Synaptogenesis in chick paravertebral sympathetic ganglia: a morphometric analysis

Developmental Brain Research, 4 (1982) 229-240 Elsevier Biomedical Press

229

Synaptogenesis in Chick Paravertebral Sympathetic Ganglia: A Morphometric Analysis K. A. HRUSCHAK, V. L. FRIEDRICH, Jr. and E. GIACOBINI Laboratories of Neuromorphology and Neuropsychopharmacology, Department t~f Biobehavioral Sciences U-154, University of Connecticut, Storrs, CT 06268 (U.S.A.)

(Accepted November 3rd, 1981) Key words: synaptogenesis - - sympathetic ganglia - - morphometry

Synaptogenesis was studied in lumbar sympathetic ganglia of chicken by light and electron microscopic morphometric methods. At 10 days in ovo, fewer than 1 ~o of the adult number of synapses are present. The total numbers of synapses and of synaptic vesicles per ganglion increase progressively with age; however, the majority of both are formed after 30 days after hatching. The average number of synaptic vesicles per synapse increases several fold after hatching. The numbers of synapses and of synaptic vesicles per ganglion increase roughly in concert with biochemical markers of presynaptic development (activity of choline acetyltransferase and levels of acetylcholine) as well as postsynaptic development (tyrosine hydroxylase; based on biochemical data reported elsewhere). The amount of acetylcholine and activity of choline acetyltransferase per synaptic vesicle at 10 days in ovo are 8 and 27 times the corresponding adult values. By I day after hatching, these ratios have fallen to near adult levels. These data are consistent with the early presence of cholinergic neuroblasts, as suggested by others, and suggest further that such cholinergic neuroblasts are eliminated, or their cholinergic properties suppressed, before hatching. INTRODUCTION The formation of synapses during development involves the development of synaptic transmission, the induction of neurotransmitter related enzymes, and the appearance of specific cell structures recognized as synapses. The interrelationships between these events are of great interest but are not known in detail la. Sympathetic ganglia have been a useful model system in the study of this question because of the pharmacological disparity between the pre- and postsynaptic elements; the postsynaptic neurons are largely catecholaminergic while the presynaptic fibers are cholinergic. Studies by Black and others 2-6 in the mouse superior cervical ganglion show that the developmental increases in choline aeetyltransferase activity, the enzyme involved in the synthesis of acetylcholine, are congruent to the developmental increases in the number of synaptic adhesions. In addition, tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine, follows a similar course

of development. Choline aeetyltransferase may not be the rate-limiting enzyme in the synthesis of acetylcholine 15 and the activity of this enzyme may therefore not be a good index for the amount of acetylcholine present during development 21. Clarification of the relationship between synapse numbers and the amount of neurotransmitter (acetylcholine) present thus requires direct determination of acetylcholine levels and of synapse numbers in the same system. Information on the development of acetylcholine content, as well as choline aeetyltransferase, acetylcholinesterase, and tyrosine hydroxylase activities, is available in the chick lumbar sympathetic gangliaS,X0,t4,tg-21, 29. Although qualitative information is available 2s, correlative quantitative morphological information on synaptogenesis has not been available for this system. Consequently, we have determined the number of synapses in the chick paravertebral ganglia during development, using quantitative electron microscopy, and the relationship of this parameter to the

230 biochemical markers, acetylcholine, choline acetyltransferase and tyrosine hydroxylase. In addition, we have studied developmental changes in the number of synaptic vesicles, a parameter not previously studied in any system. MATERIALS AND METHODS

Tissue preparation Eight White leghorn chickens from an inbred flock (specific pathogen free, SPF-U, SPAFAS, Norwich, CT) of both sexes were used for quantitative study, two each at ages 10 days in ovo, 1 day after hatching, 30 days after hatching, and adult (365+ days after hatching). The embryos were fixed by vascular perfusion without anesthesia. Embryos were removed from the shell and perfused via cannulation of the aorta with: (1) 10 ml of Ringer's variant without calcium (290 mOsM) bubbled with O~:CO2 to pH 7.0, followed by (2) 1000 ml of 2 ~ glutaraldehyde with 0.002 ~ CaClz in 0.1 M sodium cacodylate buffer, pH 7.0 (400 mOsM total of mixture) at room temperature. Fixation was continued overnight at 4 °C by immersion of the entire specimen in the same fixative. On the following day, the visceral organs, limbs and head were removed. The intact ganglia, still attached to the vertebral column, were postfixed in 2 ~ OsO4 in 0.2 M sodium cacodylate buffer, pH 7.0, for 6 h on ice. During osmication, the vertebral column was transected to yield one piece consisting of segments LI-L4 with the sympathetic chains still attached. The location of the LI segment was defined by the fusion of the seventh thoracic vertebrae synosteotically with the first lumbar vertebralT; birds with abnormal bone structure in this area were excluded from this study. Osmication was followed by overnight treatment at 4 °C with 1 ~ uranyl acetate in 0.05 M maleate buffer with 4 ~ dextrose at pH 5.2. The next day, specimens were washed in maleate buffer, dehydrated in graded ethanols and embedded in a mixture of 50.7 g Epon 812, 27.3 g NMA, 22 g DDSA and 1.5 ml DMP-30. The blocks were polymerized 48 h at 60 °C. All post-hatching birds were anesthetized, the 1and 30-days-after-hatching birds by inhalation of fluothane, and the adults by intravenous injection of sodium pentobarbital. All birds were perfused via

canulation of the aorta with: (1) Ringer's variant as above with 4 ~ dextran (molecular weight 60,400) added; (2) 0.8~ formaldehyde, 1.60~0 glutaraldehyde, 4 ~ dextran and 0.002 3o CaCl,~ in 0.15 M sodium phosphate buffer, pH 7.5 (747 mOsM total of mixture); and (3) 5% glutaraldehyde, 0.002°.o CaCI2 and 0.02~ trinitroresorcinol in 0.15 M sodium phosphate buffer, pH 7.5 (820 mOsM total of mixture). All perfusion fluids were at 38 ~C. The carcasses were then stored overnight at 4 °C in closed plastic bags and the paravertebral chains dissected free the following morning. The 1-dayafter-hatching chicks were processed as described above for embryonic tissues. The 30-days-afterhatching and adult specimens were postfixed for 6 h in 0.15 M sodium cacodylate buffer containing 7 IY,, dextrose, 0.5 ~ CaClz and 2 ~ OsO4 on ice. This was followed by overnight treatment at 4 °C with 2 0.,/, uranyl acetate in 0.1 M sodium acetate and 8 o~; dextrose, pH 5.2. Dehydration and embedding were as described above for the embryos.

Design of study As described in detail in the section o:a Results, initial examination of serial paraffin and frozen sections revealed 3 features central to the design of this study: (1) there is difficulty in defining the rostrocaudal boundaries of individual lumbar ganglia at 10 days in ovo; (2) variations of up to 100 ~ occur in the volume of individual ganglia among different specimens, even when taken from the same level of the spinal cord and from adult birds; (3) neuronal somata are irregularly distributed through the ganglia grouped in association with neuropil. Between these islands of neuropil and neuronal somas are bundles of parallel axons, myelinated after hatching. The grain of this inhomogeneity is large in the older specimens and effectively precludes sampling by randomly placed thin sectioning pyramids. On examining the ganglia in the electron microscope, we found that the groups of parallel axon bundles exclude neuronal somas and neuropil elements. Thus, there are no synapses within these axon bundles. For our analysis, we considered the ganglionic chain to be divided into two compartments: a connective compartment, consisting of the axon bundles, and a nonconnective compartment, consisting of all else. The nonconnective compart-

231 ment therefore contained all neural somas, dendrites, and synapses in the ganglia; for some purposes, we distinguished two subdivisions of the nonconnectire compartment: neuronal somas, and neuropil (consisting of satellite cells, dendrites, dispersed axons and synapses). Our scheme for estimating numbers of synapses and synaptic vesicles was as follows: (1) the average volume of chain per spinal segment was obtained from serial 15-30 /~m thick sections of several adjacent spinal levels; (2) the average volume per spinal segment of the nonconnective compartment (neuropil and neuronal somas combined) was determined by point hit counting of semithin sections and from the total volume (point 1 above); (3) the number of synapses and synaptic vesicles per unit volume of nonconnective compartment was determined from electron micrographs by stereological procedures; (4) total numbers of synapses and synaptic vesicles per spinal segment were computed from the results from points 2 and 3 above. This procedure is appropriate, because synapses do not occur within the axon bundles (connective compartment). The determination of volumes as averages over several adjacent segments substantially reduced the variability of the total volume estimates.

Sectioning and photography Lumbar ganglionic chain segments (L1-L4) embedded in Epon were sectioned serially at 15-30/~m on a sliding microtome with a heated steel knife (V.L. Friedrich, Jr., unpublished observation). Every third section was mounted for semithin and thin sectioning. Two micron sections were cut from these remounted 15-30 btm thick sections and stained with toluidine blue. Ultrathin sections were cut from one arbitrarily selected, well fixed area from one of the remounted 15-30 #m thick sections from each specimen, using a LKB microtome with a diamond knife. These ultrathin sections were placed on mesh grids, stained with uranyl acetate and lead citrate and examined and photographed with a Hitachi HU-12 electron microscope. Each ultrathin section was scanned and photographed at 3 magnifications. First, a photograph was taken at 150 × of a single whole section to be studied. This photograph was used for the determination of grid square areas. Several grid squares

containing low proportions of connective (axon bundles) were selected for further study. A montage at 3500 × was taken of each selected grid square and used to estimate the area of nonconnective (neuronal somas and neuropil) present in each square. The third series comprised 15,000 × photographs of all synapses present in each grid square studied. These photographs were used for the various counts and measurements on the synaptic profiles.

Quantitative procedures Total volumes The average volume of chain per spinal segment (Vs) was determined by planimetry of projections of the 15-30/,m serial thick sections, by:

Zi A~ Al 4

Vs

with A the area of chain (L1-L4) in section i and Al the interval between corresponding faces of the ith and the (i + 1)th section measured (this interval was not constant since some sections folded or were otherwise flawed and could not be measured).

Partial and absolute compartment volumes The 2 #m toluidine blue stained sections were examined with a Leitz microscope using a 25 × or 40 × (N.A. = 0.65 or 0.90) objective and an ocular reticule. The image of the reticule defined a counting field containing 12 test points in a rectangular array of area 0.00216 or 0.00135 mm 2. For each field, the points overlying each defined compartment were counted. Since the counting field was considerably smaller than the sections, we scanned each section by regularly displacing the field by either the length or width of the counting area, so that the test points of adjacent fields were in register without overlapping. The partial (Vv) and absolute (V) volumes of each compartment were estimated by: ~:i P(a)i A~ Vv(a)

-

Za,i P(a)l Ai

and Vs(a) = Vv(~) Vs

with Vv(a) and Vs(a) the partial and total volumes of compartment a per segment of spinal cord (a =

232 connective, neuronal somas or neuropil), the interval between the ith and the (i + 1)th section counted and P(a)i the number of point hits on compartment a in section i, summed over all microscope fields and sections from each specimen. Some sections were flawed and were not used; consequently, Ai is not constant. These equations weight hits in proportion to the volume they represent; they reduce to simple summation of hits, when section spacing is constant.

Analysis of micrographs Each synapse in the area scanned was photographed and the following were determined using a digitizing device (Model 1224, Numonics, Lansdale, PA): (1) total area of bouton profile in the micrograph; (2) path length of the synaptic adhesion in the micrograph; (3) path length of direct apposition between pre- and postsynaptic elements (synaptic apposition). In addition, we counted the synaptic vesicles in each profile. The numbers of synaptic bouton profiles analyzed for each specimen were 4, 8, 92, 18, 14, 42, 92 and 18 (two specimens each at ages 10 days in ovo, 1 day after hatching, 30 days after hatching, and 365+ days after hatching, respectively). Number of synapses Synapses were identified by the presence of both synaptic vesicles and a synaptic adhesion. At all ages, synapses were present only in the nonconnective compartment. The number of synapses per unit area of nonconnective (NA) per segment was determined by adding synapse counts from all grid squares examined for each specimen and dividing by the summed area of nonconrtective in the grid squares examined. The total number of synapses per segment (N) was estimated using 3 methods to determine the number of synapses per unit volume (Nv) of nonconnective as described below. In each case, the total number of synapses (N) was then estimated by: N == Nv V(nc) where V(nc) = Vv(nc)Vs is the volume of nonconnective and Nv is the number of synapses per unit volume of the nonconnective part. In all cases, the results are expressed as percent of adult value because the absolute values depend upon constants

which could not be determined, but whose values are expected to be reasonably independent of age. The 3 methods used to determine N are described in detail elsewhere (V.L. Friedrich, Jr. and K.A. Hruschak, in preparation); they are all based on the synaptic adhesion rather than the presynaptic bouton, since we did not photograph or count profiles which did not include the adhesion. All 3 methods are based on the assumption that the synaptic adhesion is a circular disk. (1) Disks of constant size. This procedure estimates synapses by: NA Nv--

L

where NA is the number of synapses per unit area of thin section of nonconnective, and L is the average length of synaptic adhesion profiles as seen in thin sections. This equation derives from: Nv:N2A/BA (Ref. 7, p. 91) with BA~ NAL the summed lengths of synaptic adhesion per area of thin section. This procedure assumes constant size of synaptic adhesions within each specimen. (2) Method of Fullman 1~ (adapted for circular disks by DeHoffT). The estimation given by: 8 mNA Nv

--

~2

where m is the mean reciprocal length of synaptic adhesion profiles in thin sections and NA is the number of synapses per unit area of nonconnective in thin section. This approach allows that synaptic adhesions within any specimen may be of various sizes. (3) Method of Schwartz-Saltykov 27 (adapted for circular disks by DeHoffT). This procedure estimates the number of circular disks and the distribution of their diameters, from the lengths of the sectioned profiles. This procedure is expected to be less sensitive to the loss (through failure of recognition) of small pieces of synaptic adhesions than is the Fullman procedure above.

Number of synaptic vesicles per segment The relative number of synaptic vesicles per spinal segment was estimated for each specimen as the product of the relative number of vesicles per synaptic bouton and the number of boutons per spinal

233 segment, from the Schwartz-Saltykov analysis above. The average volume per synaptic bouton was estimated as the average of the product of an unknown constant and the 3/2 power of the areas of the individual profiles in the micrographs, weighted to correct for sectioning bias. The number of vesicles per unit volume of synaptic bouton was computed as the product of another unknown constant and the number of synaptic vesicles per unit area of each bouton in electron micrographs, and the number of synaptic vesicles per bouton as the product of those two parameters. We assume that the unknown constants are the same at the 4 ages we studied; their actual values do not appear in our results, which are expressed as ratios to the average adult value. The calculation procedure and the assumptions which underlie it are described in detail elsewhere (V.L. Friedrich, Jr. and K. A. Hru~chak, in preparation). This procedure was performed separately for clear synaptic vesicles and for the small dense core vesicles in presynaptic boutons.

Total areas of synaptic adhesion and of synaptic apposition The average areas per synaptic adhesion and per synaptic apposition were calculated using the Schwartz-Saltykov procedure above. The total adhesion area and total apposition area per segment were calculated as the product of their average areas and the number of synapses per segment. RESULTS Although the paravertebral chain can be idealized as defined clusters of nerve cells (the ganglia) separated by bundles of myelinated fibers (the interganglionic connectives), our observations show that ideal to be an oversimplification in several regards. First, the paravertebral chain at 10 days in ovo consists of irregular clumps of cell bodies only infrequently separated by areas of unmyelinated fibers (Fig. la, b). Distinct ganglionic swellings are not present and it is impossible to distinguish individual ganglia. As the chick matures, distinct ganglionic swellings appear, separated by myelinated connectives. Even in the adults, however, the interganglionic connectives contain many small islands

of nerve cell bodies and associated neuropil (Fig. 2a). Thus, the ganglia, although macroscopicaUy distinct, may at times be nearly continuous microscopically. Second, the volume of the ganglionic swellings varies up to 100 % from level to level and at the same level in different specimens of the same age. Third, the ganglionic swellings themselves consist of islands of neuronal somas and associated neuropil, irregularly and asymmetricallydistributed among bundles of axons (Figs. 1, 2). This asymmetry and irregularity is in contrast with the more regular structure of the mammalian superior cervical ganglion16. Because of these features, we chose to take a region of the lumbar paravertebral chain as defined by the L1-L4 vertebrae instead of only one particular ganglion, and to average our results over these 4 segments. Our approach is thus somewhat different from that of others to the mammalian superior cervival ganglion1,16.

Fine structure of the ganglia This study has been concerned primarily with the quantitative aspects of synaptogenesis in the chick lumbar paravertebral ganglia. Some qualitative aspects of this system, however, should be mentioned. At 10 days in ovo, the neuroblasts can be distinguished from the satellite cells because the nuclei of the satellite cells contain a prominent rim of condensed chromatin at the nuclear envelope, while the nuclei of the neuroblasts do not. The majority of neuroblasts exhibit only a few dense core vesicles from 70 to 150 nm in diameter. A minority of neuronal cell bodies contain many dense core vesicles, up to a maximum density of 22.5 vesicles/#m 2. The vesicles, which are mostly round, range in size from 70 to 290 nm with core sizes of 40-250 nm. These cells correspond to the 'granule cells' described by Luckenbill-Edds and van Horn is. We encountered mitotic figures containing these large granules, indicating that these cells are still dividing at this stage. The granule cells receive synapses as do neuroblasts which do not contain the large granules (Fig. 3). We did not encounter granule-containing cells in material from the other 3 ages examined. We occasionally observed nuclear changes and electron-dense debris at 10 days in ovo, suggestive of cell death22.24.

Figs. 1,2. Sections of lumbar sympathetic chain (2/~m thick) from 10-day in ovo and adult chicken, respectively. Fig. 1.a: the L1-L5 region of the lumbar paravertebral chain from a 10-day in ovo specimen. Lightly stained regions are bundles of axons, unmyelinated at this age; dark regions are areas of neuronal cell bodies and associated neuropil. The individual ganglia (*) are irregular in shape; regions of neuronal cell bodies are in places continuous from one ganglion to the next. x 46. b: higher magnification of a. Neuronal somas and associated neuropil are grouped in islands separated by bundles of parallel axons (-0-). Dividing cells (M) are present, x 450. Fig. 2.a: a single segment (L3) from the sympathetic chain of an adult. A distinct, well-defined ganglionic swelling is lacking. The darkest regions are myelinated axons. Nerve cell bodies (n) are present throughout the segment, x 26. b: higher magnification of a. The cell bodies and neuropil are grouped together in islands. The bundles of parallel axons (-O-), which separate the islands, are myelinated at this age. x 150.

235

F~g. 3. Electron micrograph of an axosomatic synapse from a 10-day m ovo chick. The postsynaptic cell body contains numerous small and large dense-core vesicles, the largest of which is 200 nm in diameter. This is a granule-containing cel; at this stage, synapses are also found onto cells not containing these granules. N, nucleus, x 48,000. Fig. 4. Electron micrograph of these axo-dendritic synapses from an adult chicken. The presynaptic boutons (B) contain round and flattened clear vesicles as well as small dense-core vesicles. D, dendrite. × 18,000.

236 In the adult, neuronal somas range in size from 15 to 83 /~m diameter. A histogram analysis of the distribution of area measurements of over 2000 nucleated neuronal cell body profiles in 2/~m toluidine blue-stained sections gave no clear evidence of distinct cell classes based on size. The primary peak of the histogram, which is very broad, indicates a medal cell diameter of 55 #m. All synaptic boutons appeared similar in fine structure, at all ages (Figs. 3, 4). The majority of vesicles in almost all profiles were clea~ and either round (40 nm) or flat (40-60 nm by 25 nm); most profiles also contained a few round vesicles with dense cores (60-140 nm in diameter). Since the purpose of this study was quantitative, we did not survey many grids or large areas of the specimens. It remains possible that a more extensive search or the application of different preparative procedures would reveal a greater variety in presynaptic fine structure than we encountered in this study. As seen in thin sections, synaptic boutons become larger with age (areas: 10 days in ovo, 0.94 /~m'); 1 day after hatching, 0.64 Fm~; 30 days after hatching, 1.2 #m2; adult, 2.1 /zm2). There is also progressive increase in the number of clear vesicles per profile (38, 54, 164 and 197 vesicles per profile at the 4 ages, respectively) and in the number of dense cored vesicles (from 1 vesicle per profile at 10 days in ovo to 12 vesicles in the adult). By contrast, the lenght of the synaptic adhesion was relatively stable at about 0.5 k~m at all ages. At all ages studied, the majority of synapses present are the axodendritic type (70-95 ~). Axosomatic synapses are also present at all 4 ages; the highest frequency is 3 0 ~ at 10 days in ovo. Subsynaptic bars25, 26 occur in a small fraction (4 ~ ) of synaptic profiles from 30 days-after-hatching specimens. In the adult, 15 ~ of the synaptic profiles examined contained a subsynaptic bar.

Quantitative results The total volume of chain spinal segment increases progressively, from 0.008 mm 3 per segment at 10 days in ovo to 0.7789 mm 3 in the adult. All 3 compartments, somas, neuropil and connectives, show some increase in absolute volumes during each of the 3 developmental intervals examined (Fig. 5). The partial volume of the neuropil increases with

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Fig. 5. Partial and absolute compartment volumes as functions of age. Connectives are bundles of axons, myelinated at all ages except I0 days in ovo; somas are neuronal somas. Each point represents the results from one animal. The absolute volumes refer to one chain only; they are doubled to give values for both chains. age from less than 1 0 ~ at 10 days in ovo to 37.8 in the adult. By contrast, the partial volume of cell bodies is greater than 6 0 ~ at 10 days in ovo and decreases to about 20 ~ in the adult (Fig. 5). Synapses are sparse in thin sections at 10 days in ovo (758 mm2; Fig. 6a) but have increased substantially by 1 day after hatching (5259 per mm2; Fig. 6a). The value at 1 day after hatching represents a maximum; those at 30 days after hatching and in adults are lower (4610 and 3900 per mm 2, respectively). The 3 procedures for estimating the number of synapses yielded very similar developmental curves (Fig. 6b). All 3 methods indicate that less than 1 of the adult number of synapses are present at 10 days in ovo (Fig. 6b). A substantial number of synapses are formed between 10 days in ovo and 1 day after hatching. Approximately 25 ~ of the adult number of synapses are present at 1 day after hatching; however, in the period from 1 day alter hatching to 30 days after hatching, there is little change in the number of synapses. Nearly 70 ~ of the adult number of synapses are formed after 30 days after hatching. These results reveal substantial

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Fig. 6. a-d: developmentof synapses. Each point represents the results from one animal, b: results from procedures 1-3 (Materials and Methods).d" numberof clear synapticvesicles in synapticboutons. formation of synapses both before hatching (10 days in ovo to 1 day after hatching) and after hatching (30 to 365+ days after hatching). A progressive increase occurs in both total area of synaptic adhesions and total area of synaptic apposition per segment (Fig. 6c). The ratio of these 2 parameters (total area of synaptic apposition per segment to total area of synaptic adhesion per segment) remains constant at 2.2 at these 4 ages. The total number of clear synaptic vesicles (in synaptic boutons) per segment (Fig. 6d) alsoincreases progressively during the period studied. The total number of dense-core vesicles (in synaptic boutons) increases in concert with the total number of clear vesicles. The number of synapses increases substantially between 10 days in ovo and 1 day after hatching; however, the number of clear synaptic vesicles changes considerably less (Fig. 6b, d). DISCUSSION In our initial studies, we found that the ganglionic swellings are of varying size and shape, and that islands of cell bodies and neuropil stream into the interganglionic connectives in varying amounts

(Figs. 1, 2). To account for these features we chose to study a region of the sympathetic paravertebral chain spanning 4 spinal segments and to average the results over the 4 segments. The effectiveness of this approach of averaging is demonstrated by the consistent agreement we obtained in the partial and absolute compartment volumes per segment of duplicate specimens (Fig. 4). The technique of thick sectioning epoxy-embedded material allowed us to determine tissue volumes and to do electron microscopy on the same material, thus eliminating the need for shrinkage corrections (see e.g. ref. 23).

Granule-containing cells The cells containing large dense-core vesicles which we encountered at 10 days in ovo are similar to the granule cells described by Luckenbill-Edds and van Horn is in embryonic chick sympathetic ganglia. In agreement with Luckenbill-Edds and van Horn is, we did not find this type of cell in material from later stages of development. The origin and fate of this cell type are unknown. However, it is clear in our material that this type of cell receives synapses (Fig. 3). Quantitative results Few synapses are present at 10 days in ovo (about 0.04 ~ of the adult number); a substantial number of synapses are formed during the embryonic interval from 10 days in ovo to 1 day after hatching. A substantial increase in the number of synapses also occurs beyond 30 days after hatching. Prolonged growth of synapses has also been reported in the chicken ciliary ganglion11. The synapses present at 10 days in ovo contain far fewer vesicles per synapse than do those in the adult. This difference is expressed directly as a progressive increase in the number of synaptic vesicles per bouton profile in the electron micrographs, from 38 at day 10 in ovo to 197 in the adult. Biochemical correlations Our results show that the number of synapses increases during development, roughly in concert with acetylcholine, choline acetyltransferase, and tyrosine hydroxylase (Fig. 7). The previously published biochemical data show some difference in the developmental patterns for acetylcholine and

238 AChE 10090

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Fig. 7. Comparison of morphological and biochemical indices of synaptic and postsynaptic development. ACHE, acetylcholinesterase activity10,21. ChAc, choline acetyltransferase activity8,el. TH, tyrosine hydroxylase activity1°,z9. ACh, amount of acetylcholine~9,z°. The foregoing are relative enzymatic activities or ACh content per whole ganglion. Synapses, total number of synapses per segment (this paper); vesicles, total number of clear synaptic vesicles in synaptic boutons per segment (this paper). choline acetyltransferase (Fig. 7). The number of synapses and the number of synaptic vesicles both exhibit developmental patterns resembling acetylcholine more closely than choline acetyltransferase. The activity of acetylcholinesterase exhibits a markedly different pattern from any of the other parameters, reaching its maximum earlier in development than do either the morphological or the other biochemical parameters (Fig. 7). Edgar et al. 9 demonstrated the presence in 12-day in ovo chick sympathetic ganglia of neuroblasts capable of synthesizing acetylcholine in tissue culture, and have suggested that the low levels of choline acetyltransferase detected in vivo at 12 days in ovo may reflect the presence of cholinergic neuroblasts, rather than activity in presynaptic endings. To examine this hypothesis, we calculated the amounts of acetylcholine and the activity of choline acetyltransferase per synapse as functions of age (Fig. 8a). Both curves are biphasic; high initial levels per synapse are found at 10 days in ovo but the levels are dramatically lower at 1 day after hatching. Subsequent development sees an increase again of both these parameters. The early high level of acetylcholine and choline acetyltransferase per synapse, and their subsequent fall, are consistent with the early presence and subsequent death of cholinergic

neuroblasts. The increase in these parameters after 1 day after hatching probably reflects the maturation of synapses, as discussed below. In electron micrographs, we saw substantial increases in the number of synaptic vesicles per synapse profile with age, and a particularly marked increase in the number of vesicles per profile between 1 and 30 days after hatching. The increases observed in acetylcholine and choline acetyltransferase per synapse after 1 day after hatching might reflect increases with age in the number of synaptic vesicles per synapse; consequently, we also calculated acetylcholine content and choline acetylase activity per synaptic vesicle (Fig. 8B). Both parameters are extremely high at 10 days in ovo, and drop to near adult levels by 1 day after hatching. The high initial value for the acetytcholine to vesicle ratio could reflect the presence during development of a presynaptic, nonvesicular pool of acetylcholine; however, it seems most likely, in view of the data of Edgar et al. 9, that the relatively high levels of acetylcholine and choline acetyltransferase per synaptic vesicle at 10 days in ovo reflect the activity of cholinergic neuroblasts within the ganglia. If this hypothesis is correct, then the precipitous drop in both parameters (per synaptic vesicle)between 10 days in ovo and 1 day after hatching and their relative stability during subsequent development suggest the death of the hypothesized choli-

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Fig. 8. Amounts of acetylcholine (ACh) and choline acetyltransferase (ChAc) per synapse and per synaptic vesicle. A: the amounts of ACh and ChAc per synapse. B: the amount of ACh and ChAc per synaptic vesicle. The values were derived by dividing the amounts of ACh19,2°and ChAcs,21 per whole ganglion by the total number of synapses per segment (this paper) or the total number of clear synaptic vesicles in synaptic boutons per segment (this paper).

239 nergic n e u r o b l a s t s o r the suppression o f their cholinergic properties, before hatching. The available d a t a show substantial increases after hatching in the n u m b e r o f synapses a n d synaptic vesicles on the one h a n d , a n d in choline acetyltransferase activity a n d acetylcholine c o n t e n t o n the o t h e r (Fig. 7). The ratios o f acetylcholine c o n t e n t p e r synaptic vesicle a n d choline acetyltransferase activity per synaptic vesicle r e m a i n relatively constant after h a t c h i n g (Fig. 8B); in contrast, the corres p o n d i n g ratios p e r synapse change substantially (Fig. 8A). T h a t changes occur in acetylcholine content, choline acetylase activity, a n d n u m b e r o f syn a p t i c vesicles per synapse suggests a m a t u r a t i o n o f

i n d i v i d u a l synapses, i n d e p e n d e n t o f changes in the t o t a l n u m b e r o f synapses in the tissue. Thus, informa t i o n only on the n u m b e r o f synapses in tissues m a y n o t fully describe the course o f s y n a p t i c development. The present results d e m o n s t r a t e t h a t in chick s y m p a thetic ganglia, the n u m b e r o f synaptic vesicles is closely c o r r e l a t e d with acetylcholine c o n t e n t a n d choline accetyltransferase activity in the p e r i o d after

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