Summary. The true surface of rabbit spinal ganglion neu- rons has been made directly accessible to scanning electron- microscope observation after removal of ...
Cell Tissue Res (1990) 260:167-173
Research 9 Springer-Verlag 1990
Scanning electron-microscope observations of the perikaryal projections of rabbit spinal ganglion neurons after enzymatic removal of connective tissue and satellite cells E. Pannese, M. Ledda, V. Conte, P. Procacei, and S. Matsuda* Institute of Histology, Embryology and Neurocytology, University of Milan, Italy
Summary. The true surface of rabbit spinal ganglion neurons has been made directly accessible to scanning electronmicroscope observation after removal of both the connective tissue and satellite cells that normally cover it. The neuronal surface is characterized by a profusion of slender projections whose shapes have been determined and whose length and width have been quantified. Controls carried out with transmission electron microscopy demonstrate that the procedure employed in this study satisfactorily preserves neuronal structure. Key words: Neuronal surface - Perikaryal projections Dorsal root ganglia - Enzymatic digestion - Scanning electron microscopy - Rabbit (New Zealand White)
Slender projections arising from the neuronal perikaryon have been observed by many authors in electron micrographs of thin sections of sensory ganglia. Initially these projections were interpreted as fixation artifacts (Hess 1955), but they were subsequently found in the ganglia of amphibians, reptiles, birds and mammals (see Table 1) where a variety of fixatives had been used; doubt as to their existence therefore disappeared. The observations listed in Table I provided information concerning the width of perikaryal projections, but nothing about their shape or length. They were in fact carried out on single thin sections in the transmission electron microscope, a procedure that does not allow the examination of whole perikaryal projections since they almost always extend beyond the plane of a single section. As far as we know, the only work concerning the shape and length of perikaryal projections was carried out on a single cat spinal ganglion neuron employing the serial section electron microscopy technique (Pannese et al. 1983). However, this procedure is so laborious and time-consuming that it considerably limits the number of possible observations, so that it is very difficult to effect comparisons between neurons of different species and under various conditions. The recent improvement of enzymatic digestion techniques has made it possible to culture spinal ganglion neu* Present address: Ehime University, School of Medicine, Department of Anatomy, Shigenobu, Ehime 791-02, Japan Send offprint requests to: Prof. Ennio Pannese, Via S. Michele del Carso 15, 1-20144 Milano, Italy
rons in vitro for long periods (Scott 1977; Fukuda and Kameyama 1979; Silberberg and Kim 1979; Smith and McInnes 1986) and to examine the three-dimensional cytoarchitecture of spinal ganglia by scanning electron microscopy (Matsuda and Uehara 1981). A modification of these enzymatic digestion techniques was employed in the present study to remove both the connective tissue and satellite cells of spinal ganglia so as to expose the true neuronal surface. Thus, it was possible to use scanning electron microscopy, which permits direct observation of the neuronal surface, to examine a large number of neuronal projections and to determine both their shape and length. A preliminary report of this study was presented at the VIII International Symposium on Morphological Sciences (Rome, July 1988). Materials and methods Eight rabbits (New Zealand White, male, age 6 months, 2000-2400 g body weight) were anaesthetized by intraperitoneal injection of Nembutal. Fixation by vascular perfusion
Two animals were perfused with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3); the thoracic spinal ganglia were then removed, postfixed with 2% OsO4 in the same buffer and routinely processed for transmission electron microscopy. Fixation after enzymatic digestion
Six rabbits were perfused for 20 rain with an aqueous solution of the following composition: 136 m M NaC1, 2.6 m M KC1, 3.3 m M NazHPO4"12 H20, 1.4 m M KHzPO~ (pH 7.3), hereafter referred to as buffer. The thoracic spinal ganglia were removed and placed in buffer at 4 ~ C. As much as possible of the connective tissue capsule surrounding each ganglion was carefully removed under a dissecting microscope using fine needles; the operation was carried out on ice, the specimens being immersed in the buffer. The ganglia were then incubated at 37 ~ C for 2 h with constant agitation in buffer to which 0.2 mg/ml CaC12 and 1 mg/ml collagenase (Fluka AG, Buchs, Switzerland) had been added. The ganglia were rinsed in buffer at 4 ~ C for 15 min, then incubated at 37 ~ C for 1.5 h with constant agitation in the same buffer to which 2.5 mg/ml trypsin (pancreas protease, Merck, Darmstadt, FRG) had been added. The
168 Table 1. Reports of the perikaryal projections of sensory ganglia neurons (transmission electron microscopy) Species
Ganglion
Toad Spinal ganglion Gecko, lizard Spinal ganglion Fowl Spinal ganglion Mouse Spinal ganglion Rat Spinal ganglion Guinea pig Rabbit Cat
Ox Slow loris Monkey Human
Spinal ganglion Spinal ganglion Spinal ganglion Trigeminal ganglion Spiral ganglion Petrosal ganglion Spinal ganglion Spinal ganglion Trigeminal ganglion Trigeminal ganglion
References Rosenbluth 1963 Pannese 1964, Pannese et al. 1975, 1985 Pannese 1969 Kotani and Kawashima 1961 Palay 1957, Rosenbluth and Palay 1960, Cerv6s-Navarro 1960, Andres 1961, Bunge et al. 1967, Pannese 1960, Pannese et al. 1972 Hess 1955, Pannese 1960, Pannese et al. 1972 Wyburn 1958, Pannese 1960, Pannese et al. 1972, 1983 Pannese 1960, Pannese et al. 1972 Pineda et al. 1967 Adamo and Daigneault 1973 Stensaas and Fidone 1977 McCracken and Dow 1973 Ahmed 1973 Pineda et al. 1967 Beaver et al. 1965
(Perikaryal projections were also observed on the initial segment of the axon: Zenker and H6gl 1976; Pannese 1981)
ganglia were then rinsed for 40 min in the buffer (4 changes), fixed with 3% glutaraldehyde in buffered 0.1 M phosphate (pH 7.3) for 3 h, then washed in phosphate buffer (0.2 M, p H 7.3) for 30 min and postfixed at 0 ~ C for 1.5 h with 2% OsO4 in the same buffer. For transmission electron microscopy, the ganglia were dehydrated in alcohol and embedded in Epon-Araldite resin. The thin sections were conventionally stained and examined in a Siemens Elmiskop 101 electron microscope. F o r scanning electron microscopy, the ganglia were washed for 45 rain in distilled water (4 changes), immersed in aqueous 1% tannic acid for 1 h, rinsed for 45 min in distilled water (4 changes) and then immersed in aqueous 2% OsO4 for 1 h. This procedure, a variant of that proposed by Murakami (1973), was repeated 3 times at room temperature. The ganglia were then washed in distilled water for 30 rain, dehydrated in alcohol, immersed in isoamyl acetate for I h, and dried by the critical-point method. Sputter-coating was not necessary. Finally, the ganglia were observed under a scanning electron microscope (Autoscan Siemens). The shape, length and width of each neuronal projection were evaluated on stereo-pairs of micrographs (10 ~ tilt-angle difference) enlarged x 12000, by means of a mirror stereoscope with a parallax measuring unit. The means and standard deviations o f the length and width data were calculated with a Hewlett-Packard 85 computer using a standard statistical package (General Statistic Pac). Results
fibroblasts are situated between the satellite cell sheaths enveloping adjacent neurons. The presence of m a n y projections from both the neuron and the satellite cells renders the neuron-satellite cell boundary very complicated. Single sections perpendicular to the surface of the nerve cell body display neuronal projections which sometimes appear as evaginations continuous with the neuronal perikaryon, but more frequently arise from the neuronal perikaryon at another level and appear as discrete entities (Fig. 1). These entities are embedded in the satellite cell sheath or lie along the gap between the neuron and its satellite cell sheath. Neuronal projections can be identified even when they are embedded in the satellite cell sheath since their cytoplasmic matrix is less dense and their plasma membrane thicker and denser than their counterparts in the surrounding satellite cells. The ganglia treated with collagenase and trypsin appear almost completely devoid of their interstitial connective tissue. Some nerve cell bodies appear surrounded by their own satellite cells, but, instead of being directly applied to the neuronal surface as in control ganglia, the satellite cells appear sharply separated from the neuron (Fig. 2b). In the intervening gap, which is generally very large, projections arising from the surface of the neuronal perikaryon can be found (Fig. 2b). The basal lamina, which in control specimens covers the outer surface of satellite cells, is usually absent after enzymatic treatment. A faint layer of finely granular and moderately dense material, which could be a remnant o f the basal lamina, follows the outer contour
Transmission electron microscopy
Some transmission electron-microscope observations are presented here since they help in the interpretation of the results obtained by scanning electron microscopy. Ganglia not subjected to enzymatic digestion are surrounded by a connective tissue capsule and consist mainly o f neurons and enveloping cells (satellite and Schwann cells). Each nerve cell body is enveloped by its own satellite cell sheath, whose outer surface is covered by a basal lamina (Fig. 1). Collagen fibrils, microfibrils and sometimes also
Fig. 1. Nerve cell bodies completely enveloped by their satellite cell sheaths, showing a regular contour (large figure) or a regular finely wrinkled surface (inset). Rabbit spinal ganglia. In the large figure the outlines of the neuronal perikaryon, and of its projections arising at other levels and thus appearing as isolated entities, were traced with ink to make them more easily visible, ct Connective tissue; NC nerve cell; SC satellite cells; V blood vessel. Large figure: transmission electron micrograph, x 9000. Inset: scanning electron micrograph, x 1700
169
Fig. 2a, b. Satellite cell sheaths (SC) partially surround their nerve cell bodies, each being separated from its nerve cell body by a large intervening gap (g). The true surface or contour of the nerve cell bodies (NC) shows numbers of projections, several of which
jut into the gap between each nerve cell body and its satellite cell sheath (rabbit spinal ganglia). Arrows indicate a possible remnant of the basal lamina, a Scanning electron micrograph, x 5000; b transmission electron micrograph, x 10 000
Fig. 3 a, b. Nerve cell bodies (NC) completely devoid of any enveloping sheath and therefore having their true surface or contour exposed. The surface or contour is made irregular by the presence
of projections emerging from the perikarya (rabbit spinal ganglia). a Scanning electron micrograph, x 10000; b transmission electron micrograph, x 10000
172 Table 2. Length and transverse diameter of the perikaryal projections of spinal ganglion neurons 1.84% Fig. 4. The commonest shapes of perikaryal projections, as seen under the scanning electron microscope, are shown together with their frequencies 82.36%
8.05%
4.09%
3.66%
Length of projections (gin)
Mean + standard deviation 1.30 • 0.42 Range 0.41 - 2.80 Number of projections examined 1070
Transverse Mean +_standard deviation 0.18 • 0.03 diameter of Range 0.12- 0.28 projections (~tm) Number of projections examined 711 Number of neurons examined
of the satellite cells and is, at certain points in contact with these cells; elsewhere this layer is separated from the satellite cells by a clear space of varying dimensions (Fig. 2b). Other nerve cell bodies are completely devoid of any enveloping sheath; their surface is made irregular by the presence of projections emerging from the perikaryon (Fig. 3b). Many isolated profiles of projections are seen near the neuronal surface (Fig. 3b). Possible remnants of the basal lamina are rarely observed. Usually, the neuronal projections have a similar appearance to that of their counterparts in ganglia not enzymatically treated; only occasionally are their free ends enlarged and exceptionally one of these projections may appear frayed. The fine structure of the nerve cell bodies isolated by enzymatic digestion is similar to that of nerve cell bodies fixed in situ by vascular perfusion (Fig. 3 b). In particular, the nucleus is located centrally, the cisternae of the granular endoplasmic reticulum appear flattened, both free and membrane-attached ribosomes are arranged in polysomal clusters, and mitochondria exhibit their usual structure. Sometimes, however, the Golgi complex cisternae are dilated and multivesicular bodies and autophagic vacuoles seem to occur more frequently within enzymatically isolated nerve cell bodies (Fig. 3 b).
Scanning electron microscopy After mechanical removal of the connective tissue capsule and subsequent digestion by collagenase and trypsin, the ganglia appear as clusters ( 1 ~ mm in diameter) of spherical or oval corpuscles (15-50 gm in diameter) with nerve fibres and blood vessels running among them. A single process is sometimes seen to arise from such corpuscles. Some corpuscles have a regular, smooth or finely wrinkled surface and represent nerve cell bodies completely covered by satellite cells (Fig. 1, inset). Bulges beneath the surface of these corpuscles probably correspond to satellite cell nuclei. Other corpuscles exhibit a much more irregular surface and represent nerve cell bodies devoid of satellite cells and therefore having their own true surfaces exposed (Figs. 2a, 3 a). The true surface of the nerve cell body, partially (Fig. 2a) or completely (Fig. 3a) exposed, shows a large number of projections, whose commonest shapes are: finger-shaped formations of constant thickness, which end freely; projections, which at some distance from the neuronal surface divide into 2 branches like a letter T or Y, both branches ending freely; loops (resembling cup handles) attached to the neuronal surface at both ends; lamelliform projections; and ridges attached to the neuronal surface along their entire length. A few projections are irregular, possibly as a consequence of the digestion process. The various neuronal projection shapes and their frequencies are given in Fig. 4. Lengths and transverse diameters are given in Table 2.
69
Discussion
Most of the surface features of the perikaryon of rabbit spinal ganglion neurons, observable with scanning electron microscopy after removal of the envelopes covering this surface, appear very similar to those known from transmission electron-microscope studies. However, whereas examination of thin sections in a transmission electron microscope gives the impression that perikaryal projections are not very numerous, scanning electron microscopy of the true neuronal surface reveals a profusion of projections. Projections apparently similar to these have also been observed on the true surface of bullfrog sympathetic neurons (Baluk 1986). Use of the technique described here made it possible to determine the shape, length and width of more than a thousand projections, a work which would have been difficult to realize employing the serial section electron microscopy technique in view of its laborious and time-consuming nature. Lengths and widths of these projections were similar to those of the rnicrovilli of other cell types (e.g., ependymal cells and choroidal epithelial cells) as determined with the scanning electron microscope (Peters 1974). The controls carried out with transmission electron microscopy have shown that (i) the fine structure of both cytoplasm and nucleus of neurons freed from their envelopes is well preserved and (ii) the neurons maintain their plasma membranes intact. These controls demonstrate that the technique employed here is sufficiently mild. Such a conclusion is also consistent with the fact that neurons isolated from the spinal ganglia of adult mammals using similar procedures to those described here remain viable for long periods in vitro and retain electrophysiological functions similar to those observed in situ, as shown by Scott (1977) and Fukuda and Kameyama (1979). Future work will establish whether the quantitative features of neuronal projections determined by scanning electron microscopy correspond to those revealed by transmission electron microscopy.
Acknowledgements. The authors wish to thank Prof. E. Reale for his critical reading of the manuscript and Mr. F. Redaelli for photographic assistance. This research was supported in part by a grant from the National Research Council (CNR), Italy.
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