Journal of Neurocytology 14, 193-202 (1985)

Ultrastructural correlates of naturally occurring differences in transmitter release efficacy in frog motor nerve terminals A L B E R T A. H E R R E R A 1, A L A N D. G R I N N E L L 2 a n d B I R G I T W O L O W S K E 2

1Neurobiology Section, Department of Biolog&al Sciences, and Program in Neural, Informational and Behavioral Sciences, University of Southern California, Los Angeles, California, 90089, USA 2Department of Physiology and Jerry Lewis Neuromuscular Research Center, University of California, Los Angeles, California, 90024, USA

Received 1 August 1984; revised and accepted 29 October 1984

Summary

Motor nerve terminals in cutaneous pectoris muscles of the frog Rana pipiens release more transmitter and form synapses with high<r levels of effectiveness than do those in sartorius muscles. Neuromuscular junctions from these two muscles were compared in the electron microscope to search for ultrastructural correlates of differences in transmitter release and synaptic effectiveness. The following measurements were made from cross-sections of junctions with known levels of effectiveness: (a) the presence of active zones, the presumed sites of transmitter release, (b) active zone size, (c) the perimeter, cross-sectional area, height and width of nerve terminals, (d) number of mitochondria, (e) vesicle density, and (f) the extent to which Schwann cells wrap terminals. Nerve terminals in the two muscles did not differ in size, shape or vesicle density. The more strongly releasing cutaneous pectoris terminals did, however, have significantly larger active zones due to deeper invagination of the terminal into the postsynaptic gutter and lesser interposition of Schwann cell processes between presynaptic and postsynaptic membranes. Cutaneous pectoris terminals also contained more mitochondria, presumably to supply the greater energy demand imposed by high release levels.

Introduction

Several light microscopic studies have concluded that neurotransmit ter release at neuromuscula r junctions is propor t ional to nerve terminal size (Kuno et al., 1971; Bennett & Raftos, 1977; Angaut-Pet i t & Mallart, 1979; Harris & Ribchester, 1979; Grinnell & Herrera, 1980). However , there is considerable scatter in these relationships. Recent analyses of this scatter have revealed pat terns of organization suggestive of previously u n k n o w n regulatory processes. For example, different motoneurons supplying the same muscle can vary widely in the inherent effectiveness of the synapses they form, ~i.e.

0300-4864/85 $03.00 + .12 �9 1985 Chapman and Hall Ltd.

194 HERRERA, GRINNELL and WOLOWSKE

the amount of t ransmit ter released per unit nerve terminal length. There is a direct relationship be tween synaptic effectiveness and the size of a motoneuron ' s per ipheral field (Grinnell & Herrera, 1980; Grinnell & Trussell, 1983). Also, in subpopulat ions of fibres with the same input resistance, there can actually be an inverse relationship between terminal size and release (Nudell & Grinnell, 1982, 1983), as if terminals simply stop growing w h e n a certain level of synaptic effectiveness has been achieved.

Our earlier studies showed that normal motoneurons supplying different muscles of the frog could also differ in transmitter release efficacy (Grinnell & Herrera, 1980).

Terminals in the cutaneous pectoris muscle release 2-3 times as much transmit ter per nerve impulse as those in the sartorius muscle, wi thout any differences apparen t at the light microscopic level. A prel iminary investigation of ultrastructural differences be tween cutaneous pectoris and sartorius terminals (Grinnell & Herrera, 1980) was

inconclusive. In the light of several recent studies on neuromuscular junctions that correlate

presynaptic ul trastructure with release in normal muscles (Heuser et al., 1979; Verma & Reese, 1984), dur ing regenera t ion (Ko, 1984), in aging (Fahim & Robbins, 1982; Fahim et al., 1983; Banker et al., 1983), and in disease (Fukahara et aI., 1972; Fukunaga et al., 1982), we have re-examined cutaneous pectoris and sartorius terminals for ultrastructural differences using muscles with k n o w n levels of synaptic effectiveness. We find that differences in effectiveness are associated with differences in the average size of active zones, and the number of mitochondria within the nerve terminal. A prel iminary repor t

has been publ ished (Grinnell et al., 1983).

Methods

Adult Rana pipiens (body length 6-7 cm) were used. From each frog, a sartorius and a cutaneous pectoris nerve-muscle preparation was excised. The muscles were immersed in Ringer solution containing 116 mM NaC1, 2 mM KC1, 1.8 mM CaC12, 1 mM MgC12 and I mM NaHCO3 at pH 7.2 and 15 ~ C, and tested for differences in transmitter release levels. On the basis of endplate morphology, muscle contraction time, and the ability of every fibre studied to generate action potentials (Grinnell, Herrera & Trussell, unpublished observations), we concluded that most or all fibres in both sartorius and cutaneous pectoris muscles were of the fast twitch type.

Average differences in the level of transmitter release from motor nerve terminals can be rapidly and effectively assessed by measuring the fractional drop in twitch tension evoked by nerve stimulation when [Ca 2+] in the bathing medium is lowered. As [Ca z+] is lowered, tension falls as transmission at the strongest endplate on each muscle fibre becomes subthreshold for action potential generation. Previous studies (Grinnell & Herrera, 1980) have shown that for the cutaneous pectoris and the sartorius, the extent to which tension falls is a reliable indicator of differences in average transmitter release and depends only slightly on postsynaptic differences.

After finding a frog whose cutaneous pectoris and sartorius muscles showed the typical difference in synaptic effectiveness, the muscles were briefly fixed in 2% glutaraldehyde in Ringer solution and lightly stained for cholinesterase (Karnovsky, 1964). Regions of the muscle rich in surface endplates were identified using a light microscope. Pieces of muscle (about I mm • 3 ram) containing these regions were excised, postfixed in 2% glutaraldehyde (3 h) and 2% OsO4

Synapt ic u l t r a s t ruc tu re a n d t ransmi t te r release 195

Fig. 1. Cross-section through an active zone of a sartorius nerve terminal. Corresponding drawing in inset shows Schwann cell (SC), mitochondrion (M), vesicles (V), contact width (CW), and muscle fibre (MF).

(1 h), dehydrated in acetone, and embedded in Medcast (Pelco). After identifying endplate-bearing fibres in thick sections, thin sections were taken several hundred ~m apart to make it likely that each endplate would be sectioned only once. These thin sections were stained with saturated uranyl acetate and photographed in a Zeiss EM10C electron microscope at 80 kV. Prints at a final magnification of x 47 400 were analysed with a digitizing tablet and microcomputer (resolution 0.1 m m on the tablet, corresponding to about 21 K).

The following measurements were made from each section: (a) whether or not the section passed through an active zone, usually recognized by a postsynaptic fold opening into the synaptic cleft, a presynaptic density just opposite this opening, and a cluster of synaptic vesicles associated with the presynaptic density (see Fig. 1); (b) the perimeter of the nerve terminal; (c) cross-sectional area of the terminal; (d) number of mitochondria per section (M in inset of Fig. 1);

196 HERRERA, GRINNELL and WOLOWSKE

(e) cross-sectional area of mitochondria; (f) for sections passing through active zones, the number of vesicles in a 0.04 ~m 2 area immediately surrounding the active zone (square labelled V in inset of Fig. 1); (g) for sections not passing through active zones, the number of vesicles in a 0.045 ~tm 2

area adjacent to the entire presynaptic membrane in the region of synaptic contact; (h) synaptic contact width (CW in inset of Fig. 1); (i) potential contact width, measured as the amount of nerve terminal membrane within 0.2 ~m of the muscle fibre membrane; (j) Schwann cell interposition, or the extent of the potential contact width obstructed by Schwann cell processes interposed between presynaptic and postsynaptic membranes; (k) terminal width, or the diameter of the nerve terminal measured parallel to the underlying muscle fibre membrane (this would correspond to the terminal diameter measured with a light microscope); and (1) terminal height, or the diameter of the terminal measured perpendicular to the underlying fibre membrane.

Results

In previous studies (Grinnell & Herrera, 1980), when [Ca 2+] was lowered from 1.8 to 1.0 mM, nerve stimulus-evoked twitch tension in the cutaneous pectoris fell an average of only 2%, while sartorius tension fell by an average of 58%. This was shown to be due primarily to a 2-3 fold higher level of quantal transmitter release from cutaneous pectoris terminals. The pair of muscles used in the present study showed a similar mean difference in synaptic effectiveness. Tension produced by the cutaneous pectoris fell by 5%, and sartorius tension by 50% when [Ca 2+] was lowered from 1.8 to 1.0 raM.

One thin section was taken through each of 62 randomly selected junctions in the cutaneous pectoris and 48 junctions in the sartorius. This yielded 113 cross-sections of cutaneous pectoris nerve terminal branches (62 through active zones, 51 between active zones) and 87 cross-sections of sartorius terminal branches (40 through active zones, 47 between active zones). Fig. 1 shows a typical cross-section through an active zone of a sartorius junction. Table 1 shows the average measurements made from sections taken at and between active zones. For sections passing through active zones, there were no significant differences between cutaneous pectoris and sartorius in: (a) the number of synaptic vesicles in a 0.04 ~tm 2 area adjacent to the active zone, (b) perimeter of the nerve terminal seen in cross-section, (c) cross-sectional area of the terminal, (d) terminal width, (e) terminal height, (f) an index of terminal shape obtained by dividing terminal width by terminal height. Significant differences were found, however, in several important measurements made from these sections. Of particular interest were differences found in two indirect measures of active zone length: contact width and potential contact width. Contact width, or the amount of presynaptic perimeter membrane in close apposition to postsynaptic membrane and unoccluded by Schwann cell processes, averaged 33% higher at the stronger cutaneous pectoris junctions (means: cutaneous pectoris 2.20 ~m, sartorius 1.65 ~m). Potential contact width, defined as the amount of presynaptic perimeter membrane within 0.2 ~tm of muscle membrane regardless of Schwann cell interposition, was 25% higher in the cutaneous pectoris junctions (means: cutaneous pectoris 2.36 ~m, sartorius 1.89 ~m). Thus terminals in the cutaneous pectoris had a significantly higher percentage of their perimeter in synaptic contact with the

Tab

le 1

. M

orp

ho

log

ical

fea

ture

s of

cu

tan

eou

s p

ecto

ris

(CP

) an

d s

arto

rius

(S

art.

) n

euro

mu

scu

lar

jun

ctio

ns.

At

act

ive

zon

es

No

t a

t a

ctiv

e zo

nes

Act

ive

zon

e vs

. N

ot

at

act

ive

zon

e (P

val

ues

)

CP

S

art.

P

CP

S

art.

P

CP

S

art.

Ves

icle

den

sity

5.

74 +

0.

34

5.33

+

0.31

N

.S.

2.30

+

0.35

3.

50 +

0.

33

K 0

.05

N.A

. N

.A.*

(6

2)

(40)

(4

5)

(48)

P

erim

eter

(~

m)

5.23

+

0.24

4.

82 +

0.

25

N.S

. 3.

72 +

0.

25

3.59

+

0.22

N

.S.

K 0

.000

2 K

0.0

001

(53)

(3

9)

(49)

(4

7)

Cro

ss-s

ecti

onal

1.

38 +

0.

10

1.19

+

0.11

N

.S.

0.84

+

0.11

0.

70 +

0.

07

N.S

. K

0.0

001

K 0

.000

1 ar

ea (

~m

2)

(53)

(3

9)

(50)

(4

7)

Ter

min

alw

idth

1.

99 +

0.

11

1.79

+

0.11

N

.S.

1.31

+

0.10

1.

27 +

0.

08

N.S

. K

0.0

01

K 0

.000

2 (~

m)

(51)

(3

9)

(51)

(4

5)

Ter

min

alh

eig

ht

0.90

+

0.05

0.

76 +

0.

05

N.S

. 0.

75 +

0.

06

0.55

+

0.04

K

0.0

1 K

0.0

1 K

0.0

001

(~m

) (5

2)

(38)

(5

0)

(47)

W

idth

/hei

gh

t 2.

47 +

0.

19

2.68

+

0.22

N

.S.

2.07

+

0.21

2.

72

+ 0.

26

K 0

.05

N.S

. N

.S.

(52)

(3

8)

(51)

(4

7)

Co

nta

ct w

idth

2.

20

+ 0.

14

1.65

+

0.12

<

0.01

1.

26 +

0.

12

0.71

+

0.11

K

0.0

02

K 0

.000

2 K

0.0

002

(~m

) (5

1)

(39)

(5

1)

(45)

P

ote

nti

alco

nta

ct

2.36

+

0.14

1.

89

+ 0.

11

K 0

.01

1.41

+

0.13

0.

86 +

0.

12

K 0

.002

~

0.00

02

K 0

.000

2 w

idth

(~

m)

(51)

(3

9)

(51)

(4

5)

Co

nta

ct

0.42

+

0.02

0.

35

+ 0.

02

< 0.

01

0.34

+

0.02

0.

18

+ 0.

03

K 0

.000

2 K

0.0

1 K

0.0

002

wid

th&

eig

ht

(52)

(3

8)

(50)

(4

7)

SC

I/po

tent

ial

0.05

+

0.01

0.

14 +

0.

03

K 0

.01

0.16

+

0.04

0.

51

+ 0.

07

K 0

.000

2 K

0.0

002

N.S

. w

idth

(5

1)

(39)

(5

1)

(45)

N

um

ber

of

5.13

+

0.49

2.

85 +

0.

39

< 0.

0001

3.

06 +

0.

41

2.32

+

0.26

N

.S.

K 0

.002

N

.S.

mit

och

on

dri

a (5

2)

(38)

(5

0)

(47)

Not

e:

Dat

a sh

own

are

mea

n +

s.m

of

the

mea

n, w

ith

num

ber

of s

ecti

ons

in p

aren

thes

es.

Abb

revi

atio

ns:

N.S

., n

ot s

igni

fica

nt a

t 0.

05 l

evel

; N.A

., n

ot

appl

icab

le;

SCI,

Sch

wan

n ce

ll i

nter

posi

tion

. *

Com

pari

son

not

poss

ible

bec

ause

of

diff

eren

t m

easu

rem

ent

met

hods

(se

e te

xt).

198 HERRERA, GRINNELL and WOLOWSKE

muscle at active zones (mean contact width/terminal perimeter equals 0.42 for the cutaneous pectoris, 0.35 for sartorius). The stronger cutaneous pectoris terminals also contained 80% more mitochondria per active zone cross-section than sartorius terminals (mean values: cutaneous pectoris 5.13, sartorius 2.85). There was no apparent difference in the size of mitochondria.

A separate analysis of sections taken between active zones in each type of muscle is also shown in Table 1. Vesicle density between active zones was slightly lower in the cutaneous pectoris. Terminal perimeter, cross-sectional area, and width did not differ. Terminal height was smaller and the width/height ratio larger in the sartorius, indicating that sartorius terminals are slightly more flattened between active zones. The width of close synaptic contact, potential contact width, the ratio of contact width to terminal perimeter, and the fraction of the potential contact width obstructed by Schwann cells were all significantly larger in the cutaneous pectoris. When data measured at active zones are compared with data taken between active zones in the same muscle (Table 1), it is clear that both types of nerve terminal are varicose along their length. By most measures, active zone regions of the terminal were larger than regions between active zones.

Table 1 also shows a particularly striking difference between the cutaneous pectoris and sartorius in the way in which Schwann cell interposition varies along the length of the nerve terminal. In the cutaneous pectoris 5% of the potential contact width is obstructed by Schwann cells at active zones and 16% between active zones. In the sartorius the respective values are 14 and 51%. Thus sartorius terminals are much more completely wrapped by Schwann cell processes between active zones than are cutaneous pectoris terminals. Fig. 2 is a pictorial summary of these ultrastructural differences between cutaneous pectoris and sartorius neuromuscular junctions.

Discussion

Previous light microscopic studies (Kuno et al., 1971; Bennett & Raftos, 1977; Angaut-Petit & Mallart, 1979; Harris & Ribchester, 1979; Grinnell & Herrera, 1980; Nudell & Grinnell, 1982) have shown that, when all junctions in a muscle are considered, larger terminals release more transmitter. Recent studies showing the importance of the active zone in the release process suggest that this light microscopic correlation can be interpreted on the basis of an increased number of active zones in longer nerve terminals. Our previous study showed that there is a large difference in release between cutaneous pectoris and sartorius terminals but no differences in nerve terminal length or appearance as judged with the light microscope (Grinnell & Herrera, 1980). The present study shows that ultrastructural differences do exist, however. Differences in synaptic effectiveness are correlated with several measures of the width of synaptic contact. Studies of neuromuscular junctions in frog fast twitch muscles using freeze-fracture electron microscopy have established that active zone particles in the

Synaptic ultrastructure and transmitter release

CUTANEOUS PECTORIS SARTORIUS

At act ive zones lpm

199

Between act ive zones

Fig. 2. Hypothetical cross-sections through neuromuscular junctions illustrating more mitochon- dria, and longer active zones due to greater invagination and less Schwann cell interposition in cutaneous pectoris terminals. Dimensions correspond to mean values given in Table 1.

presynaptic membrane occur in rows aligned perpendicular to the long axis of the terminal and in most cases span nearly the entire terminal width (Peper et aI., 1974; Heuser et al., 1974, 1979). If this is true for the junctions we have studied, the present results suggest that active zones are about 1/3 larger in the cutaneous pectoris than the sartorius. The amount of transmitter release, and therefore synaptic effectiveness, can depend on the size of individual active zones, as well as their total number.

This greater degree of synaptic contact was not due to an overall difference in terminal size or shape, since the perimeter, area, width and width/height ratio did not differ significantly. Instead, the greater contact in the cutaneous pectoris seemed to be due to: (a) deeper invagination of the whole terminal into the postsynaptic gutter, and (b) lesser interposition of Schwann cell processes between presynaptic and postsynaptic membranes. On average, for all sections, 38% of the perimeter of cutaneous pectoris terminals was in close contact with the postsynaptic membrane compared with 26% for sartorius terminals. The percentage of potential contact width obstructed by Schwann cell processes was three times greater in sartorius than in cutaneous pectoris junctions.

Positive correlations between active zone size and release have also been seen in diseased human junctions (Fukahara et al., 1972; Fukunaga et al., 1982), in frog (Heuser et al., 1979; Verma & Reese, 1984; Ko, 1984) and toad (Davey & Bennett, 1982) neuromuscular junctions, in the exciter nerve synapses onto crab (Sherman & Atwood, 1972) and lobster (Govind & Chiang, 1979; Govind & Meiss, 1979; Meiss & Govind, 1979, 1980) muscle, and in squid (Pumplin et al., 1981) and Aplysia (Bailey & Chen, 1983) synapses.

200 HERRERA, GRINNELL and WOLOWSKE

It was somewhat surprising that the density of synaptic vesicles adjacent to active zones did not correlate with synaptic effectiveness. However, it has not been determined whether vesicles so positioned represent the immediately releasable transmitter pool. Given this uncertainty, and uncertainty regarding vesicle mobility and the effects of tissue preparation on vesicle position, definite conclusions seem unwarranted. Positive correlations between vesicle number and release have been f o u n d in the developing avian iris (Pilar et al., 1981) and in long-term habituated Aplysia (Bailey &Chen, 1983). However, in aged mice, motor nerve terminals contain fewer vesicles than in young mice, even though the older terminals release more transmitter (Fahim & Robbins, 1982; Banker et al., 1983; Kelly & Robbins, 1983).

The more strongly releasing cutaneous pectoris terminals contained significantly more mitochondria, even though they did not differ from sartorius terminals in volume. Although mitochondria sequester Ca 2+ and in this way may under some conditions significantly limit transmitter release (Alnaes & Rahamimoff, 1975), it appears that the mitochondria play a more important role in supplying the greater energy demand imposed by higher release levels.

If the amount of release apparatus at active zones is the most important determinant of release, then the relationship between these two parameters is steep. For a mean 33% difference between cutaneous pectoris and sartorius terminals in the amount of release apparatus (assuming its presence across the entire contact width at active zones and equal spacing between active zones), there is a mean difference of 2-3 fold in transmitter release per unit terminal length (Grinnell & Herrera, 1980). It is probable, however, that physiological differences accompany the morphological differences. For example, in a study of sartorius terminals of different release efficacy, more strongly releasing terminals were found to have higher intraterminal Ca 2+ levels, due both to higher resting and impulse-linked Ca 2+ influx (Pawson & Grinnell, 1984; and unpublished observations).

In our earlier physiological and morphological comparison of cutaneous pectoris and sartorius junctions, we found few ultrastructural differences (Grinnell & Herrera, 1980), while in the present study several differences were found. In the earlier work, a large number of sections were taken from a few junctions, while in the present study we made an effort to take only one section from a large number of junctions. This presumably explains the discrepancy, for if synaptic ultrastructure were relatively uniform within a junction, but quite variable between junctions, our earlier sampling procedure would be susceptible to error.

Acknowledgements We are grateful to Lisa Banner, Herman Kabe, Frances Knight and David Scott for technical assistance. This work was supported by USPHS grants NS06232 to ADG and NS18186 to AAH and by the Muscular Dystrophy Association.

S y n a p t i c u l t r a s t r u c t u r e a n d t r a n s m i t t e r r e l e a se 201

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202 HERRERA, GRINNELL and WOLOWSKE

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