Relative Numbers of Several Types of Synaptic Connections in the Substantia Gelatinosa of the Cat Spinal Cord DONALD DUNCAN AND RICARDO MORALES Department of Anatomy, The University of Texas Medical Branch, Galweston, Texas 77550
ABSTRACT The relative numbers of axo-dendritic, axo-axonic, dendroaxonic and dendro-dendritic synapses were determined by classifying and recording all such specialized contacts in sample areas of the substantia gelatinosa. The samples were taken from segments Ll-L5 of the cat spinal cord. In the glomerular complexes 97%of all synapses were recorded as axo-dendritic. In substantia gelatinosa deprived of glomerular complexes by dorsal root section, 96.5% were axo-dendritic. The remainders were about equally divided between axo-axonic, dendro-dendritic and dendro-axonic synapses. were washed in 0.1 M cacodylate buffer for two hours. The slices were trimmed down t o include the substantia gelatinosa and approximately 1 mm of surrounding tissue and placed in 1%OsO, in 0.1 M cacodylate buffer. The osmicated blocks were washed with distilled water and left overnight in 1%magnesium uranyl. Following the staining in block they were again washed in distilled water, dehydrated and embedded in Spurr's plastic mixture. Thin sections were mounted on 150-mesh copper grids and then stained additionally with uranyl acetate in 100% methanol and with lead citrate. Inspection and counting of specialized contacts between neuronal profiles were done by inspecting a grid square one strip a t a time a t x 12,000 on the viewing screen amplified x 10 with a binocular microscope. After one up or down strip was finished, the field was shifted either to the right or to the left so as to slightly overlap the one that had been examined. Whenever a specialized contact was MATERIALS AND METHODS seen, magnification on the screen was inThin sections of substantia gelatinosa were creased to x 33,000 and occasionally higher obtained from lumbar levels 1-5of six healthy before it was classified and recorded. Only adult cats. Most of the cats had been subjected contacts clearly displaying unit membranes to dorsal root rhizotomies and were sacrificed on both sides of the gap and with a t least one a t various intervals following the operation. vesicle attached or very close to the presynapThe animals were perfused via the left ventri- tic membrane were counted. The synapses cle with 3%glutaraldehyde, 0.5%sucrose and were recorded in two groups. In one group 0.025%CaC12in 0.8 M sodium cacodylate at pH were those associated with the glomerular 7.2-7.4. In three instances 3% NaCl was in- complexes; i.e., the centrally placed dorsal cluded in the perfusion mixture. After fixa'Research for this publication waB supported in part by NIH tion for 12 hours or more, slices 1-2 mm thick Grant 1 R01 NS 13171-01A1 NEUB.
In publications by a number of authors (Coimbra et al., '74; Gobel and Dubner, '69; Gobel, '71, '74, '76; Kerr, '66, "70, '71; Ralston, '65, '69, '71; Rethelyi and Szentagothai, '69; Westrum and Black, "711, statements and illustrations indicate that most of the synapses in the substantia gelatinosa are axo-dendritic characterized by synaptic vesicles on the presynaptic side and amorphous and fuzzy dense material on the postsynaptic side. In addition to the axo-dendritic synapses, axo-axonic, dendro-dendritic and dendro-axonic synaptic junctions have been described with varying degrees of emphasis as to their numbers and importance. However, none of the papers supply numerical data on the incidence of any one type in relation to the others. Because such information should add to a more complete understanding of the structure of the substantia gelatinosa, counts were made of the several types of synapses with results that are reported in this paper.
J. COMP. NEUR. (1978) 182: 601-610.
601
602
DONALD DUNCAN AND RICARDO MORALES TABLE 1
Results ofcounting and classifyingsynapses in the spinal substantia gelatinosa of the cat Cat no.
A-D
A-A
D-D
Glomerular associated synapes 9/24/74 128 0 1 12/30/15 260 2 2 1/6/76 171 0 8 1/20/76 197 0 3 5/14/76 99 1 2 2 3 5/17/76 331 Sub-totals 1,192 5 19 % of all 97.2% 0.7% 1.6% counted Non-glomerular synapses normal material 9124174 170 1 1 12/39/75 69 0 0 1/20/76 44 0 0 6 5/17/76 _154 _ 2 Sub-totals 437 3 7 % of all 97.1% 0.7% 1.5% counted Material devoid of glomeruli 3 2 1/6/76 120 1 5/14/76 158 1 -
Sub-totals
-
278
4
96.5%
1.5%
D-A
1 2 0 3 2 2 10 0.8%
1 1 0 1
3
0.7% 2 1 -
3
3
1%
1%
% of all
counted
A-D, axo-dendritic; A-A, axo-axonic; D-D, dendro-dendritic; D-A, dendro-axonic.
root terminals and the profiles in contact with them. Most of the surrounding profiles are dendrites and a few have been identified as axons (Gobel, '76). Synapses that were not associated with glomerular clusters were recorded as non-glomerular synapses. In normal material some of the contacts registered as non-glomerular undoubtedly were portions of a glomerulus cut in such a way that only a single dendrite and a portion of the central terminal were visible, but in deafferented specimens this probability was eliminated. Contacts of the axo-dendritic type vary greatly in length, contour and amounts of cytoplasmic and extracellular densities; likewise, there are appreciable differences in gap widths and in the appearance of gap material (Peters et al., '76). In this report all of the subtypes of axo-dendritic synapses were grouped together for comparison of their numbers with other types. RESULTS
Numerical results obtained in this study are summarized in table 1and the various types of
synaptic contacts that were counted are illustrated in figures 1-9.Most of the axo-dendritic synapses in the glomerular complexes are between the centrally placed terminals and the surrounding dendritic spines (fig. 2). In addition to the spines, the central terminals frequently synapse with larger profiles identified as dendritic shafts (fig. 1).Most of the spines, Gobel's ('76) type D1are rather empty looking profiles without synaptic vesicles (figs. 2, 7, 8). They greatly outnumber the D, variety which contain loose aggregates of mostly round vesicular profiles as depicted in figures 2 and 9. In one count from a single cat there were 5 D1 and 32 D,. In another field from the same cat there were 9 D, and 55 D,. In addition to the axo-dendritic synapses between central terminals and dendrites, a very occasional profile filled with a mixture of round, oval and flattened vesicles formed a synapse with a D1or D2dendritic spine and more often with dendritic shafts not associated with C terminals (fig. 4). These presynaptic endings were identified as belonging to the axons called p axons by Gobel ('76). They were not recorded separately but included in the total number of axo-dendritic synapses. As indicated in table 1very few axo-axonic synapses were encountered in the glomeruli, only five in a total of 1,226 synapses. The presynaptic element in these synapses contained many flattened vesicles (p axons) and the postsynaptic element was a central terminal (fig. 1).Dendro-dendritic synapses in the glomerular complexes were, without exception, D2-D1(fig. 2). Although more numerous than axo-axonic and dendro-axonic synapses they contributed only 1.7% of the total number counted. Likewise, the dendro-axonic synapses (fig. 3) were but a small fraction of the total. Most of the synapses that were not associated with the glomeruli were axo-dendritic and most of the presynaptic profiles contained round vesicular profiles. However, those containing numerous distinctly flattened vesicles were not inconsequential. Profiles containing flattened vesicles (fig. 4) were most evident in material that had been fixed with solutions containing 3% NaCL. Substantia gelatinosa material from one such cat, No. 5/17/76, was used to count the numbers of profiles containing nearly all round vesicles versus those with many flattened vesicles. The counts in three sample areas were 150r-l5f, 51r-5f, 31r-7f. Consequently, it was apparent that profiles
RATIOS OF FOUR TYPES OF SYNAPSES IN SUBSTANTIA GELATINOSA
containing flattened vesicles are much more numerous in neuropil that is not associated with the glomerular complexes. Recognition of axo-axonic synapses apart from the glomeruli was only by inference. When two small profiles both containing close and regularly spaced neurotubules were attached to each other by an asymmetric junction wherein the presynaptic element contained a few vesicles and the postsynaptic profile exhibited a distinct postsynaptic density the contact was recorded as a n axo-axonic synapse (fig. 5). A further suggestion of the synapse being axo-axonic was association of both profiles with a cluster of naked axons. Dendro-dendritic synapses in the non-glomerular category were easily recognized by the size of the profiles which were much larger than the unmyelinated axons and contained profiles attributed to smooth endoplasmic reticulum (fig. 6). When a larger profile containing scattered vesicles formed a distinct synapse with a smaller vesicle filled process the arrangement was recorded as dendro-axonic. Less attention was paid to specialized contacts without vesicles. It was obvious that most of them were puncta adhaerentia as described by Peters et al. ('76) for central nervous system tissue. It was equally apparent that their frequency exceeded all types of synapses except the axo-dendritic. Synaptic contacts with the cell bodies of substantia gelatinosa neurons were so few in relation to other synapses that they were not included in t h e counts. No tight or gap junctions between neural profiles were seen. Gap junctions (fig. 1) between astrocytes were numerous, about onethird of all specialized contacts in the two 150mesh squares that were counted for determining their frequency. In much smaller numbers gap junctions were located between astrocytes and neurons, between astrocytes and oligodendroglia and between oligodendroglia cells and neurons as previously reported by Morales and Duncan ('75). DISCUSSION
The results presented in this report were obtained mostly by observations undocumented by photography. Consequently, they should not be regarded as exact as to numbers and classification. Other investigators repeating the work might see and record twice as many axo-axonic, dendro-dendritic and dendro-axonic synapses as reported here. Although this
603
possibility appears unlikely to the authors, a doubling of the numbers of any or all types of the rarer forms of synapses would still leave the results in accord with the statement by Ralston ('71) who wrote with reference to the substantia gelatinosa that the "vast majority of the synapses are axo-dendritic." The criteria described by Gobel ('74, '76) for identification of profiles in the glomerular complexes of the trigeminal portion of the substantia gelatinosa were valid for the same formations in the lumbar levels. It is believed that they permit a large measure of certainty in our recognition of the four types of synapses occurring in these configurations. The D, type of dendritic spines was confirmed by repeatedly finding them attached by a narrow stalk to unmistakable dendritic shafts. The D2type a t tached by a neck to a dendritic shaft was seen in two instances but not photographically recorded. However, recent use of the modified Golgi method of Fairen et al. ('77) resulted in locating D, spines containing particles of gold. One of the spines was connected to a dendritic shaft by a relatively long neck (fig. 9). Since there was but one impregnated neuron in a considerable block of tissue, it is believed that the profiles containing both vesicles and gold particles and in synaptic contact with the central terminal of a glomerular complex are D:, spines as described by Gobel ('76). Likewise, the profiles filled with vesicles, many of which are distinctly flattened, were seen occasionally in continuity with small unmyelinated axons. Furthermore, similar profiles filled with a mixture of round, oval and flattened vesicles form typical axo-dendritic synapses in material devoid of glomerular complexes (fig. 4). Apart from the glomerular complexes most of the synapses present in the substantia gelatinosa conform with the repeatedly described characteristics of axo-dendritic synapses. In addition there are a few that are obviously dendro-dendritic because the contacting profiles are much too large to be naked axons and the scanty vesicles occupy a very small part of the total area. In addition the dendrites concerned contained bits of endoplasmic reticulum but no ribosomes (fig. 6). The number of dendro-dendritic synapses was much less than anticipated by reading the literature. It is possible t h a t some of those diagnosed as axo-dendritic were in fact dendrodendritic especially in the non-glomerular associated group, but the chances of this
604
DONALD DUNCAN AND RICARDO MORALES
mistake with the glomerular complexes is regarded as unlikely and the values for both groups were essentially the same. Presumed contacts believed to be axo-axonic synapses were as illustrated in figure 5; namely, a small unmyelinated axon displaying a pronounced postsynaptic density, a distinct substance containing gap and another small profile containing one, two, three or more presynaptic vesicles. Seven such synapses were found among a total of 738. If there was certainty that these are axo-axonic, such synapses might play a significant role in the activities within the substantia gelatinosa despite their low incidence in relation to other types. A very low incidence of axo-axonic synapses is in keeping with the opinion of Famiglietti and Peters ('72) who doubt the existence of axo-axonic synapses in the lateral geniculate nucleus and the finding of only two such synapses by Saito ('74) in an extensive study of the nucleus dorsalis of the cat. Recently a new technique that permits recognition of dendrites impregnated by the Golgi method and subsequently processed for examination with the electron microscope has been described by Fairen e t al. ('77). Application of this method to the substantia gelatinosa may dispel most of the uncertainty accompanying present attempts to distinguish between small axonic and dendritic profiles. An initial use of the method in this laboratory is illustrated in figure 9. On the basis of their numerical preponderance it might be assumed that axo-dendritic synapses in the substantia gelatinosa far outweigh all other types combined in functional importance. The difference is greatly magnified if the greater lengths of axo-dendritic membrane specializations as seen in cross sections are indications of the surface areas of the various types. If, in addition, the transmitter released by the central terminals of the glomeruli is far more powerful than that of the others the difference in energy values becomes enormous. Such considerations are well within the range of the possible. It is known that large numbers of substance P containing fibers enter the substantia gelatinosa, Hokfelt et al. ('75, '76). It is known also that substance P has the same effect on some neurons in the substantia gelatinosa as peripherally applied noxious stimuli (Randic and Miletic, '77). In addition, pharmacological experiments have shown that the depolarizing potency of substance P is 1,000-9,000 times
greater than L-glutamate, Otsuka and Konishi ('76). While such facts suggest that all else must give way to a discharging central terminal, they do not imply that the other types of synapses in the glomerular complexes are inconsequential especially during intervals between dorsal root discharges into any portion of the substantia gelatinosa. Nevertheless, assuming that the results accumulated in this study are approximately correct it appears that from the standpoint of quantitative morphology all but a small fraction of synapses are axo-dendritic both within the glomerular complexes and in the rest of the substantia gelatinosa as well. ACKNOWLEDGMENTS
The authors wish t o thank Donald B. Edwards, Earl Pitsinger and Phyllis Fletcher for valuable assistance in the preparation of this manuscript. LITERATURE CITED Coimbra, A,, P. B. Sadre-Borges and M. M. Magalhaes 1974 The substantia gelatinosa of the rat. Fine structure, cytochemistry (acid phosphotase) and changes after dorsal root section. J. Neurocytol., 3: 199-217. Famiglietti, E. V.,and A. Peters 1972 The synaptic glomerulus and the intrinsic neuron in the lateral geniculate nucleus of the cat. J. Comp. Neur., 144: 287-590. Fairen, A., A. Peters and J. Saldanha 1977 A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J. Neurocytol., 6: 311-337. Gobel, S. 1971 Structural organization of the main sensory trigeminal nucleus. In: Oral-Facial Sensory and Motor Mechanisms. R. Dubner and Y. Kawanura, eds. Appleton-Century-Crofts, New York, pp. 183-202. 1974 Synaptic organization of the substantia gelatinosa glomeruli in the spinal trigeminal nucleus of the adult cat. J. Neurocytol., 3: 219-243. 1976 Dendroaxonic synapses in the substantia gelatinosa trigeminal nucleus of the cat. J. Comp. Neur., 167: 165.176. Gobel, S., and R. Dubner 1969 Fine structural studies on the main sensory trigeminal nucleus in t h e cat and rat. J. Comp. Neur., 137: 459-494. Hokfelt, T., R. Eldi, 0. Johansson, R. Luft, G. Nilsson and A. Animira 1976 Immunohistochemical evidence for separate populations of somatostatin-containing and substance p-containing primary afferent neurons in the rat. Neuroscience, 1: 131-136. Hokfelt, T., J. 0. Kellerth, G. Nilsson and B. Pernow 1975 Substance P. Localization in the central nervous system and in some primary sensory neurons. Science, 190: 889-190. Kerr, F. W. L. 1966 The ultrastructure of the spinal tra c t of the trigeminal nerve and the substantia gelatinosa. Exp. Neurol., 16: 359-376. 1970 The fine s truc ture of t h e subnucleus caudalis of the trigeminal nerve. Brain Res., 23: 129-245. 1971 Electron microscopic observations on primary deafferentation of the subcaudal nucleus of the trigeminal nerve. In: Oral-Facial Sensory and Motor Mechanisms. R. Dubner and Y. Kawanura, eds. Appleton-Century-Crofts, New York, pp. 159-181.
RATIOS OF FOUR TYPES OF SYNAPSES IN SUBSTANTIA GELATINOSA Morales, R., and D. Duncan 1975 Specialized contacts of astrocytes with astrocytes and with other cell types in the spinal cord of t h e cat. Anat. Rec., 182: 255-265. Otsuka, M.,and S. Konishi 1976 Substance P and excitatory transmitter of primary sensory neurons. Proc. of Cold Spring Harbor Symposium, “The Synapse,” 40: 135-143. Peters, A.,S. L. Palay and H. DeF. Webster 1976 The Fine Structure of t h e Nervous System. W. B. Saunders Co., Philadelphia, London, Toronto. Ralston, H. J.,111 1965 The organization of the substantia gelatinosa Rolandi in t h e cat lumbosacral cord. Z. Zell., 671: 1-23. 1969 Dorsal root projections to the dorsal horn in t h e cat spinal cord. J. Comp. Neur., 132: 303-330. 1971 The synaptic organization of the dorsal horn of the spinal cord and t h e ventrobasal thalamus of the cat. In: Oral-Facial Sensory and Motor Mechanisms.
605
R. Dubner and Y. Kawanura, eds. Appleton-CenturyCrofts, New York, pp. 229-250. Randic, M., and V. Miletic 1977 Effect of substance P in cat dorsal horn neurones activated by noxious stimuli. Brain Research, 128: 164-169. Rethelyi, M., and J. Szentagothai 1969 The large complexes of the substantia gelatinosa. Exp. Brain Res., 7: 258-274. Rexed, B. 1954 A cytoarchitectonic atlas of the spinal cord in the cat. J. Comp. Neur., 100: 297-379. Saito, K. 1974 The synaptology and cytology of the Clarke cell in the nucleus dorsalis of the cat: an electron microscope study. J. Neurocytol., 3: 179-197. Westrum, L. E.,and R. C. Black 1971 Fine structural aspects of the synaptic organization of the spinal trigeminal nucleus (Pars interpolaris) of the cat. Brain Res., 25: 265-288.
PLATE 1 EXPLANATION OF FIGURES
All figures are from substantia gelatinosa of adult cats. Arrows point to post-synaptic profiles. 1 The central axonic terminal (C) of a glomerular complex displaying two axo-dendritic synapses and another axonic terminal (P) which is presynaptic to the central terminal (C), two gap junctions between astrocytic processes are located in the upper left-hand corner of the picture. X 50,000. 2 A dendro-dendritic synapse (D,-D,) and a portion of a glomerular central terminal
(C) are shown in this figure. X 50,000. 3 A dendro-axonic synapse (D2-C)in a glomerular complex is illustrated. Immediately to the right of the synapse there is a punctum adhaerens. X 100,000. 4
In this figure a profile (P axon) filled with round, oval and flattened vesicles is presynaptic to two dendrites. X 50,000.
5 In the center of this figure there is an asymmetric synapses tentatively identified as an axo-axonic synapse.
606
X
71,000.
RATIOS OF FOCR T Y P E S O F S Y N A P S E S IN SUBSTANTIA CELATINOSA
1’1,AlE 1
Donald I h n c a n and Ricardo Moraies
607
PLATE 2 EXPLANATION OF FIGURES
As in plate 1 all figures are from the substantia gelatinosa of normal adult cats 6 A dendro-dendritic synapse
(D-D). X 53,000
7 Showing a D, type of dendritic spine (S) attached by a narrow neck to the parent dendritic shaft (D). The two axo-dendritic contacts may be a sectional view of a ring synapse surrounding the neck of the spine. x 51,000. 8 A type D, spine connected by a neck with a dendritic shaft is In synaptic contact with a central terminal (0.X 38,200. 9 A portion of a gold-toned Golgi preparation showing a dendrite (D), neck and spine from an isolated impregnated neuron in the substantia gelatinosa. The spine contains a few synaptic vesicles (note pointed guide line) and the spines in the inset from a n adjacent section displays many vesicles. Both spines in the figure are in synaptic contact with a central terminal (C). x 42,000.
608
609
ABSTRACT The relative numbers of axo-dendritic, axo-axonic, dendroaxonic and dendro-dendritic synapses were determined by classifying and recording all such specialized contacts in sample areas of the substantia gelatinosa. The samples were taken from segments Ll-L5 of the cat spinal cord. In the glomerular complexes 97%of all synapses were recorded as axo-dendritic. In substantia gelatinosa deprived of glomerular complexes by dorsal root section, 96.5% were axo-dendritic. The remainders were about equally divided between axo-axonic, dendro-dendritic and dendro-axonic synapses. were washed in 0.1 M cacodylate buffer for two hours. The slices were trimmed down t o include the substantia gelatinosa and approximately 1 mm of surrounding tissue and placed in 1%OsO, in 0.1 M cacodylate buffer. The osmicated blocks were washed with distilled water and left overnight in 1%magnesium uranyl. Following the staining in block they were again washed in distilled water, dehydrated and embedded in Spurr's plastic mixture. Thin sections were mounted on 150-mesh copper grids and then stained additionally with uranyl acetate in 100% methanol and with lead citrate. Inspection and counting of specialized contacts between neuronal profiles were done by inspecting a grid square one strip a t a time a t x 12,000 on the viewing screen amplified x 10 with a binocular microscope. After one up or down strip was finished, the field was shifted either to the right or to the left so as to slightly overlap the one that had been examined. Whenever a specialized contact was MATERIALS AND METHODS seen, magnification on the screen was inThin sections of substantia gelatinosa were creased to x 33,000 and occasionally higher obtained from lumbar levels 1-5of six healthy before it was classified and recorded. Only adult cats. Most of the cats had been subjected contacts clearly displaying unit membranes to dorsal root rhizotomies and were sacrificed on both sides of the gap and with a t least one a t various intervals following the operation. vesicle attached or very close to the presynapThe animals were perfused via the left ventri- tic membrane were counted. The synapses cle with 3%glutaraldehyde, 0.5%sucrose and were recorded in two groups. In one group 0.025%CaC12in 0.8 M sodium cacodylate at pH were those associated with the glomerular 7.2-7.4. In three instances 3% NaCl was in- complexes; i.e., the centrally placed dorsal cluded in the perfusion mixture. After fixa'Research for this publication waB supported in part by NIH tion for 12 hours or more, slices 1-2 mm thick Grant 1 R01 NS 13171-01A1 NEUB.
In publications by a number of authors (Coimbra et al., '74; Gobel and Dubner, '69; Gobel, '71, '74, '76; Kerr, '66, "70, '71; Ralston, '65, '69, '71; Rethelyi and Szentagothai, '69; Westrum and Black, "711, statements and illustrations indicate that most of the synapses in the substantia gelatinosa are axo-dendritic characterized by synaptic vesicles on the presynaptic side and amorphous and fuzzy dense material on the postsynaptic side. In addition to the axo-dendritic synapses, axo-axonic, dendro-dendritic and dendro-axonic synaptic junctions have been described with varying degrees of emphasis as to their numbers and importance. However, none of the papers supply numerical data on the incidence of any one type in relation to the others. Because such information should add to a more complete understanding of the structure of the substantia gelatinosa, counts were made of the several types of synapses with results that are reported in this paper.
J. COMP. NEUR. (1978) 182: 601-610.
601
602
DONALD DUNCAN AND RICARDO MORALES TABLE 1
Results ofcounting and classifyingsynapses in the spinal substantia gelatinosa of the cat Cat no.
A-D
A-A
D-D
Glomerular associated synapes 9/24/74 128 0 1 12/30/15 260 2 2 1/6/76 171 0 8 1/20/76 197 0 3 5/14/76 99 1 2 2 3 5/17/76 331 Sub-totals 1,192 5 19 % of all 97.2% 0.7% 1.6% counted Non-glomerular synapses normal material 9124174 170 1 1 12/39/75 69 0 0 1/20/76 44 0 0 6 5/17/76 _154 _ 2 Sub-totals 437 3 7 % of all 97.1% 0.7% 1.5% counted Material devoid of glomeruli 3 2 1/6/76 120 1 5/14/76 158 1 -
Sub-totals
-
278
4
96.5%
1.5%
D-A
1 2 0 3 2 2 10 0.8%
1 1 0 1
3
0.7% 2 1 -
3
3
1%
1%
% of all
counted
A-D, axo-dendritic; A-A, axo-axonic; D-D, dendro-dendritic; D-A, dendro-axonic.
root terminals and the profiles in contact with them. Most of the surrounding profiles are dendrites and a few have been identified as axons (Gobel, '76). Synapses that were not associated with glomerular clusters were recorded as non-glomerular synapses. In normal material some of the contacts registered as non-glomerular undoubtedly were portions of a glomerulus cut in such a way that only a single dendrite and a portion of the central terminal were visible, but in deafferented specimens this probability was eliminated. Contacts of the axo-dendritic type vary greatly in length, contour and amounts of cytoplasmic and extracellular densities; likewise, there are appreciable differences in gap widths and in the appearance of gap material (Peters et al., '76). In this report all of the subtypes of axo-dendritic synapses were grouped together for comparison of their numbers with other types. RESULTS
Numerical results obtained in this study are summarized in table 1and the various types of
synaptic contacts that were counted are illustrated in figures 1-9.Most of the axo-dendritic synapses in the glomerular complexes are between the centrally placed terminals and the surrounding dendritic spines (fig. 2). In addition to the spines, the central terminals frequently synapse with larger profiles identified as dendritic shafts (fig. 1).Most of the spines, Gobel's ('76) type D1are rather empty looking profiles without synaptic vesicles (figs. 2, 7, 8). They greatly outnumber the D, variety which contain loose aggregates of mostly round vesicular profiles as depicted in figures 2 and 9. In one count from a single cat there were 5 D1 and 32 D,. In another field from the same cat there were 9 D, and 55 D,. In addition to the axo-dendritic synapses between central terminals and dendrites, a very occasional profile filled with a mixture of round, oval and flattened vesicles formed a synapse with a D1or D2dendritic spine and more often with dendritic shafts not associated with C terminals (fig. 4). These presynaptic endings were identified as belonging to the axons called p axons by Gobel ('76). They were not recorded separately but included in the total number of axo-dendritic synapses. As indicated in table 1very few axo-axonic synapses were encountered in the glomeruli, only five in a total of 1,226 synapses. The presynaptic element in these synapses contained many flattened vesicles (p axons) and the postsynaptic element was a central terminal (fig. 1).Dendro-dendritic synapses in the glomerular complexes were, without exception, D2-D1(fig. 2). Although more numerous than axo-axonic and dendro-axonic synapses they contributed only 1.7% of the total number counted. Likewise, the dendro-axonic synapses (fig. 3) were but a small fraction of the total. Most of the synapses that were not associated with the glomeruli were axo-dendritic and most of the presynaptic profiles contained round vesicular profiles. However, those containing numerous distinctly flattened vesicles were not inconsequential. Profiles containing flattened vesicles (fig. 4) were most evident in material that had been fixed with solutions containing 3% NaCL. Substantia gelatinosa material from one such cat, No. 5/17/76, was used to count the numbers of profiles containing nearly all round vesicles versus those with many flattened vesicles. The counts in three sample areas were 150r-l5f, 51r-5f, 31r-7f. Consequently, it was apparent that profiles
RATIOS OF FOUR TYPES OF SYNAPSES IN SUBSTANTIA GELATINOSA
containing flattened vesicles are much more numerous in neuropil that is not associated with the glomerular complexes. Recognition of axo-axonic synapses apart from the glomeruli was only by inference. When two small profiles both containing close and regularly spaced neurotubules were attached to each other by an asymmetric junction wherein the presynaptic element contained a few vesicles and the postsynaptic profile exhibited a distinct postsynaptic density the contact was recorded as a n axo-axonic synapse (fig. 5). A further suggestion of the synapse being axo-axonic was association of both profiles with a cluster of naked axons. Dendro-dendritic synapses in the non-glomerular category were easily recognized by the size of the profiles which were much larger than the unmyelinated axons and contained profiles attributed to smooth endoplasmic reticulum (fig. 6). When a larger profile containing scattered vesicles formed a distinct synapse with a smaller vesicle filled process the arrangement was recorded as dendro-axonic. Less attention was paid to specialized contacts without vesicles. It was obvious that most of them were puncta adhaerentia as described by Peters et al. ('76) for central nervous system tissue. It was equally apparent that their frequency exceeded all types of synapses except the axo-dendritic. Synaptic contacts with the cell bodies of substantia gelatinosa neurons were so few in relation to other synapses that they were not included in t h e counts. No tight or gap junctions between neural profiles were seen. Gap junctions (fig. 1) between astrocytes were numerous, about onethird of all specialized contacts in the two 150mesh squares that were counted for determining their frequency. In much smaller numbers gap junctions were located between astrocytes and neurons, between astrocytes and oligodendroglia and between oligodendroglia cells and neurons as previously reported by Morales and Duncan ('75). DISCUSSION
The results presented in this report were obtained mostly by observations undocumented by photography. Consequently, they should not be regarded as exact as to numbers and classification. Other investigators repeating the work might see and record twice as many axo-axonic, dendro-dendritic and dendro-axonic synapses as reported here. Although this
603
possibility appears unlikely to the authors, a doubling of the numbers of any or all types of the rarer forms of synapses would still leave the results in accord with the statement by Ralston ('71) who wrote with reference to the substantia gelatinosa that the "vast majority of the synapses are axo-dendritic." The criteria described by Gobel ('74, '76) for identification of profiles in the glomerular complexes of the trigeminal portion of the substantia gelatinosa were valid for the same formations in the lumbar levels. It is believed that they permit a large measure of certainty in our recognition of the four types of synapses occurring in these configurations. The D, type of dendritic spines was confirmed by repeatedly finding them attached by a narrow stalk to unmistakable dendritic shafts. The D2type a t tached by a neck to a dendritic shaft was seen in two instances but not photographically recorded. However, recent use of the modified Golgi method of Fairen et al. ('77) resulted in locating D, spines containing particles of gold. One of the spines was connected to a dendritic shaft by a relatively long neck (fig. 9). Since there was but one impregnated neuron in a considerable block of tissue, it is believed that the profiles containing both vesicles and gold particles and in synaptic contact with the central terminal of a glomerular complex are D:, spines as described by Gobel ('76). Likewise, the profiles filled with vesicles, many of which are distinctly flattened, were seen occasionally in continuity with small unmyelinated axons. Furthermore, similar profiles filled with a mixture of round, oval and flattened vesicles form typical axo-dendritic synapses in material devoid of glomerular complexes (fig. 4). Apart from the glomerular complexes most of the synapses present in the substantia gelatinosa conform with the repeatedly described characteristics of axo-dendritic synapses. In addition there are a few that are obviously dendro-dendritic because the contacting profiles are much too large to be naked axons and the scanty vesicles occupy a very small part of the total area. In addition the dendrites concerned contained bits of endoplasmic reticulum but no ribosomes (fig. 6). The number of dendro-dendritic synapses was much less than anticipated by reading the literature. It is possible t h a t some of those diagnosed as axo-dendritic were in fact dendrodendritic especially in the non-glomerular associated group, but the chances of this
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DONALD DUNCAN AND RICARDO MORALES
mistake with the glomerular complexes is regarded as unlikely and the values for both groups were essentially the same. Presumed contacts believed to be axo-axonic synapses were as illustrated in figure 5; namely, a small unmyelinated axon displaying a pronounced postsynaptic density, a distinct substance containing gap and another small profile containing one, two, three or more presynaptic vesicles. Seven such synapses were found among a total of 738. If there was certainty that these are axo-axonic, such synapses might play a significant role in the activities within the substantia gelatinosa despite their low incidence in relation to other types. A very low incidence of axo-axonic synapses is in keeping with the opinion of Famiglietti and Peters ('72) who doubt the existence of axo-axonic synapses in the lateral geniculate nucleus and the finding of only two such synapses by Saito ('74) in an extensive study of the nucleus dorsalis of the cat. Recently a new technique that permits recognition of dendrites impregnated by the Golgi method and subsequently processed for examination with the electron microscope has been described by Fairen e t al. ('77). Application of this method to the substantia gelatinosa may dispel most of the uncertainty accompanying present attempts to distinguish between small axonic and dendritic profiles. An initial use of the method in this laboratory is illustrated in figure 9. On the basis of their numerical preponderance it might be assumed that axo-dendritic synapses in the substantia gelatinosa far outweigh all other types combined in functional importance. The difference is greatly magnified if the greater lengths of axo-dendritic membrane specializations as seen in cross sections are indications of the surface areas of the various types. If, in addition, the transmitter released by the central terminals of the glomeruli is far more powerful than that of the others the difference in energy values becomes enormous. Such considerations are well within the range of the possible. It is known that large numbers of substance P containing fibers enter the substantia gelatinosa, Hokfelt et al. ('75, '76). It is known also that substance P has the same effect on some neurons in the substantia gelatinosa as peripherally applied noxious stimuli (Randic and Miletic, '77). In addition, pharmacological experiments have shown that the depolarizing potency of substance P is 1,000-9,000 times
greater than L-glutamate, Otsuka and Konishi ('76). While such facts suggest that all else must give way to a discharging central terminal, they do not imply that the other types of synapses in the glomerular complexes are inconsequential especially during intervals between dorsal root discharges into any portion of the substantia gelatinosa. Nevertheless, assuming that the results accumulated in this study are approximately correct it appears that from the standpoint of quantitative morphology all but a small fraction of synapses are axo-dendritic both within the glomerular complexes and in the rest of the substantia gelatinosa as well. ACKNOWLEDGMENTS
The authors wish t o thank Donald B. Edwards, Earl Pitsinger and Phyllis Fletcher for valuable assistance in the preparation of this manuscript. LITERATURE CITED Coimbra, A,, P. B. Sadre-Borges and M. M. Magalhaes 1974 The substantia gelatinosa of the rat. Fine structure, cytochemistry (acid phosphotase) and changes after dorsal root section. J. Neurocytol., 3: 199-217. Famiglietti, E. V.,and A. Peters 1972 The synaptic glomerulus and the intrinsic neuron in the lateral geniculate nucleus of the cat. J. Comp. Neur., 144: 287-590. Fairen, A., A. Peters and J. Saldanha 1977 A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J. Neurocytol., 6: 311-337. Gobel, S. 1971 Structural organization of the main sensory trigeminal nucleus. In: Oral-Facial Sensory and Motor Mechanisms. R. Dubner and Y. Kawanura, eds. Appleton-Century-Crofts, New York, pp. 183-202. 1974 Synaptic organization of the substantia gelatinosa glomeruli in the spinal trigeminal nucleus of the adult cat. J. Neurocytol., 3: 219-243. 1976 Dendroaxonic synapses in the substantia gelatinosa trigeminal nucleus of the cat. J. Comp. Neur., 167: 165.176. Gobel, S., and R. Dubner 1969 Fine structural studies on the main sensory trigeminal nucleus in t h e cat and rat. J. Comp. Neur., 137: 459-494. Hokfelt, T., R. Eldi, 0. Johansson, R. Luft, G. Nilsson and A. Animira 1976 Immunohistochemical evidence for separate populations of somatostatin-containing and substance p-containing primary afferent neurons in the rat. Neuroscience, 1: 131-136. Hokfelt, T., J. 0. Kellerth, G. Nilsson and B. Pernow 1975 Substance P. Localization in the central nervous system and in some primary sensory neurons. Science, 190: 889-190. Kerr, F. W. L. 1966 The ultrastructure of the spinal tra c t of the trigeminal nerve and the substantia gelatinosa. Exp. Neurol., 16: 359-376. 1970 The fine s truc ture of t h e subnucleus caudalis of the trigeminal nerve. Brain Res., 23: 129-245. 1971 Electron microscopic observations on primary deafferentation of the subcaudal nucleus of the trigeminal nerve. In: Oral-Facial Sensory and Motor Mechanisms. R. Dubner and Y. Kawanura, eds. Appleton-Century-Crofts, New York, pp. 159-181.
RATIOS OF FOUR TYPES OF SYNAPSES IN SUBSTANTIA GELATINOSA Morales, R., and D. Duncan 1975 Specialized contacts of astrocytes with astrocytes and with other cell types in the spinal cord of t h e cat. Anat. Rec., 182: 255-265. Otsuka, M.,and S. Konishi 1976 Substance P and excitatory transmitter of primary sensory neurons. Proc. of Cold Spring Harbor Symposium, “The Synapse,” 40: 135-143. Peters, A.,S. L. Palay and H. DeF. Webster 1976 The Fine Structure of t h e Nervous System. W. B. Saunders Co., Philadelphia, London, Toronto. Ralston, H. J.,111 1965 The organization of the substantia gelatinosa Rolandi in t h e cat lumbosacral cord. Z. Zell., 671: 1-23. 1969 Dorsal root projections to the dorsal horn in t h e cat spinal cord. J. Comp. Neur., 132: 303-330. 1971 The synaptic organization of the dorsal horn of the spinal cord and t h e ventrobasal thalamus of the cat. In: Oral-Facial Sensory and Motor Mechanisms.
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R. Dubner and Y. Kawanura, eds. Appleton-CenturyCrofts, New York, pp. 229-250. Randic, M., and V. Miletic 1977 Effect of substance P in cat dorsal horn neurones activated by noxious stimuli. Brain Research, 128: 164-169. Rethelyi, M., and J. Szentagothai 1969 The large complexes of the substantia gelatinosa. Exp. Brain Res., 7: 258-274. Rexed, B. 1954 A cytoarchitectonic atlas of the spinal cord in the cat. J. Comp. Neur., 100: 297-379. Saito, K. 1974 The synaptology and cytology of the Clarke cell in the nucleus dorsalis of the cat: an electron microscope study. J. Neurocytol., 3: 179-197. Westrum, L. E.,and R. C. Black 1971 Fine structural aspects of the synaptic organization of the spinal trigeminal nucleus (Pars interpolaris) of the cat. Brain Res., 25: 265-288.
PLATE 1 EXPLANATION OF FIGURES
All figures are from substantia gelatinosa of adult cats. Arrows point to post-synaptic profiles. 1 The central axonic terminal (C) of a glomerular complex displaying two axo-dendritic synapses and another axonic terminal (P) which is presynaptic to the central terminal (C), two gap junctions between astrocytic processes are located in the upper left-hand corner of the picture. X 50,000. 2 A dendro-dendritic synapse (D,-D,) and a portion of a glomerular central terminal
(C) are shown in this figure. X 50,000. 3 A dendro-axonic synapse (D2-C)in a glomerular complex is illustrated. Immediately to the right of the synapse there is a punctum adhaerens. X 100,000. 4
In this figure a profile (P axon) filled with round, oval and flattened vesicles is presynaptic to two dendrites. X 50,000.
5 In the center of this figure there is an asymmetric synapses tentatively identified as an axo-axonic synapse.
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X
71,000.
RATIOS OF FOCR T Y P E S O F S Y N A P S E S IN SUBSTANTIA CELATINOSA
1’1,AlE 1
Donald I h n c a n and Ricardo Moraies
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PLATE 2 EXPLANATION OF FIGURES
As in plate 1 all figures are from the substantia gelatinosa of normal adult cats 6 A dendro-dendritic synapse
(D-D). X 53,000
7 Showing a D, type of dendritic spine (S) attached by a narrow neck to the parent dendritic shaft (D). The two axo-dendritic contacts may be a sectional view of a ring synapse surrounding the neck of the spine. x 51,000. 8 A type D, spine connected by a neck with a dendritic shaft is In synaptic contact with a central terminal (0.X 38,200. 9 A portion of a gold-toned Golgi preparation showing a dendrite (D), neck and spine from an isolated impregnated neuron in the substantia gelatinosa. The spine contains a few synaptic vesicles (note pointed guide line) and the spines in the inset from a n adjacent section displays many vesicles. Both spines in the figure are in synaptic contact with a central terminal (C). x 42,000.
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