Bouton ultrastructure and synaptic growth in a frog autonomic ganglion

THE JOURNAL OF COMPARATIVE NEUROLOGY 281:159-168 (1989)

Bouton Ultrastructure and Synaptic Growth in a Frog Autonomic Ganglion

LAURA C. STREICHERT AND PETER B. SARGENT Neurosciences Program, Stanford University, Stanford, California 94305 (L.C.S.); Division of

Biomedical Sciences, University of California, Riverside, California 92521 (L.C.S., P.B.S.)

ABSTRACT Postmetamorphic growth in the frog, Xenopus laeuis, is accompanied by

an increase both in the size of autonomic neurons in the heart and in the number of synaptic boutons that contact their surface. To determine whether the properties of individual boutons change as their number increases, serial-section electron microscopy was used to examine bouton ultrastructure a t the end of metamorphosis and in the adult. The area of bouton contact, number of active zones per bouton, active zone size, percent of bouton area occupied by active zone, and vesicle density were examined. No differences were found between the two bouton populations for any of the parameters examined. These results support the hypothesis that boutons are structural units of synaptic growth, whereby the total area of synaptic contact increases through the addition of boutons without a change in their morphological properties.

Key words: cardiac ganglion, synapse formation, perforation, active zone, electron microscopy

As animals develop and grow, the targets of many neurons increase dramatically in size. How does the nervous system respond to these changes while concurrently maintaining effective synaptic transmission? Although there have been a number of ultrastructural studies of synaptic growth a t the neuromuscular junction (Atwood and Kwan, '76; Easter, '79; Rheuben and Kammer, '81; Walther, '81; Davey and Ben- nett, '82; Govind and Pearce, '82; Govind and DeRosa '83; Pearce et a)., '85), the growth of contacts between neurons has not been extensively characterized.

The cardiac ganglion of the frog is an excellent model system for the study of synaptic growth. The neurons within this autonomic ganglion are easily visualized within the transparent interatrial septum. The cell body of each unipolar neuron is innervated by a preganglionic axon, which ends in a number of preterminal and terminal swellings called synaptic boutons. Both the anatomy and the physiology of synapses in this system have been well characterized (McMahan and Kuffler, '71; Dennis et al., '71).

During the postmetamorphic growth of Xenopus there is a substantial increase both in ganglion cell size and in the number of synaptic boutons contacting the cell surface. The number of boutons increases in proportion to the surface area of the ganglion cell body; i.e., bouton density remains constant (Sargent, '83a,b). This suggests that the bouton is a structural "building block" of synaptic growth, whereby the total area of synaptic contact increases by an increment in bouton number without a change in the properties of indi-

vidual boutons. We have tested this hypothesis by compar- ing the fine structure of boutons a t two stages of postmetamorphic development. Our morphometric analysis supports the hypothesis that boutons are structural units of synaptic growth.

Preliminary results of this work have been published in abstract form (Streichert et al., '87).

MATERlALs AND METHODS Animals and dissection

Xenopus laevis were obtained from the laboratory breed- ing colony. The adults had a body length of 55-60 mm. Frogs completing metamorphosis had a body length of 16-19 mm and were classified as stage 66 animals according to the cri- teria of Nieuwkoop and Faber ('56).

Animals were anesthetized with 2 mM aqueous tricaine methanesulphonate (Sigma) and pithed. Interatrial septa were dissected in frog Ringer's (114 mM NaC1, 2 mM KC1, 1.8 mM CaCl,, 2 mM HEPES, pH 7.4) and pinned flat on Sylgard-lined petri dishes.

Cell size and bouton counts Cardiac ganglion cells are shaped like prolate ellipsoids,

with their long axis parallel to the plane of the interatrial

Accepted October 27,1988.

0 1989 ALAN R. LISS, INC.

160

septum. The major and minor axes of the cell were measured by using an eyepiece reticule, and the surface area was calculated by formula (Sargent, '83a).

Boutons on the cell bodies of neurons at the end of metamorphosis (stage 66) and in the adult were stained by means of the zinc iodide-osmium technique (Maillet, '62). Ganglia were stained as described in Sargent ('83a) except that stage 66 ganglia were treated at pH 5.1 rather than pH 4.6 for 2.5 hours rather than for 1 hour. Under these conditions, all boutons at stage 66 are impregnated with electron-dense reaction product, as determined by electron microscopy. (Of 306 synapses examined in six ganglia, 303 were heavily impregnated and three were lightly impregnated with stain.) All boutons in adult ganglia are also stained with this technique (Sargent, '83a). Boutons were counted at a final magnification of 1 , 2 5 0 ~ with Nomarski optics.

Electron microscopy Septa were fixed in 1 % glutaraldehyde with 0.3 % tannic

acid in 0.09 M Na phosphate buffer, pH 7.3, postfixed in 1 % osmium tetroxide in 0.09 M Na phosphate buffer, stained en bloc with 2% uranyl acetate in water, dehydrated in ethanol and propylene oxide, and embedded as wafers in Epon/Aral- dite. Areas containing cardiac ganglion cells were cut out and remounted on Beem capsules. Semithin (1 pm) sections were taken until a cluster of ganglion cells was present at the block face, following which ultrathin (80 nm) serial sections were collected with a Reichert ultramicrotome. Sections having silver interference color were placed on Formvar- coated slot grids, which were then stabilized with a light carbon coating. Tissue was viewed with a Hitachi H-600 transmission electron microscope without grid staining, and sections were photographed at a magnification of 15,000- 25 ,000~ .

Data analysis The boutons analyzed were those encountered on ran-

domly selected cell bodies. The lengths of the ganglion cell surface in contact with both boutons and active zones in individual thin sections were measured on prints of electron micrograph negatives by using a digitizing tablet and micro- computer. An average of 35 sections were contained in each series. Bouton contact area was determined by the sum of the values for bouton-soma contact multiplied by the mean value for section thickness based on diffraction color (80 nm). Active zone area was determined in a similar way. Val- ues for the contact area of perforated active zones did not include the area of the perforation(s). Contact length for missing sections was estimated by averaging the data for the immediately adjacent sections. Nearly 80% of the gaps con- sisted of only a single section, and missing sections accounted for only 11% of the total. It is improbable that such a loss of sections could have significantly affected the results.

The density of synaptic vesicles adjacent to each active zone was measured by counting the number of vesicles lying entirely within 0.2 pm of the presynaptic membrane over a length of 0.2 pm. A single measurement was made for each active zone by choosing the central 0.2 pm in the thin section having the longest active zone length.

Statistical comparisons of mean values measured in stage 66 and adult animals were made by means of the Student's t-test and Mann-Whitney U-test. Comparisons of correlation coefficients were made by using Fisher's transforma- tion (Zar, '74).

L.C. STREICHERT AND P.B. SARGENT

RESULTS Bouton morphology and density

The cardiac ganglion is situated within the interatrial septum, where several hundred parasympathetic neurons lie along the cardiac branches of the paired vagosympathetic nerves. The ganglion cells are unipolar, ellipsoidal cells with an axon which joins the intracardiac nerve trunk and pro- jects to cardiac muscle. The preganglionic axons originate in the dorsal motor nucleus of the vagus and appear to spiral around the postganglionic axon before forming synapses with both the axon and the soma of the ganglion cell via preterminal and terminal swellings called synaptic boutons. In the present study, only boutons found on the ganglion cell body were analyzed. This population represents a majority of the boutons contacting the cell; in adults, virtually all boutons contacting the ganglion cell body arise from a single axon (Sargent, '86).

During postmetamorphic growth there is a positive relationship between ganglion cell body size and number of boutons; larger- cells have proportionally more boutons than smaller ones (Sargent, '83b). At the end of metamorphosis the density of boutons per 1,000 pm2 of cell body surface area is similar to that in adults despite the approximately threefold difference in both soma1 surface area and number of boutons between those two stages of development (Table 1). This is similar to the situation in developing locust neuromuscular junctions in which the density of synaptic contacts undergoes little change while the number increases by at least an order of magnitude (Walther, '81).

The fine structure of boutons and active zones is qualitatively similar in stage 66 and adult frogs (Fig. 1). Boutons are recognized by the presence of small, clear vesicles and large, dense-cored vesicles. Active zones, the presumed sites of transmitter release (Heuser et al., '79), are characterized by dense pre- and postsynaptic specializations and by an accumulation of clear synaptic vesicles along the length of the presynaptic specialization. The dense-cored vesicles tend to be found around the perimeter of clusters of clear vesicles. All boutons examined in the cardiac ganglion have at least one active zone; approximately 60% have only one, 25% have two, and the remainder have three to five active zones.

Structures resembling puncta adhaerentia were seen in approximately 20% of the boutons examined in both postmetamorphic and adult animals (Fig. 1). These structures are characterized by symmetrical pre- and postsynaptic densities and are usually not associated with synaptic vesicles. They have a mean area of 0.05 * 0.03 pm2 (n = 9) and are considerably smaller than most active zones. These junctions may promote adhesion between the bouton and the ganglion cell (Peters et al., '76).

Morphometric analysis Synaptic boutons from postmetamorphic and adult gan-

glia were compared with regard to several parameters: bouton contact area, number of active zones per bouton, active zone contact area, percentage of bouton contact area occupied by active zone, and synaptic vesicle density. An analysis of 21 boutons from 18 postmetamorphic cells and 23 boutons from 16 adult cells revealed no significant differences for any of the parameters examined (Table 2). Thus, individual synaptic boutons are structurally similar at the two stages of growth. What distinguishes the two stages is the

SYNAPTIC ULTRASTRUCTURE AND GROWTH 161

Fig. 1. Transmission electron micrographs of synaptic boutons on cardiac ganglion cells in adult (A) and stage 66 (B) Xenopus laeuis. The extent of bouton contact with the ganglion cell (large arrows) and active zone contact (small arrows) are indicated in each micrograph. A punc-

turn adhaerens-like structure (arrowhead) is present in A. Total bouton and active zone contacts were reconstructed from serial sections as described in Materials and Methods. Scale bar equals 0.5 pm.

number of boutons: larger cells in adult animals have more boutons than smaller cells in postmetamorphic animals (Ta- ble 1).

In both stage 66 and adult frogs, there is a considerable degree of variability in the parameters measured (Table 2). For example, bouton contact area varies by up to tenfold, from approximately 1.5 to 15 pm2. The range in values is as great among boutons on the same cell as it is among boutons on different cells (data not shown). This implies that the variability cannot be attributed to differences in bouton structure from one cell to the next. When the variability is examined more closely, several interesting features of bouton structure and composition emerge. For instance, there is a significant correlation between the size of the bouton contact area and the number of active zones: larger boutons tend to have more active zones (P < .001; Fig. 2). There is also considerable variability in active zone size, which ranges from 0.04 pm2 to more than 1 pm2 in both stage 66 and adult animals. When data from all boutons are analyzed, active zone size is found to be independent of bouton

TABLE 1. Bouton Number, Cell Size, and Bouton Density in Stage 66 and Adult Cardiac Ganglia'

Stage 66 Adult ln = 7) ln - 11) P

3.5 * 1.1 10.6 * 1.4 <.001 Boutons per cell body

Loo00 urn*\

Cell body size (pm? 450 f 120 1,400 + 180 <.001 7.7 i 1.8 7.5 f 1.0 2.4 Bouton density (per

'Data shown are the means * the standard deviations, with the number of ganglia shown in parentheses (1&70 cells analyzed per ganglion). Adult data taken from Sargent ('83a). Comparisona were made by using the Mann-Whitney U-test.

size ( P > .05; Fig. 3). Thus, larger boutons do not appear to have larger active zones, only more of them.

If larger boutons have more active zones than smaller boutons, while mean active zone size remains constant, then total active zone area should increase as bouton size increases. In fact, larger boutons do have proportionately

162

TABLE 2. Bouton Ultrastructure in Stage 66 and Adult Cardiac Ganglia'

Staee 66 Adult

L.C. STREICHERT AND P.B. SARGENT

Bouton contact area (rm')

No. of active zones per bouton

Active zone area (em2)

% of bouton contact area oecupied

No. of vesicles per active zone

4.88 f 2.93 (n = 21) 1.76 t 1.06

(n = 37) 0.36 -r 0.28

(n - 37) 13.9 t 8.6 (n - 21) 13.3 * 2.5 (n - 37)

by active zone

4.56 f. 2.80 (n - 23) 1.43 f 0.71

(n - 33) 0.32 f 0.22

(n = 33) 10.3 _+ 4.5

(n - 23) 13.3 t 2.8

(n - 33) 'Each of five variables waa analyzed in 21 serially reconstructed boutons from cardiac ganglia at the end of metamorphosis (stage 66) and in 23 serially reconstructed boutons from adult ganglia. No significant differences were found between any of the variables. Compar- isons were made by using the Mann-Whitney U-test.

more total active zone area than smaller boutons (P < .001; Fig. 4). Is this correlation attributed solely to larger boutons having more active zones? The majority (62%) of the boutons examined have only one active zone, and differences in active zone number clearly cannot be a factor for this major bouton subpopulation. When boutons with only one active zone are examined, larger boutons have larger active zones (P < .01; Fig. 5, filled squares). Thus, active zone size is correlated with bouton size for this subclass of boutons, although not for the entire bouton population (Fig. 3). Since most of the small boutons have only one active zone, there is a tendency for active zone size to increase with bouton size until bouton size reaches approximately 4 pm2, beyond which an increasing number of boutons with more than one active zone are encountered. The sum of the active zone

areas continues to increase for larger boutons (Fig. 4), while individual active zone size does not (Fig. 3). For example, the total active zone area for 4.0-7.0-pm2 boutons having two active zones is similar to that of single active zone boutons of the same size (0.66 0.44 pm2, n = 7 us. 0.59 +- 0.32 Km2, n = 8; P > .05) Consequently, individual active zone size for boutons with two active zones is approximately half that for single active zone boutons in this size range. The reduction in individual active zone size which accompanies the progression from small boutons to large boutons ex- plains why the correlation between bouton size and active zone size observed for single active zone boutons is obscured when all data are pooled.

Active zones are heterogeneous in shape as well as size. Smaller active zones are generally disc-shaped; larger active zones have more complex shapes and often have one or more perforations (Fig. 6). Perforations are characterized by an interruption in the electron-dense specializations and a lack of associated synaptic vesicles (Fig. 7). Serial sections must, be analyzed to distinguish a perforated active zone from two separate active zones which might be seen in the same section.

Approximately 30% of the all active zones examined had perforations. There is a significant correlation at both stage 66 and in the adult between the number of perforations and the size of the active zone: larger active zones tend to have more perforations (Fig. 8). The perforated active zones in the cardiac ganglion resemble the annular and horseshoe- shaped postsynaptic densities found in the central nervous system (Peters and Kaiserman-Abramof, '69; Peters et al., '76; Cohen and Siekevitz, '78; Greenough et al., '78; Vrensen

A stage 66 A adult

5 I A A

A A

A

A

A

r = 0.722 P < 0.001

v

1 2 3 4 5

NUMBER OF ACTIVE ZONES Fig. 2. Bouton size is significantly correlated with active zone num-

ber. Bouton contact area (size) is plotted against number of active zones for 21 postmetamorphic boutons (filled triangles) and 23 adult boutons (open triangles). The x-values for the two stages are slightly displaced for clarity. For each stage there was a highly significant correlation

between the two variables ( P < .001); larger boutons tend to have more active zones. Neither the slopes of the regression lines nor the coefficients of correlation for the two populations were significantly different ( P .05). Regression line, r value, and P value calculated for the pooled data.

SYNAPTIC ULTRASTRUCTURE AND GROWTH

1.0

163

'

A stage 66 A adult

A

A

A

A

A

A

A A A A

A A A

A

r = 0.032 P > 0.05

A

- A

b A A A ~ A A A 4 A

f A AA A

A A A ' A A A

' A A, A A A , f 0.0 0 5 10 15

BOUTON SIZE (square microns)

Fig. 3. Active zone size is not significantly correlated with bouton size. Active zone size is plotted against bouton size (contact area) for 37 postmetamorphic active zones (filled triangles) and 33 adult active zones (open triangles). There was no significant correlation between the

two variables for either set of data (P z .05). Neither the slopes of the regression lines nor the coefficients of correlation for the two populations were significantly different (P > .05). Regression line, r value, and P value calculated for the pooled data.

2.0

L5

1.0

05

0.0

A stage 66 A adult

A

A

A

A

r = 0.677 P < 0.001

I I

0 5 10 15


Fig. 4. Bouton size is significantly correlated with total active zone area. The total active zone area, defined as the sum of all active zone areas per bouton, is plotted against bouton size (contact area) for 21 postmetamorphic boutons (filled triangles) and 23 adult boutons (open triangles). For each set of data there was a significant positive correla-

tion between the two variables (P < .001 for postmetamorphic data and P < .01 for adult data). Neither the slopes of the regression lines nor the Coefficients of correlation for the two populations were significantly different ( P z .05). Regression line, r value, and P value calculated for the pooled data.

164 L.C. STREICHERT AND P.B. SARGENT

1.5

1.0

0.5

0.0 0

A

r P

A

= 0582 < 0.01

A

5 10 15


Fig. 5. Active zone size is significantly correlated with bouton size for boutons having one active zone. Active zone size is plotted against bouton size, with data from boutons having one active zone indicated by filled squares and those having more than one active zone by open triangles. The data shown are the same as in Figure 3. The line shown is the regression line for one-active-zone boutons, and the r and P values apply to this bouton subpopulation. When all the data are analyzed, there is no significant positive correlation between the two variables (Fig. 3) in

large part because the data for multiple-active-zone boutons. Data shown have been pooled from both stages. For stage 66 single-active- zone data the correlation between the two variables wm not significant (0.1 > P > .05) while for adult data it was (P < .001). The slopes of the regression lines for single-active-zone data for the two populations were not significantly different (P > .05) nor were their coefficients of correlation (P > .05).

and Nunes Cardozo, '81; Triller and Korn, '82). Moreover, the positive relationship between active zone size and geo- metric complexity in the cardiac ganglion is a common feature of active zone structure (Peters and Kaiserman-Abra- mof, '69; Vrensen and Nunes Cardozo, '81; Nieto-Sampedro et al., '82; Dyson and Jones, '84; Pearce et al., '85; Calverley and Jones, '87).

DISCUSSION The proliferation of synaptic contacts is a characteristic

feature of developing nervous systems. In the cardiac ganglion of the frog, synaptic growth is expressed by the addition of new boutons upon growing cells. The quantitative analysis presented in this study demonstrates that the fine structure of synaptic boutons is similar at two developmental stages which demarcate a period of substantial synaptic growth. These results support the hypothesis that synaptic growth is mediated by an increase in the number of boutons rather than by changes in bouton morphology.

In the cardiac ganglion of both stage 66 and adult frogs, there is considerable variability in the size of synaptic boutons. One possible explanation for this heterogeneity is that there are different classes of boutons on the ganglion cell surface. Large boutons with two or more active zones may be qualitatively different structures than small boutons with single active zones. However, virtually all boutons which contact an individual cell body in adults arise from a single

preganglionic input (Sargent, '86). It seems probable that boutons which arise from a single axon and innervate the same target cell would be of a similar type. Differences in features such as active zone size and levels of transmitter release have been observed between different terminals of a single motor neuron in crustacea, but in that system the distinct classes of terminals are found to synapse upon different types of muscle fibers (Govind and Chiang, '79; Govind and DeRosa, '83).

An alternative explanation for the heterogeneity in bouton structure in the cardiac ganglion is that it represents a single population of boutons at different stages of growth and/or reorganization. Some of the new boutons which appear on the surface of growing ganglion cells may arise from preexisting boutons which enlarge and divide. Bouton division may occur in conjunction with changes in active zone structure, as proposed by others (Nieto-Sampedro et al, '82; Carlin and Siekevitz, '83; Dyson and Jones, '84). One strategy by which this could occur in the cardiac ganglion is shown in Figure 9. Small boutons with single active zones enlarge with a parallel increase in active zone size. Perfora- tions may eventually appear in the active zone, which subse- quently splits into two active zones. The bouton then divides, with each daughter inheriting one of the active zones. Alternatively, the bouton and its active zones might continue to grow before dividing, yielding more elaborate configurations (Fig. 9). This scheme is consistent with several findings, namely: 1) larger boutons with single active

SYNAPTIC ULTRASTRUCTURE AND GROWTH

staee 66 165

" - -

. . . a 6 8 *

Fig. 6. Two-dimensional reconstructions of serial electron microscope sections of representative active zones. Active zones were ranked according to size for both stage 66 and adult data, and every third active zone was displayed. At both stages, larger active zones tend to have perforations and more complex shapes than smaller active zones. The apparent axis of symmetry for each contact is artifactual since the reconstructions were made by centering each active zone length.

zones have larger active zones; 2) active zone size in single active zone boutons is roughly twice that in boutons of comparable size having two active zones; 3) larger boutons have more active zones, and 4) larger boutons have more total active zone area per bouton.

Immature active zones, characterized by the absence of pre- or postsynaptic specializations or synaptic vesicles (Dy- son and Jones, '84; Kunkel et al., '87), were not found in this study. It is possible that the structures interpreted to be puncta adhaerentia are actually nascent synapses which have not yet become associated with synaptic vesicles. How- ever, unlike the synaptic specializations associated with active zones, the densities associated with the puncta adhaerentia appear symmetrical. The failure to find immature synapses in the cardiac ganglion, where the number of synaptic boutons is continually increasing, is consistent with the idea that boutons arise from preexisting ones but does not rule out the possibility that some boutons also arise de nouo. New boutons may differentiate from axonal sprouts rapidly andlor a t low frequency, and the probability that they would be detected in an immature state a t the time of fixation may be small.

Active zones are plastic structures; the number, size, and/ or shape of the active zone is subject to change not only dur-

ing development (Kunkel et al., '87; Markus et al., '87), but also under a number of conditions, such as habituation and sensitization (Bailey and Chen, '83), visual training (Vrensen and Nunes Cardozo, '811, aging (Greenough et al., '78; Nieto-Sampedro et al., '82), enriched environments (Greenough et al., '78), and different hormonal conditions (Hatton and Ellisman, '82). Changes in active zone structure, including the appearance of perforations, may underlie the plasticity of synaptic connections in the developing cardiac ganglion.

The observation that perforations are more prevalent during periods of increased synapse formation and remodelling suggests that they play a role in synapse division (Nieto-Sampedro et al., '82; Carlin and Siekevitz, '83; Dyson and Jones, '84). The positive correlation between active zone size and the number of perforations in the cardiac ganglion (Fig. 8) supports the notion that perforations appear once the active zone enlarges to a threshold size or shape and predispose the active zone to divide into two smaller structures. The failure to find any boutons lacking active zones suggests that boutons do not divide or form de nouo without prior or concomitant active zone division.

Regardless of bouton size or developmental stage (postmetamorphic or adult), a constant fraction of bouton contact area tends to be occupied by active zone (Fig. 4). Although both bouton size and total active zone area differ over a wide range, they do not vary independently of one another. This could be attributed to a parallel response of bouton and active zane size to external growth signals. Another possibility is that changes in one parameter bring about changes in the other; an increase in the size of the active zone may cause the bouton itself to enlarge or, alternatively, active zones may enlarge in response to bouton growth. Regardless of the mechanism generating the covar- iance, the positive correlation between bouton size and total active zone area is likely to have functional consequences. Since larger boutons have more active zones, they are likely to release more transmitter than smaller boutons and gener- ate correspondingly larger postsynaptic currents (Walmsley et al., '85).

Synaptic structure in the cardiac ganglion differs from that of inhibitory inputs to goldfish Mauthner neurons in which almost all boutons contain a single active zone (Triller and Korn, '82). Functional studies in the goldfish system indicate that the number of boutons is equivalent to n, the number of quanta available for release (Korn et al., '82); for each impulse, either zero or one quantum is released per active zone. A similar situation exists for Ia afferents innervating spinal motoneurons in the frog (Grantyn et al., '84) and the cat (Redman and Walmsley, '83). Synaptic boutons in Clarke's column have multiple active zones, and more than one quantum can be released from each bouton per impulse (Walmsley et al., '85). Presumably, a similar situation holds for boutons in the cardiac ganglion having multiple active zones. Therefore, n corresponds to the number of active zones and not boutons per se (Korn and Faber, '87).

If individual active zones comprise the population, n, then presumably more than one quantum can be released from large boutons in the cardiac ganglion which contain multiple active zones. Walmsley et al., '85) propose that this occurs in large boutons on neurons in Clarke's column in the cat. At the neuromuscular junction, differences in active zone size are often associated with differences in transmitter release (Govind and Chiang, '79; Atwood and Marin, '83; Govind and DeRosa, '83; Herrera et al., '85; Propst and KO,

166 L.C. STREICHERT AND P.B. SARGENT

'87). If no more than one quantum can be released from a single active zone, then perhaps the properties of individual active zones determine the value of p , the probability that a quantum will be released. If so, then the substantial variability in the size of individual active zones in the cardiac ganglion raises the distinct possibility that the p values among the population, n, may be distributed rather than uniform (Brown et al., '76).

Despite the synaptic heterogeneity present in the Xeno- pus cardiac ganglion, boutons are morphologically indistin- guishable at the end of metamorphosis and in the adult. The greater number of boutons in the adult probably provide for increased transmitter release, which presumably results in proportionately more synaptic current. An increase in current would offset the decrease in input resistance accompanying cell growth, thereby maintaining a constant safety factor for transmission. Therefore, effective synaptic transmission would be sustained through a matched growth of pre- and postsynaptic elements throughout development.

ACKNOWLEDGMENTS We gratefully acknowledge the contribution of Catherine

Magill-Solc for much of the preliminary work. We thank John Kitasako for invaluable EM assistance, Michael Ad- ams, Albert Herrera, and Carla Shatz for their comments on the manuscript, and Susan Gemmell for graphics. This work

Fig. 7. A perforated active zone demonstrated with serial-section electron microscopy. The three micrographs (A-C) shown are sections 5, 7. and 11 from a set of 17 serial sections of the active zone. There is an

was by NrH grant NS 24157 to P.B.S.

interruption in both the presynaptic and postsynaptic densities seen in the middle micrograph. Scale bar equals 0.2 Gm.

15

1.0

0.5

0.0

a stage 66

r] adult

T

0 1 2

NUMBER OF PERFORATIONS Fig. 8. The number of perforations is significantly correlated with

active zone size. The size of active zones having zero, one, and two perforations is shown for both postmetamorphic and adult animals. Mean values are plotted plus 1 standard deviation, with the number of active zones analyzed indicated within each bar. Stage 66 and adult means were not significantly different from each other for active zones having zero or one perforations (Student's t-test; P > .05). Means for active

zones having one perforation were significantly different from stage- matched means having zero or two perforations (Student's t-test; P < .001 for zero US. one comparisons, P < .02 for one us. two comparison). Despite the fact that double-perforated boutons were found only at stage 66, the Mann-Whitney U-test revealed no significant differences between stage 66 and adult populations (P > .05).

SYNAPTIC ULTRASTRUCTURE AND GROWTH 167

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Fig. 9. Proposed mechanism of bouton enlargement and division in the cardiac ganglion. Possible intermediates are shown in a hypothe- sized scheme by which boutons and their active zones enlarge and divide. The extent of bouton contact is indicated by the large ovals, and the active zones, some of them perforated, are indicated by the small, filled ovals. Bouton growth is accompanied by an increase in active zone number and, a t least for single-active-zone boutons, in active zone size. Perforations appear in larger active zones, possibly predisposing them to divide. Active zone division precedes or is concomitant with bouton division, such that new boutons which lack active zones are not formed. This model is consistent with many of the findings reported in the text.

168 L.C. STREICHERT AM) P.B. SARGENT

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Bouton ultrastructure and synaptic growth in a frog autonomic ganglion