J. Cell Sd. I, 381-390 (1966) 381 Printed in Gnat Britain

THE DISTRIBUTION OF CHOLINESTERASE IN DEMONSTRATED WITH THE ELECTRON MICROSCOPE

P. R. LEWIS AND C. C. D. SHUTE Department of Anatomy, University of Cambridge

SUMMARY The thiocholine technique for cholinesterase has been successfully adapted for the demon- stration of distribution with the electron microscope. After fixation in buffered glutar- aldehyde, thin slices of tissue were taken through a histochemical procedure designed to mini- mize diffusion artifacts and cytological damage; appropriate areas were dissected out, fixed with osmium tetroxide and embedded in Araldite. This technique has been used to study the distribution of enzyme in and around known cholinergic neurons in the rat. In diaphragm muscle the intense staining of the motor end plates is confined to the actual synaptic clefts. The distribution of cytoplasmic staining was similar in all three types of cholinergic neurons studied (ventral horn cells from the cervical cord, cells from the dorsal motor nucleus of the vagus, and cells from the hypoglossal nucleus). There was intense staining of the space contained within the individual bilaminar sheets of the rough endoplasmic reticulum and occasionally in areas of the nuclear envelope. Mitochondria, lyso- somes, the smooth endoplasmic reticulum and most of the plasma membrane were unstained. In cholinergic nerve fibres staining was particularly intense at the axonal membrane and absent from the myelin sheath. In several regions of the brain known to receive an afferent cholinergic innervation many of the identifiable synaptic areas were heavily stained. Staining here was usually present round most of the presynaptic process, spreading over part of the postsynaptic process and often penetrating into the actual synaptic space. The mitochondria, microvesicles and the general cytoplasm of the synaptic processes were all unstained. In some areas, such as the hippocampus, there was intense membrane staining of fine cholinergic neuropil. The enzyme specificity of the technique is not in doubt and evidence is adduced for the view that diffusion artifacts are small. The close correlation between the electron-microscopic results and evidence from physiological and biochemical investigations is discussed.

INTRODUCTION Several attempts have been made to use histochemical techniques for cholinesterases at the electron-microscope level. The results obtained by Lehrer & Ornstein (1959), using a simultaneous coupling azo-dye technique, were not encouraging, largely because the reaction product was not sufficiently electron-dense. More satisfactory results have been obtained with the thiolacetic acid technique: the reaction product is very electron-dense and sufficiently well localized for at least some purposes (Barrnett, 1962; Zacks & Blumberg, 1961; Smith & Treherne, 1965). One serious disadvantage of any procedure based on thiolacetic acid, however, is the lack of enzyme specificity. We therefore attempted to adapt the highly specific thiocholine technique for electron microscopy. A brief outline of our method has been published as a prelimi- 382 P. R. Lewis and C. C. D. Shute nary communication (Lewis & Shute, 1964); and a method based on a somewhat different principle has been published by Karnovsky (1964). Our ultimate aim was to study cholinesterase distribution in many areas of the brain where cholinergic mecha- nisms are implicated, following on previous work at the light-microscope level (Shute & Lewis, 1963, 1965). But first it was essential to test the technique on struc- tures known, unequivocally, to be concerned in cholinergic transmission. The pro- cedure finally devised gives very good enzyme localization and adequate, though not perfect, preservation of fine cytological detail. The precise procedure used is given in some detail here because it should be directly applicable to many tissues besides brain and muscle.

MATERIALS AND METHOD Osmium tetroxide is not suitable as a primary fixative because it is so toxic to . For much of our material glutaraldehyde has been satisfactory, but it was usually necessary to fix the whole animal by perfusion before attempting to dissect out individual pieces of tissue. In some regions of the brain (for example, hippo- campus) glutaraldehyde alone did not give adequate preservation of cytological detail; such tissues were transferred after preliminary fixation in glutaraldehyde to an equiva- lent formalin solution for a few hours before proceeding to the next stage (Shute & Lewis, 1966). Aldehyde-fixed tissue sectioned on the freezing microtome did not provide material satisfactory for electron microscopy. Although many of the cytoplasmic organelles were well preserved there were numerous scattered lacunae which were obviously artifacts, presumably caused by ice-crystal formation. Such artifacts are quite un- acceptable in brain, where intercellular relationships are vitally significant. It was therefore necessary to cut thin freehand sections from the face of a chilled, but not frozen, block of aldehyde-fixed brain. In practice it proved difficult to cut sections thinner than about 150 /i without damaging them mechanically; so, in general, sections about 200-250 ft thick were cut and incubated for electron-microscopic study. A simple device facilitates the cutting of these sections. Two coverslips are mounted with balsam on an ordinary 3 in. x i\ in. glass slide with a gap of, say, 1 cm between them. A clean-cut face of the block of tissue is then firmly held against the slide and a sharp razor blade with its ends held flat against the coverslips is drawn across the block. The thickness of the coverslips determines the thickness of the tissue slice obtained. If the normal histochemical procedure, as applied routinely to frozen sections, is used for electron microscopy the results are disappointing. The deposit of copper sulphide is so electron-dense that ordinary cytological detail is obscured and there is clear evidence of diffusion artifacts. In an attempt to improve the electron-microscopic picture various modifications of the standard procedure were tried, the choice of modifications being based on the results of an earlier study with the light microscope (Lewis, 1961). Some of the modifications were aimed at improving the efficiency of the capture reaction, that is, the reaction of enzymically released thiocholine with Electron microscopy of cholinesterase 383 cupric ions to give the initial precipitate, since it is at this stage that most of the diffusion occurs. Other modifications were aimed at preserving the fine cytology: thus all solutions were buffered and made approximately isotonic, and calcium ions were incorporated into all except the incubation medium. The perfusion fluid was used at room temperature, but all the other solutions were used at about 4 °C. Albino rats were killed with an overdose of ether and immediately perfused through the heart. The composition of the perfusion fluid, which was made up and filtered immediately before use, was as follows: 16 ml 25% glutaraldehyde, 50 ml 0-2 M sodium cacodylate, 2 ml 0-2 M calcium acetate, made up to 200 ml with distilled water. The final pH was adjusted if necessary to y-o-J'S- The tissues required were dissected out, cut into convenient-sized pieces and immersed in the perfusion fluid for 4—6 h at 4 °C. If necessary they were then trans- ferred to an equivalent solution containing 4 % formaldehyde in place of glutaraldehyde for a further 4-16 h. The blocks of tissue were then washed for at least 16 h at 4°C in an isotonic buffer solution: 24 ml 0-2 M sodium cacodylate, 10 ml 0-2 M cacodylic acid, 1 ml 0-2 M calcium acetate, made up to 100 ml with isotonic sodium sulphate. Thin slices of brain were then cut, or small bundles of nerve or muscle fibres teased out, and incubated at 4°C for 2—6 h in a medium made up as follows: 100 mg of acetylthiocholine iodide (or iiomg of butyrylthiocholine iodide) was dissolved in 4-0 ml of distilled water and 7-0 ml of o-i M copper sulphate was added drop by drop. The precipitate of cuprous iodide was centrifuged down and 10 ml of the supernatant pipetted off. To this supernatant was added 50 mg glycine and the volume of M sodium acetate needed to give the desired pH. Before use the medium was made up to a total volume of 40 ml and filtered, sufficient sodium sulphate being used to give an approximately isotonic solution. Two media were used routinely, the more acid one for tissues with the highest local concentrations of cholinesterase. When required ethopropazine, at a final concentration of 2 x IO^M, was used as an inhibitor of pseudocholinesterase: 2-8 mg of the hydrochloride was dissolved, with gentle warming, in 10 ml of isotonic sodium sulphate, the solution cooled and added to 10 ml of the supernatant. The incubation media used were of the following composition (all volumes in ml):

supernatant IO 10 glycine (mg) 5° 5° M sodium acetate i 2 distilled water o 4 isotonic sodium sulphate 29 24 final pH 4-6 5

Both media contained approximately 8 mM , 12 mM copper and 17 HIM glycine. After incubation, the tissues were washed for a total of 1 h in 2 changes of the isotonic buffer and then transferred to a buffered sulphide solution of the following composition, made up and filtered immediately beforehand: about 2 g of M.A.R.

25 Cell Sci. 1 384 P- R. Lewis and C. C. D. Skute grade sodium sulphide were weighed accurately and dissolved in 0-2 N in the proportion of 45 ml for each gram of sodium sulphide; sufficient 0-2 M calcium acetate was added to give a final concentration of 2 mM and the pH adjusted, if necessary, to 5•0-5-5. After an hour in the sulphide solution the tissues were returned to the isotonic buffer overnight. The required areas of tissue were then dissected out under a binocular microscope and fixed in Dalton's or Zetterqvist's solutions, as for ordinary electron microscopy. The material was then dehydrated and embedded in Araldite. Thick sections were viewed by phase contrast and thin sections were stained with lead citrate for electron microscopy; uranyl acetate was not used as it caused some loss of the enzyme staining.

OBSERVATIONS At the myoneural junctions studied, intense staining was confined to the synaptic clefts, both primary and secondary—that is, confined to the space where the nerve and muscle membranes oppose each other in the synaptic gutter and to the ' batonnets: described by Couteaux (1947), which are rod-like spaces invaginating into the sub- stance of the muscle fibre (Figs. 1, 2). Characteristically, intense staining ceased at the end of the primary synaptic cleft, at the point where a tongue of teleoglial cell cyto- plasm is interposed between the nerve and muscle membranes. Occasionally there was some staining of the membranes of the overlying teleoglial cell and of the neuronal membrane facing it. There was very little staining of the myoplasm or neuroplasm. Occasional rosette-shaped areas of staining, 0-1-0-2 /i in diameter, were sometimes seen scattered in and around myoneural junctions; since they were not related to any morphological structure and were more obvious in over- stained preparations they are thought to be some form of diffusion artifact. In marked contrast to the results obtained with the less specific thiolacetic method, no staining of the presynaptic vesicles (Barrnett, 1962) or of the axonal mitochondria (Zacks & Blumberg, 1961) was ever seen, even in over-incubated material. The enzyme distribution in cell bodies was studied in ventral horn cells from the cervical cord, in motor cells of the hypoglossal nucleus and in cells of the dorsal motor nucleus of the vagus. All these cells are cholinergic and show essentially the same type of enzyme distribution. Most of the cholinesterase activity is associated with the rough endoplasmic reticulum. There was no activity associated with mitochondria or lysosomes. In one or two instances there was slight activity associated with what appeared to be smooth endoplasmic reticulum. The plasma membrane in general showed no enzyme activity, except in occasional areas where there was a synaptic contact. In these large motor cells, the rough endoplasmic reticulum is mostly present in the form of aggregates, some as much as 3 fi across, scattered throughout the cytoplasm. It is this type of distribution which is responsible for the characteristic pattern of Nissl staining and it also accounts for the granular appearance of the cytoplasm often seen with the light microscope in thiocholine preparations. With the electron micro- Electron microscopy of cholinesterase 385 scope, the cholinesterase activity of these aggregates is seen to be concentrated within the interval between the paired membranes (that is, within the cisternae) of the rough endoplasmic reticulum along much, but not the whole, of their length (Figs. 3-5). The precise location of the enzyme activity was particularly obvious in areas where the reticulum was slightly dilated. There appeared to be no cholinesterase activity associated with ribosomes that were free in the cytoplasm. There was no staining in the nucleus proper, but occasionally areas of staining were seen in the space between the two membranes of the nuclear envelope, a space continuous with that of the endoplasmic reticulum. This staining was most common in the ventral horn cells. These large motor cells are very richly supplied with nuclear pores which are dramatically shown up as unstained areas in those regions of the envelope which contain enzyme activity (Figs. 6, 7). The ventral horn cells contain true cholinesterase (AChE), as do most of the cells in the hypoglossal nucleus. At the caudal end of this nucleus, however, are a ventral group of cells distinguished by a high concentration of an enzyme which has all the properties of a pseudocholinesterase (ChE). This group of cells innervates intrinsic musculature of the tongue, and the intracellular distribution of ChE is essentially similar to that of AChE in the more rostral cells, except that in general a higher proportion of the rough endoplasmic reticulum shows intense staining. Thus in the cells that contain only AChE the enzyme is concentrated into small scattered areas which constitute only some 10-20% of the total endoplasmic reticulum, whereas in those containing ChE areas of high enzyme activity constitute 50% or more. In both types of cell there is a coarse stippling of enzyme staining observable throughout the rough endoplasmic reticulum (Fig. 5). The cells of the vagal nucleus contain both enzymes, with ChE predominating both in total amount and in proportion of endo- plasmic reticulum intensely stained. Enzyme distribution was investigated in several types of presumed cholinergic axons but the best evidence was obtained from the hypoglossal nerve, which is known to consist almost entirely of cholinergic motor fibres. In this nerve almost all the fibres stain for true cholinesterase, a few stain for pseudocholinesterase and a few remain unstained. In cross-section the nerve trunk presents an extremely homogeneous appearance, both in fibre diameter and thickness of myelin. There are no small or unmyelinated fibres and only an occasional fibre larger and more heavily myelinated than the majority. These occasional, large axons showed no staining for cholinesterase and were presumably sensory fibres—supplying, perhaps, spindles in the genioglossus muscle. The vast remainder of uniform, stained fibres are undoubtedly cholinergic. In the electron microscope the most obvious feature is a very intense staining of the axonal membrane (Figs. 9, 10). In other cholinergic motor fibres studied the same intense staining is associated with the axonal membrane (Fig. 8). In hypoglossal fibres the intense staining extends round most of the membrane, whereas in the others it is present only in isolated areas, often close to the internal mesaxon. In the sciatic nerve many fibres, and specifically the largest, most heavily myelinated fibres, which are known to be sensory, showed no staining. In none of the fibres was appreciable staining for true cholinesterase seen in the myelin sheath (Fig. 10). 386 P. R. Lewis and C. C. D. Shute Enzyme distribution has been studied in several regions of the brain known to receive an afferent cholinergic innervation. In the caudate, which has a particularly massive cholinergic innervation, the staining obtained with acetylthiocholine as sub- strate is associated almost exclusively with membranes, particularly with those of small, unmyelinated axonal processes and with those of the synaptic regions. Many of the identifiable synaptic areas were heavily stained. Staining was usually present round most of the presynaptic process, spreading over part of the postsynaptic process and often penetrating into the actual synaptic space. The mitochondria, the microvesicles and the general cytoplasm of the synaptic processes were all unstained. With butyryl- thiocholine as substrate, a completely different pattern of enzyme distribution was obtained. Staining was restricted to the walls of the vessels; the neural elements were unstained. The staining of blood-vessel walls occurred with the acetyl substrate if ethopropazine was omitted from the incubation medium, but not if it was included. This differential staining with the two substrates was in agreement with a previous, more detailed survey at the light-microscope level (Shute & Lewis, 1963). In the hippocampus it was less easy to distinguish synaptic areas. The most prominent enzyme staining was of very fine, unmyelinated axonal processes (o* 1-0-2 fi in dia- meter) which form a presumed cholinergic neuropil in the infrapyramidal layer. The membrane staining in the neuropil was more intense than that of the myelinated fibres in the fimbria from which it arises. In the hypoglossal nucleus and in the cervical cord most of the axodendritic and axosomatic endings on the motor cells showed little or no enzyme staining. A small proportion, however, were heavily stained. In these the enzyme had a distribution similar to that seen in the caudate, but the staining was more dramatic because the were larger. It would seem reasonable to suppose that such intense staining is indicative of cholinergic transmission. Figs. 11 and 12 show two axosomatic endings on the same cervical motor cell, the one showing no staining and the other very heavy staining. In the latter, staining is present over most of the presynaptic process and there is particularly intense, discrete staining of the synaptic area immediately under a cloud of microvesicles. There is staining in the endoplasmic reticulum, a tongue of which lies very close below the synaptic area. Again, the mitochondria and micro- vesicles are unstained.

DISCUSSION A major inherent advantage of any histochemical method based on acetylthiocholine is the high degree of specificity for cholinesterases. All the other available methods make use of substrates that are hydrolysed by a wide variety of non-specific which cannot be selectively inhibited; so the distribution of cholinesterase can be deduced only from a comparison of two adjacent pieces of tissue incubated with and without the addition of a specific inhibitor for cholinesterase. A further advantage is the existence of two substrates which can be made specific for true and pseudocholin- . Thus in the presence of ethopropazine is hydrolysed only by true cholinesterase and, in rat brain at least, only pseudocholinesterase appears to Electron microscopy of cholinesterase 387 hydrolyse butyrylthiocholine at an appreciable rate. These conclusions, arrived at from light-microscope studies (Shute & Lewis, 1963), are also undoubtedly valid at the electron-microscope level, as is confirmed by our results in the caudate, which are in excellent agreement with the biochemical measurements of enzyme activity made by Bayliss & Todrick (1956). The accuracy of enzyme localization by our technique is more difficult to assess. In the process of developing the technique many modifications of the incubation medium were tried and it was noticeable that anything which might reduce the effi- ciency of the capture reaction (Holt & O'Sullivan, 1958), such as lowering the concen- tration of copper or raising that of the glycine available to chelate with copper ions, leads to a more diffuse picture. Prolonged incubation also produced the same effect. None of these procedures, however, caused structures to stain which were not stained in the best preparations. Only one example is included in this paper and is shown in Fig. 8, in which incubation of the axon in the diaphragm muscle was too long. The staining, although more diffuse than that shown by the hypoglossal neurons (Figs. 9, 10), was still associated only with the axonal membrane. The staining was also more diffuse if the slice of tissue incubated was very thick, presumably because inward diffusion of the reagents was too slow. The occasional rosette-shaped areas of staining sometimes seen near regions of very high enzyme activity were almost certainly some form of artifact. They were a serious problem only with one particular batch of acetylthiocholine iodide. (A subsequent batch from the same manufacturer produced nearly complete inhibition of all enzyme activity.) Thus the results of varying the incubation conditions give no hint of serious diffu- sion artifacts. Furthermore, when the definitive technique is used, the very different patterns of staining seen in the caudate with the two substrates would appear to rule out the possibility of the reaction product diffusing more than a very few micra. With the butyryl substrate synaptic areas remain unstained, even those near heavily stained blood vessels; with acetyl substrate in the presence of ethopropazine the pattern is still clear-cut, but now the blood vessels remain unstained. This separate use of two substrates is a better histochemical control than the usual one of omitting substrate. The close association of staining with membranes is unlikely to be due to preferen- tial adsorption of the electron-dense deposit on to lipid structures. Staining with butyryl substrate in blood-vessel walls has a quite different type of distribution and in none of our preparations did the membranes of mitochondria, lysosomes or smooth endoplasmic reticulum show significant staining. Both in the rough endoplasmic reticulum and at the motor end plate there is staining between the apposed membranes as well as on them. Furthermore, the staining at the motor end plate virtually ceases at the point where the tongue of teleoglial cytoplasm is interposed between the nerve and muscle membranes (Fig. 1). In motor fibres, whilst the axonal membrane is intensely stained, the immediately adjacent Schwann cell membrane forming the internal mesaxon is unstained (Fig. 9). In none of these examples is it easy to see how the precise localization of staining obtained could arise from any inadequacy of the technique. The enzyme distribution seen in our material is strikingly consistent with the evidence 388 P. R. Lewis and C. C. D. Skute available from other disciplines. Thus, at the motor end plate physiological and pharmacological studies have shown that acetylcholine produces its depolarizing action only when present at the outside surface of the muscle membrane and that cholinesterase inhibitors prolong this action. This proves that the acetylcholine normally destroyed by the enzyme would otherwise remain near to but outside the muscle membrane. Thus the only place where cholinesterase could perform its normal function is in the narrow gap between the nerve and muscle membranes— precisely where the enzyme is found in highest concentration by our technique. Similarly, the distribution of cholinesterase at synaptic regions in the central nervous system is also quite consistent with its presumed function of hydrolysing excess released acetylcholine. It is interesting, however, that the enzyme is nowhere obviously not attached to membranes, and it should be emphasized that this association of staining with membranes was a constant feature seen in all the material we examined. Biochemical studies in which ultrafractionation techniques have been applied to brain homogenates have shown that cholinesterase activity is always associated with the particulate fractions (Toschi, 1959; Aldridge & Johnson, 1959; de Robertis et al., 1963; Whittaker, 1965). In the fraction containing disrupted synaptosome ghosts, which consist of the presynaptic process minus its mitochondria and microvesicles plus the adjacent area of postsynaptic membrane, cholinesterase activity is particularly high, whereas in those fractions consisting mainly of microvesicles and mitochondria it is low, in excellent agreement with the type of enzyme distribution seen in the regions of the brain which we have investigated so far. In the cell bodies of cholinergic neurons the staining was very sharply defined. The absence of staining in lysosomes and mitochondria is consistent with the fact that the thiocholine are not hydrolysed by any of the non-specific esterases. The sharp localization of intense staining in the rough endoplasmic reticulum is particularly significant in view of modern theories of protein synthesis. It suggests very strongly that cholinesterase is synthesized by the rough endoplasmic reticulum. Certainly this is where the enzyme must be stored immediately after synthesis. It is not known why the cells of the dorsal motor nucleus of the vagus and some cells in the hypoglossal nucleus should contain an enzyme with the properties of a pseudocholinesterase. These cells are undoubtedly cholinergic, and the very similar intracellular distribution seen in the electron microscope makes it very probable that this enzyme fulfils much the same sort of function as does true cholinesterase in other motor cells. The fate of the cholinesterase in the rough endoplasmic reticulum is not obvious from our electron micrographs. One possibility is that it finds its way to the axonal membrane, which is very intensely stained in cholinergic nerve fibres. Against this is the sparseness of any staining associated with the plasma membrane of the cell body. Furthermore, we have seen no unequivocal examples of a direct connexion between the rough endoplasmic reticulum and the cell membrane, though these two structures do sometimes come into very close apposition (as in Fig. 12). Such direct connexions have been postulated in other cells and our lack of success in finding them so far is no evidence against their existence: they may be very difficult to recognize under the microscope and their number per cell could be very small. Electron microscopy ofcholinesterase 389 It has been- suggested by several groups of workers that the acetylase and cholinesterase found at cholinergic terminations are synthesized in the cell body and transported down the axon. Our finding that in the axon staining of cholinesterase is concentrated at the membrane raises certain physical problems, however, if synthesis and transport of enzyme are assumed to continue throughout the life of the . Three possible explanations suggest themselves: the membrane itself may be moving down the axon; the individual molecules of enzyme may be able to move over the surface of the axon; or, the bulk of the enzyme moves down within the axoplasm in an inactive form. None of these explanations is wholly satisfying, though the third one would explain the lack of continuity in enzyme staining between the reticulum and the axonal membrane. Nevertheless, our findings at the electron-microscope level do not refute the possibility of axonal flow, and the presence of staining in the cell body and at the nerve terminals is not inconsistent with the basic idea.

We wish to thank our assistant, Miss Annette Pine, for much help with the technical pro- cedures. The work was supported by a grant from the Medical Research Council.

REFERENCES ALDRIDGE, W. N. & JOHNSON, M. K. (1959). Cholinesterase, succinic dehydrogenase, nucleic acids, esterase and reductase in sub-cellular fractions from rat brain. Biochem. J. 73, 270-276. BARRNETT, R. J. (1962). The fine structural localisation of acetyl cholinesterase at the myo- neural junction. J. Cell Biol. 12, 247-262. BAYLISS, B. J. & TODRICK, A. (1956). The use of a selective inhibitor in the estimation of pseudocholinesterase activity in rat brain. Biochem. J. 62, 62—67. COUTEAUX, R. (1947). Contribution a l'etude de la myoneurale. Revue can. Biol. 6, 563-711. HOLT, S. J. & O'SULLIVAN, D. G. (1958). Studies in enzyme cytochemistry I. Principles of cytochemical staining methods. Proc. R. Soc. B 148, 465-480. KARNOVSKY, M. J. (1964). The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. J. Cell Biol. 23, 217-232. LEHRER, G. M. &ORNSTEIN, L. A. (1959). A diazo coupling method for the electron microscopic localization of cholinesterase. J. biophys. biochem. Cytol. 6, 399-406. LEWIS, P. R. (1961). The effect of varying the conditions in the Koelle technique. Biblthca anat. a, 11-20. LEWIS, P. R. & SHUTE, C. C. D. (1964). Demonstration of cholinesterase activity with the electron microscope. J. Physiol., Lond. 175, 5-7 P. ROBERTIS, E. DE, RODRIGUEZ DE LOREZ ARNAIZ, G., SALGANICOFF, L., PELLEGRINO DE IRALDI, A. & ZIEHER, L. M. (1963). Isolation of synaptic vesicles and structural organisation of the acetylcholine system within brain nerve endings. J. Neurochem. 10, 225-235. SHUTE, C. C. D. & LEWIS, P. R. (1963). Cholinesterase-containing systems of the brain of the rat. Nature, Lond. 199, 1160-1164. SHUTE, C. C. D. & LEWIS, P. R. (1965). Cholinesterase-containing pathways of the hindbrain: afferent cerebellar and centrifugal cochlear fibres. Nature, Lond. 205, 242-246. SHUTE, C. C. D. & LEWIS, P. R. (1966). Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurones in the hippocampal formation of the rat. Z. Zellfortch. mikrosk. Anat. 69, 334-343. SMITH, D. S. & TREHERNE, J. E. (1965). The electron microscopic activity in the central nervous system of an , Periplaneta americana L,.J. Cell Biol. 26,445-465. TOSCHI, G. (1959). A biochemical study of brain microsomes. Expl Cell Res. 16, 232-255. 390 P. R. Lewis and C. C. D. Shute WHITTAKER, V. P. (1965). The application of sub-cellular fractionation techniques to the study of brain function. Prog. Biophys. molec. Biol. 15, 39-96. ZACKS, S. I. & BLUMBERG, J. M. (1961). The histochemical localisation of acetylcholinesterase in the fine structure of neuromuscular junctions of mouse and human intercostal muscle. J. Histochem. Cytochem. 9, 317-324. {Received 14 March 1966)

Unless otherwise indicated all the electron micrographs are of sections, counter- stained with lead citrate, from material incubated with acetylthiocholine as substrate in the presence of ethopropazine as a pseudocholinesterase inhibitor. Fig. i. Cross-section of a small synaptic gutter from an end plate in diaphragm muscle. Note the intense staining of the synaptic clefts and the sharp falling off in staining at the points arrowed, where the nerve and muscle membranes become separated by a teleoglial process, x 48000. Fig. 2. A larger and less intensely stained synaptic area from diaphragm muscle, x 36000. Journal of Cell Science, Vol. i, No. 3

P. R. LEWIS AND C. C. D. SHUTE (Facing p. 390) Fig. 3. Cytoplasmic staining in a ventral horn cell from cervical cord. Note lack of staining in lysosomes (/), mitochondria (in) and smooth endoplasmic reticulum (s). x 24000. Fig. 4. A high-power detail from the same field as Fig. 3, showing the intense staining of the rough endoplasmic reticulum. x 80000. Fig. 5. A high-power view of rough endoplasmic reticulum from a hypoglossal neuron (butyrylthiocholine as substrate). Note that where the enzyme staining is reduced to a coarse stippling it is obviously restricted to the interval between the paired membranes of the reticulum. x 80000. Journal of Cell Science, Vol. i, No. 3

P. R. LEWIS AND C. C. D. SHUTE Figs. 6, 7. High-power details of a cervical ventral horn cell, with the nuclear envelope cut in tangential and radial directions respectively; nuclear pores are outlined by enzyme staining, x 80000. Fig. 8. Detail of a motor fibre from diaphragm muscle. In spite of over-incubation enzyme staining is associated only with the axonal membrane (a), x 120000. Fig. 9. The internal mesaxon of a hypoglossal nerve fibre. Note that the intense staining present at the axonal membrane (a) does not extend along the mesaxon (•;») formed by the pair of membranes from the Schwann cell, x 80000. Fig. 10. Hypoglossal nerve fibre, not counterstained with lead citrate. Note virtual absence of enzyme staining in the myelin sheath, x 48000. Journal of Cell Science, Vol. i, No. 3

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a

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P. R. LEWIS AND C. C. D. SHUTE Figs, II, 12. Two axosomatic endings on the same cervical ventral horn cell. In both, the rough endoplasmic reticulum (/•) is heavily stained but the microvesicles (v) and mitochondria (;») are unstained. In Fig. u there is virtually no enzyme staining associated with the presynaptic process or the synaptic area below the microvesicles. In Fig. 12 most of the membrane round the presynaptic process is heavily stained, as is a tongue of the reticulum (arrowed) which lies closely applied to the synaptic area, x 40000. Journal of Cell Science, Vol. i, No. 3

P. R. LEWIS AND C. C. D. SHUTE