Journal of Neurocytology 7 , 1 1 9 - 1 3 3 (1978)
Postsynaptic specializations at excitatory and inhibitory cholinergic synapses HIROSHI
WATANABE
1 and G E O F F R E Y
BURNSTOCK
2
1Department ofA natomy, Toboku University, School of Medicine, Sendai 980, Japan 2Department of Anatomy and Embryology, University College London, Gower Street, London WCIE 6BT, England
Received 8 July 1976; revised and accepted 1 August 1976
Summary In serial sections of neurons in the paravertebral ganglia of the frog (Limnodynastes dumerili), the postsynaptic structures termed 'postsynaptic bar' (PSB) and 'junctional subsurface organ' (JSO)
were never observed in the same ganglion cell. Further, PSBs were found mostly in small ganglion cells (less than 22/~m), while JSOs were found mostly in large ganglion cells (up to 45 ~tm). Between 10 and 22 PSBs were located at both 'spine' and 'non-spinous' somatic synapses of the smaller ganglion cells; while 8 to 16 JSOs were located largely in the axon hillock region of the larger ganglion cells. Based on these observations, it is suggested that the two ganglion cell populations represent the B and C cell types defined according to electrophysiological data. Further, since the nerve terminals adjacent to both these postsynaptic structures appear to be cholinergic according to their vesicular conten L this provides some basis for suggesting that JSOs are associated with slow excitatory synapses, while PSBs are present at slow inhibitory synapses.
Introduction Electrophysiological studies of amphibian sympathetic ganglia have demonstrated the existence of sympathetic ganglion cells of two types, B- and C-cells, from which B- and C-fibres originate (Nishi et al., 1965). It has also been shown that the fast conduction (B-neuron) system is composed of large cells while the slow conduction (C-neuron) system is composed of small cells (Nishi et M., 1965, 1967; Honma, 1970b). Four different components o f postsynaptic potentials have been recognised in amphibian sympathetic ganglion ceils (a fast-EPSP, a slow-EPSP, a slow-IPSP and a late slow-EPSP). The fast-EPSP and the late slow-EPSP have been recorded from both B- and C-cells (Koketsu, 1969; Libet, 1970). Several authors have suggested that the slow-EPSP and slow-IPSP are recorded from different cell types; slow-EPSP from B-neurons and slow-IPSP from C-neurons (Tosaka et al., 1968; Weight and Votata, 1970; Weight and Padjen, 1973a, b). 9 1978 Chapman and Hall Ltd. Printedin GreatBritain
120
WATANABE and BURNSTOCK
The fine structure of amphibian s y m p a t h e t i c ganglia has been studied by several workers (De Robertis and Bennett, 1955; Taxi, 1961, 1967, 1976; Pick, 1963, 1970; Y a m a m o t o , 1963 ; Uchizono, 1964; F u i i m o t o , 1967; Nishi et al., 1967; Sotelo, 1968; Watanabe and Burnstock, 1976a). The existence of two types of neurons in amphibian s y m p a t h e t i c ganglia has been claimed on the basis of b o t h size (Nishi et al. 1965, 1967; Honma, 1970a) and cytoplasmic o p a c i t y (Fu]imoto, 1967). A b i m o d a l size distribution of neurons has also been reported for cultured s y m p a t h e t i c ganglia of the frog, Limnodynastes dumerili, the same species used in the present s t u d y (Hill and Burnstock, 1975). The purpose of the present s t u d y was to describe the nature of the p o s t s y n a p t i c specializations in s y m p a t h e t i c neurons of the frog and to see if these specializations could be related to the B and C neurons t h a t have been distinguished according to electrophysiological criteria. Methods Paravertebral sympathetic ganglia were excised from adult frog (Limnodynastes dumerili) in late summer (early May in Melbourne) and fixed for 45 rain at room temperature in 5% glutaraldehyde buffered to pH 7.4 with 0.1 M sodium phosphate. The specimens were then washed in buffer for 15 rain and postfixed with a buffered 1% osmium tetroxide solution for 45 rain. Tissues were washed briefly in distilled water, and block-stained in a 2% aqueous solution of uranyl acetate for 1 h. After dehydration in a graded acetone or alcohol series, the tissues were embedded in Araldite. Serial sections were cut using an LKB-III ultramicrotome and mounted on collodion film and carbon-coated single hold grids (0.5 mm). Sections were stained in an alcoholic solution of uranyl acetate followed by lead, and examined in a JEM-1OOB electron microscope.
Observations GANGLION CELLS S y m p a t h e t i c ganglion cells usually occurred in the s y m p a t h e t i c t r u n k in groups of variable size, each group being enclosed b y a thick connective tissue capsule. Inside Fig. 1. Light micrograph of a toluidine blue-stained section. Ganglion cells of various sizes are seen together with myelinated axons. A small ganglion cell (S) projects a thin axonal process (arrow). L, large ganglion cell. x 640. Fig. 2. Light micrograph showing thick axonal processes (P) originating from large ganglion cells (L). Small ganglion cells are also seen. x 640. Fig. 3a. A low magnification electron micrograph showing a large ganglion cell associated with a 'junctional subsurface organ' (JSO). Numerous mitochondria and dense bodies are present. Highly organised rough endoplasmic reticulum (ER) can be seen at the periphery of the soma. P, thick process originating from ganglion cell. x 5500. Fig. 3b. Higher magnification of the framed area in Fig. 3a. A JSO opposite a thin process of a satellite cell is associated with rough endoplasmic reticulum (arrow). G, cytoplasm of ganglion cell. x 19 000. Fig. 4. Electron micrograph showing part of the periphery of a small ganglion cell with a 'postsynaptic bar' (PSB) (arrow). A few large granular vesicles can be seen around elements of the Golgi apparatus. N, nucleus of satellite cell. x 9500.
122
WATANABE and BURNSTOCK
160 140 120 100 u
F~ I0) c 0
80 60
.,JO
E 40
Z
0
5
10
15 20 25 30 35 Size of gonglion cells ( pm }
40
45
Fig. 5. A histogram showing the size distribution of 550 sympathetic ganglion cells. The horizontal and vertical axes represent the diameter and numbers of measured ganglion cells. Thin open columns show the size distribution of 72 different cells associated with the PSB, and black columns show 68 cells associated with the JSO. The PSBs occur mostly in the small cells (less than 22 gm), while the JSOs occur in larger cells (up to 45/~m).
Fig. 6. A thick and short projection of a ganglion cell (G). Mitochondria and highly differentiated endoplasmic reticulum can be seen. x 9000. Fig. 7. A small projection (P) arising from its ganglion cell (G). The projection is partly without a satellite sheath (S). x 19 000. Fig. 8. A micrograph showing an axonal process provided with a JSO (arrow). Large numbers of neurofilaments and microtubules are present in the process. A dense innervation can be seen around the axon hillock. G, somatic cytoplasm; S, satellite sheath, x 9000. Fig. 9. An axonal process rising from a large ganglion cell (G). A well developed JSO (arrow) is formed around the axon hillock. A membrane thickening (double arrows) is evident on the other side of the axon hillock. S, thin processes of satellite cells, x 11 000. Fig. 10. An axonal process containing many microtubules and a few mitochondria. A PSB (arrow) is seen adjacent to a nerve ending which formed a synapse on the axon hillock. G. somatic cytoplasm, x 15 000. Fig. 11. A preganglionic nerve fibre which is completely enclosed by thin processes of satellite cells (S). Large granular vesicles are seen together with a smaller number of small agranular vesicles. G, cytoplasm of ganglion cell. x 13 000. Fig. 12. Enlarged nerve endings containing large numbers of small agranular vesicles. A few large granular vesicles are also seen. Somatic spines (P) project into the nerve endings. G, cytoplasm of ganglion cell. x 16 000.
124
WATANABE and BURNSTOCK
the capsule large numbers of unmyelinated and myelinated axons, clusters of small granule-containing cells and blood vessels were seen together with ganglion cells of various sizes (Figs. 1 and 2). The ganglion cells showed a spherical, elongate or polyhedral shape. The nucleus was usually placed at the periphery of the soma, distant from the axon hillock; it was spherical and usually contained one prominent nucleolus. More rarely, two or three nucleoli were observed. Light microscopic observations with serial thick sections revealed that most ganglion cells had only one large ( 1 - 4 / l m ) axon process (Figs. 1 and 2). Electron microscope observations from serial thin sections supported this finding, since only one well defined process, containing densely packed neurofilaments, microtubules and a few mitochondria, was observed arising from each ganglion cell (Figs. 8 - 1 0 ) . Various other profiles ( 0 . 2 - 4 / ~ m in diameter) arising from the ganglion cells were sometimes observed. Many of these profiles were partially free of a satellite cell sheath. Some had a cytoplasmic structure similar to that of the peripheral area of the soma, containing rough endoplasmic reticulum, mitochondria, multivesicular bodies and free ribosomes (Fig. 6). Neurofilaments and microtubules were also present. Studies of serial sections suggested that these profiles represented peripheral sections through the tips of polyhedral or elongate ganglion cells. Other profiles resembled 'somatic spines' or 'dendritic processes' (Fig. 7); they were thin and mostly remained unde~ the satellite sheath enclosing the ganglion cells. The ganglion cells contained the cytoplasmic components found in other autonomic ganglion cells, such as rough and smooth endoplasmic reticulum, Golgi apparatus, ribosomes, dense bodies and large lipid droplets (Figs. 3a and 4). Rough endoplasmic reticulum was observed chiefly in the periphery of the soma (Fig. 3a). Mitochondria, neurofilaments and multivesicular bodies were also common components of the perikaryal cytoplasm. Mitochondria, up to 4 t~m in length were often Figs. 13aand b. Semi-serial sections through the same nerve endings (E). Mixed in the nerve ending are: (a) spherical and flat-shaped small agranular vesicles and (b) small agranular vesicles together with a few granular vesicles, x 27 000. Fig. 14. A nerve ending (E) showing a rather complicated contour. A JSO lies opposite a thin satellite cell process (S). A spinous projection (P) is seen in the other nerve ending. Small agranular vesicles of varied shape and size are present in the endings, x 39 000. Fig. 15. A PSB in a somatic spine arising from a small ganglion cell (G). S, satellite sheath. • 26 000. Fig. 16. A symmetrical specialization between opposing pre- and postsynaptic membranes. x 45 000. Figs. 17a,b and c. Electron micrographs showing PSBs of various shapes. (a) A PSB composed of a series of a few small dense particles, x 39 000. (b) A doughnut-shaped PSB in a somatic spine. x 26 000. (c) An oval-shaped PSB. Note the various shaped small agranular vesicles in the nerve ending and the large granular vesicle that abuts on the presynaptic membrane (arrow). x 48 000. Fig. 18. A JSO adjacent to a postsynaptic membrane. No membrane specialization or aggregation of synaptic vesicles can be seen. M, mitochondrion, x 26 000. Fig. 19. A JSO separated from a presynaptic nerve ending by a thin process of satellite cell (S). The JSO is accompanied by an underlying cistern of endoplasmic reticulum (arrow) and a mitochondrion (M). x 26 000.
126
WATANABE and BURNSTOCK
prominent and a small number of electron dense granular vesicles (about 100 nm in diameter) were distributed throughout the soma and more often around the Golgi apparatus (Fig. 4). Ganglion cells varied in diameter from about 10/~m to 45 gin. The distribution of sizes of 550 cells is shown in Figure 5. The measurements were carried out on electron micrographs deriving from six blocks from three frogs. The diameter of elongate or polyhedral somata was expressed as the mean of long and short axes. The cell size was measured regardless of the presence or absence of the nucleus in the plane of section, since the nucleus was eccentrically situated in the soma. However, in order to avoid underestimation of the cell size, profiles of less than 5/lm in diameter were not included. A single mode of cell size is apparent at about 15 ~m. However, as described in detail later, on the basis of the distribution of the 'post-synaptic bar' (PSB) and 'junctional subsurface organ' (JSO) (see Fig. 5), there is some suggestion of two overlapping populations, since mean diameters of the cells containing PSBs and of the cells containing JSOs were 15/lm and 26/~m, respectively. No other differences in fine structure of the somata of the larger and smaller sympathetic ganglion cells were observed (Figs. 3a and 4). SATELLITE CELLS
Satellite cells surrounding the somata of ganglion cells showed a similar structure to that described previously in autonomic ganglia (Taxi, 1976; Gabella, 1976). The nucleus was mostly ellipsoidal in shape (Fig. 4). The satellite envelope was thicker around the perinuclear portion of the satellite cells. In other places, one or several layers of thin satellite processes enveloped the perikaryal surface of ganglion cells. The configuration of satellite cells was complicated by their membrane infoldings around incoming preganglionic nerve fibres (Fig. 11). Small areas of the perikaryal surface of ganglion cells directly facing the basal lamina of the satellite cell were without a covering of satellite cell processes. Similar satellite cell-free areas were commonly seen around the axon hillock (Fig. 9). Preganglionic nerve fibres loosely covered parts of these free surfaces. I N N E R V A T I O N OF TIIE G A N G L I O N CELLS
Around the ganglion cells, preganglionic nerve fibres were completely enclosed by one or several layers of thin processes of satellite cells (Fig. 11). These nerve fibres gradually lost the lamellar sheath to come into direct contact with the perikaryal surface of the ganglion cells, where they formed enlarged nerve endings (Fig. 12). In some places the nerve endings intruded deeply into the cytoplasm of ganglion cells without intervention of the satellite sheath (Fig. 13). These invaginated nerve endings occurred more often in the large ganglion cells than in the small cells. Endings were more densely distributed around the axon hillock, especially in the larger cells (Fig 8). On the basis of vesicle content, only one type of synaptic ending was found on the perikaryal surface of both large and small sympathetic ganglion cells. The nerve endings contained many small agranular vesicles together with a few large granuiar vesicles (60-100 nm) (Figs. 12 and 13). The ratio of large granular
Postsynaptic structures in frog sympathetic ganglion cells
127
vesicles to small agranular vesicles varied from one profile to another. In some sections, for example, a nerve profile contained exclusively small agranular vesicles, but examination of serial sections revealed that the same nerve ending also contained large granular vesicles (Figs. 13a and b). The large granular vesicles were more prominent in the preterminal segments of nerve fibres which were completely ensheathed by satellite cells (Fig. 11). The shape and size (30-60 nm) of the small agranular vesicles were less uniform than that of the large granular vesicles. 'Flattened' or 'elongate' small agranular vesicles were usually seen together with a larger number of spherical vesicles (Figs. 13, 14, and 16). Nerve endings containing aggregations of glycogen particles were not seen. Several types of small granule-containing cells were noted in the frog sympathetic ganglia. They did not form synapses with ganglion cells. POSTSYNAPTIC SPECIALIZATIONS
Preganglionic nerve endings formed synapses on the ganglion cells vcith a cleft of about 1 5 - 2 0 nm in width. These endings varied in shape and size: some showed an oval or elongated contour (0.5-4 tam in diameter) with a smooth presynaptic membrane (Fig. 13a); while others showed a contour invaginated by small projections arising from the postsynaptic neurons and satellite cells (Fig. 14). These 'spine synapses' with perikaryal projections, or 'somatic spines' occurred in relation to both large and small ganglion cells (Figs. 14 and 15). Dense areas were observed on both pre- and postsynaptic membranes. In most cases, the density increase was more prominent on the postsynaptic side; an aggregation of synaptic vesicles was present adjacent to the presynaptic membrane (Figs. 14 and 15). These aggregations consisted largely of small agranular vesicles, but a few large granular vesicles were sometimes also present (Figs. 17b and c). This type of membrane specialization occurred both in synapses with a smooth contour and in synapses with spines (Figs. 14 and 15). In addition to these asymmetrical membrane specializations, non-synaptic symmetrical specializations were also formed between the pre- and postsynaptic membranes (Fig. 16). Other characteristic postsynaptic structures were the 'postsynaptic bar' and 'junctional subsurface organ'. General features of these special postsynaptic and subsurface structures have been described previously (Watanabe and Burnstock, 1976a). In the present study, the distribution of these two postsynaptic structures on ganglion cells of different sizes has been examined.
Postsynaptic bar (PSB) The PSB was found mostly in ganglion cells smaller than 22/am (Fig. 5). The largest cell with a PSB was 28 tam in diameter. The PSBs were seen all round the perikaryon of one neuron, but were not formed on the axon. The PSB was present in the cytoplasm adjacent to the postsynaptic membrane showing the asymmetrical specialization, which was associated with an aggregation of synaptic vesicles on the presynaptic side (Figs. 15 and 17). Both somatic spine and non-spinous postsynaptic
128
WATANABE and BURNSTOCK
cytoplasm contained the PSB (Figs. 4 and 15). The narrow gap between the postsynaptic membrane and the outer surface of the PSB was 25--50 nm. The PSB was usually seen as an electron dense bar or disc (0.2-0.5/~m in length), and analysis of serial sections revealed that it varied in shape according to the contour of the postsynaptic site, especially in the spine synapse (Figs. 17a, b and c). In some sections, the PSB was a doughnut- or horseshoe-shape, while in others it seemed to be composed of a series of dense particles. No close topographical correlation was apparent between PSB and mitochondria o r endoplasmic reticulum. From observations on complete serial sections of four small cells (see Table 1) it was shown that such cells contained between~10 and 22 PSBs. The PSBs occupied between 52% and 71% of the total number of postsynaptic membrane specializations. Table 1
maximum diameter
cell
Number of PSBs
(gm) A B C D
18 15 25 24
x x x x
12 14 20 20
12 10 22 16
%ofpostsynaptic membrane specializations associated with PSBs 71 67 52 70
Table 2
cell
maximum diameter
Number of JSOs
mean diameter of JSO (tJm)
8 10 13 15 16
0.5 0.6 0.5 0.4 0.7
(~m) A B C D E
36 28 40 36 42
x x x x x
30 22 35 28 36
• x x • x
0.7 0.7 0.7 0.5 0.9
Junctional Subsurface Organ (JSO) The JSO occurred mainly in the larger ganglion cells up to 45/am in diameter (Fig. 5). JSOs appeared just beneath the perikaryal cell membrane, and were particularly well developed around the axon hillock (Fig. 9). Five larger cells were examined in complete serial sections (see Table 2). These cells contained from 8 to 16 JSOs. The shape of JSO varied according to the contour of the perikaryal surface (Figs. 14, 18 and 19). As has been noted elsewhere (Watanabe and Burnstock,
Postsynaptic structures in frog sympathetic ganglion cells
129
1976a), the narrow space between the JSO and the overlying cell membrane was of constant width (10-12 nm). The JSOs were close to mitochondria and endoplasmic reticulum (Figs. 9 and 19). None of the ganglion cells examined in the present study possessed both PSB and
jso. Discussion Two types of neuron in the frog sympathetic ganglion have been distinguished in the present study on the basis of distinctive postsynaptic structures and to some extent also on cell size. PSBs were observed in the smaller sympathetic ganglion cells, while JSOs were found in the larger cells. It is important to try to correlate this morphological finding with physiological observations on amphibian sympathetic ganglia. Since both the fast-EPSP and the late slow-EPSP have been recorded from both B (large) and C (small) cells (Koketsu, 1969; Libet 1970), it is not possible to relate the specific postsynaptic structures observed in either small or large neurons in the present study to these synaptic events. It has been claimed, however, that a slow-EPSP occurs in the B (large) type neuron (Tosaka et al., 1968; Weight and Votata, 1970) and a slow-IPSP in the C (small) type (Tosaka et al., 1968; Weight and Padjen, 1973a,b). This suggests that ~:he PSB, which appeared exclusively in the small cells, is involved in the generation of the slow-IPSP, while the JSO may be involved in the generation of the slow-EPSP in the large ganglion cells. It has been reported that both slow-EPSPs and slow-IPSPs can be recorded occasionally in the same cells (see Koketsu, 1969; Nakamura and Koketsu, 1972) but, while the existence of ganglion cells containing both JSO and PSB in one cell cannot be excluded, this has not been observed in the present study. The slow-PSPs have a long synaptic delay, 30-100 milliseconds for the slow-IPSP (Libet, 1967; Libet etal., 1968) and 200-300 milliseconds for the slow-EPSP (Libet, 1967, 1970). The longer synaptic delay of the slow-EPSP has been interpreted as an indication of a greater energy requirement, since metabolic inhibitors depress the slow-EPSP much earlier than the fast-EPSP or the slow-IPSP (see Libet, 1970). Further, the latency at muscarinic synapses (associated with slow-EPSP) is always longer than at nicotinic synapses (associated with fast-EPSP) (Purves, 1976). The present results allow some further speculations to be made about the relation of these physiological parameters to the topography of the junctions and, in particular, the postsynaptic specializations. For example, long synaptic delays may be related to the frequent presence of thin satellite sheaths between the presynaptic membrane and the JSO (Figs. 18 and 19; see also Watanabe and Burnstock, 1976a). The close relationship between the JSO and mitochondria or rough endoplasmic reticulum adds morphological support to the physiological interpretations, indicating the dependence on energy supply for the generation of the slow-EPSP (Libet, 1970). It is well known that fast-EPSPs and slow-EPSPs are produced by the direct action
130
WATANABE and BURNSTOCK
of ACh released from the presynaptic nerve endings (Koketsu, 1969; Libet, 1970). It has also been shown that fast-EPSPs can be recorded both from B (large type) and from C (small type) ganglion cells. Mediation of the slow-IPSP is poorly understood in amphibians. In mammalian sympathetic ganglia, various authors have suggested that the slow-IPSP is mediated by catecholamines released from either adrenergic interneurons, chromaffin cells, small intensely fluorescent cells or small granulecontaining cells (Eccles and Libet, 1961; Libet, 1970; Libet and Owman, 1974). Libet and Kobayashi (1974) came to the same conclusion for bullfrog sympathetic ganglion. On the other hand, in the present study there was no evidence for adrenergic or catecholamine-containing elements forming synapses with large or small ganglion cells, supporting earlier observations on amphibian sympathetic ganglia by fluorescence microscopy (Norberg and McIsaac, 1967; Honma, 1970a; Hill et al., 1975) and by electron microscopy (Weitsen and Weight, 1973; Watanabe and Burnstock, 1976b). Alternatively it has been suggested that ACh may be the transmitter mediating the slow-IPSP in C type cells (Weight and Padjen, 1973a,b;Weitsen and Weight, 1973; Libet and Kobayashi, 1974; Koketsu and Yamamoto, 1975). This view is supported by the present finding that all the nerve endings observed are structurally similar, and are all presumably cholinergic. It has been claimed that the late slow-EPSP, appearing in both B and C type neurons, is mediated by an unknown non-cholinergic transmitter (Nishi and Koketsu, 1968; Koketsu, 1969), but the present study did not reveal the presence of a morphologically distinct nerve ending which was clearly not cholinergic. Some 'flat' or elongated vesicles were seen, but these were usually found together with a large number of spherical vesicles in the same nerve ending. This conclusion that there is a single type of nerve ending on amphibian sympathetic ganglion cells is supported by most workers in the field (see Nishi et al., 1967; Gabella, 1976; Taxi, 1976); and to quote Taxi (1976), 'The assertion that there are several different functional types of preganglionic endings in amphibian sympathetic ganglia (Uchizono, 1964; Uchizono and Ohsawa, 1973), on the basis of various proportions of vesicles having different sizes and contents, seems highly questionable and may correspond merely to the variability of presynaptic vesicular contents'. Nerve endings with an aggregation of glycogen particles (Taxi, 1961; Yamamoto, 1963; Fujimoto, 19"67; Pick, 1970) were not seen in the present study. Seasonal differences may explain the discrep~tncy, since Mizell (1965) has reported that the liver glycogen content is higher in the autumn and decreases through winter and is lowest in the summer (see also Gabella, 1976). It seems unlikely that neurons in amphibian sympathetic ganglia can be divided into two types solely on the criterion of size; although this has been the basis of previous attempts to distinguish B and C cells according to electrophysiological properties. In any case it is a difficult task to make this type of analysis (see for example Konigsmark et al., 1970; Hendry, 1976). In the present study, cells with PSBs were about 15 btm in mean diameter, while those with JSOs were about 26 gm in mean diameter. However, mean diameters may be somewhat underestimated (see
Postsynaptic structures in frog sympathetic ganglion cells
131
Weibel, 1973). If we assume that the shape of these ganglion cells is spherical, the more accurate mean diameter (D) can be calculated by the following equation: 410 = --d (d, a mean diameter obtained from random sections). 7r According to the equation, the diameters of the ganglion cells with JSOs and PSBs are 33 ~m and 19/~m respectively. Values very close to these have been observed by Nishi et al. (1965, 1967) in toad; B- and C-neurons are 34 ~m and 18 #m in diameter respectively. A similar classification has also been proposed from the light microscopic examination of the toad sympathetic ganglion (Honma, 1970b), and it was suggested from the results of intracellular marking that the large neurons might represent the fast B conduction system and the small neurons the slow C system. Amphibian sympathetic ganglion cells have been considered by most authors to be unipolar (Uchizono, 1964; see also Pick, 1970), although 'somatic spines' (Piezzi and Rodriguez Echandia, 1968) and occasional 'dendritic processes' (Pick, 1963) have also been noted. The present light and electron microscopic examinations of serial sections confirm this finding. Recently Hill and Burnstock (1975) have reported that many frog sympathetic ganglion cells in culture are multipolar; but they suggested that the production of extra processes by cells in culture may be a response to cutting the postganglionic fibres during the initial tissue preparation. Acknowledgements Some of this work was carried out in the Department of Zoology, Melbourne University, and was supported by grants from the National Health and Medical Research Council and the Australian Research Grant Committee. We would like to thank Professor Toshi-Yuki Yamamoto, Dr. Robert Purves and Dr. Giorgio Gabella for their criticisms of the manuscript. References DE ROBERTIS, E. D. P. and BENNETT, H.S. (1955) Some features of the submicroscopic morphology of synapses in frog and earthworm. Journal of Biophysical and Biochemical Cytology 1 , 4 7 - 5 8 . ECCLES, R. M. and L1BET, B. (1961) Origin and blockade of the synaptic responses of curarized sympathetic ganglia. Journal of Physiology 1 5 7 , 4 8 4 - 5 0 3 . FUJIMOTO, S. (1967) Some observations on the fine structure of the sympathetic ganglion of the toad, Bufo vulgarisjaponicus. Archivum Histologicum Japonicum 28, 3 1 3 - 3 5 . GABELLA, G. (1976) Structure of the autonomic nervous system. London: Chapman and Hall. HENDRY, I . A . (1976) A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment. Journal of Neurocytology 5, 337-49. HILL, C.E., WATANABE, H. and BURNSTOCK, G. (1975) Distribution and morphology of amphibian extra-adrenal chromaffin tissue. Cell and Tissue Research 60, 371--87. HILL, C. E. and BURNSTOCK, G. (1975) Amphibian sympathetic ganglia in tissue culture. Cell and Tissue Research 1 6 2 , 2 0 9 - 3 3 .
132
WATANABE and BURNSTOCK
HONMA, S. (1970a) Histochemical demonstration of catecholamines in the toad sympathetic ganglia. Japanese Journal of Physiology 20, 186-97. HONMA, S. (1970b) Functional differentiation in sB and sC neurons of toad symapthetic ganglia. Japanese Journal of Physiology 2 0 , 2 8 1 - 9 5 . KOKETSU, K. (1969) Cholinergic synaptic potentials and the underlying ionic mechanisms. Federation Proceedings 28, 1 0 1 - 1 2 . KOKETSU, K. and YAMAMOTO, K. (1975) Unusual cholinergic response of bullfrog sympathetic ganglion cells. European Journal of Pbarmacology 31, 281-6. KONIGSMARK, B.W., KALYANARAMAN, V. P., COREY, P. and MURPHY, E. A. (1970) An evaluation of techniques in neuronal population estimates: the sixth nerve nucleus. Johns Hopkins Medical Journal 125, 1 4 6 - 5 8 . LIBET, B. (1967) Long latent periods and further analysis of slow synaptic responses in sympathetic ganglia. Journal of Neuropbysiology 3 0 , 4 9 4 - 5 1 4 . LIBET, B. (1970) Generation of slow inhibitory and excitatory postsynaptic potentials. Federation Proceedings 29, 1945-56. LIBET, B., CHICHIBU, S. and TOSAKA, T. (1968) Slow synaptic responses and excitability in sympathetic ganglia of the bullfrog. Journal ofNeuropbysiology 31, 383-95. LIBET, B. and KOBAYASHI, H. (1974) Adrenergic mediation of slow inhibitory postsynaptic potential in sympathetic ganglia of the frog. Journal of Neuropbysiology 37, 8 0 5 - 1 4 . L1BET, B. and OWMAN, Ch. (1974)Concomitant changes in formaldehyde-induced fluorescence of dopamine interneurones and in slow inhibitory postsynaptic potentials of the rabbit superior cervical ganglion, induced by stimulation of the preganglionic nerve or by a muscarinic agent. Journal of Physiology 237, 6 3 5 - 6 2 . MIZELL, S. (1965) Seasonal changes in energy reserves in the common frog, Rana pipiens. Journal of Cellular and Comparative Pbysiology 66, 2 5 1 - 8 . NAKAMURA, M. and KOKETSU, K. (1972) The effect of adrenaline on sympathetic ganglion cells of bullfrogs. Life Sciences 11, 1165--73. NISHI, S., SOEDA, H. and KOKETSU, K. (1965) Studies on sympathetic B and C neurons and patterns of preganglionic innervation. Journal of Cellular and Comparative Physiology 66, 19-32. NISHI, S., SOEDA, H. and KOKETSU, K. (1967) Release of acetylcholine from sympathetic preganglionic nerve terminals. Journal of Neuropbysiology 30, 1 1 4 - 3 4 . NXSHI, S. and KOKETSU, K. (1968) Early and late after-discharges of amphibian sympathetic ganglion cells. Journal of Neurophysiology 31, 1 0 9 - 2 1 . NORBERG, K . - A . and MclSAAC, R_ J. (1967) Cellular location of adrenergic amines in frog sympathetic ganglia. Experientia 23, 1052. PICK, J. (1963) The submicroscopic organization of the sympathetic ganglion in the frog (Rana pipiens). Journal of Comparative Neurology 120, 4 0 9 - 6 2 . PICK, J. (1970) The autonomic nervous system. Morpbological, comparative, clinical and surgical aspects. Philadelphia and Toronto: J. B. Lippincott Company. PIEZZI, R. S. and RODRIGUEZ ECHANDIA, E. L. (1968) Studies on the para-renalganglion of the toad Bufo arenarum Hensel. I. Its normal fine structure and histochemical characteristics. Zeitscbrift fiir Zellforschung und mikroskopiscbe Anatomie 88, 180--6. PURV ES, R. D. (1976) Function of muscarinic and nicotinic acetylcholine receptors. Nature 261, 149--51. SOTELO, C. (1968) Permanence of postsynaptic specializations in the frog sympathetic ganglion cells after denervation. Experimental Brain Research 6, 2 9 4 - 3 0 5 . TAXI, J. (1961) Etude de l'ultrastructure des zones synaptiques darts les ganglions sympathiques de la grenouille. Comptes rendus de l'Acadkmie des Sciences, Paris 252, 174-6. TAXI, J. (1967) Observations on the ultrastructure of the ganglionic neurons and synapses of the
P o s t s y n a p t i c structures in frog s y m p a t h e t i c ganglion cells
13 3
frog Rana esculenta L. In: The Neuron (edited by HYDEN, H.), pp. 221--54. Amsterdam, London and New York: Elsevier Publishing Company. TAXI, J. (1976) Morphology of the autonomic nervous system. In: Frog Neurobiology, (edited by R. LLIN,~S and w. PRECHT), pp. 93-150. Berlin: Springer-Verlag. TOSAKA, T., CH1CHIBU, S. and LIBET, B. (1968) Intracellular analysis of slow inhibitory and excitatory postsynaptic potentials in sympathetic ganglia of the frog. Journal of Neurophysiology 31, 396-409. UCHIZONO, K. (1964)On different types of synaptic vesicles in the sympathetic ganglia of amphibia. Japanese Journal of Physiology 14, 210-19. UCHIZONO, K. and OHSAWA, K. (1973) Morpho-physiological consideration on synaptic transmission in the amphibian sympathetic ganglion. A cta Physiologica Polanica 24, 205-14. WATANABE, H. and BURNSTOCK, G. (1976a) Junctional subsurface organs in frog sympathetic ganglion cells. Journal of Neurocytology 5, 125-136. WATANABE, H. and BURNSTOCK, G. (1976b) A special type of small granule-containing cell in the abdominal para-aortic region of the frog. Journal of Neurocytology 5,465-78. WEIBEL, E.R. (1973) Stereological techniques for electron microscopic morphometry. In: Principles and techniques of electron microscopy (edited by HAYAT, M.A.) Biological applications. 3, pp. 237--296. New York, Cincinnati, Toronto, London and Melbourne: Van Nostrand Reinhold Company. WEIGHT, F. F. and VOTATA, J. (1970) Slow synaptic excitation in sympathetic ganglion cells: evidence for synaptic inactivation of potassium conductance. Science 170,755-8. WEIGfIT, F.F. and PADJEN, A. (1973a) Slow synaptic inhibition: evidence for synaptic inactivation of sodium conductance in sympathetic ganglion cells. Brain Research 55,219-24. WEIGHT, F.F. and PADJEN, A. (1973b) Acetylcholine and slow synaptic inhibition in frog sympathetic ganglion cells. Brain Research 55,225-8. WEITSEN, H.A. and WEIGHT, F. E. (1973) Chromaffin cells in the frog sympathetic ganglion: morphology not consistent with role in generation of synaptic potentials. Anatomical Record 175,467. YAMAMOTO, T. (1963) Some observations on the fine structure of the sympathetic ganglion of bullfrog. Journal of Cell Biology 16, 159-70.
Postsynaptic specializations at excitatory and inhibitory cholinergic synapses HIROSHI
WATANABE
1 and G E O F F R E Y
BURNSTOCK
2
1Department ofA natomy, Toboku University, School of Medicine, Sendai 980, Japan 2Department of Anatomy and Embryology, University College London, Gower Street, London WCIE 6BT, England
Received 8 July 1976; revised and accepted 1 August 1976
Summary In serial sections of neurons in the paravertebral ganglia of the frog (Limnodynastes dumerili), the postsynaptic structures termed 'postsynaptic bar' (PSB) and 'junctional subsurface organ' (JSO)
were never observed in the same ganglion cell. Further, PSBs were found mostly in small ganglion cells (less than 22/~m), while JSOs were found mostly in large ganglion cells (up to 45 ~tm). Between 10 and 22 PSBs were located at both 'spine' and 'non-spinous' somatic synapses of the smaller ganglion cells; while 8 to 16 JSOs were located largely in the axon hillock region of the larger ganglion cells. Based on these observations, it is suggested that the two ganglion cell populations represent the B and C cell types defined according to electrophysiological data. Further, since the nerve terminals adjacent to both these postsynaptic structures appear to be cholinergic according to their vesicular conten L this provides some basis for suggesting that JSOs are associated with slow excitatory synapses, while PSBs are present at slow inhibitory synapses.
Introduction Electrophysiological studies of amphibian sympathetic ganglia have demonstrated the existence of sympathetic ganglion cells of two types, B- and C-cells, from which B- and C-fibres originate (Nishi et al., 1965). It has also been shown that the fast conduction (B-neuron) system is composed of large cells while the slow conduction (C-neuron) system is composed of small cells (Nishi et M., 1965, 1967; Honma, 1970b). Four different components o f postsynaptic potentials have been recognised in amphibian sympathetic ganglion ceils (a fast-EPSP, a slow-EPSP, a slow-IPSP and a late slow-EPSP). The fast-EPSP and the late slow-EPSP have been recorded from both B- and C-cells (Koketsu, 1969; Libet, 1970). Several authors have suggested that the slow-EPSP and slow-IPSP are recorded from different cell types; slow-EPSP from B-neurons and slow-IPSP from C-neurons (Tosaka et al., 1968; Weight and Votata, 1970; Weight and Padjen, 1973a, b). 9 1978 Chapman and Hall Ltd. Printedin GreatBritain
120
WATANABE and BURNSTOCK
The fine structure of amphibian s y m p a t h e t i c ganglia has been studied by several workers (De Robertis and Bennett, 1955; Taxi, 1961, 1967, 1976; Pick, 1963, 1970; Y a m a m o t o , 1963 ; Uchizono, 1964; F u i i m o t o , 1967; Nishi et al., 1967; Sotelo, 1968; Watanabe and Burnstock, 1976a). The existence of two types of neurons in amphibian s y m p a t h e t i c ganglia has been claimed on the basis of b o t h size (Nishi et al. 1965, 1967; Honma, 1970a) and cytoplasmic o p a c i t y (Fu]imoto, 1967). A b i m o d a l size distribution of neurons has also been reported for cultured s y m p a t h e t i c ganglia of the frog, Limnodynastes dumerili, the same species used in the present s t u d y (Hill and Burnstock, 1975). The purpose of the present s t u d y was to describe the nature of the p o s t s y n a p t i c specializations in s y m p a t h e t i c neurons of the frog and to see if these specializations could be related to the B and C neurons t h a t have been distinguished according to electrophysiological criteria. Methods Paravertebral sympathetic ganglia were excised from adult frog (Limnodynastes dumerili) in late summer (early May in Melbourne) and fixed for 45 rain at room temperature in 5% glutaraldehyde buffered to pH 7.4 with 0.1 M sodium phosphate. The specimens were then washed in buffer for 15 rain and postfixed with a buffered 1% osmium tetroxide solution for 45 rain. Tissues were washed briefly in distilled water, and block-stained in a 2% aqueous solution of uranyl acetate for 1 h. After dehydration in a graded acetone or alcohol series, the tissues were embedded in Araldite. Serial sections were cut using an LKB-III ultramicrotome and mounted on collodion film and carbon-coated single hold grids (0.5 mm). Sections were stained in an alcoholic solution of uranyl acetate followed by lead, and examined in a JEM-1OOB electron microscope.
Observations GANGLION CELLS S y m p a t h e t i c ganglion cells usually occurred in the s y m p a t h e t i c t r u n k in groups of variable size, each group being enclosed b y a thick connective tissue capsule. Inside Fig. 1. Light micrograph of a toluidine blue-stained section. Ganglion cells of various sizes are seen together with myelinated axons. A small ganglion cell (S) projects a thin axonal process (arrow). L, large ganglion cell. x 640. Fig. 2. Light micrograph showing thick axonal processes (P) originating from large ganglion cells (L). Small ganglion cells are also seen. x 640. Fig. 3a. A low magnification electron micrograph showing a large ganglion cell associated with a 'junctional subsurface organ' (JSO). Numerous mitochondria and dense bodies are present. Highly organised rough endoplasmic reticulum (ER) can be seen at the periphery of the soma. P, thick process originating from ganglion cell. x 5500. Fig. 3b. Higher magnification of the framed area in Fig. 3a. A JSO opposite a thin process of a satellite cell is associated with rough endoplasmic reticulum (arrow). G, cytoplasm of ganglion cell. x 19 000. Fig. 4. Electron micrograph showing part of the periphery of a small ganglion cell with a 'postsynaptic bar' (PSB) (arrow). A few large granular vesicles can be seen around elements of the Golgi apparatus. N, nucleus of satellite cell. x 9500.
122
WATANABE and BURNSTOCK
160 140 120 100 u
F~ I0) c 0
80 60
.,JO
E 40
Z
0
5
10
15 20 25 30 35 Size of gonglion cells ( pm }
40
45
Fig. 5. A histogram showing the size distribution of 550 sympathetic ganglion cells. The horizontal and vertical axes represent the diameter and numbers of measured ganglion cells. Thin open columns show the size distribution of 72 different cells associated with the PSB, and black columns show 68 cells associated with the JSO. The PSBs occur mostly in the small cells (less than 22 gm), while the JSOs occur in larger cells (up to 45/~m).
Fig. 6. A thick and short projection of a ganglion cell (G). Mitochondria and highly differentiated endoplasmic reticulum can be seen. x 9000. Fig. 7. A small projection (P) arising from its ganglion cell (G). The projection is partly without a satellite sheath (S). x 19 000. Fig. 8. A micrograph showing an axonal process provided with a JSO (arrow). Large numbers of neurofilaments and microtubules are present in the process. A dense innervation can be seen around the axon hillock. G, somatic cytoplasm; S, satellite sheath, x 9000. Fig. 9. An axonal process rising from a large ganglion cell (G). A well developed JSO (arrow) is formed around the axon hillock. A membrane thickening (double arrows) is evident on the other side of the axon hillock. S, thin processes of satellite cells, x 11 000. Fig. 10. An axonal process containing many microtubules and a few mitochondria. A PSB (arrow) is seen adjacent to a nerve ending which formed a synapse on the axon hillock. G. somatic cytoplasm, x 15 000. Fig. 11. A preganglionic nerve fibre which is completely enclosed by thin processes of satellite cells (S). Large granular vesicles are seen together with a smaller number of small agranular vesicles. G, cytoplasm of ganglion cell. x 13 000. Fig. 12. Enlarged nerve endings containing large numbers of small agranular vesicles. A few large granular vesicles are also seen. Somatic spines (P) project into the nerve endings. G, cytoplasm of ganglion cell. x 16 000.
124
WATANABE and BURNSTOCK
the capsule large numbers of unmyelinated and myelinated axons, clusters of small granule-containing cells and blood vessels were seen together with ganglion cells of various sizes (Figs. 1 and 2). The ganglion cells showed a spherical, elongate or polyhedral shape. The nucleus was usually placed at the periphery of the soma, distant from the axon hillock; it was spherical and usually contained one prominent nucleolus. More rarely, two or three nucleoli were observed. Light microscopic observations with serial thick sections revealed that most ganglion cells had only one large ( 1 - 4 / l m ) axon process (Figs. 1 and 2). Electron microscope observations from serial thin sections supported this finding, since only one well defined process, containing densely packed neurofilaments, microtubules and a few mitochondria, was observed arising from each ganglion cell (Figs. 8 - 1 0 ) . Various other profiles ( 0 . 2 - 4 / ~ m in diameter) arising from the ganglion cells were sometimes observed. Many of these profiles were partially free of a satellite cell sheath. Some had a cytoplasmic structure similar to that of the peripheral area of the soma, containing rough endoplasmic reticulum, mitochondria, multivesicular bodies and free ribosomes (Fig. 6). Neurofilaments and microtubules were also present. Studies of serial sections suggested that these profiles represented peripheral sections through the tips of polyhedral or elongate ganglion cells. Other profiles resembled 'somatic spines' or 'dendritic processes' (Fig. 7); they were thin and mostly remained unde~ the satellite sheath enclosing the ganglion cells. The ganglion cells contained the cytoplasmic components found in other autonomic ganglion cells, such as rough and smooth endoplasmic reticulum, Golgi apparatus, ribosomes, dense bodies and large lipid droplets (Figs. 3a and 4). Rough endoplasmic reticulum was observed chiefly in the periphery of the soma (Fig. 3a). Mitochondria, neurofilaments and multivesicular bodies were also common components of the perikaryal cytoplasm. Mitochondria, up to 4 t~m in length were often Figs. 13aand b. Semi-serial sections through the same nerve endings (E). Mixed in the nerve ending are: (a) spherical and flat-shaped small agranular vesicles and (b) small agranular vesicles together with a few granular vesicles, x 27 000. Fig. 14. A nerve ending (E) showing a rather complicated contour. A JSO lies opposite a thin satellite cell process (S). A spinous projection (P) is seen in the other nerve ending. Small agranular vesicles of varied shape and size are present in the endings, x 39 000. Fig. 15. A PSB in a somatic spine arising from a small ganglion cell (G). S, satellite sheath. • 26 000. Fig. 16. A symmetrical specialization between opposing pre- and postsynaptic membranes. x 45 000. Figs. 17a,b and c. Electron micrographs showing PSBs of various shapes. (a) A PSB composed of a series of a few small dense particles, x 39 000. (b) A doughnut-shaped PSB in a somatic spine. x 26 000. (c) An oval-shaped PSB. Note the various shaped small agranular vesicles in the nerve ending and the large granular vesicle that abuts on the presynaptic membrane (arrow). x 48 000. Fig. 18. A JSO adjacent to a postsynaptic membrane. No membrane specialization or aggregation of synaptic vesicles can be seen. M, mitochondrion, x 26 000. Fig. 19. A JSO separated from a presynaptic nerve ending by a thin process of satellite cell (S). The JSO is accompanied by an underlying cistern of endoplasmic reticulum (arrow) and a mitochondrion (M). x 26 000.
126
WATANABE and BURNSTOCK
prominent and a small number of electron dense granular vesicles (about 100 nm in diameter) were distributed throughout the soma and more often around the Golgi apparatus (Fig. 4). Ganglion cells varied in diameter from about 10/~m to 45 gin. The distribution of sizes of 550 cells is shown in Figure 5. The measurements were carried out on electron micrographs deriving from six blocks from three frogs. The diameter of elongate or polyhedral somata was expressed as the mean of long and short axes. The cell size was measured regardless of the presence or absence of the nucleus in the plane of section, since the nucleus was eccentrically situated in the soma. However, in order to avoid underestimation of the cell size, profiles of less than 5/lm in diameter were not included. A single mode of cell size is apparent at about 15 ~m. However, as described in detail later, on the basis of the distribution of the 'post-synaptic bar' (PSB) and 'junctional subsurface organ' (JSO) (see Fig. 5), there is some suggestion of two overlapping populations, since mean diameters of the cells containing PSBs and of the cells containing JSOs were 15/lm and 26/~m, respectively. No other differences in fine structure of the somata of the larger and smaller sympathetic ganglion cells were observed (Figs. 3a and 4). SATELLITE CELLS
Satellite cells surrounding the somata of ganglion cells showed a similar structure to that described previously in autonomic ganglia (Taxi, 1976; Gabella, 1976). The nucleus was mostly ellipsoidal in shape (Fig. 4). The satellite envelope was thicker around the perinuclear portion of the satellite cells. In other places, one or several layers of thin satellite processes enveloped the perikaryal surface of ganglion cells. The configuration of satellite cells was complicated by their membrane infoldings around incoming preganglionic nerve fibres (Fig. 11). Small areas of the perikaryal surface of ganglion cells directly facing the basal lamina of the satellite cell were without a covering of satellite cell processes. Similar satellite cell-free areas were commonly seen around the axon hillock (Fig. 9). Preganglionic nerve fibres loosely covered parts of these free surfaces. I N N E R V A T I O N OF TIIE G A N G L I O N CELLS
Around the ganglion cells, preganglionic nerve fibres were completely enclosed by one or several layers of thin processes of satellite cells (Fig. 11). These nerve fibres gradually lost the lamellar sheath to come into direct contact with the perikaryal surface of the ganglion cells, where they formed enlarged nerve endings (Fig. 12). In some places the nerve endings intruded deeply into the cytoplasm of ganglion cells without intervention of the satellite sheath (Fig. 13). These invaginated nerve endings occurred more often in the large ganglion cells than in the small cells. Endings were more densely distributed around the axon hillock, especially in the larger cells (Fig 8). On the basis of vesicle content, only one type of synaptic ending was found on the perikaryal surface of both large and small sympathetic ganglion cells. The nerve endings contained many small agranular vesicles together with a few large granuiar vesicles (60-100 nm) (Figs. 12 and 13). The ratio of large granular
Postsynaptic structures in frog sympathetic ganglion cells
127
vesicles to small agranular vesicles varied from one profile to another. In some sections, for example, a nerve profile contained exclusively small agranular vesicles, but examination of serial sections revealed that the same nerve ending also contained large granular vesicles (Figs. 13a and b). The large granular vesicles were more prominent in the preterminal segments of nerve fibres which were completely ensheathed by satellite cells (Fig. 11). The shape and size (30-60 nm) of the small agranular vesicles were less uniform than that of the large granular vesicles. 'Flattened' or 'elongate' small agranular vesicles were usually seen together with a larger number of spherical vesicles (Figs. 13, 14, and 16). Nerve endings containing aggregations of glycogen particles were not seen. Several types of small granule-containing cells were noted in the frog sympathetic ganglia. They did not form synapses with ganglion cells. POSTSYNAPTIC SPECIALIZATIONS
Preganglionic nerve endings formed synapses on the ganglion cells vcith a cleft of about 1 5 - 2 0 nm in width. These endings varied in shape and size: some showed an oval or elongated contour (0.5-4 tam in diameter) with a smooth presynaptic membrane (Fig. 13a); while others showed a contour invaginated by small projections arising from the postsynaptic neurons and satellite cells (Fig. 14). These 'spine synapses' with perikaryal projections, or 'somatic spines' occurred in relation to both large and small ganglion cells (Figs. 14 and 15). Dense areas were observed on both pre- and postsynaptic membranes. In most cases, the density increase was more prominent on the postsynaptic side; an aggregation of synaptic vesicles was present adjacent to the presynaptic membrane (Figs. 14 and 15). These aggregations consisted largely of small agranular vesicles, but a few large granular vesicles were sometimes also present (Figs. 17b and c). This type of membrane specialization occurred both in synapses with a smooth contour and in synapses with spines (Figs. 14 and 15). In addition to these asymmetrical membrane specializations, non-synaptic symmetrical specializations were also formed between the pre- and postsynaptic membranes (Fig. 16). Other characteristic postsynaptic structures were the 'postsynaptic bar' and 'junctional subsurface organ'. General features of these special postsynaptic and subsurface structures have been described previously (Watanabe and Burnstock, 1976a). In the present study, the distribution of these two postsynaptic structures on ganglion cells of different sizes has been examined.
Postsynaptic bar (PSB) The PSB was found mostly in ganglion cells smaller than 22/am (Fig. 5). The largest cell with a PSB was 28 tam in diameter. The PSBs were seen all round the perikaryon of one neuron, but were not formed on the axon. The PSB was present in the cytoplasm adjacent to the postsynaptic membrane showing the asymmetrical specialization, which was associated with an aggregation of synaptic vesicles on the presynaptic side (Figs. 15 and 17). Both somatic spine and non-spinous postsynaptic
128
WATANABE and BURNSTOCK
cytoplasm contained the PSB (Figs. 4 and 15). The narrow gap between the postsynaptic membrane and the outer surface of the PSB was 25--50 nm. The PSB was usually seen as an electron dense bar or disc (0.2-0.5/~m in length), and analysis of serial sections revealed that it varied in shape according to the contour of the postsynaptic site, especially in the spine synapse (Figs. 17a, b and c). In some sections, the PSB was a doughnut- or horseshoe-shape, while in others it seemed to be composed of a series of dense particles. No close topographical correlation was apparent between PSB and mitochondria o r endoplasmic reticulum. From observations on complete serial sections of four small cells (see Table 1) it was shown that such cells contained between~10 and 22 PSBs. The PSBs occupied between 52% and 71% of the total number of postsynaptic membrane specializations. Table 1
maximum diameter
cell
Number of PSBs
(gm) A B C D
18 15 25 24
x x x x
12 14 20 20
12 10 22 16
%ofpostsynaptic membrane specializations associated with PSBs 71 67 52 70
Table 2
cell
maximum diameter
Number of JSOs
mean diameter of JSO (tJm)
8 10 13 15 16
0.5 0.6 0.5 0.4 0.7
(~m) A B C D E
36 28 40 36 42
x x x x x
30 22 35 28 36
• x x • x
0.7 0.7 0.7 0.5 0.9
Junctional Subsurface Organ (JSO) The JSO occurred mainly in the larger ganglion cells up to 45/am in diameter (Fig. 5). JSOs appeared just beneath the perikaryal cell membrane, and were particularly well developed around the axon hillock (Fig. 9). Five larger cells were examined in complete serial sections (see Table 2). These cells contained from 8 to 16 JSOs. The shape of JSO varied according to the contour of the perikaryal surface (Figs. 14, 18 and 19). As has been noted elsewhere (Watanabe and Burnstock,
Postsynaptic structures in frog sympathetic ganglion cells
129
1976a), the narrow space between the JSO and the overlying cell membrane was of constant width (10-12 nm). The JSOs were close to mitochondria and endoplasmic reticulum (Figs. 9 and 19). None of the ganglion cells examined in the present study possessed both PSB and
jso. Discussion Two types of neuron in the frog sympathetic ganglion have been distinguished in the present study on the basis of distinctive postsynaptic structures and to some extent also on cell size. PSBs were observed in the smaller sympathetic ganglion cells, while JSOs were found in the larger cells. It is important to try to correlate this morphological finding with physiological observations on amphibian sympathetic ganglia. Since both the fast-EPSP and the late slow-EPSP have been recorded from both B (large) and C (small) cells (Koketsu, 1969; Libet 1970), it is not possible to relate the specific postsynaptic structures observed in either small or large neurons in the present study to these synaptic events. It has been claimed, however, that a slow-EPSP occurs in the B (large) type neuron (Tosaka et al., 1968; Weight and Votata, 1970) and a slow-IPSP in the C (small) type (Tosaka et al., 1968; Weight and Padjen, 1973a,b). This suggests that ~:he PSB, which appeared exclusively in the small cells, is involved in the generation of the slow-IPSP, while the JSO may be involved in the generation of the slow-EPSP in the large ganglion cells. It has been reported that both slow-EPSPs and slow-IPSPs can be recorded occasionally in the same cells (see Koketsu, 1969; Nakamura and Koketsu, 1972) but, while the existence of ganglion cells containing both JSO and PSB in one cell cannot be excluded, this has not been observed in the present study. The slow-PSPs have a long synaptic delay, 30-100 milliseconds for the slow-IPSP (Libet, 1967; Libet etal., 1968) and 200-300 milliseconds for the slow-EPSP (Libet, 1967, 1970). The longer synaptic delay of the slow-EPSP has been interpreted as an indication of a greater energy requirement, since metabolic inhibitors depress the slow-EPSP much earlier than the fast-EPSP or the slow-IPSP (see Libet, 1970). Further, the latency at muscarinic synapses (associated with slow-EPSP) is always longer than at nicotinic synapses (associated with fast-EPSP) (Purves, 1976). The present results allow some further speculations to be made about the relation of these physiological parameters to the topography of the junctions and, in particular, the postsynaptic specializations. For example, long synaptic delays may be related to the frequent presence of thin satellite sheaths between the presynaptic membrane and the JSO (Figs. 18 and 19; see also Watanabe and Burnstock, 1976a). The close relationship between the JSO and mitochondria or rough endoplasmic reticulum adds morphological support to the physiological interpretations, indicating the dependence on energy supply for the generation of the slow-EPSP (Libet, 1970). It is well known that fast-EPSPs and slow-EPSPs are produced by the direct action
130
WATANABE and BURNSTOCK
of ACh released from the presynaptic nerve endings (Koketsu, 1969; Libet, 1970). It has also been shown that fast-EPSPs can be recorded both from B (large type) and from C (small type) ganglion cells. Mediation of the slow-IPSP is poorly understood in amphibians. In mammalian sympathetic ganglia, various authors have suggested that the slow-IPSP is mediated by catecholamines released from either adrenergic interneurons, chromaffin cells, small intensely fluorescent cells or small granulecontaining cells (Eccles and Libet, 1961; Libet, 1970; Libet and Owman, 1974). Libet and Kobayashi (1974) came to the same conclusion for bullfrog sympathetic ganglion. On the other hand, in the present study there was no evidence for adrenergic or catecholamine-containing elements forming synapses with large or small ganglion cells, supporting earlier observations on amphibian sympathetic ganglia by fluorescence microscopy (Norberg and McIsaac, 1967; Honma, 1970a; Hill et al., 1975) and by electron microscopy (Weitsen and Weight, 1973; Watanabe and Burnstock, 1976b). Alternatively it has been suggested that ACh may be the transmitter mediating the slow-IPSP in C type cells (Weight and Padjen, 1973a,b;Weitsen and Weight, 1973; Libet and Kobayashi, 1974; Koketsu and Yamamoto, 1975). This view is supported by the present finding that all the nerve endings observed are structurally similar, and are all presumably cholinergic. It has been claimed that the late slow-EPSP, appearing in both B and C type neurons, is mediated by an unknown non-cholinergic transmitter (Nishi and Koketsu, 1968; Koketsu, 1969), but the present study did not reveal the presence of a morphologically distinct nerve ending which was clearly not cholinergic. Some 'flat' or elongated vesicles were seen, but these were usually found together with a large number of spherical vesicles in the same nerve ending. This conclusion that there is a single type of nerve ending on amphibian sympathetic ganglion cells is supported by most workers in the field (see Nishi et al., 1967; Gabella, 1976; Taxi, 1976); and to quote Taxi (1976), 'The assertion that there are several different functional types of preganglionic endings in amphibian sympathetic ganglia (Uchizono, 1964; Uchizono and Ohsawa, 1973), on the basis of various proportions of vesicles having different sizes and contents, seems highly questionable and may correspond merely to the variability of presynaptic vesicular contents'. Nerve endings with an aggregation of glycogen particles (Taxi, 1961; Yamamoto, 1963; Fujimoto, 19"67; Pick, 1970) were not seen in the present study. Seasonal differences may explain the discrep~tncy, since Mizell (1965) has reported that the liver glycogen content is higher in the autumn and decreases through winter and is lowest in the summer (see also Gabella, 1976). It seems unlikely that neurons in amphibian sympathetic ganglia can be divided into two types solely on the criterion of size; although this has been the basis of previous attempts to distinguish B and C cells according to electrophysiological properties. In any case it is a difficult task to make this type of analysis (see for example Konigsmark et al., 1970; Hendry, 1976). In the present study, cells with PSBs were about 15 btm in mean diameter, while those with JSOs were about 26 gm in mean diameter. However, mean diameters may be somewhat underestimated (see
Postsynaptic structures in frog sympathetic ganglion cells
131
Weibel, 1973). If we assume that the shape of these ganglion cells is spherical, the more accurate mean diameter (D) can be calculated by the following equation: 410 = --d (d, a mean diameter obtained from random sections). 7r According to the equation, the diameters of the ganglion cells with JSOs and PSBs are 33 ~m and 19/~m respectively. Values very close to these have been observed by Nishi et al. (1965, 1967) in toad; B- and C-neurons are 34 ~m and 18 #m in diameter respectively. A similar classification has also been proposed from the light microscopic examination of the toad sympathetic ganglion (Honma, 1970b), and it was suggested from the results of intracellular marking that the large neurons might represent the fast B conduction system and the small neurons the slow C system. Amphibian sympathetic ganglion cells have been considered by most authors to be unipolar (Uchizono, 1964; see also Pick, 1970), although 'somatic spines' (Piezzi and Rodriguez Echandia, 1968) and occasional 'dendritic processes' (Pick, 1963) have also been noted. The present light and electron microscopic examinations of serial sections confirm this finding. Recently Hill and Burnstock (1975) have reported that many frog sympathetic ganglion cells in culture are multipolar; but they suggested that the production of extra processes by cells in culture may be a response to cutting the postganglionic fibres during the initial tissue preparation. Acknowledgements Some of this work was carried out in the Department of Zoology, Melbourne University, and was supported by grants from the National Health and Medical Research Council and the Australian Research Grant Committee. We would like to thank Professor Toshi-Yuki Yamamoto, Dr. Robert Purves and Dr. Giorgio Gabella for their criticisms of the manuscript. References DE ROBERTIS, E. D. P. and BENNETT, H.S. (1955) Some features of the submicroscopic morphology of synapses in frog and earthworm. Journal of Biophysical and Biochemical Cytology 1 , 4 7 - 5 8 . ECCLES, R. M. and L1BET, B. (1961) Origin and blockade of the synaptic responses of curarized sympathetic ganglia. Journal of Physiology 1 5 7 , 4 8 4 - 5 0 3 . FUJIMOTO, S. (1967) Some observations on the fine structure of the sympathetic ganglion of the toad, Bufo vulgarisjaponicus. Archivum Histologicum Japonicum 28, 3 1 3 - 3 5 . GABELLA, G. (1976) Structure of the autonomic nervous system. London: Chapman and Hall. HENDRY, I . A . (1976) A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment. Journal of Neurocytology 5, 337-49. HILL, C.E., WATANABE, H. and BURNSTOCK, G. (1975) Distribution and morphology of amphibian extra-adrenal chromaffin tissue. Cell and Tissue Research 60, 371--87. HILL, C. E. and BURNSTOCK, G. (1975) Amphibian sympathetic ganglia in tissue culture. Cell and Tissue Research 1 6 2 , 2 0 9 - 3 3 .
132
WATANABE and BURNSTOCK
HONMA, S. (1970a) Histochemical demonstration of catecholamines in the toad sympathetic ganglia. Japanese Journal of Physiology 20, 186-97. HONMA, S. (1970b) Functional differentiation in sB and sC neurons of toad symapthetic ganglia. Japanese Journal of Physiology 2 0 , 2 8 1 - 9 5 . KOKETSU, K. (1969) Cholinergic synaptic potentials and the underlying ionic mechanisms. Federation Proceedings 28, 1 0 1 - 1 2 . KOKETSU, K. and YAMAMOTO, K. (1975) Unusual cholinergic response of bullfrog sympathetic ganglion cells. European Journal of Pbarmacology 31, 281-6. KONIGSMARK, B.W., KALYANARAMAN, V. P., COREY, P. and MURPHY, E. A. (1970) An evaluation of techniques in neuronal population estimates: the sixth nerve nucleus. Johns Hopkins Medical Journal 125, 1 4 6 - 5 8 . LIBET, B. (1967) Long latent periods and further analysis of slow synaptic responses in sympathetic ganglia. Journal of Neuropbysiology 3 0 , 4 9 4 - 5 1 4 . LIBET, B. (1970) Generation of slow inhibitory and excitatory postsynaptic potentials. Federation Proceedings 29, 1945-56. LIBET, B., CHICHIBU, S. and TOSAKA, T. (1968) Slow synaptic responses and excitability in sympathetic ganglia of the bullfrog. Journal ofNeuropbysiology 31, 383-95. LIBET, B. and KOBAYASHI, H. (1974) Adrenergic mediation of slow inhibitory postsynaptic potential in sympathetic ganglia of the frog. Journal of Neuropbysiology 37, 8 0 5 - 1 4 . L1BET, B. and OWMAN, Ch. (1974)Concomitant changes in formaldehyde-induced fluorescence of dopamine interneurones and in slow inhibitory postsynaptic potentials of the rabbit superior cervical ganglion, induced by stimulation of the preganglionic nerve or by a muscarinic agent. Journal of Physiology 237, 6 3 5 - 6 2 . MIZELL, S. (1965) Seasonal changes in energy reserves in the common frog, Rana pipiens. Journal of Cellular and Comparative Pbysiology 66, 2 5 1 - 8 . NAKAMURA, M. and KOKETSU, K. (1972) The effect of adrenaline on sympathetic ganglion cells of bullfrogs. Life Sciences 11, 1165--73. NISHI, S., SOEDA, H. and KOKETSU, K. (1965) Studies on sympathetic B and C neurons and patterns of preganglionic innervation. Journal of Cellular and Comparative Physiology 66, 19-32. NISHI, S., SOEDA, H. and KOKETSU, K. (1967) Release of acetylcholine from sympathetic preganglionic nerve terminals. Journal of Neuropbysiology 30, 1 1 4 - 3 4 . NXSHI, S. and KOKETSU, K. (1968) Early and late after-discharges of amphibian sympathetic ganglion cells. Journal of Neurophysiology 31, 1 0 9 - 2 1 . NORBERG, K . - A . and MclSAAC, R_ J. (1967) Cellular location of adrenergic amines in frog sympathetic ganglia. Experientia 23, 1052. PICK, J. (1963) The submicroscopic organization of the sympathetic ganglion in the frog (Rana pipiens). Journal of Comparative Neurology 120, 4 0 9 - 6 2 . PICK, J. (1970) The autonomic nervous system. Morpbological, comparative, clinical and surgical aspects. Philadelphia and Toronto: J. B. Lippincott Company. PIEZZI, R. S. and RODRIGUEZ ECHANDIA, E. L. (1968) Studies on the para-renalganglion of the toad Bufo arenarum Hensel. I. Its normal fine structure and histochemical characteristics. Zeitscbrift fiir Zellforschung und mikroskopiscbe Anatomie 88, 180--6. PURV ES, R. D. (1976) Function of muscarinic and nicotinic acetylcholine receptors. Nature 261, 149--51. SOTELO, C. (1968) Permanence of postsynaptic specializations in the frog sympathetic ganglion cells after denervation. Experimental Brain Research 6, 2 9 4 - 3 0 5 . TAXI, J. (1961) Etude de l'ultrastructure des zones synaptiques darts les ganglions sympathiques de la grenouille. Comptes rendus de l'Acadkmie des Sciences, Paris 252, 174-6. TAXI, J. (1967) Observations on the ultrastructure of the ganglionic neurons and synapses of the
P o s t s y n a p t i c structures in frog s y m p a t h e t i c ganglion cells
13 3
frog Rana esculenta L. In: The Neuron (edited by HYDEN, H.), pp. 221--54. Amsterdam, London and New York: Elsevier Publishing Company. TAXI, J. (1976) Morphology of the autonomic nervous system. In: Frog Neurobiology, (edited by R. LLIN,~S and w. PRECHT), pp. 93-150. Berlin: Springer-Verlag. TOSAKA, T., CH1CHIBU, S. and LIBET, B. (1968) Intracellular analysis of slow inhibitory and excitatory postsynaptic potentials in sympathetic ganglia of the frog. Journal of Neurophysiology 31, 396-409. UCHIZONO, K. (1964)On different types of synaptic vesicles in the sympathetic ganglia of amphibia. Japanese Journal of Physiology 14, 210-19. UCHIZONO, K. and OHSAWA, K. (1973) Morpho-physiological consideration on synaptic transmission in the amphibian sympathetic ganglion. A cta Physiologica Polanica 24, 205-14. WATANABE, H. and BURNSTOCK, G. (1976a) Junctional subsurface organs in frog sympathetic ganglion cells. Journal of Neurocytology 5, 125-136. WATANABE, H. and BURNSTOCK, G. (1976b) A special type of small granule-containing cell in the abdominal para-aortic region of the frog. Journal of Neurocytology 5,465-78. WEIBEL, E.R. (1973) Stereological techniques for electron microscopic morphometry. In: Principles and techniques of electron microscopy (edited by HAYAT, M.A.) Biological applications. 3, pp. 237--296. New York, Cincinnati, Toronto, London and Melbourne: Van Nostrand Reinhold Company. WEIGHT, F. F. and VOTATA, J. (1970) Slow synaptic excitation in sympathetic ganglion cells: evidence for synaptic inactivation of potassium conductance. Science 170,755-8. WEIGfIT, F.F. and PADJEN, A. (1973a) Slow synaptic inhibition: evidence for synaptic inactivation of sodium conductance in sympathetic ganglion cells. Brain Research 55,219-24. WEIGHT, F.F. and PADJEN, A. (1973b) Acetylcholine and slow synaptic inhibition in frog sympathetic ganglion cells. Brain Research 55,225-8. WEITSEN, H.A. and WEIGHT, F. E. (1973) Chromaffin cells in the frog sympathetic ganglion: morphology not consistent with role in generation of synaptic potentials. Anatomical Record 175,467. YAMAMOTO, T. (1963) Some observations on the fine structure of the sympathetic ganglion of bullfrog. Journal of Cell Biology 16, 159-70.
