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Sally Tarrant-Cobb
Cresap Neuroscience Laboratory, Northwestern University
Evanston, Illinois 60201.
The Synaptic Spinule in the Dendritic Spine:
Electron Microscopic Study of the Hippocampal Dentate Gyrus.Sally B. Tarrant and Aryeh Routtenberg
Tissue & Cell 1977 9 (3) 461-473
(Published by Longman Group Ltd. Printed in Great Britain)
ABSTRACT: The present report calls attention to a component of certain synaptic junctions which has received little attention since its description in 1962 by Westrum and Blackstad. This component, which we term the synaptic spinule, is found in dendritic spine synapses in rat telencephalon (e.g., hippocampus, caudate nucleus, temporal and frontal cortex. Its major feature is a invagination of the presynaptic terminal by the presynaptic membrane protruding into this invagination. The synaptic spinule occurs in close association with the post-synaptic density, often occurring between breaks in this specialization. Serial sections reveal that when a synaptic spinule is present, a spine apparatus is observed associated with the synaptic spinule ; in all such instances they are associated with the presynaptic membrane.
We studied the distribution of synaptic spinules in the dentate gyrus of the hippo- campus. In contrast to its presence in the molecular layer, the synaptic spinule has not been observed in terminals of the subgranular layer of the dentate hilus. It is speculated that the synaptic spinule may play a role in exchange of material at dendritic spine synaptic junctions.
Introduction
In 1962, Westrum and Blackstad studied the finer structure of the hippocampus and reported on the existence of an invagination by the presynaptic membrane into the presynaptic terminal with a parallel protrusion of the post- synaptic membrane into this invagination. Because the was seen in dendritic spine synapses,the post synaptic membrane protrusion was referred to as a spinule or small spine, and the entire morphological entity, the spinule complex. A literature search of the last 8 years reveals that other investigations have described features of similar phenomena (Hamlyn, 1962; Andres, 1964; Cotman, Taylor, and Lynch, 1973; Eccles, Ito and Szentagothai, 1967; Matthews et al., 1976; Palay and Chan-Palay, 1974; Peters and Kaiserman-Ambramof, 1969; Van Harreveld and Trubatch,1975). Close examination of the morphological features demonstrate, however, that in certain cases, the entity described differs from the synaptic spinule. For example, Westrum and Blackstad (1962), in addition to describing the spinule complex,describe a "spinelet", a small dendritic projection into mossy fiber terminals of the hippocampus. This spinelet differs, however, in an important respect from the synaptic spinule ; that is, in the spinelet the post synaptic density occurs within the evaginated portion of the dendrite. In contrast, the postsynaptic membrane of the synaptic spinule is devoid of postsynaptic density, although the spinule invariably occurs adjacent to, most generally within, the active zone of the synapse.
Since diverse perspectives and terminologies have been applied to morphological entities which appear similar, recognition of the synaptic spinule as an axonal-dendritic configuration that is a significant component has not occurred. For example, in a recent review of synaptic morphology (Jones, 1975), there is no mention of spinule-like entities associated with the synapse. In addition, Peters, Palay and Webster (1970) include illustrations of dendritic spines which contain a spinule complex in the synapse (Figs.20, 52), but they do not describe this entity in their discussion of synaptic fine structure.
In order to study this entity, we have focused our attention on the molecular and hilar portions of the hippocampal dentate gyrus and of the caudate nucleus. Our observations suggest a morphological definition of this synaptic occurrence, which we have termed the synaptic spinule to emphasize it slink with the synaptic junction. In addition to the qualitative study, we have undertaken an initial quantitative analysis of the occurrence of the synaptic spinule in the hippocampal dentate gyrus.*
* Initial reports of this work have been given (Routtenberg and Tarrant, 1975 ; Tarrant and Routtenberg,1976).
Methods
Subjects were adult male albino rats, 200-300g (Holtzman Research, Madison,Wis). Animals were administered 50 mg/kg pentobarbital sodium and perfusion was begun after 5 min in the anesthetized state. To control for effect of anesthesia, a few subjects were paralyzed with 1 mg/kg of d-turbocurarine chloride (Sigma),and a local anesthetic, Xylocaine (Astra), was applied subcutaneously to the chest area prior to making an incision. Animals in both groups were artificially respirated with a mixture of 95% O2 / 5% CO2 for 2 min prior to opening the chest cavity for intracardiac aldehyde perfusion. A Cole-Parmer Masterflex pump (no. 7014) was calibrated to deliver a continuous stream of fluid at 60 ml.min ; 250 ml of a dilute fixative containing a mixture of paraformaldehyde and glutaraldehyde (Karnovsky, 1965 ; Peters , 1970) was perfused through the left ventricle. This was followed by perfusion with 100ml of a concentrated paraformaldehyde and glutaraldehyde fixative (Karnovsky, 1965 ; Peters , 1970). Both solutions were kept at 37oC. Following decapitation, the head was immersed in cold (4oC) concentrated fixative for 12 hr before the skull was chipped away from the brain.
Small tissue cubes of brain were dissected from the molecular layer and hilus of the dentate gyrus of the dorsal hippocampus and from the ventrolateral caudate nucleus at the level of the anterior commissure. These cubes were placed in cold concentrated fixative for 1 hr, followed by two washes in 0.2M sodium cacodylate buffer solution, post-fixation in 1% osmiumtetroxide for 1-2 hr and dehydration in a graded ethanol series and propylene oxide. Blocks of tissue were embedded in Araldite 502 and prepared for sectioning. Accurate and consistent placement of the final small block for thin sectioning was determined by the use of a method described by us previously (Routtenberg and Tarrant, 1974).
Frontal and horizontal sections of the medial dorsal blade of the hippocampal dentate gyrus were studied. This region included ventral leaf granule cells, hilus, dorsal leaf granule cells, and dorsal leaf molecular layer, terminating at the hippocampal fissure. At this location, the dorsal-ventral extent of the hilus is 165:m.
Grey sections were picked up on uncoated 400 mesh copper grids. Serial sections were collected according to the recommendations of Anderson and Brenner (1971) on large slot grids which had been lightly coated with formvar and carbon. All sections were double stained with 2.5% uranyl acetate in 50% ethanol for 15min, and then with a lead citrate solution at high pH for 5min.
In order to have available a random selection of synapses for quantitative purposes, the first single grid hole of the 400 mesh grid which contained a clean unfolded area of neuropil was chosen for photographic recording. At least five electron micrographs were taken of adjacent areas of molecular layer of the hippocampus and ventro- lateral caudate neuropil from each of 12 animals at an initial magnification of x12,000, creating a montage suitable for quantitative study. A count was made, then, of instances and signs of the synaptic spinule. These signs are an interruption of post synaptic specialization, axonal and dendritic membranes in roughly parallel fashion protruding into the presynaptic terminal (Figs.1-6 and 9a-h), or a double membrane circular structure in the presynaptic terminal (Figs. 9c, d, e, f, g, h). Spine apparatus and coated vesicles were also counted. As there was no demonstrable difference in the incidence of synaptic spinules in d-turbocurarine and pentobarbital conditions, material prepared by both methods was combined for further quantitative analysis.
The hippocampal molecular layer of animals used in a previously reported study of post-mortem effects on synaptic morphology (Routtenberg and Tarrant, 1974) was re-examined for possible evidence relating the occurrence of the synaptic spinule to fixation delay. In this previous study, changes in synaptic curvature, incidence of cytoplasmic densities and thickness of postsynaptic specialization as a function of time of fixation onset, were reported. That is, perfusion had been initiated according to the following schedule:
Control ~ after 5 min in the anesthetized condition ;
0 min ~ when respiration had ceased but heart still beating ;
1 min ~ after both respiration and heart beat had stopped ;
after 5 min post-mortem ;
after 10 min post-mortem.The incidence of synaptic spinules, coated vesicles, and spine apparatus was obtained as described previously.
Results
General description
The synaptic spinule represents a distinct variation in the traditional asymmetric type 1 synaptic morphology (Gray, 1959). Examples of the synaptic spinule are shown Fig. 1-6, 9a-h. The axonal membrane component of the synaptic spinule invaginates 0.05-0.9:m into the presynaptic terminal. The postsynaptic membrane of the synaptic spinule evaginates parallel to the invaginated presynaptic axonal membrane. Thus, parallel apposed pre- and postsynaptic membranes penetrate the presynaptic terminal. In most cases, protoplasmic astrocytotic processes surround this entire dendritic spine synapse.
In the presynaptic terminal,the synaptic spinule is surrounded by presynaptic vesicles. Of special interest is the fact that the presynaptic membrane is frequently observed to have a coated vesicle attached to or fused with this membrane (Figs. 9a, b, c, h0. Synaptic vesicles fused with the presynaptic membrane in omega-formations have not been seen, although vesicles often lie immediately adjacent to the membrane. In certain sections (Figs. 4, 6), the presynaptic membrane of the spinule had a vesiculated appearance. When viewed in cross-section, the synaptic spinule appears as a spherical body with a double membrane surrounding an area of dense cytoplasm (Figs. 9c, d, e, f, g, h). The invagination can vary in size from a simple shallow protrusion (Fig. 3), to a deep asymmetric, elaborately branched process with multiple coated vesicle attachments ; two such protrusions are illustrated in Fig, 9.
Dendritic component
The spine apparatus. In the area of hippocampus and caudate nucleus investigated in this study,the synaptic spinule occurs only within asymmetric dendritic spine synapses. Several hundred spinules were studied to obtain the present qualitative description. In addition, material selected for quantitation contained 77 different synaptic spinules. In 62 of the 77, the dendritic spine contained portions of a spine apparatus. When serial sections of these same areas were studied, a spine apparatus was observed in the head or neck of every spine which terminated in a synaptic spinule (Figs. 1, 4). The dendritic spine cytoplasm is more dense within the area of the spinule (Figs. 1, 2, 4, 5, 9) and filaments can be seen within the protrusion and in the portions of the dendritic spine which partially encircle the bouton (Figs. 1, 3, 9a-h). At times a fine network of filamentous material within the dendritic spine is observed (Figs 1, 4, 5, 9a-h). Ribosome-like particles are frequently present in the vicinity of the spine apparatus and within the cytoplasm of the spinule (Figs. 2, 9a-h).
Hippocampus. In the molecular layer of the dentate gyrus, in both the ventral and dorsal leaf, the synaptic spinule occurs only within dendritic spine synapses. Measuring from the outer granule cell layer, synaptic spinules are first observed in the molecular layer at a distance of 11:m from granule cells. Synaptic spinules are observed throughout the remainder of the molecular layer. In a count of synaptic spinules in single sections of 1390:m2 of molecular neuropil from 8 animals, one synaptic spinule was observed per 27:m2. Of all synapses counted, 10.1% of them formed synaptic spinules.
Caudate nucleus. The synaptic spinule is also observed in the ventrolateral caudate. The incidence is less than in the hippocampus, one per 63:m2 in a total of 1652:m2 of material. Synaptic spinules are observed only on dendritic spines. The dendritic spines are similar in appearance to those in the hippocampus, also, there is a spine apparatus in every dendritic spine which terminates in a synaptic spinule (e.g. Fig.4). Of all synapses counted, 8.47% of them formed synaptic spinules.
Axonal Component
Hippocampus. The presynaptic terminals within the molecular layer which contain synaptic spinules share certain characteristics. They are small to medium sized (< 0.5:m), irregularly shaped and with rather dense cytoplasm. In the hippocampal molecular layer two types of vesicles populations were observed in synaptic spinule-containing terminals. One consisted of closely packed, spherical vesicles of a similar, small size (Fig.1), while the other contained vesicles which varied in size and shape (Figs. 2, 5, 6). Thus, in the molecular layer, the presynaptic terminal portion of synaptic spinules can have synaptic vesicle populations of either small (380 Å)or large (600 Å) diameter.
Caudate nucleus. Synaptic spinule presynaptic terminals in the caudate are somewhat larger (0.5-1.0:m) than hippocampal terminals and are irregularly shaped. Most have a heterogeneous, polymorphic vesicle population of about 600Å in diameter (Fig 4). No terminals with synaptic spinules were observed to contain the small 380Å homogeneous vesicles.
Hilar area terminals: lack of synaptic spinules
The hilus fascia dentata of the hippocampus demonstrates a morphology quite distinct from that of the granule and molecular layer (Laatsch and Cowan, 1967). In order to determine whether the synaptic spinule was seen in other portions of the dentate gyrus, this area was selected for study. We have found, however, no cases of synaptic spinules in the hilar area. There is one prevalent type of presynaptic terminal (Figs 7, 8), which can be distinguished from those containing synaptic spinules in the molecular layer in several respects. This terminal is larger (1-2:m in diameter), ovoid and has a clear cytoplasm (Fig. 8). The synaptic vesicles are round, of a medium size (480Å), and dispersed within the terminal. Large dense-core vesicles are observed and, in single sections, often are seen to lie close to the presynaptic membrane (Fig. 7, 8). The external dimensions of these granular vesicles vary within the same terminal from 700 to 1200Å, although the size of the dense core remains approximately 450Å. Fig. 7 also illustrates that these terminals participate in crest synapses (Akert et al., 1972) which contain subjunctional bodies in the dendritic core.
The most frequent type of synapse, within the portion of hilar layer studied, is between these synaptic terminals and a dendritic process. In longitudinal and cross sections of dendrites, these terminals can be seen covering the dendritic membrane with no intervening glial processes between individual terminals. Few dendritic spines are in evidence and when one is observed, its cytoplasm is distinctly pale and unelaborated in comparison with the dendritic spines observed in the molecular layer. No spine apparatus was observed in this region. Instead, slender dendritic processes, devoid of membranous saccules, are seen emerging from large dendrites. These processes are generally covered throughout their length with synaptic contacts as illustrated in Fig. 8.
In summary, then, the results of this initial examination of the subgranular layer of the hilar area indicates that the synaptic spinule is not an element in synaptic junctions in this cytoarchitectural region of the hippocampus.
Effect of fixation schedule upon synaptic spinule and spine apparatus
In a previous report, the effect of immersion versus aldehyde perfusion and the effect of varying post-mortem perfusion time upon synaptic morphology was studied (Routtenberg and Tarrant, 1974, Table 1). The results showed a significant effect (P<0.001) on both immersion and post-mortem fixation upon synaptic curvature, width of postsynaptic specialization and incidence of cytoplasmic densities. A re-investigation of this material for signs of the synaptic spinule revealed, in fact, a decrease in the occurrence of the synaptic spinule or spine apparatus in the hippocampus and caudate nucleus as a function of post-mortem time. There was an effect upon the number of coated vesicles, but this increase was limited to dendritic processes and cell bodies, and there was no increase in the number of coated vesicles in presynaptic terminals.
Discussion
This report calls attention to the synaptic spinule as a significant component of certain dendritic spine synapses. In this discussion we wish to consider what functional significance may be attributed to this entity on the basis of its morphological features. In our definition, the synaptic spinule is a specialized relationship of axonal and dendritic membranes immediately adjacent to a site considered to be one of nervous communication between cells, e.g. the 'active zone' of Couteaux (1961). We use the term synaptic spinule to emphasize its intimate association with the synaptic junction, and to distinguish this morphological entity from other double membrane cell invaginations, not in close proximity to the active zone.
Several basic questions concerning the synaptic spinule require consideration. First, what is the neuroanatomical distribution of the synaptic spinule? The synaptic spinule has been observed in several brain locations in rat telencephalon. The present report has focused on its distribution in the hippocampal dentate gyrus and ventro- lateral caudate nucleus. Westrum and Blackstad (1962) reported on the existence of spinule complexes in hippocampal pyramidal cell dendritic spine synapses. We have observed this entity in medial frontal cortex and entorhinal cortex, as well (Tarrant and Routtenberg, 1976). The synaptic spinule has also been observed in such diverse instances as rat parietal cortex (Peters and Kaiseman-Abramof, 1969) using chemical fixation procedures and in frog cerebrum (Van Harreveld and Trubatch, 1975) using freeze-substitution methods. It is interesting, given this ubiquity, that the synaptic spinule is not observed in synapses of the mammalian cerebellum (Palay and Chan-Palay, 1974). Thus, the synaptic spinule is present in several brain locations, but is by no means observed in all regions. We have also shown in this study that in the hippocampal dentate gyrus its presence is restricted to the molecular layer ; no instances of the synaptic spinule were observed in the subgranular layer of the hilus.
Second, what type of synaptic junctions are associated with the synaptic spinule? In all cases in this study the synaptic spinule was associated with axo-dendritic synapses made on dendritic spines (Gray, 1959, 1963). It is of interest that about one in every ten dendritic spine synapses in the molecular layer possess synaptic spinules (see also Matthews et al., 1976). Thus, synaptic spinules do not occur in every synapse in those cytoarchitectural regions where they have been demonstrated. In addition, certain brain regions rich in dendritic spine synapses such as the cerebellum appear to have no synaptic spinules at, for example, dendritic spine-parallel fiber synapses (Palay and Chan-Palay, 1974). Thus, the determinants of synaptic spinule occurrence,both with regard to its anatomical as well as its sub-cellular location, are not known. The predictability of its appearance in certain locations, but not others, may be an important clue to its functional role.
Third, what subscellular components are associated with the synaptic spinule? A significant finding in this report is the association of the spine apparatus with the synaptic spinule. This apparatus, first described in Gray (1963), has not been ascribed any function to our knowledge. Although we cannot ascribe a function to the spine apparatus either, it is worthwhile to emphasize its association with the synaptic spinule in so far as this association may provide some idea as to the role of the spine apparatus. It is of interest that in the cerebellum where no spinules are observed, there is an absence of spine apparatus. Although discussed in a different context, Peters and Kaiserman-Abramof presented micrographs (1969, Fig.5) associating the spine apparatus with the synaptic spinule (Fig. 5). Peters et al., (1970; Figs, 20, 52) have several micrographs illustrating this association as well.
Another subcellular component associated with the synaptic spinule is the coated vesicle. This association was described by Westrum and Blackstad in their initial study of the spinule complex as 'a corona (consisting of) ... short radiating dark lines or dots (which could) ... share a short distance of cell membrane with the invagination (Westrum and Blackstad, 1962, p.286)'. This description fits well the attached coated vesicles in Figs. 9a, b, c, h for example, in the present report.
In addition to the spine apparatus and coated vesicle, subcellular components always associated with synaptic junctions are obviously observed associated with the synaptic spinule. The form of these associations may be significant. Thus, the postsynaptic density typically surrounds the synaptic spinule. The description of Peters and Kaiserman-Abramof (1969) of post synaptic plaques with holes may well reflect locations for synaptic spinules and emphasize its close association to the postsynaptic density.
Fourth, is there any evidence that the synaptic spinule is artefactual? We were concerned with this problem since the initial description of Westrum and Blackstad (1962) used immersion fixation and post-mortem artefacts such as spherical electron densities, described by us as an artefact previously (Routtenberg and Tarrant, 1974), were, in fact, present in the Westrum and Blackstad report. In the report, however, we found no evidence that the synaptic spinule is derived from post-mortem artefacts.
The presence of synaptic spinules in the micrographs of other reports prepared by a variety of chemical fixation methods also supports our view that the synaptic spinule is unlikely to be an artefact due to our particular methods of tissue preparation. In addition, Van Harreveld and Trubatch (1975) have reported on the existence of morphological entities, which we define as synaptic spinules, in material prepared by freeze-substitution. Thus, both chemical and physical fixation techniques can be used to demonstrate the synaptic spinule, making it less likely to be an artefact of type of fixation. Although this question may be difficult to answer other than in relative terms, the evidence available suggests that the synaptic spinule is not an artefact.
Fifth, what is the function of the synaptic spinule? Although we can as yet bring no direct evidence to bear on the answer to this question, the morphological observations of the synaptic spinule suggest the involvement in transport of material between the pre- and postsynaptic processes. The parallel apposed pre- and postsynaptic membranes would be ideal for such transport. The coated vesicles in particular may be important for the transfer of large molecules (Roth and Porter, 1964). In this context, the spine apparatus, which we have observed in association with the synaptic spinule, may play a role either as a source or a repository of macromolecules(*)
These speculations may be relevant to views of synaptic function advanced most recently by Schmitt, Dev and Smith (1976), who note 'the emergence of knowledge of fast bidirectional transport and biochemical signaling between brain cells. Such molecular exchange may function not only to provide metabolic support for electrical activity but also to transmit information between neurons (pp.114-115).' The presence of both active zones and extended apposition of membranes in the synaptic spinule could provide for the occurrence and compartment- alization of both electrophysical and molecular events, respectively, at the same synaptic junction.
Acknowledgments
We wish to thank Paloma Larramendi, Donna Mathe and Sara Plath for technical assistance, and Betty Wells for preparing the manuscript. Support by the Alfred P. Sloan Foundation grant, N.I.H. grants NS10768 and MH25281, and N.S.F. grant BMS74 19388 to A. Routtenberg.
* It is possible that the synaptic spinule represents an active synapse and that the presynaptic invagination represents the coalescence of synaptic vesicles and the coated vesicle a device for membrane recycling (Heuser and Reese, 1973). The spine apparatus might contribute to the postsynaptic membrane as it protrudes into the presynaptic membrane invagination.
Feb 1999: With your patience, the diagrams for this report were originally done
in photoplate dots. They are proving difficult to transpose for the web. Work
is proceeding and the micrographs will be available soon. Meanwhile, pictures
of the origonal electron microscopic studies are available at
ceptualinstitute.com/genre/cobb/spinulepix.htm
They are presented in general viewing format and also in extra high resolution
format (large files 1-2 Mb) suitable for scientific evaluation.
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