Synaptic Morphology and Cytochemistry

Synaptic Morphology and Cytochemistry KARL H. PFENNINGER

With 88 figures

GUSTAV FISCHER VERLAG· STUTTGART PORTLAND-OREGaN-USA· 1973

KARL H. PFENNINGER, Dr. med. Brain Research Institute University of Zurich, Switzerland and Department of Anatomy Washington University School of Medicine St. Louis, Missouri U. S. A.

Acknowledgements Most of the experimental work in which the author was involved was carried out in collaboration with Dr. K. AxERT and Miss C. SANDRI, Brain Research Institute, Zurich, and with Dr. H. MOOR and Miss C. BERGER, Botany Department of the Federal Institute of Technology, Zurich. Their assistance, advice and criticism are gratefully acknowledged. I am particularly indebted to Dr. AxERT for his encouraging support of, and interest in, the work on synapses; it was during innumerable discussions with Dr. AxERT when many of the concepts presented in this monograph developed. The skillfull assistance in the preparation of the illustrations by Misses H. BRUPPACHER, L. DECOPPET, R. EMCH, and V. SCHNEIDER of the Brain Research Institute is gratefully acknowledged. I am also most thankful to my present host institution, the Anatomy Department of Washington University School of Medicine, St. Louis, at which this review was fmished. Especially helpful was the careful criticism of the manuscript by Dr. RICHARD P. BUNGE. I also wish to express my appreciation to Mrs. LUCILLE MILLER of the Anatomy Department for here expert help in preparing the paper for publication. The following authors kindly provided original electron micrographs for this article: Dr. F. E. BLOOM, National Institutes of Mental Health, Washington (Figs. 21 and 22). Drs. T. HOKFELT and A. LJUNGDAHL, Karolinska Institutet, Stockholm (Figs. 58 to 65). Dr. E. KAWANA, Brain Research Institute, University of Tokyo and Brain Research Institute, Zurich (Figs. 73 and 74). Dr. W. J. MEYER, University of California at Los Angeles (Figs. 19 and 20). Dr. G. D. PAPPAS, Albert Einstein College of Medicine, New York (Fig. 48). Dr.]. L. PRICE, Washington University School of Medicine, St. Louis (Figs. 27 and 28). Drs. G. RICHARDS, H. THOENEN, andJ.-P. TRANZER, F. Hoffmann-La Roche & Co. Ltd., Basel (Figs. 66 to 68). These important contributions to the monograph are greatly appreciated. The following publishers kindly gave their permission to reproduce figures which appeared in their journals or books: Academic Press, New York; Birkhaeuser Verlag, Basel; Chapman and Hall, London; Elsevier, Amsterdam. This work was supported by grants from the Swiss National Foundation for Scientific Research awarded to Dr. K. AxERT (#3-133.69 and 3-134.69) as well as a fellowship to the author.

ISBN 3-437-10303-2 cD Gwtav Fischer Verlag· Stuttgart· 1973 . Aile Rechte vorbehalten Gesamtherstellung: Druckerei Mayr, Miesbach Printed in Gennany

Contents Introduction ..

I

Cytochemical Methods in Synaptology

2

I.

2.

Conventional Methods for Electron Microscopy Osmium tetroxide and potassium permanganate .. Aldehydes .. The combined aldehyde-osmium tetroxide fixation procedure Staining of Acidic Groups. Uranyl acetate .. Ruthenium red, colloidal iron oxide and other cations

3. Staining of Basic Groups ..

Phosphotungstic acid Bismuth iodide . 4. Staining of Carbohydrates Methods using periodic acid Acidic phosphotungstic acid

2 2 2

3 4 4 4 7 7 8 10 10

12

5. Demonstration of Macromolecules by

Enzymatic Digestion. Incubation of unfixed or aldehyde-fixed specimens .. Incubation of thin sections 6. Staining of Vesicle Contents Osmium tetroxide .. Glutaraldehyde-potassium dichromateosmium tetroxide Potassium permanganate .. Zinc iodide-osmium tetroxide staining (ZIO) .. 7. Tracer Techniques and Enzyme Cytochemistry .. Radioautography and macromolecular tracers. Demonstration of enzyme activity ..

Cytochemical Results and Synaptic Function .. I.

The Greater MembraneCytochemistry of the "Fuzz Coat" . The external coat The cytoplasmic coat The concept of the greater membrane

13 13 14 14 15

15 15 16 16 16 17

2. Comparative Morphology of Synapes Presynaptic densities . The synaptic cleft The postsynaptic organelles Excitatory versus inhibitory synapses The synaptic apparatus ..

24 24 30

3I 33

33

3. "Cytology" of Intercellular Adhesiveness 36 Cytoplasmic densities in intermediate junctions and synapses Structure and cytochemistry of the synaptic cleft material 37 The mechanism of adhesion 43

4. "Cytology" of Intercellular Communication 5. Transmitter storage ..

Synaptic vesicles Large dense core vesicles .. Complex vesicles, plasmalemmal vesicles and smooth endoplasmic reticulum The origin of synaptic vesicles .

47 47 51 56 57

6. Transmitter Release .. 59 The fme structure and cytochemistry of the presynaptic membrane 59 Vesicle hypothesis or membrane control 62 theory? Transient vs irreversible vesicleplasmalemma contact The true nature of the vesicle-plasmalemma contact: Transmembraneous or membrane-bound channel? 65 Dynamics of the presynaptic membrane 66

7. Cytochemistry of electrogenic regions .. Surface carbohydrates and cytoplasmic membrane coat .. Intramembraneous structures .. Postsynaptic organelles

66 67

68 70

Synopsis.

71

Abbreviations Used

71

References

72

18 18 19 21 21

Subject Index.

Synaptic Morphology and Cytochemistry .

I

Introduction When it became evident at the tum of the century that the brain consisted of a network of individual neurons rather than a syncytium (for review see e. g. PETEIl.S et al. 1970), the contacts between the nerve cells gained especial importance with respect to the function of nervous tissue. In 1906, the physiologist SHERRINGTON named these contacts "synapses", a term which means "clasp"; at that time, SHERRINGTON could not know how appropriate this name was. Whereas scientists adopted the term it was almost fIfty years before the morphology of synaptic regions was clearly defmed by means of electron microscopy (PALADE and PALAY 1954; DE ROBERTIS and BENNETT 1954). In the meantime, it had been recognized that the synapses were the key sites where information was transferred from one neuron to another and that, in the chemical synapse, this was achieved via the transformation of electrical impulses into chemical signals. The evolution of our understanding of the role of the synapse in information processing or storage has centered in two major areas which occupy a central position in today's neurobiology: (i) The concept of transmitter release whereby the arrival of electrical signals prompts the release of a discrete quantum of neurotransmitter from the synaptic terminal within a fraction of a millisecond, and (ii) the question of origin of the remarkable contact specifIcity which plays an important role in the development of the brain and its plasticity, i. e. the structural adaptation of neuronal circuitry to special functional needs. So far as we can say today, these mechanisms

depend upon membrane interactions. These reactions are intracellular (between the transmitter vesicle and the presynaptic membrane) and inter-cellular (between the pre- and postsynaptic plasmalemma). It is evident, therefore, that knowledge of membrane properties, especially of membrane coats, is of basic importance for the understanding of these highly complex synaptic functions. Accordingly, the emphasis of this review article has been placed in this area. The data available regarding the synapses can be roughly divided into two classes, the morphologic on one hand and the physiologic and biochemical on the other hand. The former fmdings are more static whereas the latter are related more closely to dynamics. It is the aim of this review to explore the gap between the two fIelds by integrating data originating from different sources. This attempt, wherever possible, is to further understanding of the relationship between known structural components and special molecules important for function; this line of investigation is generally known as cyto-

chemistry. Weare far from a complete knowledge of synaptic cytochemistry; this publication endeavors to present the current status in this fIeld to aid in understanding synaptic mechanisms and to provide a basis for further morphological, cytochemical and biophysical research. The fIrst part surveys the main techniques used in the fIeld whereas the subsequent and major section considers the morphological and cytochemical fmdings on the chemical synapse in the light of biophysical evidence.

2 •

K. H. PFENNINGER

Cytochemical Methods in Synaptology 1. Conve~tiona1 methods for electron microscopy Osmium tetroxide and potassium permanganate The early studies employing thin section electron microscopy used potassium permanganate and osmium tetroxide as the main fIxatives. Both agents yield the typical trilaminar aspect of membranes and thus greatly influenced concepts in membraneology. KMn04 (LUFT 1956), because of its poor penetration of tissue, its unsatisfactory preservation of cellular structure and protein, is no longer used as a general fIxative. Because of its specilic staining properties of synaptic vesicles, however, it has become one of the major reagents in synaptic cytochemistry (see below). Osmium tetroxide, an agent similar to KMn0 4 in its strong oxidizing action, is one of the most important general purpose fIxatives for the preparation of nerve tissue. Though no longer frequently used as the primary fIxative because of its slow penetration into tissue blocks and its insufficient preservation of protein, it is now generally used after aldehyde treatment as a second fIxation reagent which greatly increases contrast in the same time. The fIrst report on the use of OS04 dates back to 1804, its introduction into histology to 1864 (see BARR 1954). After its introduction into electron microscopy by PALADE (1952), osmicated tissue provided the basis for uncountable studies. In spite of this long history, the mechanisms leading to the typical trilaminar aspect of membranes after OS04 treatment are still far from being understood (for review see PEARSE 1968 a ;KORN 1966 c, 1969; HOPWOOD 1969). Considerable data are available on the chemical reaction of OS04 with isolated tissue components in vitro. Some reaction takes place with amino acids (BARR 1954) in which oxidative deamination seems to be of especial importance (HAKE 1965). The most prominent reaction, however, occurs with unsaturated hydrocarbon chains (CRIEGEE 1936; CRIEGEE et

al. 1942; BARR 1954; RIEMERSMA 1963; KORN 1966a, b). These authors agree that a cyclic osmic acid ester between hydrocarbon chain and OS04 is formed fIrst; KORN (1966a, b) postulates the formation of diesters in which one molecule of osmic acid links two molecules of fatty acid. By subsequent hydrolysis, stable glycol osmates are believed to develop. Afterwards, in the case of lecithin, osmium dioxide has been reported to reach the polar end of the molecule (RIEMERSMA 1963). These observations altogether suggest a major reaction of osmium tetroxide with the lipid fum of the membrane and thereafter a precipitation of the formed osmium dioxide molecules at the polar interface between fatty acids and protein. This view, however, does not explain the observation that triple layered membranes are also found in osmium fIxed mitochondria or myelin from which all the lipid had previously been removed by acetone extraction (FLEISCHER et al. 1965; NAPOLITANO et al. 1967). This may indicate that our knowledge of the mechanisms of osmication has not reached the point which would allow interpretation of pictures obtained from osmicated tissue in terms of cytochemistry.

Aldehydes Since the introduction of glutaraldehyde as a fIxative for electron microscopy by SABATINI and collaborators (1963), aldehydes have become the most important reagents for the preservation of tissue for ultrastructural analysis. Aldehydes mainly react with protein; amino, imino, amido and guanidino groups as well as aromatic hydrogen may be the most important reactive sites (BOWES and CATER 1966; HABEEB and HmAMOTO 1968; PEARSE 1968; HOPWOOD 1969; PFENNINGER 1971 b). Formaldehyde and glutaraldehyde are the compounds most commonly used. The former is a fIxative with considerable crosslinking ability when applied in dilute solutions (only in dilute solutions is it present in its monomeric form). Formaldehyde binds to a compound containing a reactive hydrogen atom thus forming a hydroxymethyl compound which usually is reactive again. It may condense with a further

Synaptic Morphology and Cytochemistry' 3

H atom to form a methylene bridge. These bridges, however, turn out to be very unstable. Washing of tissue for 90 minutes decreases its formaldehyde content to less than 3% (for review see PEARSE 1968). Glutaric dialdehyde OHC(CH2)3CHO, in contrast, is a much more efficient crosslinking agent of proteins due to its two aldehyde groups. In addition, the bonds formed are much more stable as tested by heat and acid treatment (BOWES and CATER 1966). It is not surprising, therefore, that the introduction of glutaraldehyde for electron microscopy brought a tremendous improvement of structural preservation. In cytochemistry it must be considered however, that those reactive sites which are irreversibly bound by glutaraldehyde are no longer available for cytochemical reactions and that, due to possible conformational changes, enzyme activity (SABATINI et al. 1963) as well as antigenecity of tissue components (HABEEB and HIRAMOTO 1968) may at least partly be affected.

Acrolein or acrylic aldehyde H 2 C = CH·CHO is a bifunctional aldehyde which produces excellent morphological preservation (SABATINI et al. 1963). Because of its instability at basic pH and its toxic vapors it is not frequently used at the present time. Aldehydes often contain many impurities, especially acidic components and tend to polymerize which is especially true for glutaraldehyde at a pH exceeding 8. It is essential therefore to apply freshly prepared solutions of the purest reagents available. Aldehydes are applied in a balanced salt or in a buffer solution. Whereas isotonic and hypertonic solutions produce similarly bad structural preservation, moderately hypertonic solutions due to increased aldehyde concentration give the best results (SCHULTZ and KARLSSON 1963) in the perfused brain. This result is not fully understood. It may be due to reactions of the vascular walls to the fixative which would influence the flow of the perfusate and/or to the differential diffusion characteristics of vehicle and fixative which may cause extracellular hypotonicity if isotonic solutions are used.

Th,~ combined aldehyde-osmium tetroxide fixation procedure

Most schemes for the preparation of brain specimens involve the following steps: (i) perfusion with aldehyde, occasionally preceded by a rinse of the vascular bed with a balanced salt solution (FORSSMANN et al. 1967). 2.5 to 3% glutaraldehyde or, more frequently, mixtures of paraformaldehyde and glutaraldehyde, or 4% paraformaldehyde alone, are used in the perfusate. In the hands of many authors, paraformaldehyde mixtures seem to produce better perfusion results than plain glutaraldehyde perfusion (GRAHAM and KARNOVSKY 1966; KARNOVSKY 1967; SOTELO and PALAY 1970). In a second step, glutaraldehyde may be perfused at higher concentration (REESE and KARNOVSKY 1967; PETERS et al. 1968) or, after dissection of the specimens, small tissue blocks can be immersed in a 5 to 6.5% solution of glutaraldehyde to achieve the full crosslinking effect of the dialdehyde (PFENNINGER 1971 b). These steps are carried out at room temperature. It seems to prove helpful to inject several thousand units of heparin and sodium nitrite into the heart or into the blood stream to prevent coagulation and to induce vasodilatation for easier perfusion. (ii) washing of the tissue blocks for several hours or at least short rinsing in a buffer solution before immersion into osmium tetroxide. (iii) osmication usually in a 2% OS04 solution preferably in cacodylate or veronal acetate buffer. (iv) block staining with uranyl acetate (KARNOVSKY 1967). The detailed work by PALADE and BRUNS (1968) on plasmalemmal vesicles and by BRIGHTMAN and REESE (1969) on gap junctions have demonstrated the improvement of membrane preservation by uranyl acetate block staining. (v) dehydration and embedding preferably in Epon 812 (LuFT 1961) or Araldite (GLAUERT and GLAUERT 1958), or mixtures of both (MOLLENHAUER 1964; for review see PEASE 1964). (vi) section staining with (a) uranyl acetate (WATSON 1958) if not applied on blocks and (b)

4 . K. H. PFENNINGER

lead hydroxide (KARNOVSKY 1961) or lead citrate (e. g. REYNOLDS 1963). Osmicated material is generally treated with either uranyl acetate or lead hydroxide or citrate, or both. The additional heavy metal treatment is necessary because contrast of osmicated material without poststaining is weak. In addition to the effects of uranyl or double staining as described below, there may be a binding of lead compounds to the OS02 deposits in tissue. This conclusion is derived from the fact that lead poststaining produces very little electron density without previous osmication (Table I); after osmication however, it greatly increases contrast. To summarize, the effects of the combined aldehyde - osmium tetroxide fixation and impregnation and uranyl - lead staining procedures (OsUL) consist of (i) fixation of protein by aldehydes, (ii) fixation of lipids and impregnation by OS04' (iii) staining by uranyl and/or lead. Characteristic features of tissue treated by these methods are: Clear intercellular spaces, the tramline aspect of all membranes, and a cytoplasmic matrix containing some fuzzy material. Cytoplasmic densities attached to membranes are generally well preserved. It must be stressed that this classical picture in cytology is produced and influenced by a series of different factors also including extraction and degradation effects which are not known in detail.

2. Staining of acidic groups Cationic heavy metal complexes are candidates for the demonstration of acidic groups in tissue. Such complexes are, e. g. uranyl (WATSON 1958), ruthenium red (LUFT 1964, 1966a, b), ferric oxide (colloidal iron; HALE 1946; MOWRY 1958; GASIC and BERWICK 1962; CURRAN et al. 1965), lanthanum (DOGGENWE1LER and FRENK 1965) and colloidal thorium dioxide (REVEL 1964). Differences in the staining effect of these cationic dyes probably are due to the sizes of the molecules which influence the diffusion characteristics into tissue. Also important are the sizes of the precipitates which affect the resolution limit of the methods. The strength of the ionic binding

forces and the pH of the staining solution are other essential factors influencing the selectivity of these techniques. Uranyl acetate The best known example of these cations is uranyl UO~+. In the following, the chemistry of its application on thin sections will be dealt with in more detail. Since uranyl staining alone without previous osmication produces little contrast, it will be discussed as combined with lead hydroxide (KARNOVSKY 1961). The combined procedure is forthwith designated as UL staining. Table I provides a comparison of the staining effects of uranyl and/or lead on Eponembedded thin sections of glutaraldehyde - fixed, non-osmicated tissue. It turns out that the contrast after double staining is mainly due to the UO~+ binding to acidic groups as derived from the decrease in electron density with increasing hydrogen ion concentration. Whereas lead hydroxide staining prior to uranyl acetate has no effect, the reversed sequence leads to a tremendous increase in electron density as compared with uranyl staining alone (PFENNINGER 1971; see also HUXLEY and ZUBAY 1961). This result indicates an affmity of plumbite to uranyl by which mechanism the contrast is enhanced. A binding of uranyl ions to acidic groups has also been proposed by HODGE and SCHMITT (1960), HUXLEY and ZUBAY (1961), STOECKENIUS (1961), WOLFE, S.L. et al. (1962), and BONDAREFF (1967b). The weakening of UL staining with decreasing pH of the uranyl solution is gradual and differs from structure to structure. Nuclei are the last cell elements to loose their contrast Staining procedure

Contrast

none Lalone U alone

(+)

L,U U,L U (pH t), L

(+)

+ +

+++ +++-+ (+)

Table I. Staining effects of uranyl and lead.

Synaptic Morphology and Cytochemistry . 5 (at around pH I). Thus, a rough differentiation of the pH of acidic groups in tissue seems possible. Carboxymethylation (esterification) combined with staining of acidic groups offers a further method to differentiate anionic groups (FRAENKEL-CONRAT and OLCOTT 1945). The esterification method for electron microscopy (CURRAN et al. 1965; PFENNINGER 1971 b) is a modification of histochemical techniques described by WIGGELSWORTH (1952), FISHER and LILLIE (1954), and PEARSE (1968 b), involving methanolic dehydration of the specimen and subsequent incubation in 0.1 N HCI in absolute methanol for an extended period at 37° C. The tissue blocks subsequently are embedded or rehydrated for further treatment in aqueous solutions. The same method can also be applied on thin sections mounted on gold grids but carboxymethylation possibly is less complete than in the methods using unembedded tissue blocks (PFENNINGER 1971 b). Tissue preservation is satisfactory in both cases. The effect of blockade of carboxyl groups is different comparing various tissue elements. This is shown in Figs. 1,2,3,4 and summarized in Table 2. Note that the electron density after UL staining of nuclear chromatin is little affected by esterification whereas mitochondria become almost invisible (Fig.2, d. with the control in Fig. I). In Fig. 2, synaptic cytoplasmic densities as well as large dense core vesicles are seen to retain part of their original contrast. Fig.3 shows part of a capillary endothelium. Note that lysosomal structures and basement laminae still have a

Structure

a) nuclear chromatin b) nucleolus c) mitochondria d) Iysosomes e) postsyn. density £) basement lamina

Electron density (U, L staining) control

carboxymethylation

++++ +++ ++ +++ +++

++++ +

++

(+)

+++ (+)

++

Table 2. The effect of carboxymethylation on UL-staining.

strong affinity for the contrasting agents. In Fig. 4, the nucleolus shows some remaining contrast but its electron opacity is much decreased as compared with the nuclear chromatin. The contrast of "sphaeridia" or nuclear bodies (BUTTNER and HORSTMANN 1967) is almost entirely abolished. These results are consistent with the idea that esterification selectively blocks carboxyl groups and therefore decreases the electron density of structures rich in protein (Table 2 b, c, e) whereas phosphate - and sulfaterich elements (Table 2a, f) show little change in contrast as compared with the controls. Thus, differentiation of carboxyl vs. sulfate and phosphate residues in electron microscopy is possible.

Ruthenium red, colloidal iron oxide and other cations Ruthenium red staining has been introduced for electron microscopy by LUFT (1964, 1965, 1966). It is generally used in combination with osmium tetroxide. Treatment with this reagent produces excellent contrast but optical resolution is in the range of tens of Angstroms only and the penetration of ruthenium red into tissue is not satisfactory at all. The interior of the cells and cell organelles is stained in exceptional cases only. For this reason, i. e. the usual restriction of the staining to the extracellular space and, further, because of the affinity of the cationic ruthenium red to acidic groups, this dye has often been considered as specific for acidic polysaccharides. However the following observations obtained in our laboratory (PFENNINGER 1969) are not in agreement with this view: Whereas the staining is usually found extracellularly in the brain (BONDAREFF 1967a), we occasionally observed intracellular deposits as well when about 200 (J. thick tissue slices were incubated in the ruthenium red solution. Figs. 14 and 15 show staining of microtubules which becomes especially well visible when no section poststaining is applied (Fig. 15); the ruthenium red contrast is apparently due to the large number of acidic amino acids in microtubular protein (see SCHMITT and SAMSON 1968). Fig.47 shows part of a synapse where not only synaptic cleft material but also postsynaptic

6 . K. H. PFENNINGER

density and presynaptic dense projections and a fuzzy material around the synaptic vesicles are heavily stained. This result was more frequently obtained in tissue slices treated previously with neuraminidase. The incubation in solutions of

neuraminidase (see PFENNINGER 1971 c) does not result in a decrease of ruthenium red staining in the intercellular space. The electron density appears unchanged but the heavy metal complex has permeated the whole depth of the specimen.

Synaptic Morphology and Cytochemistry' 7 This observation may be explained by the fact that, after removal of an important part of the N-acetyl-neuraminic acid (NANA) residues in the intercellular space, the amount of ruthenium red bound to the membrane coat is diminished and thus may no longer block further diffusion of the dye. The amount of non-sialic acid or neuraminidase - resistant NANA anionic groups is still sufficient to produce an intense staining of the intercellular space (d. PFENNINGER 1971 c). Hence it is inferred that ruthenium red is a potent agent for the demonstration of anionic groups of every kind; probably due to its size, it usually does not penetrate cellular membranes and therefore gives the impression of being specific for intercellular cleft material. Lanthanum nitrate, colloidal iron oxide and colloidal thorium dioxide specificity are presumably based upon similar staining mechanisms (see e. g. CURRAN et al. 1965).

3. Staining of basic groups The demonstration of cationic groups in tissue offers special difficulties since the basic nitrogen-containing residues are the ones which react preferably with the aldehydes used for fixation. The condensation product of this reaction no longer has the original cationic properties and, therefore, cannot bind anionic heavy metal complexes (PFENNINGER 1971 b). Under certain conditions, however, the staining of basic groups with anionic dyes is possible.

Phosphotungstic acid Phosphotungstic acid (PTA) has been the subject of numerous publications concerned with its cytochemical action. The scientists who have contributed to this problem may be divided into two groups. One group, starting with PEASE (1966), claims that PTA applied in strongly acidic solution stains polysaccharides (MEYER 1969a, b, 1970; RAMBOURG 1967, 1968, 1969; MARINOZZI 1968; PEASE 1970). This view of the PTA specificity (which will be dealt with in detail in the following chapter) has been passionately attacked by the second group of authors who claim that PTA exclusively binds to cationic residues, especially basic protein (NEMETSCHEK etal. 1955; KUEHN et al. 1958, 1959; HODGE and SCHMITT 1960; BENEDETTI and BERTOLINI 1963; BLOOM and AGHAJANIAN 1966, 1968; SHERIDAN and BARRNETT 1967; SILVERMAN and GLICK 1969; GLICK and SCOTT 1970; SCOTT 1971; QUINTARELLI et al. 1971 a, b). PTA as an anionic heavy metal complex of a very high molecular weight is of course a good candidate for the demonstration of basic groups in electron microscopy. Accordingly, a large series of in vitro experiments has shown that basic substances occuring in tissue such as basic amino acids are indeed very effectively precipitated with PTA (SILVERMAN and GLICK 1969; QUINTARELLI 1971 a; SCOTT 1971). Further support for this view comes from the decrease of PTA staining after deamination (SILVERMAN and

Figs. I, 2, 3 and 4. Effects of carboxymethylation on the staining of acidic groups with uranyl acetate and lead hydroxide (UL). Fig. I: Control; UL staining without previous esterification. Note the electron density of the filamentous material (f) and mitochondrial matrices (m). n, nucleus; r, ribosomes. Figs. 2, 3 and 4: Esterification prior to UL staining. Note the decrease in the staining of most cytoplasmic organelles, particularly mitochondria (m), whereas nuclear chromatin retains most of its stainability. In neuropil (Fig.2), the cytoplasmic densities of synapses (s), but not the synaptic cleft material, and large dense core vesicles (dv) belong to those structures which are still fairly well visible. In capillary walls (Fig. 3), basement laminae (bl) stand out due to their almost unchanged electron density. In the same figure, the high contrast of lysosomal dense bodies is also evident. Fig.4 illustrates more details of the nucleus after esterification and UL staining. Note the high electron density of the chromatin whereas the staining of the nucleolus (nl) is markedly diminished. Another feature of Fig. 4 is the nuclear bodies (nb), the staining of which is extinguished. (Only the central granules seem to retain some electron density). This picture may reflect the distribution of phosphate containing compounds, particularly nucleic acid. - All electron micrographs are taken from aldehyde fixed, unosmicated cat subfomical organ. Magnifications, X 32,000 (Fig. I), X 33,000 (Fig. 2), X 10,000 (Fig.3), X 13,000 (Fig. 4) ; calibration in all figures, Ill.

8 . K:H. PFENNINGER

GLICK 1969; QUINTARELLI et al. 1971 b) or after amino acetylation (SHERIDAN and BARRNETT 1967; QUINTARELLI et al. 197Ia). The observation that staining with bromphenol blue (an anionic dye which binds stoichiometrically to positively charged groups) is decreased after PTA treatment of the tissue speaks in favor of the same hypothesis (QUINTARELLI et al. 1971 b). These observations leave little doubt that PTA demonstrates basic groups on the basis of coulombic interaction. It must be stressed, however, that all these experiments were carried out with aqueous, moderately acidic solutions. The application of PTA for the demonstration of fme structure in the brain was performed first by GRAY (1963). This lead to the first clear visualization of presynaptic dense projections. Cytochemistry of nerve tissue on the basis of PTA staining, though, was done later only when BLOOM and AGHAJANIAN (1966, 1968) applied this heavy metal complex in ethanolic solution on aldehyde fixed, non-osmicated brain tissue (d. also OGAWA et al. 1970). After application of this procedure which they called E-PTA technique, these authors fOlmd a very selective staining of synaptic densities in neuropil whereby unit membranes are spared and most cellular

elements other than the nucleus and desmosomelike junctions are almost invisible (Fig. 21). In accordance with the fmdings on PTA staining in moderately acidic solution, E-PTA staining is decreased after aminoacetylation (BLOOM and AGHAJANIAN 1968); the fmding that proteolytic activity rather than polysaccharide-degrading enzymes digest the E-PTA positive material (BLOOM and AGHAJANIAN 1968) further supports the notion that the E-PTA method demonstrates basic amino groups in tissue. The interference of aldehyde fixation with the stainability of basic amino groups has been pointed out already. The results after E-PTA staining indicate that, probably, the condensation of aldehydes with amino compounds is either incomplete or partly reversible or both. Unfortunately there are no data available indicating which kind and which proportion of cationic residues are visualized.

Bismuth iodide Bismuth iodide (BI) with the exact formula BiI; is another anionic heavy metal complex which is used in chemistry for the demonstration of different amines (POETHKE and TRABERT 1955; BOBTELSKY and COHEN 1960; ZABICKY 1968).

Fig. 5. Electrophoretic bench for testing the reaction of staining reagents with pure substrates. b is the bench supporting a strip of chromatography paper which connects two dishes containing the liquid phase and anode (A) and cathode (C), respectively. A strip of plastic (c) covers the chromatography paper. It bears two rows of large holes for the application of the staining reagent and smaller holes (arrows) for the application of four different substrates. In the present case, bismuth iodide (BI) was tested with four different polyaminoacids. The basic amino acid applied in the upper hole on the left side is the only compound reacting with BI as derived from the dense precipitate and the immobilization of an important part of BI (P). In the three other cases, the BI moves freely towards the anode.

Synaptic Morphology and Cytochemistry . 9 The introduction of bismuth iodide impregnation for electron microscopy (PFENNINGER et al. 1969 a, b) lead to the finding of several formerly unknown structural details of the synapse (see e.g. AKERT et al. 1969; AKERT and PFENNINGER 1969; AKERT et al. 1972) so that its cytochemical background was thoroughly investigated (PFENNINGER 1971 b). The first series of experiments, the precipitation of various polyamino acids and other compounds by BI was tested before and after glutaraldehyde fixation. BI was applied in a 0.2 N formic acid solution (PFENNINGER 1971 b) as used for the impregnation of tissue; the substrates were dialyzed against the same solution before BI was added. Since the reaction of the dye with the substrate does not necessarily show up as a precipitation (a problem with all test tube experiments for the investigation of cytochemical reactions) BI and organic compounds were moved against each other by electrophoresis on chromatography paper. In this experiment, the immobilization of the dark yellow BI complex by an organic compound would indicate a chemical reaction of the dye with the substrate. Fig.5 shows an electrophoretic bench (b) supporting a strip of chromatography paper which is covered with a plexiglass plate (c). Note the holes in the plate for the application of the substrates (arrows) and of a larger amount of the cytochemical reagent (rows of large holes). In the experiment displayed in Fig.5, four different substrates are tested but only the one in the upper left hand corner causes the formation of an immobilized dense band (p) of the substrate reagent complex (in these experiments, the current was about 5 rnA at 150 V). These in

vitro experiments showed that BI precipitates the basic polyaminoacids exclusively and that, after glutaraldehyde fixation, polylysine and polyhistidine retain their reactivity whereas polyarginine no longer binds BI. Since the contrast obtained after plain BI impregnation of nervous tissue is weak, thin sections are currently double stained with uranyl acetate (U) according to WATSON (1958) and lead hydroxide (L) according to KARNOVSKY'S method "B" (1961). The cytochemical effects of this method which is referred to as BlUL technique must therefore be regarded as a combination of the staining of acidic groups by UL and the BI effect proper. The influence of the individual staining components on tissue contrast and especially on synaptic densities was studied in a second series of experiments. The results are schematically demonstrated in Table 3: Lead staining alone of BI impregnated material does not increase the contrast visibly. Uranyl poststaining leads to a fair improvement of the picture when applied alone but, in combination with lead staining, increases the contrast tremendously. The increase in electron density is much bigger than one would expect from the mere addition of BI and UL contrast taken separately. This result indicates that the UL combination is not only bound to acidic groups but also to an additional compound dependent upon previous BI impregnation i. e. probably to the BI complex in tissue itself. If the hypotheses derived from the latter and from the test tube experiments are true, a stepwise reduction of tissue contrast by blockade of acidic and basic groups should be possible. These experiments represent the third step in the cytoContrast (synapses, mitochondria)

Impregnation BI

BI BI BI

BI

-

Staining

control

-

(+) (+) ++ +++++ ++ +++

L V V,L

V (pH 1.2), L V,L

Table 3. Staining effects of the BIVL technique.

ammoacetylation

carboxymethylation

+ +

+++ (+)

10 •

K. H. PFENNINGER

chemical investigation of the BIUL method. As summarized in Table 3, blocking of acidic groups by carboxymethylation or, more completely, by application of uranyl acetate staining at pH 1-I.2Ieads to a moderate decrease in BIUL contrast whereas aminoacetylation especially in combination with esterification extinguishes BIUL contrast of most tissue components almost completely (d. Fig. I I in PFENNINGER 1971 b). These results are consistent with the idea that, after blocking of the acidic groups, the BIUL method is specific for the demonstration of basic tissue components. As suggested by the test tube experiments, guanidino groups seem to be irreversibly blocked by glutaraldehyde, but at least part of the e:-amino and imidazole residues are free (or have been liberated by acid hydrolysis) to bind the BI heavy metal complex. Without blockade of acidic residues, the BIUL technique is a powerful means for the demonstration of all polar groups. Fig.6 demonstrates the hypothetical staining mechanisms of the BIUL procedure. These conclusions are in good agreement with the similarity E-PTA and BIUL staining which is especially striking if thin sections of E-PTA material are double stained (OGAWA 1970). Another approach to the staining of basic groups has been undertaken by SELIGMAN and collaborators (1968) using osmium tetroxide containing reagents with multiple acidic groups. The resolution of this method, however, seems rather limited. The technique has not been applied so far in neurocytology.

4. Staining of carbohydrates The cytochemical techniques described in the following (d. RAMBOURG 1971) presuppose that all polysaccharides remain at their original site during fixation; this assumption is questionable, though, since aldehydes and OS04 probably participate in little reaction with this class of macromolecules. For the better preservation of cell surface acidic polysaccharides, BEHNKE (1968) suggested the use of alcian blue - containing glutaraldehyde as a primary fixative.

z

...

iii

'"

c1ft

".

c Z

6 Z

. i 6 N

i2 1ft

0

BASIC

..

.

n

lI:

:0

~

CD

0)(

...-<

"'" ?5 ... ",

!:j! 1ft!: rn

ACIDIC

Fig.6. Scheme 'Summarizing the proposed staining mechanism of the BIUL procedure. The BI; complex is bound to basic amino groups. This reaction is irreversibly blocked in guanidino groups by glutaraldehyde and in primary amino groups by amino acetylation (the blockade of imidazole groups may be reversible). Uranyl ions are subsequently bound to BI but have an affmity to acidic groups as well. This reaction, however, can be blocked by application of U stain at low pH or, less completely, by carboxymethylation. To increase contrast, poststaining with plumbite (lead hydroxide) is pedormed; this heavy metal complex has an affmity to the uranyl ions already bound to tissue (from PFENNINGER 1971 b).

A survey of the different attempts which have been made to demonstrate polysaccharide in electron microscopy is given in Table 4. The problem of using cationic heavy metal complexes for the staining of acidic polysaccharides has been dealt with in the chapter on the demonstration of anionic groups. The main disadvantage of these methods is insufficient selectivity; it can be improved, however, by the simultaneous application of carbohydrate - digesting enzymes as outlined in Chapter 5 (BONDAREFF and SJOSTRAND 1969).

Methods using periodic acid The oxidation of 1,2-glycols by periodic acid to aldehydes is a well known reaction which provided the basis for various light microscopical techniques. The resulting aldehyde groups are

Synaptic Morphology and Cytochemistry . Reacting residues

Staining mechanisms and reagents

}

-COO-SO-.

uranyl acetate (+ neuraminidase) ruthenium red lanthanum nitrate ionic colloidal thorium dioxide colloidal iron oxide

+HCl polyhydroxyl

References BONDAREFF & SJOSTRAND (1969) LUFT (1964) DOGGENWEILER & FRENK (1965) REVEL (1964) GASIC & BERWICK (1962) CURRAN et al. (1965) PEASE (1966), MEYER (1969a, b)

? A-PTA (aqueous, strongly acidic)

+Cr0 3 1,2-glycol: -HCOH-HCOH-

-

I

II

-

l f

I.

RAMBOURG (1967,1969)

oxidation to aldehydes by HIO.

2. aldehyde demonstration A. Schiff reagent (P.A.S.) + PTA

THIERY (1967)

B. Reduction of silver: Ag-methenamine

DETTMER & SCHWARZ (1953), CHURG et al. (1958), MARINOZZI (1961), THIERY (1964), RAMBOURG & LEBLOND (1967)

C. Condensation with hydrazine or hydrazide (- NH - NH z) a) thiocarbohydrazide, OsO. (reduction) b) thiocarbohydrazide, Ag-proteinate (reduction) c) p-monofluorophenylhydrazine, ammonium sulfide, OsO.

HANKER (1964), SELIGMAN (1965) THIERY (1967) BRADBURY & STOWARD (1968)

Table 4. Methods for the demonstration of carbohydrate in electron microscopy.

demonstrated either by the reduction of silver (GOMORI 1937, 1946), or by treatment with the Schiff reagent (P. A. S. staining; McMANUS 1946, 1948; HOTCHKISS 1948). These methods have been modified in various ways for electron microscopy (the lettersA,B, C refer to Table 4): (A) TillERY (1967) tried to increase the contrast in the electron microscope of P. A. S. staining by subsequent treatment with PTA. Although he observed some improvement of the electron microscopic picture, and the resulting distribution of electron density was consistent with other polysaccharide staining procedures it is not known whether this PTA poststaining really enhances P. A. S. contrast or stains other tissue components. (B) Since precipitations of reduced silver are visible in the electron microscope, various authors applied GOMORI'S technique (1937, 1946) more or less directly for ultrastructural studies (DETTMER and SCHWARZ 1953; CHURG et al. 1958; MARINOZZI 1961;

THIERY 1964; RAMBOURG and LEBLOND 1967). (C) The group of SELIGMAN (HANKER et al. 1964; SELIGMAN et al. 1965) introduced another method for the demonstration of aldehyde using hydrazine (-NHNHz)-containing compounds. The reagents they used, thiocarbohydrazide or thiosemicarbazide, reduced OS04 in a further step of the procedure to produce electron opacity. THIERY (1967) modifIed this technique by the use of reduction of silver from silver proteinate to demonstrate bound thiocarbohydrazide. BRADBURY and STOWARD (1968) applied p-monofluorophenylhydrazine hydrochloride instead of the hydrazides and produced electron contrast by subsequent treatment with ammonium sulfide and finally with osmium tetroxide. As derived from theoretical considerations, the methods under (B) and (C) in Table 4 are likely to be very specific for carbohydrates. The practical evaluation of the staining selectivity,

12 •

K. H. PFENNINGER

however, is merely based on the observation of electron density in structures of well known carbohydrate content such as glycogen particles and basement laminae. A careful comparison of the staining patterns of the methods (A), (B), (a) and (b) (THIERY 1967) has shown that the periodic acid-thiocarbohydrazide-silver proteinate method probably yields the best results concerning contrast, specificity and structural details. All these methods suffer, however, from the rather coarse particulate character of their reaction products so that electron optical resolution is very limited. Among the techniques discussed in this section, the period acid - silver methenamine method is the only one which has been applied so far to investigate polysaccharide distribution in the brain (RAMBOURG and LEBLOND

1967).

Acidic phosphotungstic acid PEASE (1966) initiated this technique by staining with aqueous acidic PTA (A-PTA) of thin sections from tissue which was prepared by "inert dehydration" without previous fixation and hydroxypropylmethacrylate embedment. The resulting high contrast of goblet cell mucus, basement laminae, extracellular material associated with brush borders, etc. prompted this investigator to conclude that this method is specific for carbohydrates. As a theoretical basis for the staining, he proposed the formation of multiple hydrogen bonds between phosphotungstate radicals and chains of polysaccharide units. Pease's method was later considerably improved (MEYER 1969 a, b, 1970) using aldehyde and/or OS04 fixed and Epon - embedded specimens. A further and seemingly essential improvement of the technique consists in the lowering of the pH of the PTA solution to below 1 by hydrochloric acid (see also PEASE 1970). The resulting pictures are characterized by good tissue preservation and extensive staining of basement laminae, glycogen, intercellular spaces, and the sacs of the Golgi apparatus (MEYER 1970). About simultaneously, RAMBOURG (1967, 1968, 1969 a, b, 1971) and MAIuNOZZI (1968) modified the A-PTA method, too. The former used aldehyde-fixed, glycolmethacrylate-embedded

material for staining with a very acidic mixture of PTA and chromic acid (pH 0.3); the latter author applied the same embedding medium but no chromic acid. RAMBOURG and MARINOZZI also contributed to the body of evidence favoring a polysaccharide specificity of the A-PTA staining. It was demonstrated that blockade of hydroxyl groups in tissue by sulfatation (MARINOZZI 1968) or reversibly by acetylation (MAIuNOZZI 1968; RAMBOURG 1968) inhibits the typical A-PTA staining whereas deamination seems not to affect the contrasting mechanism (MARINOZZI 1968). Further, the mixture of chromic acid and phosphotungstate has been shown to precipitate glycogen and glycoprotein whereas many other biological substances seem not to react with the staining reagent (RAMBOURG 1968). In spite of the fact that these data are quite coherent and that the distribution of A-PTA contrast is in full agreement with the fmdings obtained by the periodic acid - aldehyde reagent methods, the carbohydrate specificity of A-PTA seems to be contradicted by the data pointing to a specificity for basic residues of PTA staining as described above. The discrepancy of the results is due to the fact that the conditions under which PTA is applied on the tissue are significantly differentwhieh apparently has not been recognized by many investigators. Only recently, it was found that, indeed, PTA precipitability of amines vs. polyalcohols differs markedly with decreasing pH, i. e. that polysaccharides can be precipitated at very low pH only (SCOTT 1971). It is to be stressed, though, that the reaction of the dye is not necessarily indicated by a precipitation of the substrate in vitro and that, alternatively, a precipitation reaction in vitro does not prove a staining reaction in situ. At the present state of knowledge, staining reactions of PTA are not fully understood. As a possible explanation it could be argued that, under moderately acidic conditions, the PTA anions are bound to basic groups in tissue by coulombic forces; the evidence for this mechanism is certainly good. On the other hand, at very low pH, phosphotungstate may become increasingly protonated so that the interactions with cationic

Synaptic Morphology and Cytochemistry' 13 groups become less important and hydrogen bonding to polyalcohols may [mally prevail. At least, the cytochemical aspect of the two staining procedures (E-PTA or moderately acidic PTA vs. A-PTA) differs markedly: E-PTA stains nuclei and mitochondrial matrices but not the Golgi apparatus, basement laminae or glycogen whereas A-PTA gives best contrast in the basement laminae, with glycogen and in Golgi cisterns but not with nucleoprotein. The staining pattern of synapses is also quite different (see below). This is evident when comparing Figures 22 and 24 (see also MEYER 1969 b).

5. Demonstration of macromolecules by enzymatic digestion Macromolecules in general are bearing a variety of different functional groups most of which they have more or less in common. Thus, their selective staining seems almost impossible except when applying immunocytochemical techniques. The selective degradation properties of certain enzymes offer an easier but far less specific method for the characterization of macromolecular components in tissue. This is done by observing the effect of different digestive enzymes on the stainability of the structures of interest. This technique was introduced by LEDUC and BERNHARD (1961, 1962) and has meanwhile been modified in different ways by various authors (MARINOZZI 1964; MONNERON and BERNHARD 1966; BLOOM and AGHAJANIAN 1968; BONDAREFF and SJOSTRAND 1969; BARRANTES and LUND 1970; PFENNINGER et al. 1970; PFENNINGER 1971 c). The last four investigations are devoted especially to the cytochemical characterization of synaptic structures. Problems of interpreting enzymatic digestion experiments should be considered as follows: The substrates are not in solution but bound to other macromolecules (e. g. "structuralprotein"). The interaction with other substances in situ may produce conformations which block the access of the enzyme to the site of its lytic action. On the other hand, a marked effect of the enzyme incubation on stainability may be mimicked when the enzyme is bound to a structure and

thus blocks its affmity to the heavy metal stain. Therefore, the preparation of good control experiments is imperative. Incubation in the enzyme solution with an inhibitor added or in a solution of, e. g. heat-inactivated enzyme may be performed. Small molecular weight inhibitors, as e. g. diisopropyl fluorophosphate for hydrolytic enzymes, are preferable to macromolecular inhibitors since they diffuse easily throughout the tissue. When no inhibitors are available or inactivation is hard to control, albumin or similar "inert" proteins may be used to replace the enzyme in the control solution. It is worthy to note that only the purest enzyme preparations available are suitable for these experiments since the organs they are extracted from in general contain a series of different lytic activities.

Incubation of unfixed or aldehyde fixed specimens The incubation of specimens in the enzyme solution can be performed at different steps of the tissue preparation procedure. This is shown in Table 5 (the letters A, B, C refer to this table). The digestion of unftxed, tiny tissue blocks (A) has the advantage of submitting the structures of interest, i. e. the macromolecules they contain, in the most natural state to the action of the enzyme. However, tissue may undergo important uncontrolled alteration when it is cut into small pieces and especially during the extended Incubation

Preparation procedure dissection of tissue

A

~

aldehyde fixation ~

B

washing in buffer -+

+

impregnation (OsO4jBIjE-PTA)

+

C

dehydration, embedding, thin sectioning -+

~

section staining

Table 5. Possibilities of enzymatic degradation in the course of tissue preparation for electron microscopy.

14 . K. H. PFENNINGER

period of incubation (cell death, lytic enzymes in tissue). Further, penetration of the specimen by the enzyme may be incomplete since unfixed tissue cannot be sliced fmely enough and since cells, as long as they are living, may impede the passage of the enzymes through their membranes. These disadvantages are avoided if aldehyde fixed tissue blocks are sliced (with or without a freezing microtome) and then incubated (B). Unless very potent lytic enzymes are used for digestion, preservation of structures in these specimens is more or less satisfactory. An extended period of washing between fixation and incubation is essential to avoid inactivation of the enzyme by aldehyde remainders in tissue. Possible artefacts introduced by the previous aldehyde fixation are the following: (i) The reaction with aldehydes changes chemical properties and conformation of substrates in tissue. The susceptibility of the substrate to enzyme activity may thus be partially or completely lost. (ii) Fragments of degraded substrates may remain in situ because of their linkage to intact tissue components by the aldehyde. Therefore, the effect of enzymatic degradation may not be visible. (iii) A more or less tight meshwork of proteins produced by the crosslinking action of aldehydes may block the access of the enzyme to the structure of interest.

Incubation of thin sections The treatment of thin sections (C) has a series of advantages. Cells and cell organelles on the surface of the section are fully exposed to the enzyme solution, and the action of the enzyme, if present, is very homogeneous throughout the whole specimen. Further, subsequent sections can be treated with different solutions and control media for comparison of the same structure after exposure to various treatments. However, (i) heavy metal treatment before the embedment must be avoided since most of these agents are effective enzyme inhibitors and heavy metal precipitates within macromolecules probably make them resistent to degradation. Osmication can be performed if, in thin sections, the OsOz deposits in tissue are reoxidized and washed out with HzO z. Though, the strong oxidation by

OS04 and HzO z alters the susceptibility of the tissue components towards lytic enzyme action and their affmity for later heavy metal staining. (ii) Retention of substrate fragments in situ is more likely to occur in thin sections than in method (B) because of the three-dimensional meshwork of the polymerized embedding medium. (iii) This meshwork can completely inhibit the action of enzymes especially when epoxy resins are used. In our hands, the methods (A) and especially (B), i. e. the digestion of unfixed or aldehyde prefixed tissue slices turned out to be the most fruitful attempt towards elucidating macromolecular characteristics of synaptic densities. The application of the following enzymes provided useful data: Neuraminidase, trypsin, chymotrypsin, peptidases, pepsin, pronase, papain. A detailed description of the methodology is given in the original literature (BLOOM and AGHAJANIAN 1968; PFENNINGER 1971 c).

6. Staining of vesicle contents The staining of the contents of synaptic vesicles is of special interest since they are the main candidates for the site of transmitter storage. An ideal staining reagent for transmitter substances would allow the identification of different nerve terminals and thus would be an important tool for regional neurocytochemistry; further, it would provide the possibility of localizing the neurotransmitter substances under different functional states and thus help the better understanding of transmitter pathways. It is not surprising therefore, that considerable effort has been made to develop techniques which would allow the acquisition of these data. No techniques are as yet available for the detection of acetylcholine or amino acid transmitters such as glycine or y-amino butyric acid (GABA). However, the biogenic amines noradrenaline, dopamine and s-OH-tryptamine can now be visualized in electron micrographs. The considerable body of evidence for the specificity of the methods has recently been reviewed in excellent papers (TRANZER et al. 1969; HOKFELT 1970, 1971; for methodology see especially

Synaptic Morphology and Cytochemistry . 15 BLOOM 1970; HOKFELT and LJUNGDAHL 1972). It is not the aim of this chapter to repeat all these data in detail. The main lines of evidence will be briefly summarized in the following. All cytochemical techniques for biogenic monoamines make use of strongly oxidizing heavy metal compounds such as potassium permanganate, osmium tetroxide and potassium dichromate. In the case of OS04 and KMn04, the staining mechanism seems to be dependent upon an oxidation of the phenolic hydroxyl groups of the amines which leads to the precipitation of the reduced heavy metal dioxides (HOKFELT and LJUNGDAHL 1972). It is important to note that these precipitations are probably not stoichiometric reactions so that a quantitative estimation of the transmitter content of the vesicles cannot be carried out on the basis of the amount of electron dense deposit.

Osmium tetroxide Osmium tetroxide applied as primary fixative produces electron dense granules in adrenergic fibers of the peripheral nervous system and the pineal (DE ROBERTIS and PELLEGRINO DE bALDI 1961; LEVER and ESTERHUIZEN 1961; GRILLO and PALAY 1962; RICHARDSON 1962). A considerable improvement of the technique was achieved by the introduction of glutaraldehyde prefixation (COUPLAND et al. 1964; BLOOM and BARRNETT 1966b). Glutaraldehyde probably reacts with the amino group of the monoamines and help~ (heir retention within the vesicular lumen, possibly by the formation of polymers (BLOOM 1970; HOKFELT and LJUNGDAHL 1972) so that the amount of substrate available to precipitate the heavy metal reagent is increased. However, positive staining reactions are confmed to the peripheral nervous system.

Glutaraldehyde-potassium dichromate-osmium tetroxide The combination of aldehyde fixation and potassium dichromate treatment with or without intermediate osmication (WOOD 1966, 1967; JAIM-ETCHEVERRY and ZIEHER 1968; TRANzER and THOENEN 1968; TRANzER and SNIPES 1968) not only yields good tissue preservation but also

more reliable staining of monoaminergic vesicles. The vesicle staining, however, is more or less restricted to the peripheral nervous system and the pineal.

Potassium permanganate In 1966, RICHARDSON applied KMn04 for the staining of adrenergic fibers (RICHARDSON 1966). This technique was further developed and applied in the central nervous system by HOKFELT (1967, I968a, b). Although the tissue preservation after KMn0 4 staining is not ideal and the penetration of the reagent is so poor that very small tissue slices or blocks must be used, the potassium permanganate method has many advantages. KMn0 4 is the strongest oxidative reagent of the three heavy metal compounds cited above; this is also visible in test tube experiments with pure monoamines (HOKFELT and JONSSON 1968; BLOOM 1970; HOKFELT 1971 ; HOKFELT and LJUNGDAHL 1972). Accordingly, this method produces the most reliable staining of monoaminergic vesicles in the peripheral as well as in the central nervous system (Figs. 60, 62; HOKFELT 1971). As mentioned above, considerable difficulty causes the demonstration of vesicle contents in monoaminergic terminals of the CNS, especially when the osmium staining methods are applied. This can be overcome by the increase of the amine level either by the inhibition of monoamine oxidase, or by in vitro-incubation in, or the in vivo-intraventricular injection of, transmitters. Not only the true transmitters have been used for this purpose (TRANzER and THOENEN 1967a; HOKFELT 1968 b) but also so-called "false" transmitter substances such as a-methyl noradrenaline (BONDAREFF 1966; HOKFELT 1968b) or s-OH-dopamine (TRANzER and THOENEN 1967b; RICHARDS and TRANzER 1970). After the loading of nerve terminals with amines, dense cores within the vesicles are found much more constantly (Figs. 66, 68). The results of the OS04 methods and the KMn0 4 technique are quite consistent especially in the peripheral nervous system. The evidence that they stain the transmitter and not other substances within the synaptic vesicles is

16 • K. H. PFENNINGER based upon the following observations: (i) The reagents give a strong reaction with the substrates in the test tube. (ii) The staining of electron dense granules within the vesicles parallels the fluorescence of nerve terminals as demonstrated by a technique of well established specificity (FALCK et aI. 1962). This is not only true concerning the regional distribution of the cytochemical reactions but also concerning pharmacological experiments, especially the depletion of the transmitter stores (Figs. 58-61). (iii) The loading of the terminals (after their depletion) with natural or "false" transmitter substances leads to the appearance of dense cores in the vesicles. (iv) The presence of a dense core in the vesicles parallels the presence of radioautographic silver grains after the injection of labeled monoamines (Fig. 63).

Zinc iodide - osmium tetroxide staining (ZIO) Iodide salt - osmium tetroxide solutions were used in light microscopy for the demonstration of peripheral autonomic nerve fibers for the first time by CHAMPY (1913) and in modified form by various authors, especially MAILLET (1962). STOCKINGER and GRAF (1965) applied the ZIO mixture for the preparation of electron microscopic specimens. However, the most important features of the ZIO method were described later by AKERT and SANDRI (1968). It was observed that synaptic vesicles in nerve terminals of the central and the peripheral nervous system are heavily stained with an electron dense precipitate. Only after the combination with aldehyde pre-fixation by MARTIN et aI. (1969), however, and its further modification by KAWANA et aI. (1969), did the ZIO method become a reliable tool for the study of the nervous system. The new technique also provides fairly good tissue preservation. Within the nerve terminals, a high proportion of the vesicles is stained as well as the cisternae of the smooth endoplasmic reticulum (AKERT et al. 1971 a; STELZNER 1971). In some cases, rough and smooth endoplasmic reticulum, the Golgi apparatus in the perikaryon as well as the nuclear cistern are stained (AKERT et al. 1971a; STELZNER 1971).

These fmdings are not constant though. The main staining reaction in the nervous system takes place with a component in the vesicles (Figs. 29, 56). The cytochemical background of the technique is far from being known. ZIO is certainly not specifiC for a neurotransmitter since it stains quite a variety of different vesicles (but not the large dense core vesicles) (AKERT and SANDRI 1968; KAWANA et al. 1969; AKERT et al. 197Ia). The more or less selective demonstration of the contents of different synaptic vesicles suggests the possibility of an ubiquitous, still unidentified substance which occurs mainly within the vesicular lumen.

7. Tracer techniques and enzyme cytochemistry Radioautography and macromolecular tracers Radioautography is widely used in biological research for the investigation of localization, transport, and turnover of small and large molecular substances. In the nervous system, the application of suspect and known transmitter substances is of special interest (see, e. g. WOLFE, D.E. et aI. 1962; DECARRIES and DROZ 1970). This technique is mainly based on the specific uptake by the neuron of the biogenic amine or its precursors. It must be pointed out, however, that many factors such as the passage of barriers (e. g. the blood brain barrier) and metabolization influence the fmal distribution of the labeled compound; furthermore, resolution of radioautographic techniques can be no greater than 80 to 150 nm (SALPETER et aI. 1969) which makes an exact localization of the labeled compound, e. g. in the vesicles, impossible (see also BLOOM 1970; HOKFELT and LJUNGDAHL 1972). Macromolecular tracers such as ferritin or horseradish peroxidase do not penetrate membranes. They are useful for studying pinocytosis and the fate of pinched-off plasmalemmal vesicles. Whereas the ferritin molecules are directly visible in the electron microscope, peroxidase containing tissue has to be incubated in a

Synaptic Morphology and Cytochemistry· 17 solution contammg a suitable substrate and H 20 2 • The oxidized reaction product reduces osmium tetroxide to insoluble OS02' These precipitates mark the presence of peroxidase (GRAHAM and KARNOVSKY 1966; REESE and KARNOVSKY 1967). As compared with ferritin, the peroxidase technique yields more electron density so that the tracer is easier to localize. This technique has recently reached considerable importance in the investigation of vesicle formation and turnover in nerve terminals (HEUSER 1971; HOLTZMAN et a1. 1971).

Demonstration of enzyme activity The general principle of these techniques is as follows: The tissue is incubated with a substrate which is as specific as possible for the enzyme in concern and which yields a reaction product stainable with a heavy metal complex so that an insoluble, electron opaque precipitate is formed at the site of enzyme activity. In neurocytochemistry, enzymes of the transmitter pathways are of particular interest. The main goals in the development of techniques for the localization of such enzymes are similar to those for the demonstration of the transmitter itself: (i) regional cytochemistry, i. e. the defmition of specific pathways in the brain, and (ii) determination of the exact site of transmitter synthesis or degradation for the better under-

standing of turnover and transport of these substances. Acetylcholine esterase (AChE) was the first enzyme which was successfully localized (KOELLE and FRIEDENWALD 1949). The specificity of the methods now available and the significance of the results, however, are still a controversial subject. From the enormous amount of literature on the topic, just a few references may be quoted to help in the search of the original articles: KOELLE 1963, 1969; BLOOM and BARRNETT 1966a; BRZIN et a1. 1966; LEWIS and SHUTE 1966; KASA 1968; KOKKO et a1. 1969; GWYN and FLUMERFELT 1971. The demonstration of choline acetyltransferase by BURT (1970) seems to offer a more specific technique for localizing cholinergic systems. The method, however, has so far been worked out for light microscopy only. A similar, very promising attempt has been made in the GABA (y-aminobutyric acid) system by the light microscopical demonstration of brain succinic semialdehyde dehydrogenase, a GABA degradative enzyme (SIMS et a1. 1971). Thus, methodologyfor regional cytochemistry in the brain has made a good start; on the other hand, conclusions on the mechanisms of synaptic function can be derived from such studies only when more specific and more precise techniques for the localization of enzyme activities have been developed.

18 . K. H. PFENNINGER

Cytochemical results and Synaptic function 1. The greater membrane cytochemistry of the "fuzz-coat" Early biological applications of electron microscopy using KMn0 4 and OS04 as fixating and contrasting agents revealed cellular membranes as sharply defined trilaminar structures. Their uniform appearance initiated the unit membrane concept (ROBERTSON 1959). This theory had not only the advantage of a common structural basis for all membranes but also seemed to present direct visualization of the lipid bilayer model of DANIELL! and DAVSON (1935). Meanwhile, however, it has been understood that the trilaminar aspect of membranes in electron micrographs does not necessarily imply a protein - lipid - protein sandwich structure (see e. g. KORN 1969; STOECKENIUS and ENGELMAN 1969; HENDLER 1971) and, further, that the unit membrane concept in its strict sense is not compatible with the tremendous variety of

membranes of different cells and cell organelles. Recent progress in membrane research has indicated that, indeed, the membrane structure must be more complex as previously assumed: a-helical protein has been found withi:l membranes (e. g. WALLACH and GORDON 1968); membrane sublmits can be demonstrated under special conditions (see e. g. ROBERTSON 1966; FERNANDEZ-MoRAN 1967) and particles, probably of protein nature, can be seen in freeze fractured membranes of different origin (MOOR and MUEHLETHALER 1963; AKERT et al. 1969; TILLACK et al. 1970, 1971). A series of observations suggests that the membrane as seen in ordinary electron micrographs is not complete: (i) Presence of an intercellular space of more or less constant width in cell appositions as well as at attachment sites (desmosome-like structures). (ii) Staining of the intercellular space and of a coat on free cell surfaces with different cytochemical methods (PEASE 1966; RAMBOURG et al. 1966; REVEL and ITo 1967; BLOOM and AGHAJANIAN 1968; AKERT and PFENNINGER 1968; PFENNINGER

Fig. 7. Synaptic and non-synaptic plasmalemma in the rat spinal cord after BIUL treatment. The synaptic site is characterized by the well stained presynaptic dense projections (dp), postsynaptic density (po) and synaptic cleft mat::;:ial (sc). These appositions to the unit membrane (not visible in this picture) are continuous with the cytoplasmic (cc) and the external (ec) membrane coats of the nonspecialized plasmalemma. This is particularly well visible on the dendritic side (d) at the transition between the synapse and the apposition. The continuity of the nonsynaptic membrane coat with the synaptic densities indicates that these structures are specialized membrane coat formations. - Magnification, X lIO,OOO; calibration, 0.1 fJ-. Fig.8. An apposition between two cells in the neuropil of the cat subfornical organ. The tissue has been treated with BI and subsequently with OsO 4; double staining with UL has been performed on thin sections. Thus the "tramlines" of the unit membranes (urn) as well as the cytoplasmic (cc) and the external membrane coats are visualized. The latter fill the intercellular space (is) more or less homogeneously. - Magnification, X 185,000; calibration, 0.1 (.I..

Synaptic Morphology and Cytochemistry . 19

Figs.9, 10 and II. Gap junctions in the cat subfornical organ. OsUL staining (Fig.9) demonstrates the close approximation and the thickened cytoplasmic leaflets of the unit membrane. The arrow points to the 20 Agap separating the two plasmalemmae. In contrast, BIUL preparation (Fig. 10) stains the very thin layer of remaining external membrane coat material (arrow). The thickened cytoplasmic membrane coats are now most prominent. Fig. I I displays a gap junction after esterification and UL staining of nonosmicated material. Note the fairly high electron density especially of the cytoplasmic coat indicating the presence of a significant number of noncarboxyl acid residues. The arrow points to the faintly visible central membrane coat layer. Magnifications, X 200,000 (Fig. 9), X 180,000 (Fig.IO), X 210,000 (Fig. II); calibration in all figures, o. I !J.. 1971 b). (iii) Staining of substances apposed to the cytoplasmic side of the membrane (AKERT and PFENNINGER 1969; PFENNINGER 1971 b). The latter cytochemical fmdings are illustrated in Figs.7, 8, 19, 20: Fig. 7 shows a synaptic contact and the adjacent non-specialized plasmalemma. Especially to the right of the synapse, the dendritic membrane is seen as a trilaminar structure the dense layers of which are in continuity with the postsynaptic density and the synaptic cleft substance, respectively. This means that the dense lines of BIUL treated membranes are not identical with but adjacent to the dense layers in OS04 stained membranes (d. also Fig.23). This is also evident in Fig. 8 which shows the apposition of two membranes after BI and subsequent OS04 impregnation and double staining. The tramlines of the unit membrane are easily recognized. In addition, some electron dense material is visible in the intercellular space and most prominent are the electron dense layers covering the cytoplasmic side of the unit membranes. Figs. 19 and 20 show the A-PTA staining in the intercellular space which is consistent with the staining pattern obtained by other teclmiques

which are believed to be specific for carbohydrates (see e.g. THIERY 1967; RAMBOURG and LEBLOND 1967).

The external coat The pr~sence of matelial attached to free cell surfaces (REVEL and ITo 1967; PFENNINGER 1971 b) and on synaptic membranes after opening of the contact (PFENNINGER et al. 1970, 1971 d; PFENNINGER 1971 c) indicate that the stained material in the intercellular space is indeed part of the membrane and not a freely precipitated secretion product of the cells (Figs. 50,53,55). Even in gap junctions (BRIGHTMAN and REESE 1969) where the intercellular space is reduced to 20 A (Fig.9), there is a thin layer of outer membrane coat present (Fig. 10), probably belonging to both of the adjacent cells. The intraperiod line in myelin was formerly regarded as the fusion product of the outer plasmalemmal leaflets of the glial or Schwann cell. Meanwhile, it was found to consist of two individual elements (ELFVIN 1961; DI CARLO 1967; REVEL and HAMILTON 1969), separated by a thin membrane

20 •

K. H. PFENNINGER

coat stainable with the PTA-chromic acid mixture (RAMBOURG 1969), with ruthenium red (PFENNINGER 1969) or with BIUL (PFENNINGER et al. I969a, PFENNINGER 1971 b). The BIUL staining of central nervous system myelin is illustrated in Figs. 12 and 13. The thin layer of ruthenium red precipitate in myelin is shown in Figs. 14 and 15; it is more distinct when the thin

sections are not post-stained. Probably the only points of the cell surface where the external coat is interrupted are tight junctions which seem to serve as sealing units of the intercellular space (BRIGHTMAN and REESE 1969). Thus, an outer membrane coat appears to be a more or less universal feature of the animal cell (see also SCHMITT and SAMSON 1969).

Synaptic Morphology and Cytochemistry .

The cytoplasmic coat The cytoplasmic membrane coat which was demonstrated in non-specialized plasmalemmal areas with the BlUL technique only (AKERT and PFENNINGER 1969; PFENNINGER et al. 1969a; PFENNINGER 1971 b) seems to be a similarly constant membrane component at least in the nervous system. The axon hillock is characterized by a thick cytoplasmic osmiophilic layer (PALAY et al. 1968; PETERS et al. 1968). Under special conditions, i. e. excitation of the nerve, a dense layer has also been found on the inner face of the axolemma after plain osmication (PERACCHIA and ROBERTSON 1968, 1971). These are further indications for the presence of a cytoplasmic coat. The cytoplasmic coat is modified in synapses and desmosome-like structures and also in gap junctions. At the latter sites, it is slightly thicker than usual (Fig. 10). The chemistry of this coat seems to be considerably different from the cytoplasmic coat in synapses and desmosome-like structures (see below). It turns out to be resistent to the most powerful proteolytic digestion by pronase (PFENNINGER 1971 c) and its UL staining is not much diminished by carboxymethylation. The latter result indicates the presence of a large proportion of non-carboxylic acidic groups such as phosphate or sulfate residues (Fig. II).

The concept of the greater membrane In view of these observations, REVEL and ITo (1967) and LEHNINGER (1968) introduced the

21

term "the greater membrane" for the membrane as a whole including not only the trarnlines which were called "inner zone" but also the adjacent coat. Fig.16 represents one possible interpretation of the cytochemical, biochemical and biophysical fmdings in apposed membranes of the nervous system (d. SCHMITT and SAMSON 1969). Most probably, the dense unit membrane (urn) layers produced by osmium tetroxide are located at the interface between lipid and protein; it is not known to what extent protein and lipid staining are involved. The inner zone of the membrane is shown to contain oc-helical protein and lipid arranged in a bilayer; the proteins on the membrane surfaces are shown to have a subunit organization. BruL as a potent stain for both, acidic and basic groups, probably stains at least the outer part of these protem layers with their branches extending into the cytoplasm (thinner cytoplasmic coat) and into the extracellular space (thicker external coat). The A-PTA staining reflects the high density of carbohydrates in the intercellular space. The sugar derivatives are either linked to lipid (ganglioside) or to protein. They bear many anionic N-acetyl-neuraminic acid (NANA) terminals. It must be emphasized that, in addition to NANA, there are many other anionic groups probably mostly of peptidic nature as indicated by the resistance to neuraminidase but the susceptibility to proteolytic digestion of the major proportion of stainable acidic groups. Further, there is also a considerable amount of

Figs. 12, 13, 14 and 15. Fornix myelin of the cat after BIUL staining is shown in Figs. 12 and 13 at X 110,000 and X 21 5,000 magnification. Despite the fact that this myelin has not been treated with OsO 4' the periodicity of layers is well visible. Note the clear-cut staining of the major dense lines (arrows) as well as the intraperiod lines. The inner and the outer surface of the myelin sheath are characterized by a particularly thick electron dense layer (asterisks). a, axon. Calibration in both figures, 0.1 fL. Figs. 14 and 15 display ruthenium red staining of cat fornical myelin with UL poststaining at left and without poststaining at the right side. Note the thick intraperiod lines (arrows) due to deposits of ruthenium red in Fig. 14. This becomes more evident when no UL poststaining is applied as in Fig. 15. The arrows point at the thin layers of the ruthenium reagent which are partly even darker than the major dense lines in the same picture. Note also the clear-cut demonstration of microtubules (mt) in the axons (a). This finding demonstrates that intracellular staining can be obtained with ruthenium red and that the reagent is not specific for polysaccharides but stains all structures rich in acidic groups. Note also the intensive staining of the axonal cytoplasmic membrane coat (cc). Figs. II, 12, 13 and 14 illustrate that staining procedures which reveal membrane coats do also contrast membrane components in myelin. This finding suggests that the unit membranes of myelin are not really fused but separated by thin layers of coat material. - Magnification, X 240,000 (Fig. 14), X 220,000 (Fig. 15); calibration 0.1 fL in both figures.

K. H. PFENNINGER

22 •

CYTOPLASM

Os04

BIUL

fA' PTA

z

cr

cr

CD

::IE

w

::IE

Cr w

...cr

w cr

I'

I

:.11 ~l:I::.:

LEGEND: cationic groups anionic groups (not NANA) •.

NANA

00 polysaccharid"

membrane lipid .,.......J

CYTOPLASM

protein

l

Fig. 16. Schematical representation of molecular structure and staining of plasmalemma. The left part of the graph shows the apposition of two cell membranes. The membrane structure is shown to consist essentially of a lipid bilayer, but the presence of intramembraneous protein (particularly in ex-helical form) is schematically indicated by the "springs". The outer protein layers of the membrane are shown to have a globular subunit structure. The emphasis, however, is not on the arrangement of these elements of the inner zone of the membrane, but on the membrane coats. The proteins of the membrane are shown to bear many polar groups and a number of proteinaceous and carbohydrate tails standing out into the intercellular space. Some of the sugar derivatives of the external membrane coat belong to gangliosides which have their roots in the inner zone of the membrane. In contrast to the external membrane coat, the cytoplasmic coat consists of a thin layer containing polar protein but no cytochemically demonstrable carbohydrates. The inner zone of the membrane consisting of the lipid and the protein subunits plus the cytoplasmic and external membrane coats are called "the greater membrane". The right side of the graph represents the electron microscopical visualization of the various membrane layers. The unit membrane (um) "tramlines" visible after osmication probably represent just the interfaces of lipid and protein. The BIUL technique most likely reveals all polar proteinaceous components of the inner zone as well as of the cytoplasmic and external membrane coats. In contrast, the A-PTA method and other techniques specific for carbohydrates visualize the sugar derivatives which are located in the intercellular space exclusively. In electron micrographs, the intercellular space is well defined by the outer osmiophilic layers of the unit membranes whereas, in terms of molecular structure of the membrane, the situation is not quite clear. In reality, the intercellular space is probably limited by the hydrophobic layers of the membranes. NANA, N-acetyl neuraminic acid.

basic residues present as indicated by bismuth iodide staining. Acidic and basic groups, probably of peptidic nature, but no visible amounts of carbohydrates are also present on the cytoplasmic side of the membrane. Membrane coats are of especial importance where one membrane interacts with another,

intracellularly or extracellularly. In the latter case, i. e. in intercellular junctions, recognition and connectivity mechanisms are involved which are probably based on special features of the membrane coat. The former case, represented e. g. in interactions of vesicles with the plasmalemma, is of particular meaning in the mechanism of transmitter release.

Fig. 17. Synapse at high magnification after aldehyde and OS04 fixation. The nerve terminal is filled with clusters of synaptic vesicles (sv). The synaptic site is defined by a row of faintly stained dense projections (dp) and by the postsynaptic density (po). The synaptic membranes running in parallel to each other bound the synaptic cleft (sc) which contains a little fuzzy material only. - Cat subfornical organ. Magnification, X 230,000; calibration, o. I fL. Fig. 18 shows a synapse after BIUL preparation. The synaptic site is much more prominent than in Fig. 17 due to the well stained presynaptic dense projections (dp), the postsynaptic density (po) and the synaptic cleft material (sc). The large arrow points to an area where the synaptic cleft material appears to consist of two dense layers separated by a thin electron lucent gap. Note that the unit membranes are not stained. Instead, two wide electron lucent gaps (small arrows) are visible between the synaptic cleft material and the cytoplasmic densities. The membranes of synaptic vesicles (sv) are not visible, either. However, the shape of the vesicles is roughly outlined by fuzzy, slightly electron dense material which surrounds them.-Rat spinal cord; magnification, X 220,000; calibration o. I fL.

24 . K. H. PFENNINGER

2. Comparative morphology of synapses As pointed out in the introduction, this review deals primarily with chemically transmitting interneuronal junctions so that the term "synapses", unless otherwise specified, is used for these junctions exclusively. Whereas KMn0 4 treatment and plain osmication of synapses reveal just clusters of synaptic vesicles in association with a membrane running in parallel to an apposed plasmalemma (d. GRAY 1966), the characteristic membrane specializations of the synapse show up only after aldehyde fixation and especially after the application of cytochemical methods such as E-PTA and BIUL (GRAY 1963; BLOOM and AGHAJANIAN 1966; PFENNINGER et al. 1969 b).

Presynaptic densities The «classical» chemical synapse contains a series of not sharply outlined electron dense patches attached to the presynaptic membrane which protrude into the cytoplasm of the bouton (Figs. 7, 17, 18, 21, 22, 35, 36). They were described first by GRAY (1963) and are generally known as presynaptic dense projections. They are only occasionally visible in glutaraldehydeOsUL material (Figs. 17 and 24) but are consistent fmdings in BIUL or E-PTA treated neuropil. Sections in parallel to the synaptic cleft crossing the presynaptic dense projections reveal that they form a triagonal meshwork of dense patches and interconnecting filaments (Figs. 24, 37, 38, 79). The interspaces between the dense projections are hexagonally arranged and have

the size of the associated synaptic vesicles (AKERT et al. 1969; PFENNINGER et al. 1969b; AKERT and SANDRI 1970). This is shown schematically in Fig.25. In a given species and anatomical region (cat subfornical organ), the center-to-center distance iv of two closely approached 380 Asynaptic vesicles amounts to 450 A; a fuzzy coat of the vesicles seems to prevent closer approximation. This 450 A interval is taken as a basis for calculating the periodicity of a hexagonal lattice built of these vesicles. The resulting center-tocenter distance of the rosettes turns out to be 780 A which is very close to the actual centerto-center distance ddp of the associated presynaptic dense projections (800 A). The whole assembly consisting of vesicles and dense projections has been termed "the presynaptic vesicular grid" (AKERTet al. 1969). In the spinal cord, a considerably smaller dense projection arrangement has been found in addition to the 800 A pattern. The tighter meshwork probably belongs to terminals containing smaller vesicles (AKERT et al. 1972) (d. Figs. 37 and 38). As derived from the continuity of the presynaptic dense material with the cytoplasmic coat of the axolemma (see Fig.7), dense projections are a presynaptic "fuzz coat" specialization. They are found in a wide range of different synapses such as e. g. spine synapses (Fig. 26b) (see GRAY 1969), dendro-dendritic synapses (Figs.27, 28) (!tALL et al. 1966; PRICE 1968; PRICE and POWELL 1970; FAMIGLIETTI 1970) and some insect synapses (SCHURMANN 1971). Other but very similar presynaptic structures are found in primitive nervous systems in coelenterates (WESTFALL 1970, 1971)

Figs. 19 and 20. A-PTA staining of neuropil (specimens were aldehyde-fixed, embedded in Vestopal-W or Epon without osmication, and thin sections were treated with acidic PTA). The survey picture (Fig. 19) shows axon hillock (ah) and initial segment of a pyramidal neuron (n). Note the accumulation of dense precipitate at the initial segment (arrow). Two synaptic contacts with intense staining of the cleft material are circled. Myelinated axons (ax) are also visible; note the thin but well defmed A-PTA-positive layer in the intercellular space between axolemma and myelin sheath. - Rat cerebral cortex; magnification, X20,000; calibration, 1 fl. Fig. 20. Is an electron micrograph from the same material at X47,000 magnification. Presynaptic nerve terminals are labeled pr l> pr2 and pr3' The A-PTA staining is more or less confmed to intercellular spaces; it is particularly intense in synaptic clefts (sc). In contrast, cytoplasmic densities such as postsynaptic web (po) and dense projections (arrows) have minimal binding capacity for A-PTA. Note the striking difference between the staining pattern of this technique which apparently is specific for carbohydrates and the E-PTA method which demonstrates basic amino groups (Figs. 21 and 22). - Calibration, 0.5 fL (from MEYER, 1970).

Synaptic Morphology and Cytochemistry . 2S

or in the snail (CHALAZONITIS 1969). The presynaptic spicules described by JONES (1970) in Octopus synaptosomes are even less different from the classical dense projections. On the presynaptic membranes of neuromuscular junctions, small densities are located opposite to the

origin of junctional folds (Figs. 26e, 29, 30) (AKERT et al. 1969), thus probably forming bands along which the vesicles are clustered (COUTEAUX and P:EcOT-DECHAVASSINE 1970). Similarly, certain insect synapses (LAMPARTER et al. 1969; BOECKH et al. 1970), have a presynap-

26 •

K. H. PFENNINGER

tic apparatus consisting of bars opposite to the intercellular space separating the adjacent postsynaptic elements. A thin flat electron opaque band is regularly found on top of the presynaptic bars. The whole structure thus is T-shaped in

cross-sections (Figs. 26f, 31, 32). The synaptic vesicles form dense clusters around these presynaptic structures (Fig. 31). Although receptor synapses in the retina or in the organ of Corti (see e. g. WERSALL et al. 1965; GRAY and PEASE

sc

22

Synaptic Morphology and Cytochemistry . 27

OsUl

BIOsUl

dv

sv

BIUl

dv

pr

sc po A-PTA

E-PTA

Fig.23. The appearance of the synaptic apparatus after various staining procedures. The OsUL method primarily reveals the unit membranes and the postsynaptic density (po); presynaptic dense projections (pr), and synaptic cleft material are only faintly visible. The latter often forms a layer in the middle between preand postsynaptic membranes. In contrast, the BIUL technique visualizes the cytoplasmic and external coats of synaptic and nonsynaptic plasmalemma. Presynaptic dense projections are particularly well stained. Another striking feature is the distinction of three layers of cleft material, i. e. two electron opaque sheets separated by a thin electron lucent gap. The combination BIOsUL reveals both, unit membranes as well as cytoplasmic and external membrane coats. A-PTA stains the synaptic cleft (sc) with high contrast whereas cytoplasmic densities have almost no affinity for the reagent. The E-PTA method is characterized by fairly selective demonstration of the complete synaptic apparatus. sv, synaptic vesicles; dv, large dense core vesicles (for the sake of clarity, the unit membrane structure of the vesicles is not displayed). (Modified after PFENNINGEll. 1971b).

Figs.21 and22. E-PTAstaining of neuropil (21 day old rat, cerebellar vermis, folium VII). The survey picture displays the strikingly selective staining of synaptic membrane complexes. E-PTA is believed to reveal basic amino groups. Thus, this picture indicates the particularly high concentration of stainable basic amino groups in the synaptic densities, especially on the cytoplasmic sides. FUIther, this method offers a good basis for quantitative work on the number and distribution of synaptic contacts. Fig. 22 shows two synaptic contacts at higher magnification. Note the particularly dense cytoplasmic densities (presynaptic dense projections, dp; postsynaptic web, po). The synaptic cleft material (sc) is visible as a more or less homogeneous band of slightly weaker contrast. The unit membranes are not visible in this material. Therefore, the synaptic cleft material and the cytoplasmic densities appear to be separated by a wide electron lucent gap (d. Figs. 18 and 23). Magnifications, X 31,000 and X 154,000; calibrations I {L (Fig.21) and 0.1 {L (Fig.22). Unpublished pictures contributed by Dr. F. E. BLOOM.

28 •

K. H. PFENNINGER

1971) are much different from the other synapses,

they are not devoid of a specific presynaptic organelle. Their characteristic feature consists of the presence of a so-called synaptic ribbon

vertically oriented with respect to the synaptic cleft around which the synaptic vesicles are aggregated (Fig.26g). Although the synaptic ribbon is cytochemically similar to the presynap-

Fig.24. Tangential section through the dense projections of an OsUL prepared synapse of the cat subfornical organ. In some cases as in this figure, dense projections are well visible even in osmicated material. This picture shows seven dense projections which are triagonally arranged. Note the synaptic vesicles (sv) sitting between the dense projections. - Magnification, X 103,000; calibration, 0.2 fL.

THE VESICULAR GRID

ddp: me su,cd calculated

i..,

:

d. :

800 A 180 A 450 A 380 A

Cdata from glul I B J malerutl)

25

Synaptic Morphology and Cytochemistry . 29 tic dense projections (BUNT 1971) it differs markedly from the other presynaptic structures in that it is not in direct connection with the presynaptic membrane and therefore very probably not a membrane coat derivative. However III these receptor junctions (GRAY and PEASE G

1971) and especially in another sensory cell synapse (in the ampullary sense organ in the electric fish), small electron dense bands attached to the presynaptic membrane have been found opposite to the edge of the synaptic ribbon (MULLINGER 1969). This observation may indiH

00°00

/~@~ o

o

(

f

Fig.26. Comparative morphology of the synapse. A represents a putative "basic synaptic type" with a dense projection meshwork as presynaptic specialization, the postsynaptic web and the synaptic cleft of well defined size. All the other synapses can be regarded as modifications of A (see text). B, spine synapse; note the presence of a spine apparatus. C, synapse with subjunctional bodies. In D, the subsynaptic web is replaced by a so-called subsurface cistern. E, neuromuscular junction; note that nerve terminal and muscle cell are separated by a basement lamina and that presynaptic densities are present opposite to the junctional folds. F, insect synapse with a "T"-shaped presynaptic density opposite to the intercellular space separating the postsynaptic elements. G, retinal rod synapse. The characteristic feature is the so-called synaptic ribbon which apparently is no membrane coat formation. However, presynaptic densities attached to the plasmalemma are found just opposite to the ribbon. H, dendra-dendritic reciprocal synapse. I, crest synapse. K, serial synapse. Fig.25. Schematical representation of the arrangement of dense projections and the synaptic vesicles adjacent to the presynaptic membrane: The presynaptic vesicular grid. Whereas the distribution of the dense projections is triagonal, the vesicles form hexagonal rosettes surrounding the densities. Thus, both structures form a most "economical" distribution of vesicles and dense projections in 2:1 ratio. The reconstruction of this pattern is primarily based on measurements of the vesicular diameter and the grid constant of the dense projection arrangement. The diameter of the vesicles (d y ) in synapses of the cat subfornical organ is in the range of 380 A. However, their closest approximation (center to center distance, i y ) amounts to 450 A. Hexagonal distribution of such vesicles results in an arrangement with a center to center distance of rosettes of 780 A. This value is very close to the actual distance between dense projections (ddp = 800 A) in the same material (from AKERT et al. 1969).

30 • K. H. PFENNINGER

Figs. 27 and 28. Dendro-dendritic reciprocal synapses in rat olfactory bulb. Fig. 27 displays a typical synaptic pattern between a granule cell (G) and a mitral cell (M) dendrite. The arrows indicate the direction of transmission in the various synaptic contacts as derived from the presence of postsynaptic densities. Synapses of the mitral cells onto the granule cells are characterized by a thick subsynaptic density and by the association with spherical synaptic vesicles whereas the granule cell dendrites are filled with flattened vesicles clustering around a synapse with a thin postsynaptic density. (Magnification, X 35,000; calibration 0.5 fL). In addition to these features, Fig.28 demonstrates presynaptic dense projections (asterisks) in both parts of the reciprocal synapse. Magnification, X 60,000; calibration, 0.5 fL. Fig. 27 (PRICE 1968) and Fig.28 (unpublished) were provided by Dr.]. L. PRICE.

cate that the receptor cell synapses are closely related to the basic synaptic type as visualized in Fig.26a although they look strikingly different at the first glance. It should be emphasized that, in contrast to all nerve terminals described above, no special presynaptic organelles except for the vesicles have been described in the socalled free nerve terminals in smooth muscle or in the closely related chromaffm cells of the adrenal medulla.

The synaptic cleft Another feature of the synapse is the welldefmed cleft which differs in width in different types of junctions but which is very constant in a

given junction. Routine preparations (OsUL) in general demonstrate a more or less clear space (Fig. 19) in which, sometimes, one or two very thin lines can be seen running in the middle between pre- and postsynaptic membrane (d. also PAPPAS and WAXMAN 1972). In some cases, filaments crossing the synaptic cleft (Fig.49) have also been observed (VAN DER Laos 1963; DE ROBERTIS 1964; GRAY 1966). Despite this appearance which suggests the presence of little material, the synaptic cleft is far from being empty. The cleft material can be visualized only by special cytochemical procedures (see below). It is important to note that the neuromuscular junctions differ from all inter-neuronal synapses

Synaptic Morphology and Cytochemistry . 31

Fig.29. ZIO preparation of an endplate in the rat diaphragm. Note consistent staining of these cholinergic synaptic vesicles (sv). Their shape is not always spherical but sometimes polygonal probably due to crystal formation of the precipitated staining reagent (arrows). m, mitochondria; jf, junctional fold. Note the vesicles clustering around a patch of slightly electron dense material located opposite to a junctional fold. Magnification, X 52,000; calibration, 0·5 fL. Fig. 30. Endplate in the rat diaphragm after BlUL preparation. The presynaptic densities (dp) opposite to the junctional folds (jf) are regularly found. In this picture, further, dense material comparable to thepostsynaptic density (po) is found attached to the sarcolemma at the junctional site. Some fuzzy material occasionally outlines the synaptic vesicles (sv). - Magnification, X 54,000; calibration 0·5 fL. in that they contain a thick basement lamina separating pre- and postsynaptic membranes from each other.

Postsynaptic organelles The postsynaptic density, postsynaptic organelle, or subsynaptic web (DE ROBERTIS et al. 196ra; VAN DER Loos 1963) is the most prominent feature of many vertebrate and invertebrate synapses. Similar cytoplasmic densities are also seen attached to the junctional folds of the neuromuscular contact (see Figs. 30 and 26e). In dendritic spines, another typical postsynaptic structure is the so-called spine apparatlls (GRAY 1959, 1965) which consists of a stack of flattened cisterns located at some distance from the postsynaptic density (Fig. 26 b). The postsynaptic density is by far not as certain a synaptic feature as often assumed.

Whereas the postsynaptic organelle is usually easily visible and of considerable thickness in synapses containing spherical vesicles, its width is reduced to a narrow, sometimes almost invisible band in those synapses which are associated with flattenable vesicles (GRAY 1969) (Figs. 27, 28, 36, 56). In other cases, no postsynaptic density is seen at all, but, instead, a socalled subsurface cistern (ROSENBLUTH 1962) is found immediately beneath the postsynaptic membrane (Fig. 26d). Modifications of the postsynaptic density are shown in Figs.33, 34 and 26c and i. A layer of triagonally arranged spherical electron dense structures is present at a short distance beneath a thick postsynaptic density (TAXI 1965; MILHAUD and PAPPAS 1966). These so-called subjunctional bodies are not membrane-limited and seem somehow connected to the postsynaptic web by thin electron

32 •

K. H. PFENNINGER

dense strands (Fig.34). The structural aspect is even more striking in the so-called crest synapses (AKERT et al. 1967a, b) where two presynaptic endings form typical junctions with the opposed membranes of a postsynaptic plasmalemmal fold and where the subjunctional bodies are

sandwiched between the two postsynaptic densities. The width of the crest amounts only to about 1300-1500 A (Figs.33, 34). The function of this peculiar arrangement of pre- and postsynaptic elements is unknown. Less complex synaptic arrangements involving more than one

Synaptic Morphology and Cytochemistry . 33 nerve terminal are represented in so-called serial synapses (GRAY 1969) (Figs. 26k, 27). They show that the axolemma even of a bouton is also capable of forming a postsynaptic density.

Excitatory versus inhibitory synapses The comparison of synapses of inhibitory and excitatory function is an especially important topic in the comparative morphology of synapses. GRAY (1959) fIrSt described two types of synapses in cerebral cortex: Type I is characterized by a broad and large disc-like postsynaptic organelle and a relatively wide synaptic cleft whereas the postsynaptic density of type II synapses is so narrow that it often is hard to see and tends to form two or more small patches as seen in cross sections. Type II further is characterized by a narrow synaptic cleft (Figs. 35 and 36). It turned out later that this differentiation of synaptic types can be made in many different brain areas (GRAY 1969). COLONNIER (1968) termed these synaptic types asymmetrical (corresponding to type I) and symmetrical (type II). This terminology was chosen because the narrow type II-postsynaptic density often is almost invisible when preservation of paramembraneous densities is not ideal. Gray's terminology definitely is preferable since a chemical synapse never is symmetrical as derived from the unilateral presence of presynaptic dense projections (AKERT and SANDRI 1970) (see also Fig. 36). Under special conditions of aldehyde fixation, synapses can further be distinguished by the

shape of their vesicles (UCHlZONO 1965, 1968; BODIAN 1966) in thin sections of osmium tetroxide or ZIO impregnated neuropil (Figs. 27, 56). Whereas type I synapses contain the usual spherical vesicles (S-type), nerve terminals forming type II synapses in general contain flattened (F-type vesicles). Only recently, it was found that, in the spinal cord, the type II or F-type synapses accumulate specifically the putative inhibitory transmitter glycine (MATUS and DENNISON 1971; d. HOKFELT and LJUNGDAHL 1971). This fmding supports Uchizono's hypothesis that the F-type synapses are inhibitory at least in the spinal cord. AKERT and collaborators (1972) found two sizes of dense projection meshworks in the spinal cord (Figs.37 and 38) whereby the pattern with the smaller holes probably belongs to the F-type terminals which, in freeze fractured preparations (Fig. 57), turn out to contain spherical but smaller vesicles (see below). Thus, the putative inhibitory synapses are morphologically characterized by "miniaturized" structural elements as derived from the smaller vesicles, the dense projection meshwork with a shorter periodicity, as well as the narrow synaptic cleft and postsynaptic density. A comparison of the different sizes is given in Table 6.

The synaptic apparatus It can be inferred from these comparative studies that, at the synaptic site, the membrane coats invariably form an entity consisting of the three following elements: (i) Presynaptic den-

Figs. 3I and 32. Synapses with a "T"-shaped presynaptic element in the brain of the fly (calliphora). Note the clustering of the synaptic vesicles (sv) around the horizontal plate of the "T" (arrow) in Fig. 3I. Fig. 32 shows a bouton with synaptic vesicles (sv) terminating on two postsynaptic elements (P1 and P2)' The "T"-shaped presynaptic density consisting of a broad vertical "stalk" and a thin horizontal plate sits right opposite to the intercellular space separating P1 and P2' po, postsynaptic density. - MagnifICation of both figures, X IIO,OOO; calibration, 0.2 fl. Figs. 33 and 34. Crest synapses in the subfornical organ of the cat. Two nerve terminals (pr1 and pr2) are in synaptic contact with a narrow fold, the so-called crest of the postsynaptic element. The BIUL preparation in Fig.33 demonstrates, particularly well, the presynaptic dense projections (dp) and the broad postsynaptic densities (po) between which a layer of so-called subjunctional bodies (sb) is sandwiched. Note also the intense staining of the material in the intercellular space at the synapse. Fig. 34 shows a synaptic crest after OsUL preparation. Note the globular subjunctional bodies (sb) which appear to be linked somehow to both postsynaptic densities by a material of filamentous nature. Dense projections (dp) are faintly visible. - Magnification of both figures, X 110,000; calibration, 0.2 fl.

34 . K. H. PfENNINGER

sities (mostly as a triagonal meshwork of dense projections) which represent by far the most specific synaptic feature; (ii) synaptic cleft of

well-defmed width and characteristic cytochemistry (see below); (iii) postsynaptic density of varying width and shape (in some cases

Figs.35. 36, 37 and 38. Gray type I and type II synapses in the rat spinal cord after BIUL preparation. The comparison of the cross section in Figs.35 and 36 shows that both synapses have presynaptic dense projections (dp) but that the synaptic cleft (sc) and particularly the postsynaptic density (po) are thinner in type II than in type I. Tangential sections through the dense projection meshwork (dp) are visualized in Figs. 37 and 38. The pattern of the type I synapse is wider and leaves interspaces for larger vesicles than the meshwork of the type II synapse displayed in Fig.38. The arrows in Fig.37 point at filaments interconnecting the dense projections.Magnification of all figures, X IIO,OOO; calibration 0.2!J..

Synaptic Morphology and Cytochemistry' 35

BLOOM et al. (1970) proposed the term "synaptic

replaced by a subsurface cistern). In order to account for the unity of these three elements, Structure

apparatus" for the whole complex. Excitatory (S-Type)

Synaptic vesicles aldehyde-OsO 4 freeze-fracturing Dense projections (center-to-center distance) Synaptic cleft (width, BIUL) Postsynaptic density

Inhibitory (F-Type)

spherical spherical, 480 AI)

elongated spherical, 390 AI)

800 A

600 A

160 A 200-500 A

13 0A 100-200 A

Measures are approximate 1) Average Diameter Table 6. Excitatory versus inhibitory synapses in the spinal cord.

DESMOSOME· LIKE

STRUCTURES

VS SYNAPSES

o

w

>

o

~ z

Macula adhaerens - - Zonula adhaerens

z Q

Synapse type

- - - Synapse type

~

cc(

a:

C)

w

C U

~ ~

~

200A

Fig.39. Schematic representation of the differences between desmosome-like structures and synapses. The cleft is widened at type I but not at type II synapses. A wide intercellular space is also found at maculae and zonulae adhaerentes. The middle dense line in the cleft (which is particularly well visible at the synapse after tryptic degradation and OsUL staining) is also a striking feature of maculae adhaerentes but it has not been described in the zonula adhaerens. The cytoplasmic densities are the most striking features of both, desmosome-like structures and type I synapses. In native material, cytoplasmic densities of zonulae adhaerentes cannot be distinguished from type I postsynaptic densities. However, in maculae adhaerentes, they are characterized by a band of higher density running in parallel to the junctional membranes. A similar subunit pattern is found after tryptic degradation and OsUL preparation of zonulae adhaerentes: Two layers of electron dense material are running at some distance in parallel to the plasmalemma. This fmding clearly distinguishes the intermediate junction from the type I synapse where trypsin treatment removes OsUL staining (sometimes, a few fllaments remain attached to the pre- and the postsynaptic membranes). These cytochemical differences between the two classes of intercellular contacts are probably of great functional significance.

36 . K. H. PFENNINGER

3. Cytology of intercellular adhesiveness The mechanical resistance of various tissues suggests the presence of special attachment devices interconnecting the cells. Tissues which are often exposed to mechanical forces as e. g. epidermis, indeed, are rich in intercellular junctions of desmosome-like type (FARQUHAR and PALADE 1963, 1965). The adhesiveness of synaptic membranes becomes most strikingly apparent during the preparation of brain subcellular fractions. The contacts are found to resist homogenization and centrifugation procedures so that presynaptic terminals are usually found with postsynaptic membranes attached, structures called synaptosomes (GRAY and

WmTTAKER 1960, 1962; DE ROBERTIS et al. 1961 a, b). It may be worthwhile to look for common structures in desmosome-like junctions and synapses as a basis for the connectivity mechanism.

Cytoplasmic densities in intermediate junctions and synapses The most striking structure which synapses and desmosome-like structures have in common is the cytoplasmic density and the subsynaptic web, respectively. Morphologically, the synapse can therefore be regarded as a modification of an intermediate junction. This view becomes less valuable, though, when considering the variability of the postsynaptic organelle or its absence in certain synaptic contacts as outlined above,

Fig.40, 41 and 42. Desmosome-like junctions. Fig.40 shows an intermediate junction between ependymal cells in the cat subfornical organ after QsUL preparation. Note the widening of the intercellular space at the junctional site, the slightly electron dense cleft material and the more or less homogeneous cytoplasmic densities (cd). - Magnification, X 98,000; calibration, 0.2 fL. Fig.4I, shows a similar junction after trypsin treatment which followed aldehyde fixation. Note that the cytoplasmic densities are no longer homogeneous; instead, two electron dense layers run in parallel to the plasmalemma (arrows). The intercellular space seems to be free of material and the contact is partially opened (asterisk). - Magnification, X 52,000; calibration 0.2 fL. Fig. 42 displays a desmosome-like junction in the rat heart papillary muscle after BIUL treatment. The intercellular space (is) is filled with electron dense material forming two layers separated by a narrow gap (asterisks). The unit membranes are not stained but, instead, a wide electron lucent space is seen between intercellular material (is) and cytoplasmic densities (cd). The cytoplasmic densities are more or less homogeneous. However, a thin layer immediately adjacent to the unit membrane space appears to be slightly less electron opaque than the major, more cytoplasmically located part of the densities (arrow). - Magnification, X 240,000; calibration, 0.1 fL.

Synaptic Morphology and Cytochemistry . 37

and especially when comparing the cytochemical properties of the two kinds of intercellular junctions (see Fig. 39). The cytoplasmically attached paramembraneous material in desmosome-like junctions often shows bands of alternating density running in parallel to the plasmalemma (FARQUHAR and PALADE 1965) (Fig. 39). Even in those intermediate junctions which usually are characterized by paramembraneous structures of uniform density, similar subunit patterns become visible after tryptic degradation (Figs. 39, 40 and 41) (PFENNINGER 1971 c). A similar organization of the cytoplasmic density has not been observed in the synapse neither in native tissue nor after treatment with various proteolytic enzymes. In contrast, proteolytic activity removes the postsynaptic filaments as seen after OsUL preparation more or less completely from the membrane (Figs.43, 44). In BlUL-treated tissue, the situation is somewhat different. In both junctions, the cytoplasmic densities and the subsynaptic web appear as more or less homogeneous bands (Figs. 18, 42) which can be digested with pepsin and pronase but which are resistent to trypsin (PFENNINGER 1971 c) (Fig.45). The same result was obtained regardless whether the digestion experiments were carried out with unfixed or aldehyde-prefixed tissue slices. In similar specimens, but in connection with E-PTA impregnation, BLOOM and AGHAJANIAN (1968) found the same result with pepsin but described an important fading of the contrast in postsynaptic densities also after tryptic degradation. Possible explanations for this difference may be: (i) E-PTA and BIUL have similar but not identical staining mechanisms and (ii) a trypsin-induced loss of cytoplasmic paramembraneous material which is indicated by a slight decrease of BIUL staining may be sufficient to reduce the weaker E-PTA contrast to almost invisibility. Neither pronase nor trypsin seem to have a degradation effect on the postsynaptic density if applied on thin sections (BARRANTES and LUNT 1970). The results of the various digestion experiments are summarized in Table 7. A-PTA (MEYER 1970) and periodic acid-silver methenamine (RAMBOURG and LEB-

LOND 1967) staining are very faint in cytoplasmic junctional densities (Figs. 20, 23). Thus the postsynaptic density is cytochemically characterized as follows: It consists of a fine meshwork of ftlaments attached to the postsynaptic "unit membrane" which are mainly of proteinaceous nature as derived from their susceptibility to proteolytic digestion. They seem to contain very little carbohydrate. The proteins are highly polar containing both acidic as well as basic groups (UL, BlUL, E-PTA staining). On the basis of the differential digestion by trypsin, two proteinaceous compartments may be distinguished; a trypsin -labile fraction containing more acidic groups and a trypsin - resistant portion which is visualized only after BIUL staining and therefore contains many basic groups. The resistance of part of the UL staining to esterifICation (Fig.2) is suggestive of phosphate and/or sulfate groups. The postsynaptic density has the BlUL-positive, trypsin - resistant, but pronase-labile compartment in common with intermediate junctions. In both cases, this fraction looks homogeneous. However, intermediate junctions are clearly distinguished from synapses by the periodicity of the trypsinizcd, OsUL-stained filaments attached to their plasmalemma. Cellular filaments are often seen to insert in the cytoplasmic density of desmosomes (FARQUHAR and PALADE 1965). This observation suggests that the inner membrane coat specialization somehow helps to strengthen the cells and the tissue mechanically. Analogous observations have not been made on the synapse so that a similar hypothesis seems unjustified for the postsynaptic density. Structure and cytochemistry of the synaptic cleft material The cleft material is morphologically less striking than the cytoplasmic densities. However, it must be the main site of the connectivity mechanism as it consists of the two opposed external membrane coats with their most distal parts meeting in the middle. As mentioned above, one of the few electron dense structures

38 . K. H. PFENNINGER

seen in the synaptic cleft of OsUL material is a thin and faint, eventually double, line which runs in the middle between the two plasmalemmas (Fig. 17) (d. PAPPAS and WAXMAN 1972). This sheet becomes better visible if glutaral-

43

dehyde-prefixed tissue slices are subject to tryptic degradation (Fig. 44) (PFENNINGER 1971 c). In ruthenium red - OS04 material (Fig. 47), the corresponding structure is regularly found but it is visualized best by the saccharated iron oxide

Synaptic Morphology and Cytochemistry . 39

I

Experiment esterification bromalin trypsin

chymotrypsin peptidases pepsin pronase hyaluronidase neuraminidase helix digest. juice DNase, RNase

}

f l

Staining

Ref.

UL OsUL OsUL OsUL UL E-PTA BIUL (Os)UL BIUL (Os)UL E-PTA BIUL OsUL OsUL BIUL (Os)U(L) U(L) OsUL E-PTA

5 1 1 3,5

5 3 5 5 5 3,5

3 5 1 5 5 4,5 4,5 1 2

I

Synaptic cleft

Cytoplasmic densities

+++

++

+ +++a +++ ++ to +++ + +++a + +++b +++ +++b + +++b +++b

presyn. ++/postsyn. + +++ +++ ++ to+++ + +++ + +++ +++ +++ + +++ +++

++

+

-

-

-

-

-

-

-

Key: -

no effect minimal/++ marked decrease of staining intensity +++ staining abolished middle dense line persisting in aldehyde-prefixed specimens, but not in unfixed material a where the synaptic contact is opened b synaptic contact opened

+

Reference

Specimens used for incubation

1. BARRANTEs and LUNT (1970) 2. BLOOM in : BLOOM et al. (1970) 3. BLOOM and AGHAJANIAN (1968) 4. BONDAREFF and SJOSTRAND (1969) 5. PFENNINGER (1971 c)

Araldite-Epon-embedded thin sections

}

Aldehyde-fixed tissue slices Unfixed synaptosomes Aldehyde-fixed or unfixed tissue slices

Table 7. Cytochemical characterization of synaptic densities: effects of enzymatic digestion and esterification.

Figs.43. 44. 45 and 46. The effect of tryptic degradation on synaptic densities. Fig.43 (trypsin treatment before aldehyde fixation) shows a presynaptic nerve terminal (pr) containing numerous synaptic vesicles (sv) and a large dense core vesicle (dv) in the immediate neighborhood of a dendritic element (d). Remainders of faintly stained material (asterisks) remind of the former synaptic densities. The synaptic cleft (arrow) appears to be cleared out and the junction is opened. In a large granulated vesicle (dv), the core is partially deteriorated. - Magnification, X 71,000. The synapse depicted in Fig. 44 has been subject to tryptic degradation eifter aldehyde fixation but has also been prepared with the OsUL method. Note the filamentous remainders of the density on the postsynaptic membrane (asterisks). In comparison with Fig.43, the main difference consists in the presence of a middle dense line between the pre- and the postsynaptic membrane (arrow) as well as in the preservation of the synaptic contact. pr, presynaptic nerve terminal; d. dendrite; m, mitochondrium. Figs. 45 and 46 are derived from the same material as in Fig. 43 (tryptic digestion before aldehyde fixation) but were prepared with the BIUL method. As in Fig.43, tryptic degradation leads to the opening of the synaptic contact as indicated by the isolated postsynaptic density (po) in Fig. 45 and the more or less isolated presynaptic sites of the nerve terminals (pr) which are identified by a series of dense projections (dp). In contrast to the OsUL material, dense projections and postsynaptic density still stand out with good contrast. The synaptic cleft material. too. is clearly preserved. It is present as a somewhat thinned (particularly on the presynaptic side) coat (mc) on both junctional membranes. - Magnification, X 82.000 (Fig.45) and X 120,000 (Fig.46); calibration in all figures. 0.2 fL.

40 . K. H. PFENNINGER

Fig. 47. Ruthenium red staining of the synapse. The particular feature of this picture is that not only the synaptic cleft (sc) but also presynaptic dense projections (dp) and the postsynaptic density (po) are stained. The synaptic vesicles (sv) seem to be packed in less dense, but also ruthenium red-positive material. The arrow points at a line of greater density running in the middle between pre- and postsynaptic membranes. Cat subfornical organ, magnification X 155,000; calibration, 0.2!-t. Fig.48. Synapse in the rat cerebral cortex after local injection of saccharated iron oxide. Note the specific accumulation of electron dense material in the synaptic cleft but not in the other intercellular spaces. The electron dense particles form a layer right in the middle between pre- and postsynaptic membranes (arrow). po, postsynaptic density. - Magnification, X 96,000; calibration, 0.2 !-t. (From PAPPAS and PURPURA 1966). method (PAPPAS and PRUPURA 1966) as shown in Fig. 48. In osmicated material, VAN DER Loos (1963), DE ROBERTIS (1964) and GRAY (1966) described ftlamentous material crossing the synaptic cleft (Fig. 49). In contrast, A-PTA (PEASE 1966; MEYER 1969 a, b, 1970) and E-PTA staining (BLOOM and AGHAJANIAN 1966, 1968) reveal an intensive homogeneous staining throughout the synaptic cleft (Figs.20, 22, 23). Similar findings have been obtained with the periodic acid - silver methenamine technique (RAMBOURG and LEBLOND 1967) and the PTAchromic acid method (RAMBOURG 1969). Still another morphological aspect of the synaptic cleft is visualized by the BIUL method (PFENNINGER et al. 1969; AKERT and PFENNINGER 1969). The cleft material appears to consist of two layers of electron dense material which are

separated by a very thin electron lucent gap about 20 A wide (Figs. 18, 23). Electron microscopy of partially opened (PFENNINGER et al. 1970; PFENNINGER 1971 c) and BIUL stained synaptic contacts makes the topic of synaptic cleft fine structure even more controversial. Fig. 50 shows that bundles of extremely thin filaments (thickness near the resolution limit of the technique) arise from both the pre- and the postsynaptic external membrane coats to cross the widened synaptic gap and to interconnect the synaptolemmas. These filaments which may be uncoiled by the separation of pre- and postsynaptic membranes are believed to represent the real structural subunits of the synaptic cleft material (Fig. 49). After certain preparation procedures the filaments may be clumping to form the thicker

Synaptic Morphology and Cytochemistry . 41

r E.

FI

E S

UC URE OF THE SY APTIC CLEFT D

OsUL

-....

°11 ,,-

A

DE

LOOS

F,"m --====1

DE ROBERT IS

m

BIUL F

E- PTA

Fig. 49. The structural appearance of the synaptic cleft after various preparative procedures. In osmicated tissue, VAN DER Laos (1963), DE ROBERTIS (1964) and GRAY (1966), observed structures crossing the cleft which are schematically represented in A, Band C. On the other hand, aldehyde osmium preparations often reveal a structural organization in parallel to the synaptolemmae (D) which consists in a thin electron dense line running in the middle of the intercellular space. This is particularly striking after trypsin treatment of aldehyde fixed tissue or after saccharated iron oxide injection into neuropil. The BIUL preparation (E) stresses this "horizontal" organization by contrasting two separate layers of thickened external membrane coat. Thus, the binding region of pre- and postsynaptic membrane coats has no affinity for BIUL but is osmiophilic and binds saccharated iron oxide. The E-PTA method (as well as the A-PTA technique) yields a more or less homogeneous staining of the cleft material (F). Partly opened synaptic junctions (center) reveal further structural details. Some single, probably uncoiled filaments (f), or strands thereof, arising from the synaptic external membrane coats remain attached to each other and reveal the filamentous nature of the cleft material. The superimposition of these threads is believed to generate the compact aspect of the outer synaptic membrane coats (ic) in BIUL and PTA preparations whereas clumping and partial loss of the filaments could lead to the thicker structures crossing the intercellular space (A, B, C). urn, unit membranes; pr, presynaptic dense projections; po, postsynaptic web. (From PFENNINGER 1971 c).

strands observed by VAN DER Loos (1963), DE ROBERTIS (1964), and GRAY (1966). After E-PTA, BIOL, UL, and A-PTA staining, the high number of filaments superimpose in the depth of a thin section to form homogeneous bands (PFENNINGER 1971 c). The layers of the synaptic cleft material, i. e. the two BIUL positive sheets and the osmiophilic dense layer in the middle may be explained as follows. The synaptic intercellular space can be subdivided into at least three different sheets: the two pre- and postsynaptic proximal layers and the central lamina; they reflect the proximal part of preand postsynaptic membrane coat filaments and their very short distal segments meeting each other in the middle of the synaptic cleft. Cytochemically, this central layer is characterized by osmiophilia and a distinct binding capacity for

saccharated iron oxide and further, by the lack of stainable acidic or basic groups as derived from its BIUL negativity. The two proximal sheets, in contrast, are rich in basic and acidic groups (E-PTA, BIUL; BLOOM and AGHAJANIAN 1968, PFENNINGER 1971 b) whereby most of the anionic residues are of the carboxyl type since esterification inhibits UL staining completely (Fig.2). Methods specific for the demonstration of carbohydrates indicate the presence of these compounds throughout the whole synaptic cleft (PEASE 1966; RAMBOURG and LEBLOND 1967; RAMBOURG 1969; MEYER 1970). This is illustrated in Figs. 20 and 23. Part of the sugar residues are sialic acid as indicated by the decrease of UL staining after neuraminidase treatment (Table 7) (BONDAREFF and SJOSTRAND 1969; PFENNINGER 1971 c).

42 •

K. H. PFENNINGER

Fig. 50. Synaptic contact in the cat subfornical organ after treatment with 0.5M MgCl2 and subsequent aldehyde andBIUL preparation. The presynaptic area can be identified by the presence of dense projections (dp). The contact between the terminal (pr) and the postsynaptic element (po) is opened. Note the clearly visible two halves of the synaptic cleft material which are represented by the coats (mc) of the synaptolemmas. In this synapse, some bundles of thin filaments arise from the membrane coats and interconnect preand post- synaptic elements (arrows). m, mitochondrium. - Magnification, X 150,000; calibration, 0.1 fL. Experiments with proteolytic enzymes provide further results on synaptic cleft cytochemistry. It has been mentioned already that trypsin treatment of glutaraldehyde - prefixed tissue slices eliminates the staining of the synaptic cleft (except for the thin middle dense line) as seen after OsUL. The synaptic contact, however, persists (Fig.44). But, if unfrxed tissue slices are incubated in the same enzyme the middle dense line is no longer visible and the synaptic junctions are opened (Figs.43, 45, 46). Experiments with trypsin and UL staining alone again show a clear-cut degradation effect in the synaptic cleft, whereas BlUL staining of the

same material reveals somewhat weakened but still visible synaptic external membrane coats. Fig.45 displays a more or less isolated postsynaptic membrane with its clearly visible postsynaptic density and the synaptic external membrane coat attached. Fig. 46 shows a presynaptic nerve terminal with clearly visible dense projections and, again, some of the presynaptic external membrane coat attached. Both pictures are taken from neuropil which was incubated in trypsin before aldehydefrxation; the preservation of the outer coat is more consistent if aldehyde - prefixed tissue is incubated. Other enzymes such as chymotrypsin, peptidase and the mixture of trypsin and carboxypeptidase have the same effect as trypsin whereas pepsin and pronase completely degrade the synaptic cleft material even after glutaraldehyde fixation. The middle dense line in the OsUL preparations as well as the BlUL positive cleft material disappear. These [mdings (PFENNINGER 1971 c) are consistent with the observations made by BLOOM and AGHAJANIAN (1968) except for the fact that E-PTA does not reveal the trypsin - resistent portion of synaptic cleft material. A similar discrepancy between BlUL and E-PTA staining has been discussed above in connection with the effect of trypsin on the staining properties of the postsynaptic density. In enzymatic digestion experiments on thin sections, BARRANTES and LUNT (1970) reported only a partial loss of OsUL stainability of the synaptic cleft after treatment with either trypsin or even pronase. In this work, the surprisingly small effect of pronase on synaptic cleft material may be due to the epoxy resin embedment before application of the enzyme. The differential action of the proteolytic enzymes on synaptic cleft material (see also Table 7) is interpreted as follows. Tryptic degradation removes only part of the synaptic cleft material as indicated by the persisting BlUL staining, by the dense line still running in the middle of the synaptic cleft, and further, by the fact that the synaptic connectivity is still maintained in prefixed tissue. The last observation is probably explained by a glutaraldehyde crosslinkage of pre- and postsynaptic BlUL - positive

Synaptic Morphology and Cytochemistry . 43 membrane coat remainders. The trypsin - sensitive portion (fraction I) bears mainly acidic groups as derived from the tremendous decrease in UL contrast after treatment with this enzyme; it may well be a glycoprotein. The trypsin - resistent remainder (fraction II) is visible only after BIUL but not after UL staining and thus seems to bear a large proportion of basic residues. The sensitivity of the cleft fraction II to pepsin and pronase indicates that it is of proteinaceous nature. Thus the synaptic cleft material which has also been termed "synaptin" (SCHMITT 1969) turns out to consist of a highly polar, mostly proteinaceous material which is also rich in sugar derivatives. These cytochemical fmdings are in good agreement with biochemical investigations of synaptosome fractions which indicate a high density of acidic as well as basic amino acids in insoluble synaptosomal proteins (COTMAN et al. 1968), and of N-acetyl-neuraminate bearing glycoproteins and gangliosides (WOLFE 196I; BRUNNGRABER et al. 1967). These biochemical studies are of course not restricted to the synaptic cleft material but analyzed the whole synaptic apparatus. However, the cytoplasmic synaptic densities are cytochemically similar to the cleft material with the exception of carbohydrate content (the cytochemical properties of presynaptic dense projections will be discussed below).

The mechanism of adhesion The experiments with proteolytic enzymes indicated that synaptic proteinaceous material is participating in the mechanism of connectivity. The observation that these proteins contain a large amount of polar groups and that the middle layer where the two opposed membrane coats meet cannot be stained with acidophilic or basophilic dyes may suggest a blockade of the most distal polar residues by mutual binding. This could be the mechanism of synaptic adhesiveness. This hypothesis was tested by incubating fresh nervous tissue in solutions of increasing ionic strength whereby different mono- and divalent cations and anions were used. The calcium complexing agent ethylenediamine tetraacetate (EDTA) was also applied

(PFENNINGER et al. 1970; PFENNINGER 1971 c). Fig.51 shows the results of this study. The percentage of opened synaptic contacts is plotted in the ordinate while the abcissa indicates the ionic strength (right side) or the salt concentration (left side). Note that the removal of calcium ions by EDTA even at high concentration (0.1 M) has very little effect on synaptic connectivity. The separation of the junctions follows the increase of the ionic strength regardless which kind of salt has been applied; the only exception is NaCI0 4 which acts about twice as powerful as all the other salts at the same ionic strength. CaClz, LiBr and (NH4)zS04 which are not listed in Fig. 5I dissociate synaptic contacts similarly to NaCI or MgClz. Similar experiments have meanwhile been carried out on synaptosome fractions (PFENNINGER et al. 1971 d). The results are quite consistent with the experiments of incubated tissue slices. Fig.52 is a survey picture of a control, i. e. a synaptosome fraction of the guinea pig cortex stained with BIUL. Note the presence of several intact synaptic junctions (arrows). Figs. 53, 54 and 55 are taken from synaptosomes which have been resuspended in 0.8 M or 1.2 M MgS0 4. The result of the treatment has been estimated by counting and classification (intact/partially split/fully opened contact) of 100 to 150 presynaptic areas (identification by the presence of a row of presynaptic dense projections). In the controls, more than 40% of the synaptic contacts were intact (as in native tissue), while about the same number of junctions were partially opened; about 20% of the presynaptic areas showed no postsynaptic membrane attached. Treatment with 1.2 M MgS0 4 decreased the number of intact junctions to 5-10% whereas the number of isolated presynaptic areas (Figs. 54 and 55) reached 50%. The remaining presynaptic membranes belonged to junctions which were clearly opened but where pre- and postsynaptic membranes were not fully independent from each other (Fig. 53). Within the synaptosome suspensions in MgS0 4, further, a high number of dark membrane-limited elements of elongated shape were visible. These structures may represent isolated postsynaptic membranes; probably,

44 . K. H. PFENNINGER DISSOCIATIO

. o

,.

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OF S

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AP IC CO

.")

a

.

:~:

AC S

",0<.

t .... o tI\ot .... 1 1 1\

ot.d1

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••

"

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.I

IS

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:. /: ~:/:~. , . ,.1.7 ... ,,--:--,:7, ~

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Fig. 51. The effect of high ionic strength on synaptic contacts. Upper part ofthe figure: quantitation of synaptic contacts into three groups (intact/altered/dissociated) after incubation in different salt solutions (unfixed material used for incubation; isoosmolar conditions at 3 osmoles; pH 7.2 to 7.4; 60 minutes at 4° C). Countings of synaptic contacts are based on at least 60 clearly identifiable and ideally sectioned synapses in every case. Results are given in percent of total number of classified synapses. Note the increase of dissociated and the decrease of intact synapses with enhanced ionic strength as well as the lack of a clear-cut EDTA effect. Lower part: the number of dissociated junctions is plotted as a function of the salt concentration (left) and of ionic strength (right). Mono- as well as di-valent cations similarly split synaptic contacts with increasing ionic strength. However, NaCl0 4 opens the junctions about twice as effectively as the other salts. This indicates that basic groups (to which perchlorate ions have a strong affmity) play an important role in the mechanism of adhesiveness. Hence it is inferred that pre- and post-synaptic membranes are bound to each other by polyionic interactions. (From PFENNINGER 1971 c). the free ends of the liberated postsynaptic membranes frequently meet and fuse and thus engulf the postsynaptic density (Fig. 55). These observations, the lack of an effect by EDTA and the action of high ionic strength on synaptic adhesiveness both in incubated tissue slices and in synaptosomes, indicate that the mechanism of synaptic adhesion is of polyionic nature rather than based on coordination com-

plexes involving Ca 2 + and carboxyl groups. The powerful action of NaCI0 4 which is known to bind strongly to amino groups (OHLENBUSCH et al. 1967) stresses the role of these cationic residues of the cleft material in the mechanism of adhesion. The relatively high number of not or only partially disconnected pre- and postsynaptic membranes may be due to the lack of mechanical forces pulling pre- and postsynaptic

Figs. 52, 53, 54 and 55. The effect of high ionic strength on synaptosomes of the guinea pig cerebral cortex. The control, a sediment oftheP2 B fraction without incubation, is shown inFig. 52. Note the many presynaptic nerve terminals and intact synaptic junctions (arrows). - Magnification, X 22,000; calibration I fL. Treatment with high ionic strength leads to the opening of a large proportion of synaptic contacts. Fig. 53 displays a split synaptic contact; however, pre- (pr) and post-synaptic (po) elements are not fully dissociated. Note that a wide gap (arrow) separates the two external membrane coats which usually are immediately adjacent to each other. dp, dense projections. - Magnification, X 70,000; calibration 0.2 fL. Fig. 54 displays a nerve terminal (pr) with a completely isolated presynaptic membrane which is identified on the basis of dense projections (dp). Magnification, X 63,000; calibration, 0.2 fL. In its upper part, Fig. 55 shows another isolated presynaptic area (pr, dp) with a well-visible external membrane coat (mc). The elongated membrane-bound structure, which is filled with a homogeneous electron opaque material (lower part of the figure), is believed to be an isolated postsynaptic element (po). Note the presence of a well-defmed outer membrane coat (mc). - Magnification, X 94,000; calibration, 0.2 fL.

Synaptic Morphology and Cytochemistry . 4S membranes apart after the ionic binding mechanism has been weakened by high salt concentration. An additional mechanism for adhesiveness which would be resistant to high ionic strength seems less likely but cannot be excluded.

The problem of the attachment of the pre- to the postsynaptic membrane also involves questions of contact selectivity and recognition. The exceedingly high number of specific synaptic contacts in the brain and the complexity of

46 . K. H. PFENNINGER neuronal circuitry are only two examples which strongly suggest the presence of a recognition mechanism which would enable nerve terminals to interact with select groups of neurons exclusively. Presently, there are no data available proving the notion of a coded neuronal surface as recognition mechanism. However, the fmding of a polyionic binding mechanism between preand postsynaptic membrane provides a possible basis for highly selective interactions. A mosaiclike pattern of acidic and basic groups on the surface of a nerve terminal would stick to a complementary pattern only (PFENNINGER 1971 c). This model is even more attractive in view of the presence of the two putative "synaptin" fractions, the mainly acidic type I glycoprotein and the type II protein rich in basic residues. The intercellular space of desmosome-like structures is structurally and cytochemically similar to the synaptic cleft. In OsUL material, a middle dense line is often found to be very prominent (Figs. 39,40) (FARQUHAR and PALADE 1965); BIUL reveals the two thickened external membrane coats separated by a thin gap (Fig. 42). The contact, further, is susceptible to proteolytic activity. These data suggest that, at least in rough terms, the intercellular spaces at the synapse and at desmosome-like junctions have similar features. A main difference, however, seems to lay in the connectivity mechanism. SEDAR and FORTE (1946) and BENEDETTI and EMMELOT (1967) suggested the complex binding of Ca++ by the carboxyl groups of juxtaposed sialic acid residues as the mechanism of adhesiveness for maculae and zonulae adhaerentes. If further investigations confJ.rm this difference in the mechanism of binding in synapses vs. desmosomes, these fmdings are not only most significant for the distinction of the two types of junctions but also point to differential surface characteristics of the cell at specific sites.

4. Cytology of intercellular communication There are few data available which allow a correlation between cytological fmdings and

intercellular communication. Since information transfer is the most important function of the synapse it has been tried to discuss this topic briefly from the cytological point of view (despite the fact that it is primarily a field of speculations in our days). Most of the ideas about intercellular communication have developed from theoretical considerations assuming that there must be an exchange of information from cell to cell to provide growth control for the formation of complex organisms. There are supposedly two forms of intercellular communication. One type serves to control growth and development of a tissue. It is probably found in every organism consisting of more than one cell. The other type of information transfer - rather than controlling a group of cells within a tissue - serves the whole organism at a higher level by providing the relationship with the environment and the coordination of thefunctions of the different organs. This function is performed by the endocrine and the nervous systems. Although intercellular communication may take place between simply apposed cells, the intercellular junctions are the main candidates for information transfer. The gap junction (BRIGHTMAN and REESE 1969) is a well investigated specialized junction where not only small but also macromolecules are known to cross the intercellular space through channels which interconnect the lumina of the adjacent cells (ROBERTSON et al. 1963; LOEWENSTEIN 1966; BENNETT et al. 1967; PAYTON et al. 1969; Me NUTT and WEINSTEIN 1970). It is the only example where substances have been demonstrated to travel from one cell cytoplasm into the other (LOEWENSTEIN 1966; PAYTON et al. 1969) and thus could transfer information. The wide spread occurance of gap junctions may suggest that these cellular formations are possibly designed to serve control of growth (and other functions?) of tissue. On the other hand, gap junctions seem to serve intercellular communication at a higher level in some cases. They are found in the brains of mainly lower vertebrates and invertebrates (ROBERTSON et al. 1963; BENNETT et al. 1967), birds (BRIGHTMAN and

Synaptic Morphology and Cytochemistry • 47

REESE 1969), and even mammals (SOTELO and PALAY 1970) where they interconnect and electrically couple axons and dendrites or axons and axons, etc. In the brain, they are, therefore, called "electrical synapses" or "electrotonic junctions" (for review see PAPPAS and WAXMAN 1972). The fact that intermediate junctions (zonulae adhaerentes) and desmosomes (maculae adhaerentes) are symmetrical implies that the adjacent cells are modifying their external and cytoplasmic membrane coats in exactly juxtaposed areas. This is hard to understand without the transfer of information at these sites. However, there is no evidence for a local exchange of substances and there are no data available either indicating that, as an alternative mechanism, the linkage of the outer membrane coats of the neighboring cells somehow serves intercellular communication. In the synapse, the situation is different. Intercellular communication providing e. g. the relationship with the environment (sensory pathways) and the functional coordination of the organs (autonomic nervous system) is the function of the synapse. As compared with the electrotonic junctions, chemical synapses have an additional, most important feature: the flow of information is polarized or unidirectional, i.e., it travels only from the terminal of one neuron to the postsynaptic cell and not in the opposite direction. While there is an enormous litf'rature about this form of intercellular commulllcation (see e. g. PERKEL and BULLOCK 1968) very little is known about the other type of information transfer at synaptic sites. As in intermediate junctions, the specialization of the coats of the pre- and postsynaptic membrane at exactly opposed sites suggests an information transfer in addition to the classical synaptic transmission. The generally accepted view that nerve cells no longer divide after the formation of synaptic contacts, further points in this direction. The socalled trophic effects of neurons (see e. g. GUTH 1969) which occur not only in the periphery but also within the central nervous system (see e. g. SZENTHAGOTHAI and HAMORI 1969 ; COWAN 1970) are additional important observations supporting the notion of information transfer

for growth control (and other functions?) at synaptic sites. Whether the structural similarities between intermediate junctions and synapses indicate a common cytological basis for this putative form of intercellular communication is far from being known.

5. Transmitter storage In the early 1950S, the release of transmitter substances in distinct quanta (for review see KATZ 1969) and the presence of synaptic vesicles in nerve terminals (PALADE and PALAY 1954; DE ROBERTIS and BENNETT 1954, 1955), were discovered at about the same time. This lead to the obvious hypothesis that neurotransmitters are stored within vesicles the size of which determines the quanta. In fact, this theory implies three hypotheses: (i) The storage of transmitter in vesicles at all, (ii) the release of the transmitter out of the vesicles and (iii) the depletion of a vesicle at once which means that the content of one vesicle equals the amount of one quantum of transmitter. While the second and third hypotheses will be discussed later, the ftrst assumption is the topic of this chapter. Detailed morphological analysis distinguishes the following types of vesicles in nerve terminals: (i) synaptic vesicles, (ii) large dense core or large granulated vesicles, (iii) coated or complex vesicles, (iv) plasmalemmal vesicles, (v) smooth endoplasmic reticulum.

Synaptic vesicles The synaptic vesicles (sv) represent the vast majority of spherical or elliptic profiles in the nerve terminal (exceeding 90% of the vesicular components). They are visible not only after chemical fixation and thin section procedures but also after physical fixation as demonstrated in freeze-cleaved preparations (MOOR et al. 1969). Their staining properties are listed in Table 8. Within a single class of nerve terminals, synaptic vesicles are more or less uniform in size, i. e. the histogram of their diameters forms a unimodal curve (MOOR et al. 1969). Different types of nerve terminals may be characterized by vesicles of different size classes ranging from 300

48 . K. H. PFENNINGER

Vesicles and type of terminals

Shape and size AldehYde-os0 4

Synaptic vesicles

cholinergic monoaminergic glycine-containing 1) non-identified

spheric, ~ 500 A spheric, ~ 500 A flattened spheric or flattened

Staining of contents

Aldehyde-FE

ZIO

spheric, ~ 500 A spheric, ,...;, 500 A spheric, ~400 A spheric, 300-600 A

+ + + +

\

Large dense core vesicles

cholinergic monoaminergic non-identified

spheric, 900-1200 A spheric, 900-1200 A

I

UL, BIUL, E-PTA

I

KMn0 4 /Os0 4 , KZ CrZ 0 7

+ + + +

+

1) Rat spinal cord.

Table 8. Synaptic and large dense core vesicles. up to 600 A (LARRAMENDl et al. 1967). As mentioned above, a more conspicuous difference lies in the shape of vesicles. Especially after paraformaldehyde fixation, the vesicles associated with Gray type II synapses are of elongated shape in thin sectioned material (Figs. 27, 56) (UcmzoNo 1965, 1968; BODlAN 1966; GRAY 1969). It was recognized soon that the elongated conformation of the vesicles is dependent upon certain conditions of the aldehyde prefixation (LUND and WESTRUM 1966; WALBERG 1966; BODlAN 1970; VALDIVIA 1971). This notion, however, did not really help to solve the problem whether the elongated shape is the natural state of the vesicles or whether flattening of originally spherical vesicles occurs as a selective artefact due to a special sensitivity of certain vesicular classes. Freeze-cleaving sheds some light on this problem in that it reveals ellipsoid profiles in nerve terminals only in rare instances even after paraformaldehyde-glutaraldehyde prefixation. Instead, a smaller class of spherical vesicles is regularly found (d. Figs. 56 and 57) (d. LARRAMENDl et al. 1967). In addition, BIUL staining visualizes dense projection meshworks of different periodicity. The smaller pattern has holes of appropriate size for the smaller vesicles found in freeze-etching (AKERT et al. 1972). These data speak in favor of the spherical shape as the natural conformation of the vesicles so that the term flattenable vesicles seems most appropriate for the elongated profiles, i. e. disc-

shaped vesicles in putative inhibitory synapses. Whether the cylindrical vesicles observed e. g. in the goldfish spinal cord (DENNISON 1971) are also of spherical shape in vivo is unknown. The distinction of different classes of synaptic vesicles raises the question whether they are characteristic of nerve terminals using different transmitter substances. At least in part, this seems to be the case as derived for instance from the exclusive accumulation of labeled glycine in F-type terminals in the spinal cord (MATUS and DENNISON 1971). But a generalization of such data to the whole brain and to different species seems impossible, as e. g. nerve terminals with /lattenable vesicles in the pontine raphe nucleus of the rat seem to accumulate labeled 5-0Htryptamine (BLOOM and COUCH 1970). In direct connection with the previous problem is the question whether the synaptic vesicles are the transmitter storage sites. The following investigations are relevant to this topic: (i) In the biochemical assay, isolated synaptic vesicles were found to contain the major portion of neurotransmitter. This fmding was obtained in adrenergic (VON EULER and HILLARP 1956; GEFFEN and LIVETT 1971) as well as in cholinergic systems (DE ROBERTIS et al. 1963; Mc INTOSH 1963; WHITTAKER et al. 1963, 1964; ISRAEL and GAUTRON 1969; MARCHBANKS 1969). (ii) As pointed out above, the relatively low resolution of radioautography does not allow the exact localization of neurotransmitters. In mono-

Synaptic Morphology and Cytochemistry . 49

Fig. 56. Nerve terminals in rat spinal cord anterior hom, ZIO preparation. Synaptic vesicles in both terminals are heavily impregnated. Note that the spherical vesicles in terminal S belong to a synapse with a broad postsynaptic thickening (po) whereas the terminal F containing elongated or flattened vesicles forms a synapse with a narrow postsynaptic density (po). - Magnification, X 80,000; calibration, 0.2 (1.. Fig. 57. Two nerve terminals from the same tissue as in Fig. 56 but as seen after freeze-etching. Both boutons synapsing with the postsynaptic element (d) contain spherical vesicles. However, the average diameter of vesicles in pr, amounts to about 470 A whereas the ones in pr2 have an average size of 390 A only. The smaller vesicles in pr2 are believed to be identical with the elongated vesicles seen after aldehyde fixation and osmication or ZIO staining. -Magnification, X 42,000; calibration, 0·5 (1..

50 .

K. H.

PFENNINGER

aminergic systems, however, other cytochemical procedures such as KMn0 4' OS04' and OS04KZCrZ07 are available for specific and exact

localization of the amine (for references and discussion of staining specificity see methodology). These methods consistently produce

Synaptic Morphology and Cytochemistry' 51 electron dense precipitates within the synaptic vesicles of monoaminergic nerve terminals in the peripheral and the central nervous system (DE ROBERTIS and PELLEGRINO DE IRALDI 1961; LEVER and ESTERHUIZEN 1961; GRILLO and PALAY 1962; RICHARDSON 1962; BLOOM and BARRNETT 1966b; RICHARDSON 1966; HOKFELT 1968a, b; TRANZER et al. 1969 and others). This is illustrated in Figs. 60-63 and 66. No similar data could be obtained for acetylch::>line and amino acid transmitters since specific cytochemical methods for their localization are as yet not available. (iii) The fmding of dopamjne-~-oxidas~(DBO), the enzyme performing the fmal step in noradrellaline syllthesis within synaptic vesi::les of peripheral adrenergic nerve (POTTER and AXELROD 1963; STJARNE and LISCHAJKO 1967; DE POTTER 1971 ; SMITH 1971 a) is additional, indirect evidence for the synaptic vesicles as major transmitter stores. (iv) The following physiological and pharmacological experiments provide further data relevant to this topic. Monoaminergic nerve terminals treated with depleting agents nch as e. g. reserpine (Figs. 60 and 61) no longer show granules in synaptic vesicles (see e. g. DE ROBERTIS and PELLEGRINO DE IRALDI 1961; HOKFELT 1968). The exogeneous amine level in brain can be increased by intraventricular injection of, or in vitro incubation of the tissue in, an amine solution. This procedure leads to the appearance of dense precipitates in monoaminergic synaptic vesicles which have previously been depleted with reserpine or did not contain the dense granule (Figs. 67 and 68) (BONDAREFF 1966; TRANZER and THOENEN 1967b; HOKFELT 1968; RICHARDS and TRANZER 1970). Such reloading of depleted vesicles is inhibited by the action of cocain which blocks membrane transport of the amines (HOKFELT 1968). Incubation

in metaraminol, which is a drug probably displacing the endogeneous catecholamine from its storage site, and which shows only a weak reaction in vitro with KMn0 4 (HOKFELT and LJUNGDAHL 1972) also produces agranular vesicles. Electrical stimulation of adrenergic nerves previously treated with noradrenaline synthesis inhibitors is another experiment resulting in clear vesicles (HOKFELT 1967). This is illustrated in Figs.64 and 65. All these lines of evidence which are also in good agreement with the fluorescence microscopical (Figs. 58-61) and radioautographical (Figs. 62 and 63) data available speak in favor of the synaptic vesicles as the transmitter storage site (for review see also BLOOM 1970; HOKFELT 1970; HUBBARD 1970; GEFFEN and LIVETT 1971). In view of the fact that transmitters are secretory products of the neuron, the storage of amines in vesicles is consistent with the notion that all cellular s~cletory products are packed in membranebound particles. Analyses of subcellular fractions reveal a considerable portion of the transmitter in the supernatant (see e. g. KOPIN 1966; ISRAEL and GAUTRON 1969; MARCHBANKS 1969). These observations lead to the conclusion that there may be a cytoplasmic transmitter storage site in addition to the vesicular pool. In view of the fact that these data are derived from preparations which had undergone homogenization and disruption of nerve terminals and that there are no cytochemical data available favoring this hypothesis, the problem of the existence and role of a cytoplasmic transmitter storage site is still open.

Large dense core vesicles Large dense core vesicles or large granular vesicles (ldv) were discovered by GRILLO and

Figs. 58, 59, 60 and 61. Cytochemical demonstration of catecholamines with the Falck-Hillarp technique (Figs. 58 and 59) and the KMn0 4 method (Figs. 60 and 61), and the effect of reserpine treatment. All specimens are from the periventricular region of the rat hypothalamus. In the untreated animal (Fig. 58 and 60), the amine content is indicated by the high density of fluorescent dots, the so-called varicosities or, at the electron microscopical level, by terminals containing granulated vesicles. Note that small as well as large vesicles in the amine-positive terminal (asterisk) contain the electron dense precipitate. After reserpine treatment (10 mg(kg, i. p., 24 hours before death of the animal), the fluorescent dots have disappeared (Fig. 59) and nerve terminals containing granulated vesicles are no longer found (Fig.61). v, third ventricle; s, synapses. - Magnifications, Figs. 58 and 59, X 300; Figs. 60 and 61, X 20,000; calibration I fl. (Figures provided by Dr. T. HOKFELT).

52 • K. H. PFENNINGER

Figs.62, 63, 64 and 65. Demonstration of norepinephrine in the rat iris dilator muscle. Fixation by 3% ice cold KMnO•. In Fig.63, one (gr) out of three nerve terminals contains clearly granulated synaptic vesicles whereas the two other terminals (agr), presumably of cholinergic nature, do not contain the KMnO. precipitate. - Magnification, X 38,000. Fig.63 is a picture from the same material except for the fact that it was incubated in 10- 5 M 3H-norepinephrine for 20 min and that, after KMnO. impregnation, radioautography was performed. Note that the autoradiographic grains are accumulated over nerve terminals containing granulated vesicles (arrow) and not over terminals containing clear vesicles (agr). - Magnification, X24,000. Figs.64 and 65 are from rats treated with 500 mg/kg H 44/68, i. p., for 4 hours (an inhibitor of norepinephrine synthesis). Granulated vesicles are stilI visible in many nerve terminals (gr). However, after preganglionic electric stimulation (20/sec, 90 min) all the vesicles appear depleted (Fig.65). Note that treatment with norepinephrine synthesis inhibitor does not deplete the vesicles but that, after electrical stimulation, there is obviously no transmitter left or resynthesized to refill the vesicles. - Magnifications, X28,000 (Fig. 64), X27,000 (Fig.65); calibration in all figures, I (J.. (Figs.62 and 63 were provided by Drs. T. HOKFELT and A. LJUNGDAHL from unpublished experiments; Figs. 64 and 65 were contributed by Dr. T. HOKFELT).

PALAY (1962) as a second class of vesicles in terminals of the autonomic nervous system. They have now been described in a large variety of different adrenergic and cholinergic and other nerve terminals in the peripheral and the central

nervous system (for review see also TRANZER et al. 1969; BLOOM 1970; BLOOM et al. 1970; HOKFELT 1970; FILLENZ 1971). It is to be emphasized that they are not typical organelles of monoaminergic terminals (FUXE et al. 1965).

Synaptic Morphology and Cytochemistry . 53

Fig. 66. Cat iris after treatment with the "false transmitter" 5-0H-dopamine (4 times 20 mg/kg, i. p., over a period of 48 hours). Note that, after "simple" 3% glutaraldehyde and 2% OS04 treatment, small as well as large vesicles in some nerve terminals (gr) are loaded with an electron dense precipitate whereas other nerve terminals (agr), presumably the cholinergic elements, do not contain the substance. - Magnification, X 50,000; calibration, 0.5 fL. (From TRANZER and THOENEN, 1967b).

54 . K. H. PFENNINGER

Figs. 67 and 68. Nerve terminals in the rat hypothalamus after glutaraldehyde and OsO 4 fixation. In the control material (Fig. 67), all nerve terminals contain clear empty vesicles (agr) whereas, after 5-0H-dopamine injection into the ventricular lumen, some endings (gr), presumably the catecholaminergic ones, are loaded with the "false" transmitter. This is reflected in the electron dense precipitate filling most of their vesicles. The application of "false" transmitters serves as a tool for the identification of monoaminergic terminals. Further, it suggests that transmitter substances are stored in synaptic vesicles. - Magnification, X 56,000; calibration 0.5!J.. (Fig.68 from TRANZER et al. 1969; Fig.67, unpublished, provided by Drs. RICHARDS and TRANZER). The large dense core vesicles are distinct from the synaptic vesicles not only by their size of about 900 to 1200 A but also by their particular staining characteristics as illustrated in Table 8 and in Figs.69, 70 and 71. Most striking is the dense core which is separated from the vesicular membrane by a narrow electron lucent gap. It is visible after staining for acidic as well as for basic groups (UL, E-PTA, BruL). The UL staining is partly resistent to carboxymethylation (Fig. 71) whereas proteolytic enzymes such as trypsin and pronase destroy the dense core (Fig.70) (PFENNINGER 1969). Another important feature of the dense granule is its resistence to reserpine treatment (BONDAREFF 1965; TAXI 1965; HOKFELT 1966; VAN ORDEN et al. 1966). ZIO staining further distinguishes large dense core and

synaptic vesicles significantly (AKERT and SANDRI 1968; AKERT et al. 1971 a). Although this method has been found to stain all kinds of synaptic vesicles (Figs. 29, 56) tested so far (KAWANA et al. 1969; AKERT et al. 1971 a), it does not generate the typical black precipitate within the ldv (Fig. 69). Methods for the demonstration of monoamines were applied to determine whether the large dense core vesicles store norepinephrine or other biogenic amines. KMn0 4 (Fig.60) (HOKFELT 1968) and the aldehyde-dichromate combination with or without subsequent osrnication QAlM-ETCHEVERRY and ZIEHER 1969; TRANZER et al. 1969; FILLENZ 1971) iudicate that, within monoaminergic nerve terminals, ldv can accumulate biogenic monoamines especially

Synaptic Morphology and Cytochemistry . 55

Figs.69, 70 and 71. Large dense core vesicles (dv) under different experimental conditions. Fig.69 shows a nerve terminal in the rat vas deferens after ZIO staining. Note that the large dense core vesicles are devoid of the black precipitate that fills most synaptic vesicles (sv). The large dense cores are of the usual electron opacity encountered after OsUL preparation. - Magnification, X 170,000. Fig. 70 shows a nerve terminal from the cat subfornical organ treated with trypsin and then fixed with glutaraldehyde and OS04 (d. also Fig.43). Note that the core of large dense core vesicles sometimes appears to be darker than usual (arrow) but, in most cases, is irregularly shaped, i. e. partly digested (asterisk). - Magnification, X 125,000. Fig.71 displays dense core vesicles from the cat subfornical organ after carboxymethylation and UL staining (without osmication). The electron density of the cores is reduced but there is still some staining visible indicating the presence of non-carboxylic acidic groups. - Magnification, X 120,000; calibration in all figures, 0.1 fL. Figs.69 to 71 stress the cytochemical difference between large dense core vesicles and synaptic vesicles. They indicate that the dense core of the larger vesicles is at least partly of proteinaceous nature and contains many acidic groups, a significant amount of which is of phosphate or sulfate nature (E-PTA and BIUL staining further indicate the presence of many basic residues).

after their exogeneous administration (Fig. 66) (TRANZER and THOENEN 1967b; RICHARDS and TRANZER 1970). These data could also be confirmed by biochemical assay of subcellular fractions (BISBY and FILLENZ 1970). As concluded from the cytochemical observations, the usually visible electron dense core in large granulated vesicles is of higWy polar, probably proteinaceous, nature. The esterification experiment further indicates the presence of a considerable amount of phosphate and/or sulfate groups. There are no data available suggesting large amounts of carbohydrates. The

cytochemical similarity of the dense core of large granulated vesicles and presynaptic dense projections has been mentioned earlier (PFENNINGER et al. 1969b). The ability of large dense core vesicles in monoamine nerve terminals to accumulate transmitter may indicate that they are an additional storage site or that they have something to do with transmitter metabolism. It has been suggested (SMITH 1971 a, b) that large dense core vesicles are particles speciflC for carrying the proteins dopamine-~-oxidase and chromogranin which are released together with the adrenergic neurotransmitter. Despite the

56 . K. H. PFENNINGER fairly detailed data on the large dense core vesicles in monoaminergic nerve terminals, it is impossible at the present state of knowledge to defme their common role in the function of various types of synapses.

Complex vesicles, plasmalemmal vesicles and smooth endoplasmic reticulum

Complex vesicles (coated vesicles, spiny vesicles; ROTH and PORTER 1964) have been observed in nerve terminals long ago (GRAY 1961, 1963, 1966), but received special attention only recently (KANASEKI and KADOTA 1969; GRAY and WILLIS 1970; GRAY and PEASE 1971). In general, they form only a small but varying

proportion of the vesicular components in the boutons. This may not only be due to the fact that fIxation procedures are not always adequate to demonstrate the vesicle shell or coat but may as well reflect functional differences. The characteristic feature of the complex vesicle consists in an array of bars extending out radially from the membrane to form hexagons and pentagons all around the vesicle (KANASEKI and KADOTA 1969). This is shown in Figs. 73 and 74. ROTH and PORTER (1964) as well as KANASEKI and KADOTA (1969) ascribed a role in membrane vesiculation to the bristles of the membrane coat. As demonstrated further in Fig.73, the complex vesicles are often seen to be in fusion with the plasmalemma and, in rare instances even with

Fig.72. Nerve terminals (pr l and pr2) in the cat subfomical organ 30 min after intraventricular injection of horseradish peroxidase. Note that one of the terminals (pr2) contains a number of vesicles (of the size of synaptic vesicles) filled with the electron dense precipitate typical for the peroxidase reaction. sv, synaptic vesicles; dv, large dense core vesicle; d, dendrite. - Magnification, X 52,000; calibration, 0·5 fL. Figs. 73 and 74. Complex vesicles in Deiter's nucleus of the cat. Two complex vesicles (cv) are in close relation to the presynaptic membrane in Fig.73 (arrows). Fig.74 shows a series of complex vesicles (cv) at some distance from a synapse (s). Note the polygonal subunits of the shells. - MagnifIcation, X 80,000 (Fig. 73), X 110,000 (Fig. 74); calibration in both figures, 0.2 fL. (By courtesy of Dr. E. KAWANA).

Synaptic Morphology and Cytochemistry . 57 the presynaptic membrane. Since the shell alone apparently without the vesicle inside, and fragments thereof, are also found in the presynaptic nerve terminal, GRAY and WILLIS (1970; d. also DOUGLAS et al. 1971) proposed a process of vesicle formation via pinching off of complex vesicles and subsequent loss of the shell (hypothesis 4 in Fig. 75). In this context, it is also interesting to note that, occasionally, coated vesicles share ZIG - positivity with the synaptic vesicles (AKERT et al. 1971 a). However, complex vesicles are also found in fusion with cisterns of the smooth endoplasmic reticulum and occur in different sizes within the same nerve terminal (GRAY and WILLIS 1970) which makes it more difficult to understand their role in synaptic function. Plasmalemma I vesicles (PALADE and BRUNS 1968) exist in presynaptic nerve terminals as derived from the direct observation of fission or fusion sites in thin sectioned (ANDRES 1964; WESTRUM 1965; BIRKS 1966) and freeze-fractured (AKERT et al. 1969, 1971 b) material as well as from the pinocytotic uptake of exogeneously administered macromolecular tracers such as HYPOTHETICAL

ECHANIS S OF

horseradish peroxidase and ferritin (BRIGHTMAN 1967; PFENNINGER 1969; ZACKS and SAITO 1969; HOLTZMAN 1971; HOLTZMAN et al. 1971; HEUSER 1971). The latter result is illustrated in Fig. 72. Note that the vesicles containing the tracer do not differ significantly in size from the clear synaptic vesicles. To some extent, plasmalemmal vesicles may be identical with coated vesicles the shell of which is not always easy to detect. Plasmalemmal vesicles are a very common cytological fmding not restricted at all to the nervous system. However, at the site of the nerve terminal, vesicle fission may play an important role in the formation of the synaptic vesicles (see below). Smooth endoplasmic reticulum (sER) is present in small amounts in the axon and in the nerve terminals. It is quoted in this section for its possible relationship to synaptic vesicles and since, in cross sections, it may sometimes have the appearance of vesicles instead of cisternae.

The origin of synaptic vesicles The discussion of complex and plasmalemmal vesicles as well as smooth ER in the nerve terminal leads us to the problem of synaptic sY APTIC VESICLE FORMATION

Fig. 75. The origin of synaptic vesicles. For discussion of the various hypotheses see text. 1. perikaryal synthesis and transport of the vesicles to the terminal. 2. formation out of microtubules. 3. plasmalemmal vesicle fission. 4. fission of complex vesicles and subsequent loss of the shell. 5. fission of complex vesicles from plasmalemma, fusion with smooth endoplasmic reticulum, subsequent budding of vesicles from smooth endoplasmic reticulum (6). 7. fission of complex vesicles from large dense core vesicles (d. neurosecretory and chromaffin granules) which are in the process of releasing their contents.

58 . K. H. PFENNINGER

vesicle formation. The two major hypotheses include (i) vesicle formation in the perikaryon and subsequent transport to the nerve terminal and (ii) local formation in the bouton (for review see also BLOOM et al. 1970). As a possible site of local origin of synaptic vesicles, almost every organelle occuring in the nerve terminals has been taken into consideration (Fig.7S). Whereas the budding of synaptic vesicles from the end of microtubules (hypothesis 2 in Fig.7S) (PELLEGRINO DE IRALDI and DE ROBERTIS 1968) seems very unlikely since vesicles are membraneous structures in contrast to the microtubules, the fission of vesicles from the axolemma as "regular" plasmalemmal vesicles (WESTRUM 1965; BIRKS 1966) or as complex vesicles (GRAY and WILLIS 1970) and their further transformation into synaptic vesicles would be easier to understand (hypotheses 3 and 4 in Fig.7S). Indeed, many of the vesicles which have taken up exogeneously administered peroxidase (BRIGHTMAN 1967; PFENNINGER 1969) seem morphologically not distinct from synaptic vesicles (Fig.72). However, the differential chemical composition of vesicular membranes and the nerve terminal plasmalemma (WmTTAKER 1966; LAPETINA et al. 1968) is a significant observation not supporting the idea of the origin of the vesicles by pinocytosis. In the adrenal medulla, coated vesicles bud from the membrane of chromaffm granules which are in the process of releasing their contents (GRYNSZPAN-WYNOGRAD 1971); the analogous observation has been obtained in neurosecretory terminals of posterior pituitary glands with respect to the formation of the small clear "synaptic vesicles" (NAGASAWA et al. 1970, 1971; DOUGLAS et al. 1971; d. also AMSTERDAM et al. 1969). SMITH (1971 b) proposed a similar mechanism of vesicle formation in nerve terminals from ldv (hypothesis 7 in Fig. 7S). However, electron micrographs showing steps of such a process have not been published so far. A further possibility of vesicle formation in nerve terminals is the pinching off from sER cisternae (hypothesis 6 in Fig.7S). This proposition has been made on the basis of the common ZIO staining for sER cisterns and synaptic vesicles in

the nerve terminal (AKERT et al. 1971 a). At about the same time, HOLTZMAN et al. (1971) and HEUSER (1971) detected changes in the number of peroxidase-labeled synaptic vesicles with varying activity of the nerve. In peroxidase pulselabeling experiments, Heuser observed the uptake of the macromolecular tracer into complex vesicles and subsequently into the sER. Later, the label seems to appear within the synaptic vesicles (hypothesis S and 6 in Fig.7S) and, after repetitive, exhaustive stimulation of the nerve, can be released therefrom. Heuser concludes that, in the case of the muscle endplate, vesicles may be formed from sER which, in turn, receives membrane from the axolemma. The whole mechanism would thus consist of a recycling of vesicular membrane after its fusion with, and incorporation into, the axolemma. There are several lines of evidence speaking in favor of a perikaryal synthesis of synaptic vesicles. The notion of the Golgi apparatus as the vesicle factory is consistent with the well-known key role of this organelle in the formation of most secretory products. Accordingly, the structural (GRAY 1970) and cytochemical (ZIO staining; AKERT et al. 1971 a; STELZNER 1971) similarity of vesicles near the Goigi apparatus and in the respective nerve terminals have been interpreted to reflect the role of the Golgi apparatus in vesicle formation. The most significant data supporting the notion of a perikaryal vesicle synthesis, however, originate from studies on axoplasmic transport (for review see e. g. BARONDES 1967). Vesicles indistinguishablefrom the synaptic ones are long known to occur in axons preferentially in connection with microtubules. This is especially striking in the lamprey nerves as demonstrated by SMITH et al. (1970). Ligation of the nerve leads to proximal accumulation of the vesicles. In monoaminergic neurons, this accumulation of KMn0 4 -positive vesicles parallels an increase in monoamine fluorescence (for review see DAHLSTROM 1971). The local application of mitosis inhibitors which are known to be bound to microtubular protein produces the same results as ligation (HOKFELT and DAHLSTROM 1971). These observations taken together are good evidence for the perikaryal formation

Synaptic Morphology and Cytochemistry . 59 of vesicles and their subsequent transport to the nerve terminal (hypothesis I in Fig. 75). At present, the data as outlined above do not allow exclusion of one of the two alternatives of synaptic vesicle formation; even both mechanisms may occur concomitantly. However, it is still possible that vesicles of the different origins form two classes of as yet unknown characteristics and that only one of them serves the storage of the transmitter. The synthesis of neurotransmitters (for review see e. g. HALL 1972) occurs to the largest extent in the nerve terminals as indicated by the high amine concentration and the presence of large amounts of the synthesizing enzymes. As mentioned before, the last step in noradrenaline synthesis requires the presence of synaptic vesicles which contain DBO. In contrast, the enzymes performing the previous steps in catecholamine synthesis seem to occur in the terminal axoplasm in a non-particle-bound form (STJARNE and LISCHAJKO 1967). Lower amine concentrations are also found in the perikaryon as indicated by fluorescence microscopy (FALCK et al. 1962) and by radioautography (DESCARRIES and DROZ 1970). In connection with the data known about axoplasmic transport of the amines, this indicates that part of the catecholamine synthesis occurs in the perikaryon. In cholinergic systems, the main synthesizing enzyme, choline acetyltransferase, is freely soluble in the axoplasm (for review see MARCHBANKS 1969).

6. Transmitter release Although problems of transmitter release have been reviewed only recently (BLOOM et al. 1970; HUBBARD 1970) this topic appears today in a slightly different light for new cytological data have recently been obtained.

The fine structure and cytochemistry of the presynaptic membrane As the transmitter has to cross the presynaptic axolemma during the release process, it is this membrane which we will look at first. It has been pointed out that the presynaptic membrane

is characterized by dense structures attached to it, in the typical case by a triagonal array of dense projections. The hollows between these cytoplasmic membrane specializations form hexagons and are of the appropriate size for the vesicles to fit into them. The whole assembly consisting of dense projections and vesicles in a most economically arranged 2: I ratio has been called "the presynaptic vesicular grid" (AKERT et al. 1969). This pattern which is clearly distinct from the tight honeycomb type distribution of plasmalemmal vesicles in certain areas of endothelial cells (PFENNINGER 197Ia; PFENNINGER et al. 1972) suggests a special role of the dense projections in the release process. Digestion experiments have confirmed GRAY'S former suggestion (1966) that the matrix of presynaptic dense projections is of filamentous nature (PFENNINGER 1971 c). The fact that dense projections exhibit especially good contrast after E-PTA or BIUL staining (Figs. 18, 22, 23) (GRAY 1963; BLOOM and AGHAJANIAN 1966; PFENNINGER et al. 1969a, b; PFENNINGER 1971 b) indicates a high density of basic groups. The presence of significant amounts of acidic residues is suggested by plain UL and ruthenium red staining (Fig. 47) (PFENNINGER 1969, 1971 b). The staining with UL of part of these anionic groups is resistent to esterification indicating phosphate and/or sulfate groups (Fig.2) whereas A-PTA (MEYER 1969 a, b) and periodic acid silver methenamine staining (RAMBOURG and LEBLOND 1967) produce only minimal dense projection staining (Fig. 20) and thus speak against the presence of a significant amount of carbohydrate. Digestion experiments with proteolytic enzymes reveal the proteinaceous nature of dense projections whereby, similar to the postsynaptic density, a trypsin-labile component can be distinguished from a trypsin-resistant but pronase-sensitive fraction which is demonstrated by BIUL staining only (Table 7) (PFENNINGER 1971 c). Thus, in these crude experiments presynaptic dense projections appear to be cytochemically similar to the postsynaptic specialization of the inner membrane coat. However, the inconsistent visualization of the dense projections in contrast to the postsynaptic density in OsUL material

60 • K. H. PFENNINGER may indicate cytochemical differences which, with the techniques as yet available, cannot be clearly demonstrated. During the formation of a synapse, dense projections may develop from a more plate-like presynaptic density by focalization of the dense material (AGHAJANIAN and BLOOM 1967; AKERT and PFENNINGER 1969; JONES and REVELL 1970; BLOOM 1972); this hypothesis implies the presence of a desmosome-like structure as a precursor of the synaptic junction. However, this may be an oversimplified view since synaptic and desmosomal cytoplasmic densities have been shown to be considerably different (see above). In the course of the development of the synapse, vesicles and dense projections seem to appear about simultaneously (BUNGE et al. 1967; JONES and REVELL 1970). Not only cytochemical procedures but also freeze-fracturing has been applied to study the structure of the presynaptic membrane (AKERT et al. 1972; PFENNINGER 1971 a; PFENNINGER et al. 1971a, b, 1972). It is important to note that this method does not reveal the inner and outer surfaces of membranes but that it splits membranes down their middle and thus visualizes the structure inside the membrane (BRANTON 1966; TILLACK and MARCHESI 1970; PINTO DA SILVA and BRANTON 1970; WEHRLI et al. 1970). This is also true for the membranes of nervous tissue and especially for the presynaptic membrane (PFENNINGER et al. 1972).

The freeze fractured presynaptic membrane is characterized as follows: (i) Its outer membrane leaflet is almost always attached to the underlying postsynaptic membrane which is significant of the synaptic adhesiveness (Figs. 76, 81 and 82). (ii) Frequently, it shows a slight elevation toward the inside of the nerve terminal (Fig. 78), and (iii) the idented area is covered by a number of particles which are more concentrated than in the nonsynaptic surrounding. The most peculiar feature of the presynaptic membrane is (iv) the presence of a varying number of small protuberances in the outer leaflet (Figs. 76, 78 and 81) or pits of corresponding size in the inner leaflet (Fig. 77). These pits or protuberances which have also been called synaptopores (PFENNINGER et al. 1971 b, 1972; AKERT et al. 1972) can be observed to be arranged in a regular manner. Optical diffraction analysis and statistical evaluation of the intervals between synaptopores have shown that they occupy a varying proportion of nodal points of a hexagonal pattern which is identical with the pattern of synaptic vesicles (PFENNINGER 1971 a; PFENNINGER et al. 1972). This pattern becomes directly visible in those cases where the number of synaptopores is high. In Fig.78, the upper part shows one complete hexagon whereas in the lower part, three adjacent hexagons can be identified. Note that these hexagons would fit into the interspaces between the dense projections in Fig. 79 which is given at about the same

Figs.76, 77. 78 and 79. Features of the presynaptic membrane as seen after freeze-etching. Fig. 76 shows a presynaptic membrane (pr) attached to the underlying postsynaptic membrane (po). The presynaptic membrane is characterized by a cluster of small protuberances which partly have a crater-like structure (arrow). Synaptic vesicles (sv) are seen in the cytoplasm adjacent to the presynaptic membrane. Note that the cytoplasmic side of the external leaflet of the presynaptic membrane is exposed in this picture. - Magnification, X 74,000. Fig. 77 displays another nerve terminal containing numerous synaptic vesicles (sv), a large dense core vesicle (dv), and a mitochondrium (m). The exposed membrane face which represents the outer view of the inner plasmalemmalleaflet contains a slightly indented area (pr) characterized by numerous pits identical in size with the protuberances seen in Fig. 76. Cross fractures of membrane reveal its trilaminar aspect; the asterisk indicates the transition between intact, trilaminar and split, single layered plasmalemma whose face is exposed. - Magnification, X 63,000. Fig.78. Outer leaflet of the presynaptic membrane (pr; cytoplasmic view) attached to an underlying postsynaptic element (po). Note the high density of protuberances forming three adjacent hexagonal rosettes in the lower part and one hexagonal rosette in the upper part of the picture (arrows). These rosettes of protuberances fit into the interspaces between the dense projections (dp) as seen in Fig. 79. On the other hand, the triagonally arranged dense projections fit into the centers of the protuberance rosettes. - Fig. 78 : Rat spinal cord; magnification, X 100,000. Fig. 79: Cat subfornical organ, BIUL; X 120,000; the arrows indicate several axes of the triagonal dense projection meshwork. Calibration in all figures, 0.2 (l.

Synaptic Morphology and Cytochemistry . 61

62 • K. H. PFENNINGER

sc

80

po

Fig. 80. Three-dimensional reconstruction of cytochemical and freeze-etch data on the presynaptic membrane. On the left side, dense projections (dp) can be seen surrounded by hexagonal rosettes of synaptic vesicles (sv). On the right side of the picture, the membrane is split and the outer membrane leaflet is shown (cytoplasmic view). Not only membrane particles (asterisks) but also presynaptic protuberances or "synaptopores" are visible. The latter are hexagonally arranged in a pattern reflecting the array of the synaptic vesicles. It is concluded therefore, that the protuberances sit right underneath vesicles and therefore represent attachment sites of the vesicles to the presynaptic membrane. sc, synaptic cleft; po, postsynaptic density. (From PFENNINGER et al. 1972). magnification as Fig. 78. On the other hand, one dense projection taken from Fig.79 would fit into the center of the hexagons in Fig.78. Thus it appears that the sites where the synaptic vesicles touch the presynaptic membrane are not only characterized by the lack of the cytoplasmic membnne coat (AKERT and PFENNINGER 1969) but also by the ability to form small protuberances (Fig. 80). These humps very often have a smooth closed top but sometimes appear to be of crater-like structure with an opening of irregular shape. The outer diameter of the protuberances is 200 A at their base. The pits on the complementary side of the fracture often contain a few particles which may have been ruptured away from the opposite side, i. e. from the top of the protuberances thus leaving there a hole. Synaptopores are somewhat similar to plasmalemmal vesicle fission or fusion sites (PALADE and BRUNS 1968) as seen after freeze fracturing (NICKEL and GRIESHABER 1969). However the synaptic structures are not only much smaller in

size (200 vs 350 A) but they are also different in morphology. The pits never show the fracture line of the outer membrane leaflet turning inward to continue as the inner vesicular leaflet, a consistent feature of pinocytotic openings. Preliminary physiological experiments comparing active (Fig.81) and resting synapses (Fig. 82) indicate that the number of synaptopores per square unit of presynaptic membrane increases with enhanced activity (PFENNINGER et al. 1971 c). It is concluded, therefore, that synaptopores are synapse-specific, transient attachment sites of the synaptic vesicles to the presynaptic membrane which may represent the morphological basis of transmitter release (PFENNINGER 1971 a; PFENNINGER et al. 1972).

Vesicle hypothesis or membrane control theory? The different morphological and cytochemical observations on the presynaptic membrane may be interpreted in the following way. The fact

Synaptic Morphology and Cytochemistry • 63

Figs. 81 and 82. The effect of barbiturates on the presynaptic protuberances. In the control (Fig. 81) presynaptic membranes (pr) bear clusters of synaptic protuberances of varying shape and size. The effect of barbiturate treatment is illustrated in Fig. 82. It shows three adjacent presynaptic membranes (pr); they all contain an area distinct from the surrounding membrane face (arrows). There are, however, almost no protuberances visible; the ones which can be identified are smaller in size than those in the control. po, postsynaptic element; sv, synaptic vesicles. This experiment is suggestive of the fact that the presynaptic attachment sites are dependent upon synaptic activity and therefore may playa role in the mechanism of transmitter release. - Rat spinal cord; magnification X 35,000; calibration, 0·5 fL.

that synaptic vesicles are arranged in a particular manner in the immediate neighborhood of the presynaptic membrane and that they interact with this part of the axolemma strongly supports the vesicle hypothesis, i. e. the theory that the transmitter is released out of the vesicles directly into the synaptic cleft. This is in good agreement with the notion of synaptic vesicles as the main transmitter storage sites as outlined above. Dense projections have been interpreted by GRAY (1966) to represent devices guiding the vesicles towards specialized sites in the presynaptic membrane. Rather than in terms of mechanics, the role of presynaptic dens:: projections could also be interpreted biochemically. They could influence the vesicles in a way which makes the

transmitter readily releasable or they could play a role in the loading of vesicles with transmitter (AKERT and PFENNINGER 1969). The observation that, in quite a series of different nerve terminals, the newly synthesized transmitter is released first (KOPIN 1966; KOPIN et al. 1968; COLLIER 1969) is explained by the presence of two different transmitter pools separated by a diffusion barrier. Hypothetically, there could exist a free cytoplasmic pool and the vesicular pool; but there might as well exist two different fractions of vesicles. Indeed, the presynaptic dense projections separate the synaptic vesicles closest to the presymptic membrane clearly from the large number of vesicles more or less freely distributed in the terminal axoplasm.

64 • K. H. PFENNINGER

In studies on subcellular fractions from electric organs, it has been observed that the free transmitter pool is decreased when the specimens are taken from stimulated tissue (DUNANT et aI. 1971). This fmding is interpreted to favor the membrane control theory, i.e. the notion that the transmitter is released from a cytoplasmic pool and that a gating mechanism in the membrane is responsible for the release in quanta. However, no data from other sources are available which would support this view. It is even contradicted by the fact that monoamines cannot be released easily upon stimulation from terminals treated with reserpine and MAO inhibitors despite the large amount of transmitter present in the cytoplasm (MALMFORS 1969). As an alternate possibility, the changes in the amount of free transmitter in subcellular fractions could be explained as follows. The vesicles which are attached to the presynaptic membrane for the release of their content could be specially sensitive to disruption during the homogenization of the tissue; after stimulation, an increasing proportion of these vesicles are depleted. Transient versus irreversible vesicleplasmalemma contact As to the nature of the contact between the vesicle and the presynaptic membrane, a first question is whether the interaction is of transient nature or whether, in the course of transmitter release, a fusion of the two membranes leads to the incorporation of the vesicular membrane into the presynaptic axolemma (so-called reverse pinocytosis). In the latter case, exhausting stimulation of the synapse should cause a decrease in the number of synaptic vesicles. Numerous attempts have been made to test this hypothesis. However, the results are contradictory: all the three possibilities, decrease, increase and lack of a change in number of synaptic vesicles, have been reported to occur after stimulation (BmKs et aI. 1960; BURNSTOCK and MERRILEES 1964; HUBBARD and KWANBUNBUMPEN 1968; JONES and KWANBUNBUMPEN 1968, 1970; CRAGG 1969; PARDUCZ and FEHER 1970; BmKs 1971; FRIESEN

and KHATTER 1971; HEUSER 1971; JONES and BRADFORD 1971). This problem is complicated by the well known fact that the number of synaptic vesicles is varying tremendously from nerve terminal to nerve terminal even under non-experimental conditions. As a further problem, the irreversible incorporation of the vesicular membrane into the presynaptic plasmalemma would require a continuous rearrangement of the dense projection meshwork as well as of the external membrane coat mediating synaptic adhesiveness. Calculations comparing the membrane surface of the synaptic vesicle with the surface area of a CNS synapse of average size (Fig. 83) show that, after the depletion of zoo to 300 vesicles, the presynaptic membrane would be replaced completely by vesicular membrane. Studying the same problem in the neuromuscular junction, BITTNER and KENNEDY (1970) calculated an unimaginably rapid growth of the terminal axolemma as a consequence of the hypothetical addition of vesicular membrane with every transmitter quantum released. In order to maintain steady state conditions, a tremendous lipid turnover for the transformation of the vesicular into the chemically different presynaptic membrane as well as intensive membrane recapturing would be necessary. However, the number of plasmalemmal vesicle fission (or fusion) sites in nonsynaptic areas is small as compared with the number of presynaptic attachment sites (AKERT et aI. 1971 b; PFENNINGER et al. 197Z) and, further, the turnover rates are not only low for synaptosome lipid and protein (VON HUNGEN et aI. 1968; LAPETINA et aI. 1969, 1970) but they are also different for lipid of synaptic vesicles vs. the nerve terminal membrane lipid (half life of 30 vs. 61 days; LAPETINA et al. 1970). These lines of evidence speak against the idea that the release of the transmitter content of a vesicle is followed byits irreversible incorporation into the presynaptic membrane. The more likely interpretation is the reutilization of the vesicles and a transient vesicle-plasmalemma interaction (at least at the interneuronal synapse).

Synaptic Morphology and Cytochemistry' 65

s~_

f"y·20

s

y.

5,y• .Il05 -

R • O.l}'

25"",

41\'1'lv _

2

.llOIy

Sg,. 'lfR2. 1.51 T .~. _ _ 1.5_-190-300 Ssv ,ODS .DOl - -

T,'turnover rate' of presyn membrane

Fig. 83. Calculation of the turnover of the presynaptic membrane (synapse of average size in the rat spinal cord anterior horn) based on the assumption that the membrane of synaptic vesicles is incorporated into the axolemma after the release of their contents. The surface Ssv of the respective synaptic vesicles (sv) is in the range of .005 to .008 !J.2. The membrane of one vesicle increases the plane surface of presynaptic membrane by a disc with a radius rs of twice the vesicular radius rsv • The average plane surface of the presynaptic region Spr is approximately I.5!J.2. Hence, it is completely replaced by vesicular membrane after the release of about 190 to 300 vesicular contents. T, "turnover rate" of presynaptic membrane as a function of vesicular contents released.

The true nature of the vesic1eplasmalemma contact: Transmembraneousormembrane-bound channel? The true nature of the vesicle-plasmalemma contact is an additional problem. An attachment with a transmembraneous channel and a fusion site forming a membrane-bound channel are two alternatives. The comparison of transmitter release with secretory processes in other cells can easily be made. The secretory process common to most cells seems to consist in a fusion of the membrane of the secretory granule with the plasmalemma so that the former opens directly into the intercellular space (PALADE 1959; FARQUHAR 1961; IcHIKAWA 1965). This process has been called exocytosis. A similar mechanism could account for the depletion of synaptic vesicles especially since transmitter discharge is calcium dependent (KATZ and MILEDI 1965 b; KATZ 1969; BLAUSTEIN et a1. 1972) as all the other secretory processes seem to be (DOUGLAS 1966). Though rarely seen in synapses of the CNS, the opening of vesicles towards the synaptic cleft is

more frequently observed at neuromuscular junctions (BmKs et al. 1960; COUTEAUX and P:EcoT-DECHAVASSINE 1970; NICKEL and POTTER 1970). The release of protein from adrenergic nerve (chromogranin and DBO; SMITH 1971 a) and from endplates (horseradish peroxidase; HEUSER 1971) further favors the concept of exocytosis as the mechanism transmitter release (however, macromolecules could as well pass small transmembraneous channels as e. g. in gap junctions; LOEWENSTEIN 1966). The following two observations, though, seem not readily compatible with the exocytosis model as outlined above. The time course of transmitter release is extremely rapid, i. e. it takes place within less than I msec (KATZ and MILEDl 1965 a). It is not easy to understand that the transformation of membranes necessary for the fusion of the vesicle with the presynaptic plasmalemma and the subsequent generation of a relatively wide channel should occur in such a short period. The second inconsistency concerns the fmding of the vesicle attachment sites. In spite of the fact that the axolemma is capable of forming typical plasmalemmal vesicle fission and

66 • K. H. PFENNINGER

fusion sites (in nonsynaptic areas) it produces vesicle-plasmalemma junctions of different, specialized nature only at the synapse. Their small size and especially the lack of a fracture line of the outer plasmalemmal leaflet in the center of the pits probably argue against the continuity of vesicular and presynaptic membrane to form a membrane-bound channel. The more likely interpretation of these structures may be the formation of a close attachment with a transmembraneous channel interconnecting the vesicular lumen with the synaptic cleft. Possibly, this channel is similar to the "tubules" crossing the intercellular space in gap junctions. This hypothesis has the advantage that the gaiting of the proposed opening could be effected by conformational changes which could occur within very short time periods (PFENNINGER 1971 a; PFENNINGER et al. 1972). It cannot be excluded, however, that the pits and protuberances seen in freeze fractured presynaptic membranes represent only an intermediate preparatory step immediately before the fusion of vesicular and presynaptic membranes. Furthermore, we must not forget that fIxation, embedding and also freeze fracturing may influence the aspect especially of structures involved in such rapid processes in some unknown manner. Dynamics of the presynaptic membrane As derived from his physiological studies, KATZ (1969) proposed the presence of special sites in the presynaptic membrane and the collision of the vesicles with these sites as the event initiating transmitter release. An increase in the number of reactive sites with enhanced activity of the nerve would increase the statistical probability of their collision with the vesicles and thus lead to enhanced release. This theory can readily be applied to the cytological fmdings presented in this chapter. The "collision sites" are characterized by the lack of a cytoplasmic membrane coat (the holes in the dense projection meshwork) and by their ability to form attachment sites for the vesicles (synaptopores). The vesicles, rather than moving around freely, may sit within the holes between the dense projections to be reused for an extended

time period. As they are located in the immediate neighborhood of the attachment sites, the collision is unlikely to be a statistical event but, instead, the formation of "active synaptopores" could occur statistically. Such dynamics are suggested by the presence of incomplete hexagonal patterns of protuberances, i. e. the variation in the proportion of active sites, and by the simultaneous fmding of different structural aspects of synaptopores (closed, smooth surfaced vs. open protuberances). These observations suggest that (i) every preformed site undergoes a cycle ranging from the inactive state (no protuberance visible) to the close contact with the transmitter vesicle and (ii) that these changes are not synchronized. At every moment, some vesicle-plasmalemma junctions are in the process of formation to become ready for the discharge of the transmitter. In analogy to the concept of KATZ, enhanced activity of the nerve would increase the rate of formation of active collision sites (d. Figs.81 and 82) and thus increase the number of transmitter quanta released (PFENNINGER 1971 a; PFENNINGER et al. 1971 c). Hypothetically, the vesicles may undergo an "aging process" which, after a vesicle has been reutilized many times, would lead to its fusion with, and incorporation into, the presynaptic membrane. Such a process of reverse pinocytosis which would occur at a much slower rate than the vesicle attachment for transmitter release could account for, and be part of, HEUSER'S (1971) observations and membrane recycling hypothesis. The recapturing of membrane, indeed, seems to be a time consuming process as indicated by the fact, that the number of vesicles labeled with the exogeneously administered enzyme is particularly high after alternating stimulation and recovery periods (HOLTZMAN et al. 1971).

7. Cytochemistry of electrogenic regions The mechanisms of recognition and connectivity which the postsynaptic membrane is participating in have been discussed in a previous chapter. Further specific properties of the post-

Synaptic Morphology and Cytochemistry . 67

synaptic membrane are (i) their reactiVity towards selected transmitter substances and (ii) the generation of an electrical signal (for review see also BLOOM et al. 1970). The transmitter sensitivity of the postsynaptic membrane requires the presence of specific receptors. In the past years, the biochemical characterization of the cholinergic receptor which is present in the electric organs in large quantity has been considerably advanced (KASAl and CHANGEUX 1971; SMYTHIES 1971) especially since the introduction of affinity labeling techniques by KARLIN (1969). In adrenergic systems, physiological experiments indicate a role of adenylcyclase and cyclic AMP in the mechanism of postsynaptic reception (see RALL and GILMAN 1970). Cytochemical approaches to investigate these problems succeeded in the visualization of receptors at the cellular level (SALPETER 1969) but have failed so far to give insight into their localization and function at the membrane level.

Surface carbohydrates and cytoplasmic membrane coat The problem of electrogenecity of the postsynaptic membrane appears in a different light from the above discussion which is due to the fact that the axon hillock and the nodes of Ranvier have somewhat similar properties (Fig. 84). Comparative cytochemistry of these three regions reveals the following common features: MEYER (1970) showed a particularly intensive A-PTA staining of the outer membrane coat at the axon hillock (Fig. 19), the nodes of

Ranvier and within the synaptic cleft (Fig.20). This result indicates the presence of considerable amounts of sugar derivatives at these sites and may be of special significance with respect to electrical activity, i.e. the movement of ions across the membrane and in the intercellular space; the presence in these carbohydrates of many acidic groups binding reversibly cations may be of special importance (see SCHMITT and SAMSON 1969). Another significant observation has been made on the cytoplasmic coat of electrogenic membranes. PALAY et al. (1968) and PETERS et al. (1968) reported that, in osmicated tissue, the cytoplasmic leaflet of the so-called unit membrane appears much thicker at the axon hillock than in other areas of the neuron. This "undercoating" which certainly is part of the cytoplasmic fuzz coat has also been described in Ranvier's nodes (ELFVIN 1961; PETERS 1966). In the postsynaptic membrane, it is not clear whether the postsynaptic web covers this "undercoating" or whether the postsynaptic density represents an enormously developed "undercoating" of a similar kind; the osmiophilic "undercoating" typical for the axon hillock and the node of Ranvier, however, may as well be lacking at the synapse. A thickened cytoplasmic unit membrane leaflet has also been reported to occur in neuronal membranes of helix neuropil (CHALAZONITIS and CHAGNEUX 1968). PERACCHIA and ROBERTSON (1968, 1971) described a similar but more striking staining of the cytoplasmic membrane coat in the squid giant axon

ELECTROGENIC REGIONS OF THE NEURON

IS

R

84 Fig. 84. Electrogenic regions of the neuron. The three plasmalemmal areas, the postsynaptic membrane (S), the initial segment (IS), and the axolemma at the node of Ranvier (R) are capable of generating an excitatory (or inhibitory) postsynaptic potential or a spike potential, respectively. The common cytochemical feature of these regions is an external membrane coat particularly rich in carbohydrates (d. MEYER 1970).

68 . K. H. PFENNINGER provided that fixation took place during electrical stimulation of the nerve. Their experiments further indicate that this osmiophilia is dependent upon sullide groups. All these data suggest that not only carbohydrates of the external fuzz coat but also components of the cytoplasmic part of the greater membrane may play an important role in the electrical activity of the nerve. Acetylcholine esterase (AChE), the enzyme inactivating acetylcholine at an incredibly fast rate (see e. g. LEUZINGER 1969) is mainly present at cholinergic synapses but it also is found on the surface of non-cholinergic synaptic and nonsynaptic neuronal membranes and even in myelinated axons (RODRIGUEZ DE LORES ARNAIZ et al. 1967; WHITTAKER et al. 1964; SMITH and TREHERNE 1965; BLOOM and BARRNETT 1966a; BRZIN 1966; LEWIS and SHUTE 1966; KAsA 1968; KOELLE 1969). AChE thus appears to be a common feature of excitable membranes. Experiments carried out by KARLIN (1967) indicate that AChE is distinct from the cholinergic receptor. But its role in electrical activity of the nerve is far from being known.

Intramembraneous structures It seems reasonable to assume that membranes of electrogenic regions are especially rich in transmembraneous channels for ionic flux. Thus intramembraneous structure at these sites is of great interest. In those rare instances where the cleavage plane splits the postsynaptic membrane (d. PFENNINGER et al. 1972), freeze fracturing has recently revealed the presence of a large number of membrane particles within the postsynaptic membrane (SANDRI et al. 1972). This

Fig. 85. Freeze-fracture through a postsynaptic membrane (po). The cytoplasmic leaflet of the presynaptic membrane (external view) is marked by an asterisk. Note that the postsynaptic region is characterized by an accumulation of large membrane particles (arrows). pr, presynaptic nerve terminal; sv, synaptic vesicles. - Pigeon optic tectum; magnification, X 76,000; calibration, 0.2 fl.. fmding is especially striking in the outer membrane leaflet, which, in non-synaptic areas, is usually poor in membrane particles (Fig. 85). Whereas the axon hillock has not been identified yet in freeze fractured neuropil, the intramembraneous structure of the node of Ranvier and

Figs.86, 87 and 88. Freeze-etching of the node and paranode of Ranvier. In Fig. 86, part of the relatively smooth but particle-bearing nodal axolemmal outer leaflet, (al; inner view) is seen; within the adjacent paranodal area, this membrane leaflet exhibits a characteristic crystal-like pattern of circular bands and diagonal markings (large arrow). The outer plasmalemmalleaflets of the glial folds (gm) engaged in the paranodal glio-axonal contact are characterized by circular rows of large particles. g and g', intact and crossfractured glial loops; ax, axon; small arrow, inner axolemmalleaflet (outer view). Fig. 87 displays a paranodal region at lower magnification (x 38,000). The glial loops (g) generate more or less circular impressions into the axolemma (al; outer leaflet). In this region, the axolemma bears its typical paranodal markings. gm, glial membrane (inner leaflet). Fig. 88 illustrates, in more detail, that the markings in the paranodal outer axolemmalleaflet consist of circular (or spiral) bands which are composed of diagonal rows of small subunits (arrows) molded into the membrane "matrix". There are also some larger particles visible which sit in the grooves separating the bands. my, myelin sheath. Magnification, X 80,000 (Figs. 86, 88); calibration, 0.5 fI. in all figures. Rat spinal cord anterior hom. (LIVINGSTON et al. 1972a, 1973).

Synaptic Morphology and Cytochemistry . 69

the paranode is illustrated in Figs. 86-88

(LIVING-

STON et al. 1972, 1973). In those areas where the

cytoplasm-containing folds of the Schwarm cell touch the axonal membrane, the outer leaflet of the axolemma is characterized by a series of

circular bands (Figs. 87, 88). These bands bear the imprint of rows of globular subunits arranged at an angle of about 60° with respect to the axis of the band (Fig. 88). Interestingly enough, the outer leaflet of the Schwarm cell membrane

70 . K. H. PFENNINGER

which is in contact with the axolemma is characterized by circular (or spiral) chains of large particles which are in register with the axolemmal bands (Fig. 86). Next to this specialized glial-axonal junction, the nodal axoplasm contains a fairly high density of randomly distributed particles. It has been mentioned before that, in the membranes of the red blood cell, particles could be correlated with glycoproteins (TILLACK et al. 1970, 1971). This exciting result possibly applies for some of the particles of the glycoprotein-rich neuronal membranes as well. It is tempting to ascribe to the particles in the Ranvier's node and the postsynaptic membrane the role of channels for ions. This functional aspect, however, is the subject of speculation. Postsynaptic organelles

The functional meaning of the most important postsynaptic organelle, the density, has been discussed in connection with the problems of

synaptic adherence, intercellular communication, and the structure of the plasmalemma at the axon hillock and the node of Ranvier. It remains to be emphasized that the postsynaptic web seems not to be an indispensable synaptic structure. The spine apparatus (GRAY 1959, 1961) is structurally similar to the so-called subsurface cisterns (ROSENBLUTH 1962) in that it is composed of a stack of flattened membrane sacs separated by dense plates. GRAY and WILLIS (1970) reported strong BIUL staining of the cisternal contents indicating the presence of much cationic material (UL staining in combination with OS04 produces no electron density). Interestingly enough, PALAY et al. (1968) and PETERS et al. (1968) described structures composed of flattened cisterns similar to the spine apparatus in the axon hillock. From the location of this organelle one would argue that these cisterns are in some way concerned with the excitability of the adjacent plasmalemma.

Synaptic Morphology and Cytochemistry . 71

Synopsis The chemical synapse is an intercellular contact where bioelectric information in unidirectionally transferred from one nerve cell to another by the quantal release of neurotransmitter. Synapses are further characterized by a high degree of contact specificity. Cytochemical studies reveal that, at the synaptic region, the inner and the external plasmalemmal coats are specialized to form the so-called synaptic apparatus consisting of the presynaptic dense projections, the cleft material and the postsynaptic density. The paramembraneous densities consist primarily of highly polar protein (with both acidic and basic residues); the synaptic cleft is distinguished from the cytoplasmic densities by its carbohydrate content. The proteinaceous material, and probably the carbohydrates as well, provide the molecular and the structural basis for the mechanism of adhesiveness; this is of polyionic nature rather than covalent or effected by complex binding of calcium. It is a possibility that the two crude subfractions of the cleft material which can be distinguished cytochemically (a putative glycoprotein and a compartment rich in basic residues) represent the complementary pairs for the formation of ionic bridges and that they also participate in a process of recognition between the pre- and the post-synaptic membrane. At least certain neurotransmitters are stored to the greatest extent within the synaptic vesicles. This can be shown not only in biochemical assay but also in situ by means of cytochemical procedures specific for the demonstration of monoamines. In some cases, nerve terminals containing different transmitters are characterized by vesicles of different size. A further difference lies in the variable tendency of the vesicles to become elongated under certain conditions of aldehyde fixation and subsequent osmication. The origin of synaptic vesicles is not clear; there is evidence for both, perikaryal and local (i. e. in the terminal) vesicle formation. It is not known, however, whether vesicles of both ongms participate in transmitter storage and release.

A considerable body of evidence supports the vesicle hypothesis of transmitter release, i. e. the theory that neurohumors are released from vesicles directly into the synaptic cleft. Part of the vesicles are located close to the presynaptic membrane. They are arranged to form a hexagonal pattern fitting into the interspaces of the triagonal dense projection meshwork. The presynaptic dense projections appear to be a most important synaptic feature for they are regularly observed at almost all synaptic sites. Their role, however, in the release process is still unknown. The sites where the vesicles touch the presynaptic membrane are characterized by the lack of a cytoplasmic membrane coat and by the ability to form a specific type of contact with the vesicles. The formation of these attachment sites is dependent upon synaptic activity. The attachment sites could represent a preparatory stage leading to the fusion of the vesicular with the presynaptic membrane (transmitter release by a mechanism of reverse pinocytosis). At present it seems more likely, though, that these intimate vesicle-plasmalemma contacts which were also called "synaptopores" contain a transmembraneous channel for the release of the transmitter. At least in synapses of the central nervous system, the vesicle-plasmalemma contacts seem to be reversible and vesicles may be reused several times. Eventually, irreversible fusion with, and incorporation into, the plasmalemma may serve to eliminate "aged" vesicles. The exact localization of receptors in the postsynaptic membrane and the meaning of the postsynaptic density and other subjunctional structures are not known. The postsynaptic membrane shares a carbohydrate-rich external coat, a thickened cytoplasmic coat, and an aggregation of intramembraneous particles with other"electrogenic" regions of the neuron such as the axon hillock and the node of Ranvier.

Abbreviations used AChE acetylcholinesterase A-PTA aqueous, acidic phosphotungstic acid 81

bismuth iodide impregnation

72 . K. H. PFENNINGER BIUL

bismuth iodide impregnation followed by double staining with uranyl acetate and lead hydroxide

OsUL osmication followed by uranyl and lead double staining

dopamine-(3-oxidase

PTA

phosphotungstic acid

EDTA ethylenediamine tetraacetate

sER

smooth surfaced endoplasmic reticulum

E-PTA ethanolic phosphotungstic acid

sv

synaptic vesicles

FE

U

uranyl acetate staining

UL

uranyl and lead double staining (usually lead hydroxide=plumbite)

ZIO

zinc iodide-osmium tetroxide impregnation

DBO

freeze-fracturing-etching

GABA y-aminobutyric acid

L

lead hydroxide=plumbite staining (in a few cases also used for lead citrate)

ldv

large dense core vesicles

NANA N-acetyl-neuraminic (sialic) acid

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