Journal o f Neurocytology 5, 551-563 (1976)
Acetylcholinesterase activity of synaptic structures in the spinal trigeminal nucleus L E S N I C K E. W E S T R U M and S T E V A N H. B R O D E R S O N Department of Neurological Surgery and Department of Biological Structure, University of Washington, School of Medicine, Seattle, Washington 98195 U.S.A.
Received 17 February 1976; revised 2 April 1976; accepted 27 April 1976
Summary The electron microscope has been used to study the localization of acetylcholinesterase (ACHE) activity in the spinal trigeminal nucleus of normal cats with special emphasis on the distribution near synaptic structures. Reaction product is found around both round and flattened synaptic vesicle-containing axon terminals, particularly in synaptic clefts and often specifically associated with the presynaptic, or less frequently the postsynaptic membrane. The presence of reaction product at these specific sites suggests that these are areas of high AChE activity and that acetylcholine may be important in neurotransmission in these regions.
Introduction There have been a number of studies of the distribution and ultrastructural localization of acetylcholinesterase (ACHE) in the adult vertebrate central nervous system, including synapses (see e.g., Lewis and Shute, 1966; Shute and Lewis, 1966; K~sa, 1968, 1971; Kokko et al., 1969; Haj6s et al., 1970; McDonald and Rasmussen, 1971; Brown and Palay, 1972; Leonieni and Rechardt, 1972; Bridges et al., 1973; Csillik et al., 1973; Kreutzberg and T6th, 1974; Ritter et al., 1974). The authors used the techniques of Koelle and Friedenwald (1949), Karnovsky and Roots (1964), K~isa and Csillik (1966), Lewis and Shute (1966) or individual modifications of these (see e.g., Brzin et al., 1966; Koelle et al., 1974b). Recent reviews by K~sa (1971, 1975), Koelle (1971), Friedenberg and Seligman (1972), Pfenninger (1973), Silver, (1974), Tsuji (1974) and Koelle et al. (1975) have dealt with the various methods, their modification, advantages and particularly the significance of these in relation to synaptic structures in both the central and peripheral nervous systems. We describe here the electron microscopic localization of the AChE reaction product in the adult cat spinal trigeminal nucleus following the method of Karnovsky and Roots (1964). Special emphasis will be placed on the distribution at or close to synaptic structures. In vitro studies and preliminary electron micro9 1976 Chapman and Hall Ltd. Printed in Great Britain
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scopical observations have previously b e e n published (Westrum and Broderson, 1973; B r o d e r s o n et al., 1974). Since p r i m a r y d e a f f e r e n t a t i o n results in synaptic changes (Westrum and Black, 1971; Westrum, 1973, 1974) and s u b s e q u e n t s p o n t a n e o u s , m e m b r a n e - r e l a t e d , physiological h y p e r a c t i v i t y ( A n d e r s o n et al., 1971), the c u r r e n t studies will serve as controls for an investigation o f possible changes in e n z y m e activity or its distribution in the same d e a f f e r e n t a t e d preparations. Materials and m e t h o d s
Tiss~le preparation Normal adult cats were anaesthetized with Halothane and perfused for 20 rain with 1.5 1 of a mixture of fresh 4% formaldehyde and 0.5% purified glutaraldehyde in a 0.2 M phosphate buffer (pH 7.2). The brain stem was removed immediately and immersed in the perfusion solution at 4 ~ C. After 3 h, 2 mm wafers containing the spinal trigeminal nucleus were washed for 12--18 h in several changes of cold 0.2 M phosphate buffer (pH 7.2) containing 0.54% dextrose. Subsequently, the tissue was sectioned into 4 0 - 8 0 #m slices with either a Sorvall TC-2 tissue sectioner or a Vibratome.
Histochemical medium The mixture consisted of 2 mM substrate, 5 mM sodium citrate, 0.3 mM cupric sulfate and 0.5 mM potassium ferricyanide in 50 mM sodium hydrogen maleate buffer, pH 6 (Karnovsky and Roots, 1964). The substrate was acetylthiocholine iodide (AThCh) (National Biochemical Corporation). The inhibitors tetraisopropylpyrophosphoramide (iso-OMPA) and eserine (physostigmine) (Sigma Chemical Company) were employed in control experiments in addition to exposure of the tissue to the incubation medium without substrate throughout the processing.
Histochemical experiments Tissue was incubated in the histochemical medium without substrate for 30 min and transferred to medium containing substrate for 45 min or 189 h. In control experiments, sections were placed in reaction medium, without substrate, containing 10-4 M eserine or 10-4 M iso-OMPA. After 30 min the tissue was transferred to incubation medium containing substrate and iso-OMPA or eserine for 45 man or 189 h. All reactions were earned out at 4 or 25 C. Incubated tissue was rinsed in the phosphate buffer for 30 min at 4 ~ C, osmicated in the same buffer for 89 h and prepared by usual methods for electron microscopy (Westrum and Black, 1971). Ultrathin sections were either stained for 2 min with lead citrate alone, or examined unstained. Additional controls included light microscopical preparations with the same histochemical method to demonstrate motor end plates in diaphragm and tongue muscle from similar preparations. 9
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Observations T h e fine structural preservation o f m o s t o f the tissue is generally g o o d and c o m p a r a b l e to t h a t achieved following the m e t h o d s for o r d i n a r y e l e c t r o n micros c o p y (Westrum and Black, 1971). T h e usual extracellular and intracellular c o m p a r t m e n t s o c c u r t h r o u g h o u t and m e m b r a n e s u b s t r u c t u r e is readily resolvable 9 O n l y very near the surface o f the b l o c k is there m e m b r a n e d i s r u p t i o n with some areas showing surface damage to a d e p t h o f a b o u t 5 - 1 0 / a m . T h e latter is possibly due to the tissue sectioning.
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The reaction product resulting from the method is seen in all of the blocks following both incubation times (45 min and ll/~h), at 4 and 25 ~ C, and is completely eliminated by including eserine in the medium. The distribution is somewhat uneven throughout the thickness of the blocks, but shows only slight differences between preparations exposed to iso-OMPA and those without inhibitors. In each case the reaction product is similar in appearance, occurring as electron-dense deposits (even in unstained sections) composed of particles or granules measuring about 2 5 - 5 0 A in diameter. With the exception of the cell bodies, the product is primarily extracellular, usually occurring in irregular, interrupted accumulations, often filling for variable lengths the intercellular space between apposing membranes. Sometimes, however, the deposits are seen selectively closer to one of the membranes (see below). Since iso-OMPA is known to inhibit non-specific cholinesterase activity (pseudocholinesterase or butyrylcholinesterase) in the concentrations used here, these preparations were studied in most detail and compared with those not exposed to an inhibitor. Reaction product is seen around all of the identifiable neuronal profiles in variable amounts and occasionally between glial processes (Figs. t and 2). The main intracellular distribution is within the nuclear envelope (Fig. 4) and the cisternae of the granular or rough endoplasmic reticulum (RER; e, Fig. 3). Less of the reaction product is seen in the RER of some of the smaller cells of iso-OMPA-treated tissue (Fig. 4). Basement membranes (basal laminae) reacted in most of the uninhibited preparations (b, Fig. 1), but less frequently in iso-OMPA-treated sections (b, Fig. 2). Microtubules, ribosomes, mitochondria and usually Golgi complexes (g, Figs. 3 and 4) were apparently devoid of product. A few large cuboidal crystals (1000 ~ or more in size) appeared randomly distributed over parts of the tissue sections, including the control and eserine-treated sections, and therefore are considered not to be reaction product (see e.g., c, Figs. 4, 6, 7 and 12). A particularly consistent and sometimes heavy reaction is seen around unmyelinated axons (a, Fig. 7), axon terminals, and especially in or near the synaptic cleft (Figs. 5 - 8 and 10). Occasionally thinly myelinated axons have material along the surface of the axolemma, but not within the myelin (Fig. 11). Heavily myelinated axons are usually devoid of reaction product. Both of the two major groups of axon terminals that occur in this region frequently show activity. Axon terminals with either predominantly round synaptic vesicles (R terminals, Figs. 5 and 6) or those with mostly flattened or pleomorphic vesicles (F terminals, Figs. 7 and 8) have the material on the surface and within the synaptic cleft. As is the case for conventional preparations, the contacts of F terminals appear to have a narrower synaptic cleft and less dense postsynaptic thickenings than R terminals (compare Figs. 5 and 8, 6 and 7, also see below). There does not appear to be any reaction product specifically associated with the synaptic vesicles, microtubules or other cytoplasmic structures of the axons or terminals. Within the synaptic cleft the product may irregularly fill the cleft and abut against both the pre- and postsynaptic membranes (Fig. 5). More frequently, however, the
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material is p r e f e r e n t i a l l y a c c u m u l a t e d u p o n one o f the m e m b r a n e s , m o r e o f t e n on the p r e s y n a p t i c m e m b r a n e (Figs. 6 - 8 ) . This selective d i s t r i b u t i o n is m o s t obvious at F terminal contacts (Figs. 7 and 8) and, in regions d e e p e r in the b l o c k w h e r e the reaction p r o d u c t is absent f r o m o t h e r places on the terminal, some o f the p r o d u c t m a y still persist at the p r e s y n a p t i c m e m b r a n e (Fig. 8). T h e r e a c t i o n material in the synaptic cleft is often, b u t n o t always, a c c u m u l a t e d at or n e a r the area o f synaptic m e m b r a n e thickenings and p r e s y n a p t i c vesicle aggregation (Figs. 5 - 8 ) . T h e membrane specializations are s o m e t i m e s less dense t h a n following usual m e t h o d s , owing to the necessarily brief staining, b u t t h e y are usually easily discernible. S o m e examples are also seen w h e r e the r e a c t i o n p r o d u c t is absent f r o m sites with these specializations (x, Fig. 6). In addition, there m a y be material in regions w i t h o u t the synaptic specializations b u t adjacent to t h e m (x, Fig. 7). O n l y i n f r e q u e n t l y is the entire synaptic cleft filled with the reaction p r o d u c t . A p p a r e n t l y n o n - s y n a p t i c
All figures are electron micrographs taken from the cat spinal trigeminal nucleus (pars interpolaris) and except where indicated, are treated with iso-OMPA as described in the text. Unless otherwise indicated, the scale bars represent 0.25 ~tm. Fig. 1. Uninhibited preparation of a capillary segment showing reaction material in a part of the basal lamina (b) and between surrounding glial processes (arrow). C is capillary lumen. Fig. 2. Area similar to Fig. 1, but from an inhibited preparation showing absence of product on basal lamina (b), but some material persisting between glial profiles (arrows). C is capillary lumen. Fig. 3. Uninhibited preparation of a part of a neuron to demonstrate reaction product in RER (e) and relative absence of material from the Golgi complex (g). Arrow indicates product around a flat vesicle terminal (F) contacting the soma. Bar represents 1 #m. Fig. 4. A small cell with less RER which mostly lacks reaction product as does the Golgi complex (g), but which has product persisting in the nuclear envelope (arrows). N is the nucleus and c is crystalline contaminate. Bar represents 1/ma. Fig. 5. A round-vesicle terminal (R) contacting a dendrite (d) with reaction product filling the synaptic cleft at the site of presynaptic vesicle aggregation and asymmetric membrane specialization (arrow). Fig. 6. An R terminal showing irregular distribution of product within the synaptic cleft (arrows) and with absence of material at the site of part of a specialization (x). c is contaminant. Fig. 7. A fiat-vesicle (F) terminal contacting a dendrite (d) with reaction material accumulated at the presynaptic membrane opposite the membrane specialization (arrows). Product is seen at other sites on the terminal including near the contact site (x). Close by small unmyetinated axons (a) are partially surrounded by the material, c are crystals of contaminant. Fig. 8. An F terminal with reaction product selectively on the presynaptic membrane opposite a presumed symmetric contact site (arrow). Fig. 9. Reaction product is shown partially filling a nonsynaptic, symmetrical junction (arrow) between an F terminal and a presumed dendrite (d). Fig. 10. An axo-axonic synaptic contact with an F terminal presynaptic to an R terminal and demonstrating reaction product at the contact (arrows). Uninhibited preparation. Similar distribution is seen also in iso-OMPA-treated material. Fig. 11. A thinly myelinated axon with reaction product localized upon the axolemma (arrows), Fig. 12. A transversely sectioned dendrite (d) without a synaptic contact here, showing reaction product irregularly distributed over its surface (arrows) at regions opposed by glial profiles (g/). c is contaminant.
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appositions or desmosomoid clefts may contain small amounts of the material (arrow, Fig. 9). There are no obvious differences between uninhibited preparations and those treated with iso-OMPA, in the distribution of the reaction product in and around the synapses. In addition to both R and F classes of terminals showing the above reactions, all types of synaptic contacts or appositions are involved. Synapses onto dendrites of all sizes (Figs. 5-8), cell bodies (Fig. 3) and axo-axonic contacts (Fig. 10) may each show the distribution described above. Although precise estimates have not been made, it does appear that F terminals onto dendrites more consistently show activity, especially in the synaptic cleft and on the presynaptic membrane, than do the nearby R terminals. The reaction product may also occur on the surface of dendrites without synapses in the section, or between glial and neuronal profiles (Fig. 12; see also Fig. 11 in Broderson et al., 1974). Discussion
The general distribution of the reaction products resulting from the AChE enzyme activity is similar in many respects to that described by others. The present findings confirm the occurrence of the material around blood vessels and in neuronal cell bodies, particularly in the RER and nuclear envelope. The reduction of the amount of activity around blood vessels in the iso-OMPA preparations correlates well with the work of Brown and Palay (1972), who also describe the persistence of the reaction in small amounts even with the inhibitor (see also Kreutzberg and Kaiya, 1974). Most previous authors agree that the cisternae of the RER are the principal ceil body sites of AChE activity, but there is some variability in the findings with respect to the nuclear envelope and especially the Golgi complex (Brzin et aL, 1966; Lewis and Shute, 1966; Kokko et al., 1969; Pannese et al., 1974). The frequent absence of the reaction product from the Golgi complexes here confirms these reports. The reduction in the amount of material in the RER of many of the cells in iso-OMPA-treated preparations suggests that the material was non-specific cholinesterase (see e.g., Ballard and Jones, 1971) and may be indicative of a particular cell population in this area (see e.g., Csillik et aL, 1973). The latter possibility obviously requires additional studies. The occurrence of reaction products of AChE activity in association with other cytoplasmic organelles, as reported by some workers in different regions, could not be confirmed here. Microtubules, ribosomes, smooth reticulum, vesicles (including dense-cored and synaptic vesicles) and mitochondria have all been identified as structures displaying reaction product (Barrnett, 1962; Miledi, 1964; Schlaepfer and Torack, 1966; Kokko et al., 1969; de Lorenzo et al., 1969; K~sa, 1971; Mazza et al., 1973; Pannese et al., 1974). However, none of these structures contained the product in the present study. AChE activity has frequently been observed in association with axons, usually at the outer surface of the axolemma, although deposits within the cytoplasm and in
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relation to certain organelles have been reported in some cases (Bloom and Barrnett, 1966; de Lorenzo et al., 1969; Tennyson and Brzin, 1970; Villegas and Villegas, 1974). Intracellular axoplasmic localizations were not apparent in our preparations. The thinly myelinated axons more commonly have the reaction material than heavily myelinated ones, in the present study. This may be indicative of less adequate penetration of chemicals through the thicker myelin or a selective distribution of the enzyme on this class of axon. It has been pointed out, however, that although axons may show such reaction product, they may not, in fact, be cholinergic in function (Gwyn and Flumerfelt, 1971; Koelle, 1971; Barajas et al., 1974). Localization of the enzyme activity around axon terminals and particularly within the synaptic clefts was of particular interest in this investigation. A number of workers in recent years have provided considerable information about the distribution of AChE at motor end plate, and at both peripheral and central synapses (Barrnett, 1962; Smith and Treherne, 1965; Bloom and Barrnett, 1966; Brzin et al., 1966, 1975; Lewis and Shute, 1966; Davis and Koelle, 1967; K~sa, I968, 1971; Koelle, 1969, 1971; Kokko et al., 1969; Haj6s etal., 1970;McDonald and Rasmussen, 1971; Brown and Palay, 1972; Bogusch, 1973; Csillik et al., 1973; Koelle et al., 1974a; Ritter et aI., 1974; Tsuji, 1974; see also review by Pfenninger, 1973). These reports primarily indicate an extracellular distribution around the surface of the axon terminals, usually with variable amounts of the product in the cleft. A few reports, however, describe product in postsynaptic cytoplasm (Shute and Lewis, 1966; Ter~iv~iinen, 1969) or localization within presynaptic vesicles of peripheral terminals (Bloom and Barrnett, 1966; Barajas et al., 1974). Our observations generally confirm the reports of extracellular distribution of AChE activity, but do not support reports of intraceUular localization at terminals. In this study the synaptic cleft localization of AChE activity is variable. Sometimes there is complete filling of the cleft as reported also by others (Brzin et al., 1966; Lewis and Shute, 1966; Kokko etal., 1969; Brown and Palay, 1972), and other times there is an irregular distribution within the cleft of the reaction product (see e.g., Brzin et al., 1966; Lewis and Shute, 1966; Kokko et al., 1969). Occasionally, there is no reaction product in the synaptic cleft, although there may be product elsewhere on the terminal, in the particular section (see also Bridges et al., 1973; Barajas et al., 1974). Serial sections would be required to determine if product does occur in clefts of such terminals at another level (see e.g., Pannese et al., 1974). Of particular interest is the finding here that the reaction product frequently is preferentially accumulated at the presynaptic membrane and less so at the postsynaptic one. Similar distribution of reaction product at presynaptic membranes is also described by K/tsa (1968), Koelle (1969) and Ritter et al. (1974). Others, however, have reported comparatively greater AChE activity at postsynaptic membranes (Bloom and Barrnett, 1966; Davis and Koelle, 1967; Koelle, 1969, 1971) or an equal distribution at both pre- and postsynaptic membranes, but
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sometimes with absence of product in the cleft (Shute and Lewis, 1966; Koelle, 1971; Bogusch, 1973; Koelle et al., 1974b). Some investigators observed reaction product specifically in the cleft near vesicle accumulations (see e.g., Smith and Treherne, 1965; Lewis and Shute, 1966), and although this is occasionally seen in our preparations, it is not a consistent finding. In addition, K~isa's (1968) report of AChE reaction product upon 'intersynaptic fibrils' could not be confirmed in our preparations. The irregular, extracellular distribution of reaction product in the cleft with preferential localization at the presynaptic membrane (discounting problems such as postfixation diffusion), might be interpreted as revealing the sites of the greatest transmitter enzyme activity, especially in the iso-OMPA-inhibited preparations. The absence of material at particular sites may suggest lack of enzyme, or of its activity, or amounts too small to give a visible reaction product, each possibly the result of the enzyme being depleted to variable degrees. However, it cannot be unequivocally shown that the absence of reaction product definitely indicates absence of enzymatic activity as is the case for all such procedures. The presence of reaction product in certain areas could also represent an AChE distribution which might provide a barrier against transmitter accumulation at those sites (i.e., 'cross-talk' or leakage of transmitter; see e.g., Barajas et al., 1974). In this respect, the frequent occurrence of reaction products around flat-vesicle terminals, not usually thought associated with cholinergic systems, may mean that acetylcholine (ACh) is involved in the functions of the F terminals or that the enzyme is there as a safeguard against the presence of the transmitter or its activity. Non-cholinergic nerves and terminals have been associated with AChE activity and previous reports have also shown reaction product in relation to flat vesicle terminals (Koelle, 1971; McDonald and Rasmussen, 1971; Pfenninger, 1973; Ritter et al., 1974). The possibility that some of these F terminals are former R terminals in which the vesicles have flattened during the incubations, must be considered (Bodian, 1970). However, although the brief section staining does not always permit easy identification of the type of postsynaptic thickening, the synaptic clefts are of the dimensions of F terminal contacts. In addition, it is not possible to make accurate determinations of proportions of classes of terminals showing this reaction, as the reaction is somewhat irregular in deeper parts of the blocks. The latter would be useful to compare ratios of F to R terminals in these preparations with those seen in ordinary preparations (Westrum, 1974). In any case, the clear and relatively consistent deposition of product on presynaptic membranes, especially in regions otherwise totally devoid of reaction product, is indicative of AChE and a possible role for ACh at these sites.
Acknowledgements This work was supported in part by USPHS Grants NS-09678 and NS-04053 from the National Institute of Neurological and Communicative Disorders and Stroke
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(USPHS/DHEW). The authors gratefully acknowledge this support and wish to thank Maria Glaser, Ann Harris and Doris E. Ringer for valuable technical and secretarial assistance. References ANDERSON, L.S., BLACK, R.G., ABRAHAM, J. and WARD, A. A., JR. (1971) Neuronal hyperactivity in experimental trigeminal deafferentation. Journal ofNeurosurgery 3 5 , 4 4 4 - 5 2 . BALLARD, K.J. and JONES, J. V. (1971) The fine structural localization of cholinesterase in the carotid body of the cat. Journal of Physiology (London) 219, 7 4 7 - 5 3 . BARAJAS, L., SILVERMAN, A.J. and MULLER, J. (1974) Ultrastructural localization of acetylcholinesterase in renal nerves. Journal of Ultrastructure Research 49, 297-311. BARRNETT, R. J. (1962) The fine structural localization of acetylcholinesterase at the myoneural junction. Journal of Cell Biology 12, 2 4 7 - 6 2 . BLOOM, F. E. and BARRNETT, R. J. (1966) Fine structural localization of acetylcholinesterase in electroplaque of the electric eel. Journal of Cell Biology 29, 4 7 5 - 9 5 . BODIAN, D. (1970) An electron microscopic characterization of classes of synaptic vesicles by means of controlled aldehyde fixation. Journal of Cell Biology 44, 1 1 5 - 2 4 . BOGUSCH, G. (1973) Zur Lokalisation der Cholinesterase-aktivit~t in der motorischen Endplatte mit der Kupferferrocyanidemethode. Histocbemie 33, 3 9 - 4 6 . BRIDGES, T. E., FISHER, A. W., GOSBEE, J. L., LEDERIS, K. and SANTOLAYA, R. C. (1973) Acetylcholine and cholinesterase (assays and light- and electron microscopical histochemistry) in different parts of the pituitary of rat, rabbit and domestic pig. Zeitscbriftfur Zellforschung und mikroskopiscbe Anatomic 136, 1 - 1 8 . BRODERSON, S. H., WESTRUM, L. E. and SUTTON, A. E. (1974)Studies of the direct coloring thiocholine method for localizing cholinesterase activity. Histochemistry 40, 13--23. BROWN, W. J. and PALAY, S. L. (1972) Acetylcholinesterase activity in certain glomeruli and Golgi cells of the granular layer of the rat cerebetlar cortex. Zeitscbrift fz'~r Anatomie und Entwicklungsgescbicbte 137, 317--34. BRZIN, M., TENNYSON, V.M. and DUFFY, P.E. (1966) Acetylcholinesterase in frog sympathetic and dorsal root ganglia. A study by electron microscope cytochemistry and microgasometric analysis with the magnetic diver. Journal of Cell Biology 31, 2 1 5 - 4 2 . BRZIN, M., TENNYSON, V.M. and DETTBARN, W.-D. (1975) Cytochemical localization of cholinesterase activity at the giant synapse of the squid. Histochemistry 43, 3 0 5 - 1 1 . CSILLIK, B., TOTH, L. and KARCSU, S. (1973) Acetylcholinesterase activity of Renshaw elements and Renshaw bulbs. A light- and electron-histochemical study. Journal of Neurocytology 2,441--55. DAVIS, R. and KOELLE, G. B. (1967) Electron microscopic localization of acetylcholinesterase and non-specific cholinesterase at the neuromuscular junction by the gold-thiocholine and gold-thiolacetic acid methods. Journal of Cell Biology 34, 1 5 7 - 7 1 . FRIEDENBERG, R.M. and SELIGMAN, A.M. (1972) Acetylcholinesterase at the myoneural junction: cytochemical ultrastructure and some biochemical considerations. Journal of Histochemistry and Cytocbemistry 20, 7 7 1 - 9 2 . GWYN, D. G. and FLUMERFELT, B. A. (1971) Acetylcholinesterase in non-cholinergic neurones: A histochemical study of dorsal root ganglion cells in the rat. Brain Research 34, 193--98. HAJ~S, F., PRIYMAK, E.K. and KERPEL-FRONIUS, S. (1970) The electron microscopic demonstration of acetylcholinesterase activity in some cholinergic and non-cholinergic synapses of the rat brain. Acta Histocbemica 35, 114--22. KARNOVSKY, M.J. and ROOTS, L. (1964) A 'direct-coloring' thiocholine method for cholinesterases. Journal of Histochemistry and Cytocbemistry 12, 2 1 9 - 2 2 .
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K.3iSA, P (1968) Ultrastructural localization of acetylcholinesterase in the cerebellar cortex with special reference to the intersynaptic organelles. Histocbemie 14, 161-7. K3,SA, P. (1971) Ultrastructural localization of choline acetyltransferase and acetylcholinesterase in central and peripheral nervous tissue. Progress in Brain Research 34, 3 3 7 - 4 4 . K~ SA, P. (1975) Histochemistry of choline acetyltransferase. In Cbolinergic Mechanisms (Edited by WASER, P. G.), pp. 271--81, New York: Raven Press. KA.SA, P. and CSILLIK, B. (1966) Electron microscopic localization of cholinesterase by a copper-lead-thiocholine technique. Journal ofNeurocbemistry 13, 1345-9. KOELLE, G.B. (1969) Significance of acetylcholinesterase in central synaptic transmission. Federation Proceedings 28, 9 5 - 1 0 0 . KOELLE, G. B. (1971) Current concepts of synaptic structure and function. Annals of the New York Academy of Sciences 183, 5 - 2 0 . KOELLE, G.B. and FRIEDENWALD, J.S. (1949) A histochemical method for localizing cholinesterase activity. Proceedings of the Society for Experimental Biology and Medicine 70, 617-22. KOELLE, G. B., DAVIS, R. and KOELLE, W. A. (1974a) Effects of aldehyde fixation and of preganglionic denervation on acetylcholinesterase and butyrocholinesterase of cat autonomic ganglia. Journal of Histochemistry and Cytochemistry 22, 2 4 4 - 5 1 . KOELLE, G.B., DAVIS, R., KOELLE, W.A., SMYRL, E.G. and FINE, A.V. (1975) The electron microscopic localization of acetylcholinesterase and pseudocholinesterase in autonomic ganglia. In Cbolinergic Mecbanisms (edited by WASER, P. G.), pp. 251--55, New York: Raven Press. KOELLE, G.B., DAVIS, R., SMYRL, E.G. and FINE, A.V. (1974b) Refinement of the bis-(thioacetoxy)aurate (1) method for electron microscopic localization of acetylcholinesterase and non-specific cholinesterase. Journal of Histocbemistry and Cytocbemistry 22, 2 5 2 - 5 9 . KOKKO, A., MAUTNER, H.G. and BARRNETT, R.J. (1969) Fine structural localization of acetylcholinesterase using acetyl-/3-methylthiocholine and acetylselenocholine as substrates. Journal of Histocbemistry and Cytocbemistry 17, 6 2 5 - 4 0 . KREUTZBERG, G.W. and KAIYA, H. (1974) Exogenous acetylcholinesterase as a tracer for extracellular pathways in the brain. Histocbemistry 42, 2 3 3 - 3 7 . KREUTZBERG, G.W. and TOTH, L. (1974) Dendritic secretion: A way for the neuron to communicate with the vasculature. Naturwissenschaften 61, 37-9. LEONIENI, J. and RECHARDT, L. (1972) The effect of dehydration on the ultrastructure of cholinesterase activity of the subcommissural organ in the rat. Zeitscbrift fur ZellJbrscbung und mikroskopiscbe Anatomie 133, 377-87. LEWIS, P.R. and SHUTE, C. C. D. (1966) The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. Journal of Cell Science 1, 381-90. DE LORENZO, A. J. D., DETTBARN, W. D. and BRZIN, M. (1969) Fine structural localization of acetylcholinesterase in single axons. Journal of Ultrastructure Researcb 28, 2 7 - 4 0 . MCDONALD, D. M. and RASMUSSEN, G. L. (1971) Ultrastructural characteristics of synaptic endings in the cochlear nucleus having acetylcholinesterase activity. Brain Research 28, 1 - 1 8 . MAZZA, J.P., HANKER, J.S. and DIXON, A.D. (1973) Ultrastructural localization of cholinesterase activity in the trigeminal ganglion of the rat. Journal of Anatomy (London) 115, 65-78. MILEDI, R. (1964) Electron-microscopical localization of products from histochemical reactions used to detect cholinesterase in muscle. Nature 204, 2 9 3 - 9 5 . PANNESE, E., LUCIANO, L., IURATO, S. and REALE, E. (1974) The localization of acetylcholinesterase activity in the spinal ganglia of the adult fowl studied by electron microscope histochemistry. Histochemistry 39, 1 - 1 3 . PFENNINGER, K.H. (1973) Synaptic morphology and cytochemistry. Progress in Histocbemistry and Cytocbemistry 5, 1 - 8 6 .
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RITTER, V. J., WENZEL, J., DANIEL, H. and DUWE, G. (1974) Zur elektronenmikroskopischen Darstellung Acetylcholinesterase in Neurones und Synapsen des zentralen und peripheren Nervensystems. Acta histocbemica 49, 1 7 6 - 2 0 3 . SCHLAEPFER, W. W. and T O R A C K , R. M. (1966) The ultrastructural localization of cholinesterase activity in the sciatic nerve of the rat. Journal of Histochemistry and Cytocbemistry 14, 369-78. SHUTE, C. C. D. and LEWIS, P.R. (1966) Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurones in the hippocampal formation of the rat. Zeitscbrift fi~r'Zellforscbung 69, 3 3 4 - 4 3 . SILVER, A. (1974) The Biology ofCbolinesterases, p. 596. Amsterdam: North Holland. SMITH, D.S. and TREHERNE, J.E. (1965) The electron microscopic localization of cholinesterase activity in the central nervous system of an insect, Periplaneta americana. Journal of Cell Biology 26, 4 4 5 - 6 5 . TENNYSON, V. M. and BRZIN, M. (1970) The appearance of acetylcholinesterase in the dorsal root neuroblast of the rabbit embryo. Journal of Cell Biology 46, 6 4 - 8 0 . T E R A V A I N E N , H. (1969) Localization of acetylcholinesterase in the rat myoneural junction. Histocbemie 17, 1 6 2 - 6 9 . TSUJI, S. (1974) On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activity. Histocbemistry 42, 9 9 - 1 1 0 . VILLEGAS, G. M. and VILLEGAS, J. (1974) Acetylcholinesterase localization in the giant nerve fiber of the squid. Journal of Ultrastructure Researcb 46, 1 4 9 - 6 3 . WESTRUM, L. E. (1973) Early forms of terminal degeneration in the spinal trigeminal nucleus following rhizotomy. Journal of Neurocytology 2, 189-215. WESTRUM, L. E. (1974) Electron microscopy of deafferentation in spinal trigeminal nucleus. In Advances in Neurology, Vol. 4 International Symposium on Pain (edited by BONICA, J. J.) pp. 53-60. New York: Raven Press. WESTRUM, L. E. and BLACK, R. G. (1971)Fine structural aspects of the synaptic organization of the spinal trigeminal nucleus (pars interpolaris) of the cat. Brain Researcb 2 5 , 2 6 5 - 8 7 . WESTRUM, L. E. and BRODERSON, S. H. (1973) Electron microscopy of cholinesterase activity of synaptic structures in spinal trigeminal nucleus. Journal of Cell Biology 59, 362.
Acetylcholinesterase activity of synaptic structures in the spinal trigeminal nucleus L E S N I C K E. W E S T R U M and S T E V A N H. B R O D E R S O N Department of Neurological Surgery and Department of Biological Structure, University of Washington, School of Medicine, Seattle, Washington 98195 U.S.A.
Received 17 February 1976; revised 2 April 1976; accepted 27 April 1976
Summary The electron microscope has been used to study the localization of acetylcholinesterase (ACHE) activity in the spinal trigeminal nucleus of normal cats with special emphasis on the distribution near synaptic structures. Reaction product is found around both round and flattened synaptic vesicle-containing axon terminals, particularly in synaptic clefts and often specifically associated with the presynaptic, or less frequently the postsynaptic membrane. The presence of reaction product at these specific sites suggests that these are areas of high AChE activity and that acetylcholine may be important in neurotransmission in these regions.
Introduction There have been a number of studies of the distribution and ultrastructural localization of acetylcholinesterase (ACHE) in the adult vertebrate central nervous system, including synapses (see e.g., Lewis and Shute, 1966; Shute and Lewis, 1966; K~sa, 1968, 1971; Kokko et al., 1969; Haj6s et al., 1970; McDonald and Rasmussen, 1971; Brown and Palay, 1972; Leonieni and Rechardt, 1972; Bridges et al., 1973; Csillik et al., 1973; Kreutzberg and T6th, 1974; Ritter et al., 1974). The authors used the techniques of Koelle and Friedenwald (1949), Karnovsky and Roots (1964), K~isa and Csillik (1966), Lewis and Shute (1966) or individual modifications of these (see e.g., Brzin et al., 1966; Koelle et al., 1974b). Recent reviews by K~sa (1971, 1975), Koelle (1971), Friedenberg and Seligman (1972), Pfenninger (1973), Silver, (1974), Tsuji (1974) and Koelle et al. (1975) have dealt with the various methods, their modification, advantages and particularly the significance of these in relation to synaptic structures in both the central and peripheral nervous systems. We describe here the electron microscopic localization of the AChE reaction product in the adult cat spinal trigeminal nucleus following the method of Karnovsky and Roots (1964). Special emphasis will be placed on the distribution at or close to synaptic structures. In vitro studies and preliminary electron micro9 1976 Chapman and Hall Ltd. Printed in Great Britain
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scopical observations have previously b e e n published (Westrum and Broderson, 1973; B r o d e r s o n et al., 1974). Since p r i m a r y d e a f f e r e n t a t i o n results in synaptic changes (Westrum and Black, 1971; Westrum, 1973, 1974) and s u b s e q u e n t s p o n t a n e o u s , m e m b r a n e - r e l a t e d , physiological h y p e r a c t i v i t y ( A n d e r s o n et al., 1971), the c u r r e n t studies will serve as controls for an investigation o f possible changes in e n z y m e activity or its distribution in the same d e a f f e r e n t a t e d preparations. Materials and m e t h o d s
Tiss~le preparation Normal adult cats were anaesthetized with Halothane and perfused for 20 rain with 1.5 1 of a mixture of fresh 4% formaldehyde and 0.5% purified glutaraldehyde in a 0.2 M phosphate buffer (pH 7.2). The brain stem was removed immediately and immersed in the perfusion solution at 4 ~ C. After 3 h, 2 mm wafers containing the spinal trigeminal nucleus were washed for 12--18 h in several changes of cold 0.2 M phosphate buffer (pH 7.2) containing 0.54% dextrose. Subsequently, the tissue was sectioned into 4 0 - 8 0 #m slices with either a Sorvall TC-2 tissue sectioner or a Vibratome.
Histochemical medium The mixture consisted of 2 mM substrate, 5 mM sodium citrate, 0.3 mM cupric sulfate and 0.5 mM potassium ferricyanide in 50 mM sodium hydrogen maleate buffer, pH 6 (Karnovsky and Roots, 1964). The substrate was acetylthiocholine iodide (AThCh) (National Biochemical Corporation). The inhibitors tetraisopropylpyrophosphoramide (iso-OMPA) and eserine (physostigmine) (Sigma Chemical Company) were employed in control experiments in addition to exposure of the tissue to the incubation medium without substrate throughout the processing.
Histochemical experiments Tissue was incubated in the histochemical medium without substrate for 30 min and transferred to medium containing substrate for 45 min or 189 h. In control experiments, sections were placed in reaction medium, without substrate, containing 10-4 M eserine or 10-4 M iso-OMPA. After 30 min the tissue was transferred to incubation medium containing substrate and iso-OMPA or eserine for 45 man or 189 h. All reactions were earned out at 4 or 25 C. Incubated tissue was rinsed in the phosphate buffer for 30 min at 4 ~ C, osmicated in the same buffer for 89 h and prepared by usual methods for electron microscopy (Westrum and Black, 1971). Ultrathin sections were either stained for 2 min with lead citrate alone, or examined unstained. Additional controls included light microscopical preparations with the same histochemical method to demonstrate motor end plates in diaphragm and tongue muscle from similar preparations. 9
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Observations T h e fine structural preservation o f m o s t o f the tissue is generally g o o d and c o m p a r a b l e to t h a t achieved following the m e t h o d s for o r d i n a r y e l e c t r o n micros c o p y (Westrum and Black, 1971). T h e usual extracellular and intracellular c o m p a r t m e n t s o c c u r t h r o u g h o u t and m e m b r a n e s u b s t r u c t u r e is readily resolvable 9 O n l y very near the surface o f the b l o c k is there m e m b r a n e d i s r u p t i o n with some areas showing surface damage to a d e p t h o f a b o u t 5 - 1 0 / a m . T h e latter is possibly due to the tissue sectioning.
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The reaction product resulting from the method is seen in all of the blocks following both incubation times (45 min and ll/~h), at 4 and 25 ~ C, and is completely eliminated by including eserine in the medium. The distribution is somewhat uneven throughout the thickness of the blocks, but shows only slight differences between preparations exposed to iso-OMPA and those without inhibitors. In each case the reaction product is similar in appearance, occurring as electron-dense deposits (even in unstained sections) composed of particles or granules measuring about 2 5 - 5 0 A in diameter. With the exception of the cell bodies, the product is primarily extracellular, usually occurring in irregular, interrupted accumulations, often filling for variable lengths the intercellular space between apposing membranes. Sometimes, however, the deposits are seen selectively closer to one of the membranes (see below). Since iso-OMPA is known to inhibit non-specific cholinesterase activity (pseudocholinesterase or butyrylcholinesterase) in the concentrations used here, these preparations were studied in most detail and compared with those not exposed to an inhibitor. Reaction product is seen around all of the identifiable neuronal profiles in variable amounts and occasionally between glial processes (Figs. t and 2). The main intracellular distribution is within the nuclear envelope (Fig. 4) and the cisternae of the granular or rough endoplasmic reticulum (RER; e, Fig. 3). Less of the reaction product is seen in the RER of some of the smaller cells of iso-OMPA-treated tissue (Fig. 4). Basement membranes (basal laminae) reacted in most of the uninhibited preparations (b, Fig. 1), but less frequently in iso-OMPA-treated sections (b, Fig. 2). Microtubules, ribosomes, mitochondria and usually Golgi complexes (g, Figs. 3 and 4) were apparently devoid of product. A few large cuboidal crystals (1000 ~ or more in size) appeared randomly distributed over parts of the tissue sections, including the control and eserine-treated sections, and therefore are considered not to be reaction product (see e.g., c, Figs. 4, 6, 7 and 12). A particularly consistent and sometimes heavy reaction is seen around unmyelinated axons (a, Fig. 7), axon terminals, and especially in or near the synaptic cleft (Figs. 5 - 8 and 10). Occasionally thinly myelinated axons have material along the surface of the axolemma, but not within the myelin (Fig. 11). Heavily myelinated axons are usually devoid of reaction product. Both of the two major groups of axon terminals that occur in this region frequently show activity. Axon terminals with either predominantly round synaptic vesicles (R terminals, Figs. 5 and 6) or those with mostly flattened or pleomorphic vesicles (F terminals, Figs. 7 and 8) have the material on the surface and within the synaptic cleft. As is the case for conventional preparations, the contacts of F terminals appear to have a narrower synaptic cleft and less dense postsynaptic thickenings than R terminals (compare Figs. 5 and 8, 6 and 7, also see below). There does not appear to be any reaction product specifically associated with the synaptic vesicles, microtubules or other cytoplasmic structures of the axons or terminals. Within the synaptic cleft the product may irregularly fill the cleft and abut against both the pre- and postsynaptic membranes (Fig. 5). More frequently, however, the
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material is p r e f e r e n t i a l l y a c c u m u l a t e d u p o n one o f the m e m b r a n e s , m o r e o f t e n on the p r e s y n a p t i c m e m b r a n e (Figs. 6 - 8 ) . This selective d i s t r i b u t i o n is m o s t obvious at F terminal contacts (Figs. 7 and 8) and, in regions d e e p e r in the b l o c k w h e r e the reaction p r o d u c t is absent f r o m o t h e r places on the terminal, some o f the p r o d u c t m a y still persist at the p r e s y n a p t i c m e m b r a n e (Fig. 8). T h e r e a c t i o n material in the synaptic cleft is often, b u t n o t always, a c c u m u l a t e d at or n e a r the area o f synaptic m e m b r a n e thickenings and p r e s y n a p t i c vesicle aggregation (Figs. 5 - 8 ) . T h e membrane specializations are s o m e t i m e s less dense t h a n following usual m e t h o d s , owing to the necessarily brief staining, b u t t h e y are usually easily discernible. S o m e examples are also seen w h e r e the r e a c t i o n p r o d u c t is absent f r o m sites with these specializations (x, Fig. 6). In addition, there m a y be material in regions w i t h o u t the synaptic specializations b u t adjacent to t h e m (x, Fig. 7). O n l y i n f r e q u e n t l y is the entire synaptic cleft filled with the reaction p r o d u c t . A p p a r e n t l y n o n - s y n a p t i c
All figures are electron micrographs taken from the cat spinal trigeminal nucleus (pars interpolaris) and except where indicated, are treated with iso-OMPA as described in the text. Unless otherwise indicated, the scale bars represent 0.25 ~tm. Fig. 1. Uninhibited preparation of a capillary segment showing reaction material in a part of the basal lamina (b) and between surrounding glial processes (arrow). C is capillary lumen. Fig. 2. Area similar to Fig. 1, but from an inhibited preparation showing absence of product on basal lamina (b), but some material persisting between glial profiles (arrows). C is capillary lumen. Fig. 3. Uninhibited preparation of a part of a neuron to demonstrate reaction product in RER (e) and relative absence of material from the Golgi complex (g). Arrow indicates product around a flat vesicle terminal (F) contacting the soma. Bar represents 1 #m. Fig. 4. A small cell with less RER which mostly lacks reaction product as does the Golgi complex (g), but which has product persisting in the nuclear envelope (arrows). N is the nucleus and c is crystalline contaminate. Bar represents 1/ma. Fig. 5. A round-vesicle terminal (R) contacting a dendrite (d) with reaction product filling the synaptic cleft at the site of presynaptic vesicle aggregation and asymmetric membrane specialization (arrow). Fig. 6. An R terminal showing irregular distribution of product within the synaptic cleft (arrows) and with absence of material at the site of part of a specialization (x). c is contaminant. Fig. 7. A fiat-vesicle (F) terminal contacting a dendrite (d) with reaction material accumulated at the presynaptic membrane opposite the membrane specialization (arrows). Product is seen at other sites on the terminal including near the contact site (x). Close by small unmyetinated axons (a) are partially surrounded by the material, c are crystals of contaminant. Fig. 8. An F terminal with reaction product selectively on the presynaptic membrane opposite a presumed symmetric contact site (arrow). Fig. 9. Reaction product is shown partially filling a nonsynaptic, symmetrical junction (arrow) between an F terminal and a presumed dendrite (d). Fig. 10. An axo-axonic synaptic contact with an F terminal presynaptic to an R terminal and demonstrating reaction product at the contact (arrows). Uninhibited preparation. Similar distribution is seen also in iso-OMPA-treated material. Fig. 11. A thinly myelinated axon with reaction product localized upon the axolemma (arrows), Fig. 12. A transversely sectioned dendrite (d) without a synaptic contact here, showing reaction product irregularly distributed over its surface (arrows) at regions opposed by glial profiles (g/). c is contaminant.
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appositions or desmosomoid clefts may contain small amounts of the material (arrow, Fig. 9). There are no obvious differences between uninhibited preparations and those treated with iso-OMPA, in the distribution of the reaction product in and around the synapses. In addition to both R and F classes of terminals showing the above reactions, all types of synaptic contacts or appositions are involved. Synapses onto dendrites of all sizes (Figs. 5-8), cell bodies (Fig. 3) and axo-axonic contacts (Fig. 10) may each show the distribution described above. Although precise estimates have not been made, it does appear that F terminals onto dendrites more consistently show activity, especially in the synaptic cleft and on the presynaptic membrane, than do the nearby R terminals. The reaction product may also occur on the surface of dendrites without synapses in the section, or between glial and neuronal profiles (Fig. 12; see also Fig. 11 in Broderson et al., 1974). Discussion
The general distribution of the reaction products resulting from the AChE enzyme activity is similar in many respects to that described by others. The present findings confirm the occurrence of the material around blood vessels and in neuronal cell bodies, particularly in the RER and nuclear envelope. The reduction of the amount of activity around blood vessels in the iso-OMPA preparations correlates well with the work of Brown and Palay (1972), who also describe the persistence of the reaction in small amounts even with the inhibitor (see also Kreutzberg and Kaiya, 1974). Most previous authors agree that the cisternae of the RER are the principal ceil body sites of AChE activity, but there is some variability in the findings with respect to the nuclear envelope and especially the Golgi complex (Brzin et aL, 1966; Lewis and Shute, 1966; Kokko et al., 1969; Pannese et al., 1974). The frequent absence of the reaction product from the Golgi complexes here confirms these reports. The reduction in the amount of material in the RER of many of the cells in iso-OMPA-treated preparations suggests that the material was non-specific cholinesterase (see e.g., Ballard and Jones, 1971) and may be indicative of a particular cell population in this area (see e.g., Csillik et aL, 1973). The latter possibility obviously requires additional studies. The occurrence of reaction products of AChE activity in association with other cytoplasmic organelles, as reported by some workers in different regions, could not be confirmed here. Microtubules, ribosomes, smooth reticulum, vesicles (including dense-cored and synaptic vesicles) and mitochondria have all been identified as structures displaying reaction product (Barrnett, 1962; Miledi, 1964; Schlaepfer and Torack, 1966; Kokko et al., 1969; de Lorenzo et al., 1969; K~sa, 1971; Mazza et al., 1973; Pannese et al., 1974). However, none of these structures contained the product in the present study. AChE activity has frequently been observed in association with axons, usually at the outer surface of the axolemma, although deposits within the cytoplasm and in
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relation to certain organelles have been reported in some cases (Bloom and Barrnett, 1966; de Lorenzo et al., 1969; Tennyson and Brzin, 1970; Villegas and Villegas, 1974). Intracellular axoplasmic localizations were not apparent in our preparations. The thinly myelinated axons more commonly have the reaction material than heavily myelinated ones, in the present study. This may be indicative of less adequate penetration of chemicals through the thicker myelin or a selective distribution of the enzyme on this class of axon. It has been pointed out, however, that although axons may show such reaction product, they may not, in fact, be cholinergic in function (Gwyn and Flumerfelt, 1971; Koelle, 1971; Barajas et al., 1974). Localization of the enzyme activity around axon terminals and particularly within the synaptic clefts was of particular interest in this investigation. A number of workers in recent years have provided considerable information about the distribution of AChE at motor end plate, and at both peripheral and central synapses (Barrnett, 1962; Smith and Treherne, 1965; Bloom and Barrnett, 1966; Brzin et al., 1966, 1975; Lewis and Shute, 1966; Davis and Koelle, 1967; K~sa, I968, 1971; Koelle, 1969, 1971; Kokko et al., 1969; Haj6s etal., 1970;McDonald and Rasmussen, 1971; Brown and Palay, 1972; Bogusch, 1973; Csillik et al., 1973; Koelle et al., 1974a; Ritter et aI., 1974; Tsuji, 1974; see also review by Pfenninger, 1973). These reports primarily indicate an extracellular distribution around the surface of the axon terminals, usually with variable amounts of the product in the cleft. A few reports, however, describe product in postsynaptic cytoplasm (Shute and Lewis, 1966; Ter~iv~iinen, 1969) or localization within presynaptic vesicles of peripheral terminals (Bloom and Barrnett, 1966; Barajas et al., 1974). Our observations generally confirm the reports of extracellular distribution of AChE activity, but do not support reports of intraceUular localization at terminals. In this study the synaptic cleft localization of AChE activity is variable. Sometimes there is complete filling of the cleft as reported also by others (Brzin et al., 1966; Lewis and Shute, 1966; Kokko etal., 1969; Brown and Palay, 1972), and other times there is an irregular distribution within the cleft of the reaction product (see e.g., Brzin et al., 1966; Lewis and Shute, 1966; Kokko et al., 1969). Occasionally, there is no reaction product in the synaptic cleft, although there may be product elsewhere on the terminal, in the particular section (see also Bridges et al., 1973; Barajas et al., 1974). Serial sections would be required to determine if product does occur in clefts of such terminals at another level (see e.g., Pannese et al., 1974). Of particular interest is the finding here that the reaction product frequently is preferentially accumulated at the presynaptic membrane and less so at the postsynaptic one. Similar distribution of reaction product at presynaptic membranes is also described by K/tsa (1968), Koelle (1969) and Ritter et al. (1974). Others, however, have reported comparatively greater AChE activity at postsynaptic membranes (Bloom and Barrnett, 1966; Davis and Koelle, 1967; Koelle, 1969, 1971) or an equal distribution at both pre- and postsynaptic membranes, but
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sometimes with absence of product in the cleft (Shute and Lewis, 1966; Koelle, 1971; Bogusch, 1973; Koelle et al., 1974b). Some investigators observed reaction product specifically in the cleft near vesicle accumulations (see e.g., Smith and Treherne, 1965; Lewis and Shute, 1966), and although this is occasionally seen in our preparations, it is not a consistent finding. In addition, K~isa's (1968) report of AChE reaction product upon 'intersynaptic fibrils' could not be confirmed in our preparations. The irregular, extracellular distribution of reaction product in the cleft with preferential localization at the presynaptic membrane (discounting problems such as postfixation diffusion), might be interpreted as revealing the sites of the greatest transmitter enzyme activity, especially in the iso-OMPA-inhibited preparations. The absence of material at particular sites may suggest lack of enzyme, or of its activity, or amounts too small to give a visible reaction product, each possibly the result of the enzyme being depleted to variable degrees. However, it cannot be unequivocally shown that the absence of reaction product definitely indicates absence of enzymatic activity as is the case for all such procedures. The presence of reaction product in certain areas could also represent an AChE distribution which might provide a barrier against transmitter accumulation at those sites (i.e., 'cross-talk' or leakage of transmitter; see e.g., Barajas et al., 1974). In this respect, the frequent occurrence of reaction products around flat-vesicle terminals, not usually thought associated with cholinergic systems, may mean that acetylcholine (ACh) is involved in the functions of the F terminals or that the enzyme is there as a safeguard against the presence of the transmitter or its activity. Non-cholinergic nerves and terminals have been associated with AChE activity and previous reports have also shown reaction product in relation to flat vesicle terminals (Koelle, 1971; McDonald and Rasmussen, 1971; Pfenninger, 1973; Ritter et al., 1974). The possibility that some of these F terminals are former R terminals in which the vesicles have flattened during the incubations, must be considered (Bodian, 1970). However, although the brief section staining does not always permit easy identification of the type of postsynaptic thickening, the synaptic clefts are of the dimensions of F terminal contacts. In addition, it is not possible to make accurate determinations of proportions of classes of terminals showing this reaction, as the reaction is somewhat irregular in deeper parts of the blocks. The latter would be useful to compare ratios of F to R terminals in these preparations with those seen in ordinary preparations (Westrum, 1974). In any case, the clear and relatively consistent deposition of product on presynaptic membranes, especially in regions otherwise totally devoid of reaction product, is indicative of AChE and a possible role for ACh at these sites.
Acknowledgements This work was supported in part by USPHS Grants NS-09678 and NS-04053 from the National Institute of Neurological and Communicative Disorders and Stroke
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(USPHS/DHEW). The authors gratefully acknowledge this support and wish to thank Maria Glaser, Ann Harris and Doris E. Ringer for valuable technical and secretarial assistance. References ANDERSON, L.S., BLACK, R.G., ABRAHAM, J. and WARD, A. A., JR. (1971) Neuronal hyperactivity in experimental trigeminal deafferentation. Journal ofNeurosurgery 3 5 , 4 4 4 - 5 2 . BALLARD, K.J. and JONES, J. V. (1971) The fine structural localization of cholinesterase in the carotid body of the cat. Journal of Physiology (London) 219, 7 4 7 - 5 3 . BARAJAS, L., SILVERMAN, A.J. and MULLER, J. (1974) Ultrastructural localization of acetylcholinesterase in renal nerves. Journal of Ultrastructure Research 49, 297-311. BARRNETT, R. J. (1962) The fine structural localization of acetylcholinesterase at the myoneural junction. Journal of Cell Biology 12, 2 4 7 - 6 2 . BLOOM, F. E. and BARRNETT, R. J. (1966) Fine structural localization of acetylcholinesterase in electroplaque of the electric eel. Journal of Cell Biology 29, 4 7 5 - 9 5 . BODIAN, D. (1970) An electron microscopic characterization of classes of synaptic vesicles by means of controlled aldehyde fixation. Journal of Cell Biology 44, 1 1 5 - 2 4 . BOGUSCH, G. (1973) Zur Lokalisation der Cholinesterase-aktivit~t in der motorischen Endplatte mit der Kupferferrocyanidemethode. Histocbemie 33, 3 9 - 4 6 . BRIDGES, T. E., FISHER, A. W., GOSBEE, J. L., LEDERIS, K. and SANTOLAYA, R. C. (1973) Acetylcholine and cholinesterase (assays and light- and electron microscopical histochemistry) in different parts of the pituitary of rat, rabbit and domestic pig. Zeitscbriftfur Zellforschung und mikroskopiscbe Anatomic 136, 1 - 1 8 . BRODERSON, S. H., WESTRUM, L. E. and SUTTON, A. E. (1974)Studies of the direct coloring thiocholine method for localizing cholinesterase activity. Histochemistry 40, 13--23. BROWN, W. J. and PALAY, S. L. (1972) Acetylcholinesterase activity in certain glomeruli and Golgi cells of the granular layer of the rat cerebetlar cortex. Zeitscbrift fz'~r Anatomie und Entwicklungsgescbicbte 137, 317--34. BRZIN, M., TENNYSON, V.M. and DUFFY, P.E. (1966) Acetylcholinesterase in frog sympathetic and dorsal root ganglia. A study by electron microscope cytochemistry and microgasometric analysis with the magnetic diver. Journal of Cell Biology 31, 2 1 5 - 4 2 . BRZIN, M., TENNYSON, V.M. and DETTBARN, W.-D. (1975) Cytochemical localization of cholinesterase activity at the giant synapse of the squid. Histochemistry 43, 3 0 5 - 1 1 . CSILLIK, B., TOTH, L. and KARCSU, S. (1973) Acetylcholinesterase activity of Renshaw elements and Renshaw bulbs. A light- and electron-histochemical study. Journal of Neurocytology 2,441--55. DAVIS, R. and KOELLE, G. B. (1967) Electron microscopic localization of acetylcholinesterase and non-specific cholinesterase at the neuromuscular junction by the gold-thiocholine and gold-thiolacetic acid methods. Journal of Cell Biology 34, 1 5 7 - 7 1 . FRIEDENBERG, R.M. and SELIGMAN, A.M. (1972) Acetylcholinesterase at the myoneural junction: cytochemical ultrastructure and some biochemical considerations. Journal of Histochemistry and Cytocbemistry 20, 7 7 1 - 9 2 . GWYN, D. G. and FLUMERFELT, B. A. (1971) Acetylcholinesterase in non-cholinergic neurones: A histochemical study of dorsal root ganglion cells in the rat. Brain Research 34, 193--98. HAJ~S, F., PRIYMAK, E.K. and KERPEL-FRONIUS, S. (1970) The electron microscopic demonstration of acetylcholinesterase activity in some cholinergic and non-cholinergic synapses of the rat brain. Acta Histocbemica 35, 114--22. KARNOVSKY, M.J. and ROOTS, L. (1964) A 'direct-coloring' thiocholine method for cholinesterases. Journal of Histochemistry and Cytocbemistry 12, 2 1 9 - 2 2 .
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