1979 quantitative comparison of low and high output neuromuscular synapses from a motoneuron of the lobster homarus americanus

However, since the m e a n surface area of synapses is significantly smaller in the high-output terminal than in the low-output one, the total synaptic surface area between the two types

Cell Tissue Res 198, 455-463 (1979)

Research

9 by Springer-Verlag 1979

Quantitative Comparison

of Low- and High-output Neuromuscular Synapses

C.K G o v i n d and D.E Meiss

Scarborough College, University of Toronto, West Hill, Ontario, Canada

Summary Representative examples of low- and high-output neuromuscular

synapses between m o t o n e u r o n and distal accessory flexor muscle of the lobster were selected on the basis o f their mean quantal content, and subsequently analysed by serial section electron microscopy The high-output terminal has twice as m a n y synapses as the low-output terminal However, since the m e a n surface area of synapses is significantly smaller in the high-output terminal than

in the low-output one, the total synaptic surface area between the two types of

terminals is similar Also, though the high-output terminal possesses a greater

n u m b e r o f presynaptic dense bodies than its low-output counterpart, the mean

n u m b e r per synapse is similar for the two terminals The terminals, however, differ significantly in the size o f their dense bodies Thus both the mean and total surface area o f these bodies is greater in the high-output terminal than in the low-output one Moreover, the mean ratio o f dense b o d y area to synaptic area is significantly greater for the high-output terminal than for its low-output counterpart This difference in dense body area parallels the difference in quantal content o f synaptic transmission between the low- and high-output terminals and supports the hypothesis that presynaptic densities represent the ultrastructural correlates o f transmitter mobilization and/or release

Key words: Neuromuscular synapses - Presynaptic density - Ultrastructure - Serial sections - Crustaceans

Crustacean neuromuscular synapses are often regarded as model systems for synapses in the C N S o f vertebrates because o f the multiple converging inputs from a single m o t o r axon and the interaction o f both excitatory and inhibitory axons

Send offprint requests to." C K Govind, Scarborough College, University of Toronto, West Hill, Ontario

M1C 1A4, Canada

* Supported by grants from the National Research Council and Muscular Dystrophy Association of Canada to C.K Govind D.E Meiss is a post-doctoral fellow of the Muscular Dystrophy Association of Canada We thank Eva Yap-Chung for her expert and unfailing technical assistance

0302-766X/79/0198/0455/$01.80

456 C.K Govind and D.E Meiss

(Katz, 1966) The analogy may be extended further as the multiple terminals arising from a single axon vary in their properties of transmitter release Thus single crustacean excitatory motoneurons such as those to the opener muscle in crayfish (Bittner, 1968), the stretcher muscle in crabs (Sherman and Atwood, 1972; Govind

et al., 1973) and the accessory flexor muscle in lobsters (DeRosa and Govind, 1978; Meiss and Govind, 1979a, b) give rise to a wide spectrum o f synapses ranging from low-quantal release to high-quantal release types This diversity in physiological properties o f synapses from a single axon must have an underlying ultrastructural basis

Likely factors in this respect are the size and number o f synaptic contacts which have been shown to differ in terminals of the single axon to the stretcher muscle of

Hyas (Sherman and Atwood, 1972) where high-quantal release terminals have more numerous and larger synapses than their low-quantal release counterparts More recently, however, a better correlation has been found between presynaptic dense bodies and transmitter output with the use of serial section electron microscopy which has permitted a quantitative analysis of the ultrastructural parameters Thus terminals that release large amounts of transmitter such as those formed by the fast axon of Pachygrapsus (Atwood and Jahromi, 1978) invariably have presynaptic dense bodies in all o f their individual synapses whereas dense bodies are present in only half o f the synapses in a crab pyloric muscle (Atwood et al., 1978) which release

a relatively smaller a m o u n t o f transmitter Moreover, comparison o f terminals from two fibers of the proximal accessory flexor muscle of the lobster reveal significant differences in the number and size of presynaptic dense bodies which correlate with the different amplitudes o f the excitatory postsynaptic potentials (EPSPs) o f these fibers (Govind and Chiang, 1979) A more rigorous test of the relationship between presynaptic densities and transmitter output would be to section serially terminals for which the quantal output of synaptic transmission had been determined The present paper reports on such a study in the distal accessory flexor muscle o f the lobster by comparing the quantitative ultrastructure of physiologically identified low- and high-output terminals

Materials and Methods

Adult lobsters (Homarus americanus) weighing approximately 500 g each, were purchased locally and held in Instant Ocean at 10-12 ~ C The first pair of walking legs was used The posterior surface of the distal accessory flexor muscle (DAFM) was exposed in the meropodite and held in cool (10 ~ C) lobster saline (Meiss and Govind, 1979b) The excitor axon to the DAFM was stimulated with short duration ( < 0.1 msec) rectangular pulses via platinum wire electrodes placed on the main leg nerve The resulting excitatory post-synaptic potentials (EPSPs) were monitored intracellularly with 10-15 Mr] microelec-

trodes filled with a saturated solution of the dye methyl blue in 1 M KAc At the end of the experiment the

dye was iontophoresed into the fiber (Thomas and Wilson, 1966) for identification during fixation for electron microscopy Simultaneous with EPSP recordings, the synaptic currents were monitored extracellularly at discrete foci with 2-4 Mf~ microelectrodes filled with 2 M NaC1 solution Conventional procedures were followed for amplifying, displaying and photographing the electrical signals After the synapses were characterized physiologically the muscle was fixed at room temperature (20-22 ~ C) for electron microscopy The entire muscle was immersed for 1.5 h in a 0.1 M Sorenson's phosphate buffer solution containing 3~ glutaraldehyde; 1% formaldehyde; 3% NaC1; 4% sucrose and 0.01 M CoC12 Next the muscle was washed for 0.5 h in 0.1 M Sorenson's buffer solution containing 8%

sucrose (pH 7.4) During this period the dye-marked fiber was isolated and subsequently post-fixed for

1 h in 2% osmium tetroxide Following a brief (5-10rain) wash in the buffer solution, the fiber was embedded in an Epon-araldite mixture Thin serial sections were mounted on Formvar coated single slot grids They were stained with uranyl acetate and lead citrate and examined with a Zeiss 9S or a Siemens

102 electron microscope A record was kept of the thickness of each section Measurements of the number and surface area of synapses were made from serial micrographs with a final magnification of 26,400 The surface area of dense bodies was measured from micrographs magnified to x 132,000 and

• 198,000 A detailed description of the methods for obtaining the surface area of synapses and dense bodies cut in serial section appears elsewhere (Govind and Chiang, 1979)

Results

Selection of Low- and High-Output Synapses

The single excitatory axon to the D A F M gives rise to physiologically diverse synapses which are regionally distributed such that those on proximal muscle fibers release comparatively little transmitter (low-output types) whereas those on distal muscle fibers release greater a m o u n t s (high-output types) (Meiss and Govind, 1979b) F o r the present study the quantal content of synaptic transmission was obtained for terminals on a proximal and a distal muscle fiber On each muscle fiber, a focal synaptic site was located with an extracellular electrode, and 200 300 consecutive ERSPs were recorded at 1 Hz stimulation in trains of 20 impulses with inter-train intervals of 2 min to minimize possible synaptic depression (Fig 1) The mean quantal content (m) calculated by means o f the failure method (Del Castillo and Katz, 1954; Dudel a n d Kuffler, 1961) was 0.27 and 5.29 at each o f the synaptic sites on the proximal and distal fibers respectively These representative examples o f low- and high-output, synapses were subsequently fixed for electron microscopy and serially sectioned

Quantitative Comparison of Low- and High-Output Synapses

The fine structure o f neuromuscular terminals and synapses on the proximal and distal fibers o f the D A F M is shown in Figs 2 4 The terminal regions are in contact with granular sarcoplasm o f the muscle (Fig 2), and this criterion is used to distinguish them from axonal regions (Atwood and Morin, 1970) Within the terminals, synapses were identified according to conventional criteria o f densely stained pre- and postsynaptic membranes, presynaptic vesicles, and dense bodies (Gray, 1963) Analysis of single sections reveals no qualitative differences between the low- (Figs 2, 3) and high-output (Fig 4) neuromuscular terminals In fact, they resemble other lobster (Govind and Chiang, 1979) and crustacean (Hoyle and McNeil, 1968; A t w o o d et al., 1977, 1978) neuromuscular terminals It is only when these terminals are serially sectioned and quantitatively analysed that differences between them are seen (Table 1)

The low- and high-output terminals serially sectioned for a c o m p a r a b l e length (14.17 lam and 15.36 ~tm respectively), show that over this length the latter terminal has twice as m a n y synapses as the former (37 versus 18; Fig 5) This is also evident

f r o m the fact that the high-output terminal has an average o f 2.40 synapses for each

458 C.K Govind and D.E Meiss

Fig 1 Electrical recordings from high-output neuromuscular terminal of distal accessory flexor muscle

of lobster at a fast time sweep A showing a single response and at a slow time sweep B showing multiple responses at 1 Hz stimulation Upper trace, extracellularly recorded synaptic potentials (ERSPs) for calculating quantal content; lower trace, intracellulady recorded excitatory post-synaptic potentials (EPSPs) for monitoring nerve activity Calibration: Vertical, upper trace 400 gV; lower trace 10 mV: Horizontal, (A)20ms; (B) 4s

micrometer o f muscle fiber compared to 1.26 synapses for the low-output counterpart Despite the the two-fold difference in the number of synapses, their total surface area is similar in the two types o f terminals, the reason being that the mean size o f individual synapses is significantly smaller (about one half) in the high- output terminal than in the low-output one Consequently, the total synaptic area is similar in the two types o f terminals and cannot account for the differences in quantal output between them

However, comparison o f the presynaptic dense bodies between low- and high- output terminals reveals differences that correlate with the differences in quantal output between the two terminals There are fewer dense bodies in the low-output terminal compared to the high-output one (9 versus 22; Fig 5) However, since synaptic number follows a similar unequal ratio between the two types o f terminals, the mean number o f dense bodies per synapse is not significantly different The distribution of the dense bodies is also similar in both types o f terminals (Fig 5), with about a third o f the synapses having one dense body and more than half not possessing a dense body The only difference in dense body distribution is that the high-output terminal has more synapses with two or more dense bodies than its low-output counterpart (Fig 5)

Fig 2 Low-output terminal (T) with single long synapse (between long arrows) associated dense body (D) and synaptic vesicles (V) Terminalin contact with granular sarcoplasm (G) of muscle and connective tissue (C) Scale mark: 0.25 ~tm Magnification: x 26,400

Fig 3 High power micrograph of low-output synapse (between long arrows) showing darkly stained pre- and post-synaptic membranes, synaptic vesicles (V) and bar-type presynaptic density (D) T terminal; G granular sarcoplasm Scale mark: 0.25 gin Magnification: x 198,000

Fig 4 High-output terminal (T) showing synapses (between long arrows) with presynaptic densities (D) with lateral (bar-type) and cross-sectional configurations V synaptic vesicles; G granular sarcoplasm Scale mark: 0.25 ~un Magnification: x 112,000

Table 1 Quantitative comparison of synapses and presynaptic dense bodies between low- and high-

output neuromuscular terminals produced by the single motor axon to the distal accessory flexor muscle

of the lobster

Low-output High-output P Terminal Terminal (Students'

t-test) Quantal content of synaptic transmission (m) 0.27 5.29

Length of muscle fiber serially sectioned (l~m) 14.17 15.36

Number of synapses per lam of fiber length 1.26 2.40

Total surface area of synapses (sq lam) 14.20 14.82

Mean surface area of synapses (sq ~tm) 0.79+ 0.12 0.42+ 0.07 1%

(.~+_ S.E.M.)

Mean number of dense bodies per synapse 0.50+ 0.19 0.59+ 0.14 NS

(.~+ S.E.M.)

% of synapses without dense bodies 61 57

% of synapses with one dense body 33 32

% of synapses with two or more dense bodies 6 11

Total surface area of dense bodies (sq pm) 0.20 1.62

Total dense body area as per cent of total 1.4 10.9

synaptic area

Mean surface area of dense bodies (sq ~tm) 2%

(X+ S.E.M.)

Mean ratio of dense body area to synaptic area 5%

(for all synapses) (X+ S.E.M.)

Mean ratio of dense body area to synaptic area 1%

(for synapses with dense bodies only)

(.~+ S.E.M.)

0.022+ 0.004 0.074 _ 0.012 0.021 _ 0.009 0.120+ 0.031 0.054+ 0.018 0.277+ 0.048

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0 6 0 8 l o 1:2 14 1 6 1:8 2 0 2'2 2 4 2 6

SYNAPTIC AREA (um 2) Fig 5 Histogram to show size distribution of 18 low-output A and 37 high-output B serially sectioned synapses from distal accessory flexor muscle of lobster Occurrence of presynaptic dense bodies shown

by closed circles within each synapse

The disparity between the two terminals is even more striking when the size of the dense bodies is considered Thus the mean surface area of individual dense bodies is significantly larger in the high-output terminal This fact, combined with their greater number in this terminal, results in an eight-fold increase in total dense body area in the high-output terminal compared to the low-output one This comparison involves absolute values which are determined by the length of the terminals sampled In order to compensate for differences in length sampled for the low and high-output terminals, the dense body area is considered as a function of the synaptic area in two respects Firstly, when the total dense body area is viewed

as a percentage of the total synaptic area, there is an eight-fold increase in favour of the high-output terminal Secondly, when the mean ratio of dense body area to synaptic area is taken, a statistically significant increase in this ratio is seen for the high-output terminal This ratio is determined both for all synapses and for synapses with dense bodies only Clearly, the high-output terminal allocates a greater proportion of its synaptic area to dense bodies than the low-output terminal

Discussion

The physiological diversity of synapses arising from a single motoneuron is well established in crustacean muscle (reviewed by Atwood, 1976) The ultrastructural correlates of this diversity have been studied only recently Thus highly facilitating terminals from the lone excitor axon to the spider crab stretcher muscle have a higher density of synaptic vesicles than poorly facilitating terminals in randomly sectioned tissue (Sherman and Atwood, 1972) A quantitative study based on serial section electron microscopy reveals that the average size (surface area) of excitatory synapses is significantly smaller than that of inhibitory ones in the crayfish opener muscle (Jahromi and Atwood, 1974) As the inhibitory terminals release relatively greater amounts of transmitter than the excitatory ones at low frequencies of stimulation, a relationship between transmitter output and synaptic size was suggested However, subsequent studies have not confirmed this relationship, e.g., the quantal content is similar in synapses of the gastric mill muscles GM 8 b, GM 9 (Atwood et al., 1977) and pyloric muscle P1 (Atwood et al., 1978) of the blue crab though the former has considerably smaller synapses than the latter Furthermore, synapses of the fast axon in Pachygrapsus (Atwood and Jahromi, 1978) are considerably smaller in size than GM8b, GM9, and P1 synapses in blue crabs even though their quantal output is greater A better correlation is found between transmitter release and presynaptic dense bodies Consequently, whereas dense bodies were found in all synapses of the fast axon of Pachygrapsus they occurred in only 75~ of the GM8b, GM9 and 50~ of the PI synapses of blue crabs These comparisons, however, are between separate species and not as persuasive as comparisons within a single species

When low- and high-output synapses are compared in the proximal accessory flexor muscle (PAFM) of the lobster (Govind and Chiang, 1979), dense bodies occur in only 40~ of the low-output synapses but in 60~ of the high-output ones Furthermore, the mean ratio of dense body area to synaptic area is significantly

462 C.K Govind and D.E Meiss

greater in the high-output terminals than in the low-output terminal This parallels

a similar difference in the size of the intracellularly recorded EPSP between the two muscle fibers As both fibers have a similar membrane input resistance, the difference in EPSP size is attributable to differences in quantal output of transmitter In the present study on the lobster DAFM a refinement was added in that the quantal content was determined at synaptic foci and these very same regions were serially sectioned As in the PAFM, a significant increase was found in the ratio of dense body to synaptic surface area in the high-output terminal as compared to its low-output counterpart Consequently, the present study reiterates the correlation between transmitter output and amount ofpresynaptic dense bodies

at neuromuscular terminals of the lobster

The accessory flexor muscle of crustaceans is bipartite consisting of a proximal (PAFM) and a distal (DAFM) head situated at either end of the meropodite segment and connected to each other by a long slender tendon (Cohen, 1963; Govind et al., 1978) Since both heads are innervated by a common excitatory axon

it is interesting to compare how the differentiation of presynaptic dense bodies characteristic of low- and high-output terminals is achieved in each muscle In the PAFM where the mean size of the dense bodies is similar in low- and high-output terminals (0.014 vs 0.02 sq lam respectively), the mean number of densities per synapse is significantly higher in the high-output terminal than in its low-output counterpart (0.73 vs 0.42 respectively; Govind and Chiang, 1979) On the other hand, in the DAFM, where the mean size of the dense bodies is different in low- and high-output terminals (0.022 vs 0.074 respectively), their mean frequency is similar

in low- and high-output synapses (0.50 vs 0.59 respectively; Table 1) Consequently differences in surface area of dense bodies between the two types of terminals are due to differences in their numbers in the PAFM and in their size in the DAFM If indeed these findings are substantiated, they would suggest that two different mechanisms may occur in a single axon to achieve differentiation of presynaptic dense bodies

Presynaptic dense bodies seem ubiquitous in synapses of both vertebrates (Pfenninger, 1973) and invertebrates (Wood et al., 1977) Though they vary considerably in form, they are associated with the presynaptic membrane and are surrounded by synaptic vesicles Therefore, they have been assigned some role in the transmitter release mechanism They could act as focal points to which synaptic vesicles are attracted and held until their contents are released into the synaptic cleft (Wernig and Stirner, 1977), or as local storage sites (Finlayson and Osborne, 1975) Their occurrence in the lobster DAFM tends to corroborate the view that they are involved in the release of transmitter, especially since differences in the relative surface area of these bodies correspond to differences in quantal output This is still circumstantial evidence for their possible role in transmitter release, and further studies are required in which these bodies are "altered" in response to experimental manipulation of quantal transmitter output Such studies have revealed changes in the frequency of synaptic vesicles (e.g., Ceccarelli et al., 1973) but not in the presynaptic densities which do not appear to be as transient as vesicles and consequently may change only over long periods of time such as during growth

References

Atwood, H.L.: Organization and synaptic physiology of crustacean neuromuscular systems Prog Neurobiol 7, 291-391 (1976)

Atwood, H.L., Jahromi, S.S.: Fast axon synapses of a crab leg muscle J Neurobiol 9, 1-15 (1978) Atwood, H.L., Morin, W.A.: Neuromuscular and axo-axonal synapses of the crayfish opener muscle J Ultrastruct Res 32, 351-369 (1970)

Atwood, H.L., Govind, C.K., Jahromi, S.S.: Excitatory synapses of blue crab gastric mill muscles Cell Tissue Res 177, 145-158 (1977)

Atwood, H.L., Govind, C.K., Kwan, I.: Non-homogeneous excitatory synapses of a crab stomach muscle J Neurobiol 9, 17-28 (1978)

Bittner, G.D.: Differentiation of nerve terminals in the crayfish opener muscle and its functional significance J Gen Physiol 51, 731-758 (1968)

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

Cohen, M.J.: The crustacean myochordotonal organ as a proprioceptive system Comp Biochem Physiol 64, 41-54 (1963)

Del Castillo, J., Katz, B.: Quantal components of the endplate potential J Physiol (Lond.) 124, 560-

573 (1954)

DeRosa, R.A., Govind, C.K.: Transmitter output increases in an identifiable lobster motoneuron with growth of its muscle fibers Nature (Lond.) 273, 67~678 (1978)

Dudel, J., Kuffler, S.W.: The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction J Physiol (Lond.) 155, 514-529 (1961)

Finlayson, L.H., Osborne, M.P.: Secretory activity of neurons and related electrical activity Adv Comp Physiol Biochem 6, 165-258 (1975)

Govind, C.K., Chiang, R.G.: Correlation between presynaptic dense bodies and transmitter output at lobster neuromuscular synapses by serial section electron microscopy Brain Res 161, 377-388 (1979)

Govind, C.K., Atwood, H.L., Lang, F.: Synaptic differentiation in a regenerating crab-limb muscle Proc Natl Acad Sci U.S.A 70, 822-826 (1973)

Govind, C.K., Meiss, D.E., She, J., Yap-Chung, E.: Fiber composition of the distal accessory flexor muscle in several decapod crustaceans J Morphol 157, 151-160 (1978)

Gray, E.G.: Electron microscopy of presynaptic organelles of the spinal cord J Anat 97, 101-106 (1963)

Hoyle, G., McNeil, P.A.: Correlated physiological and ultrastructural studies on specialized muscles Ic Neuromuscular junctions in the eyestalk levator muscles of Podophthalmus vigil (Weber) J Exp Zool 167, 523-550 (1968)

Jahromi, S.S., Atwood, H.L.: Three-dimensional ultrastructure of the crayfish neuromuscular apparatus J Cell Biol 63, 599~13 (1974)

Katz, B.: Nerve, Muscle and Synapse New York: McGraw Hill Book Co (1966)

Meiss, D.E., Govind, C.K.: Multiterminal innervation: non-uniform density along single lobster muscle fibers Brain Res 160, 163-169 (1979a)

Meiss, D.E., Govind, C.K.: Regional differentiation of neuromuscular synapses in a lobster receptor muscle J Exp Biol In Press (1979b)

Pfenninger, K.H.: Synaptic morphology and cytoehemistry Progr Histochem Cytochem 5, 1-86 (1973)

Sherman, R.G., Atwood, H.L.: Correlated electrophysiological and ultrastructural studies of a crustacean motor unit J Gen Physiol 59, 586-615 (1972)

Thomas, R.C., Wilson, V.J.: Marking single neurons by staining with intraceUular recording microelectrodes Science (Wash.) 151, 1538-1539 (1966)

Wernig, A., Stiruer, H.: Quantum amplitude distributions point to functional unity of the synaptic 'active zone' Nature (Lond.) 269, 820-822 (1977)

Wood, M.R., Pfenninger, K.H., Cohen, M.J.: Two types of presynaptic configurations in insect central synapses: an ultrastructural analysis Brain Res 130, 22-45 (1977)

Accepted February 13, 1979