w o r k was submi t t ed by C . K . M . in par-

tial fu l f i l lment of the r e q u i r e m e n t s for a Ph .D . de-

gree in the D e p a r t m e n t of A n a t o m y , Un ive r s i t y of Il-

l inois at Chicago , Med ica l Sciences Cen te r .

REFERENCES

1 Adams, J. C., Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141-145.

2 Birks, R. I., The fine structure of motor nerve endings at frog myoneural junctions, Ann. N.Y. Acad. Sci., 135 (1966) 8-19.

3 Birks, R. I., The relationship of transmitter release and storage to fine structure in a sympathetic ganglion, J. Neu- rocytol., 3 (1974) 133-160.

4 Ceccarelli, B., Grohovaz, F. and Hurlbut, W. P., Freeze- fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium, J. Cell Biol., 81 (1979) 178-192.

5 Ceccarelli, B. and Hurlbut, W. P., The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction, J. Physiol. (Lond.), 247 (1975) 163-188.

6 Ceccarelli, B., Hurlbut, W. P. and Mauro, A., Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 499-524.

7 Cooke, J. D. and Quastel, D. M. J., The specific effect of potassium on transmitter release by motor nerve terminals and its inhibition by calcium, J. Physiol. (Lond.), 228 (1973) 435-458.

8 Droz, B., Renewal of synaptic protein, Brain Research, 62 (1973) 383-394.

9 Fertuck, H. C. and Salpeter, M. M., Localization of the acetylcholine receptor by II;5-1abelled alpha-bungarotoxin binding at mouse motor end plates, Proc. nat. Acad. Sci., 71 (1974) 1376-1378.

10 Gage, P. W. and Quastel, D. M. J., Dual effect of potassium on transmitter release, Nature (Lond.), 206 (1965) 625-626.

11 Gennaro, J. F., Nastuk, W. L. and Rutherford, D. T., Re- versible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction, J. Physiol. (Lond.), 280 (1978) 23%247.

12 Graham, R. C. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proxi- mal tubules of the mouse kidney: ultrastructural cytoche- mistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302.

13 Heuser, J. E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 315-344.

14 Holtzman, E., Teichberg, S., Abrahams, S. J., Citkowitz, D., Crain, S. M., Kawai, N. and Peterson, E. R., Notes on synaptic vesicles and related structures, endoplasmic reticulum, lysosomes and peroxisomes in nervous tissue and the adrenal medulla, J. Histochem. Cytochem., 21 (1973)

W e wish to thank Dr . N o r m a n Rush fo r th of Case

Wes te rn R e s e r v e Un ive r s i t y for his inva luable he lp

on the statist ical analyses.

This w o r k was suppor t ed , in par t , by N I H G r a n t

NS-16610 and by N S F G r a n t B N S 8004688.

349-385. 15 Hubbard, J. I. and Laskowski, M. B., Spontaneous trans-

mitter release and ACh sensitivity during glutaraldehyde fixation of rat diaphragm, Life Sci., 11 (1972) 781-785.

16 Lentz, T. L. and Chester, J., Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker, Neuroscience, 7 (1982) %20.

17 Liley, A. W., The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction, J. Physiol. (Lond.), 134 (1956) 42%443.

18 Litchy, W. J., Uptake and retrograde transport of horseradish peroxidase in frog sartorius nerve in vitro, Brain Re- search, 56 (1973) 37%381.

19 Macintosh, F. C, and Collier, B., Neurochemistry of cho- linergic terminals. In E. Zaimis (Ed.), Hand. Exp. Phar- macol., Neuromuscular Junction, Vol. 42, Springer, Berlin, 1976, pp. 99-228.

20 Meldolesi, J. Borgese, N., DeCamilli, P. and Ceccarelli, B., Cytoplasmic membranes and the secretory process. In G. Poste and G. L. Nicolson (Eds.), Membrane Fusion, Elsevier, North-Holland, 1978, pp. 509-627.

21 Meshul, C. K. and Pappas, G. D., Synaptic vesicles and transmitter release at the neuromuscular junction, Soc. Neurosci. Abstr., 8 (1982) 280.

22 Pappas, G. D. and Rose, S., Localization of calcium depos- its in the frog neuromuscular junction at rest and following stimulation, Brain Research, 103 (1976) 362-365.

23 Pederson, T. C. and Aust, S. D., The mechanisms of liver microsomal lipid peroxidation, Biochim. biophys. Acta, 385 (1975) 232-241.

24 Rose, S. J., Pappas, G. D. and Kriebel, M. E., The fine structure of identified frog neuromuscular junctions in relation to synaptic activity, Brain Research, 144 (1978) 213-239.

25 Rutherford, D. T. and Gennaro, J. F., A mechanism for horseradish peroxidase induced membrane damage in recycling membrane systems, J. Cell Biol., 83 (1979) 438a.

26 Rutherford, D. T., Nastuk, W. L. and Gennaro, J. F., Horseradish peroxidase-induced alteration of neuronal membrane cycling, Fed. Proc., 38 (1979) 1399.

27 Teichberg, S. and Holtzman, E., Axonal agranular reticu- lure and synaptic vesicles in cultured embryonic chick sympathetic neurons, J. Cell Biol., 57 (1973) 88-108.

28 Tremblay, J. P. and Philippe, E., Morphological changes in presynaptic terminals of the chick ciliary ganglion after stimulation in vivo, Exp. Brain Res., 43 (1981) 439-446.

29 Waxman, S. G., Waxman, M. and Pappas, G. D., Coordi- nated micropinocytotic activity of adjacent neuronal membranes in mammalian central nervous system, Neurosci. Lett., 20 (1980) 141-146.

30 Wittenberg, J. B., Noble, R. W., Wittenberg, B. A. Anto- nini, E., Brunori, M. and Wyman, J., Studies on the equili- bria and kinetics of the reactions of peroxidase with ligand, J. biol. Chem., 242 (1967) 626-634.

the relationship of pinocytosis and synaptic vesicles at the frog neuromuscular junction

Documents