Schlosser (1955) who, in addition, give data for the choline acetylase concentration

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1 718 J. Physiol. (I956) I34, 7I8-728 CHOLINE ACETYLASE IN THE CENTRAL NERVOUS SYSTEM OF MAN AND SOME OTHER MAMMALS BY CATHERINE 0. HEBB AND ANN SILVER From the Institute of Animal Physiology, Babraham Hall, Babraham, Cambridge (Received 25 July 1956) The first systematic survey of choline acetylase in the central nervous system was made by Feldberg & Vogt (1948). Their results on the nervous system of the dog have since been confirmed in a number of particulars by Zetler & Schlosser (1955) who, in addition, give data for the choline acetylase concentration of certain parts of the human brain. In a comparable survey of the cholinesterases in the canine nervous system Burgen & Chipman (1951) noted that the ratio of the highest to the lowest brain concentration of ChEI (true or specific cholinesterase) is nearly 400:1, while the spread of values found by Feldberg & Vogt (1948) for choline acetylase gives a ratio of only 42:1. The difference suggests that cholinesterase is less evenly distributed in the nervous system than choline acetylase, but another possible explanation is that the method used to measure choline acetylase was not sufficiently sensitive to give a true quantitative estimate of the enzyme in all situations. It is now known that choline acetylase requires acetyl-coenzyme A (acetyl- CoA) as a substrate. In Feldberg & Mann's (1946) method used by the authors quoted this is generated mainly from citrate by the action of enzymes other than choline acetylase which are present in the tissue tested. The concentration of these enzymes may be too low, however, to permit formation of acetyl-coa in the amounts required for full activity of the choline acetylase present; and it has been found that much higher rates of ACh production are demonstrable in some tissues of mammals and birds if they are tested in a coupled enzyme which ensures an adequate supply of acetyl-coa (Hebb, 1955). Because of these considerations we decided to re-examine the distribution of choline acetylase in the nervous system of the dog. During the course of the investigation there was an opportunity to measure the choline acetylase of some samples of human brain as well. This led us to extend the study to a

2 CHOLINE ACETYLASE IN CENTRAL NERVOUS SYSTEM 719 number of other mammals so that differences in choline acetylase concentration associated with the evolutionary development of the brain might be evaluated. METHODS Experimental. The material used in these experiments included: (1) samples of brain removed from six human patients during neurosurgical operations; (2) post-mortem samples of two human brains obtained hr after death; (3) brains removed from rabbits, cats, dogs, pigs and sheep immediately after death produced under anaesthesia (pentobarbitonie and/or cyclopropane) by haemorrhage or by destruction of the medulla oblongata; (4) brains of guinea-pigs, rabbits, pigs and sheep which were stunned and bled to death without anaesthesia; and (5) a few tissues of bullocks and horses obtained from the slaughter-house within 20 min-3 hr after death. In these and previous experiments it has been found that the choline acetylase activity was the same in a given part of the nervous system whether the animal had or had not been anaesthetized before death. After removal the tissues were dissected and each sample cooled to 0-3 C. They were then made into acetone powders. The procedures used in preparing and extracting the acetone powders and the subsequent determination of their choline acetylase activity have been fully described in recent papers from this laboratory (Hebb & Waites, 1956; Hebb & Smailman, 1956). Choline acetylase activity will be expressed here as,ug or mg of ACh (as the chloride) synthesized per g of acetone powder per hr. Cerebral cortical areas in man have been enumerated according to Brodman maps reproduced by Kappers, Huber & Crosby (1936), and in the dog according to Klempin (1921). Cortical areas in other species were identified by reference to Winkler & Potter (1911, 1914), Kappers et al. (1936) and Wilkie (1937). Control of po8t-mortem changes. In two control experiments, one on a dog and one on a pig brain, the right and left halves of the cerebrum when first removed were separated from one another; acetone powders were then made from one half following the usual procedure, but the other half was stored for either 1 day (dog brain) or 2 days (pig brain), at the end of which times acetone powders were made from these as well. In the first of these experiments the half-brain was stored at 3-5 C for 24 hr; in the second the tissue was kept at room temperature for a preliminary period of 6 hr, then stored for a further period of 42 hr at 3-5 C. Acetone powders were made in each case from four corresponding regions of the fresh and stored portions of tissue. These experiments, which were done in order to determine whether any significant loss of choline acetylase would occur post-mortem, showed that the rates of synthesis of ACh by extracts of fresh and stored tissues agreed within the error of the method (± 10%). The data from these experiments have not been tabulated separately; but the figures obtained from stored tissues appear in Tables 1 and 2 of the results, in brackets beside the values obtained from the corresponding fresh tissues. RESULTS Range of variation of choline acetylase concentration in the nervous system The variation in the choline acetylase concentration of the adult mammalian nervous system was found to be much larger than any previous investigation had shown. The lowest concentrations observed in the optic nerves and dorsal spinal roots, each synthesizing less than significant amounts of ACh (0-25,ug/ g/hr), agreed with earlier estimates, but the concentrations in some other regions of the nervous system were times higher. Among the highest values observed were those obtained for the caudate nucleus, which synthesized between 10 and 15 mg ACh/g/hr, and for the anterior roots and the retina

3 720 CATHERINE 0. HEBB AND ANN SILVER which in a few species had an activity of the same order. The spread of values, assuming that 25,ug/g/hr represented the true rate of synthesis by the sensory nerves mentioned above, was such that the ratio of the highest to the lowest activities was between 450: 1 (cat) and 700: 1 (sheep). In man the corresponding ratio was 540: 1. TABLE 1. Choline acetylase activity expressed as ugach/g/hr determined in certain regions of the nervous system of the dog and of man. Results obtained by earlier workers (Feldberg & Mann, 1946; Feldberg & Vogt, 1948; Zetler & Schlosser, 1955) are shown in the last three columns under their initials Choline acetylase activity V Values reported earlier Species Region of nervous system Dog Cortical area 17 Cortical area 4 Cortical area 51 Ammon's horn Olfactory bulb Olfactory tract Retina Optic nerve Superior colliculus Inferior colliculus Thalamus Hypothalamus Caudate nucleus Cerebellar cortex Cerebellar peduncles Ventral spinal roots Dorsal spinal roots Man Parietal cortex Caudate nucleus Thalamus Hypothalamus Superior colliculus Present values 1,325 3,000 3,750 2,600 1,150 3, ,200-2,400 1,400 3,100 2,000 13,000-13, , ,000-13, F.& M. F.& V. Z.&S Table 1 shows for a representative list of tissues the quantitative difference between the present results and those obtained by earlier workers. The rate of synthesis by the retina, optic nerve and dorsal spinal roots of the dog which we observed are about the same as those reported earlier; but for all the other tissues listed our values are much higher. A few control tests in the present series of experiments showed that the differences between the values of the two series were dependent upon the addition of liver enzyme (for the production of acetyl-coa). If this were omitted the rates of synthesis in all tissues were of the same order as in the earlier series. It can be assumed that in those tissues in which the rates were the same for both systems the formation of acetyl-coa was not rate-limiting. This might be expected since it was true only of tissues which had low choline acetylase activity. The addition of liver enzyme not only increased the sensitivity ofthe method but also its reliability. Feldberg & Vogt (1948) noted variations of as much as

4 CHOLINE ACETYLASE IN CENTRAL NERVOUS SYSTEM % in the choline acetylase activity determined for individual tissues. On the other hand, we found that in any one species repeated estimations of anatomically well-defined tissues such as the anterior roots and caudate nucleus usually agreed within + 15%. Regional variations of enzyme in the nervous system The data already discussed give some indication of how the concentration of enzyme varied from one part of the nervous system to another. The remaining data given in Tables 2-7 provide a more detailed picture of the anatomical distribution of choline acetylase in any given species and of the variations and resemblances between species. The cerebral cortex, represented by areas 4, 17, 28 and 51 (in some species only) and Ammon's horn, exhibited species variations which paralleled one another in direction and which in broad outline appeared to be related to the degree of cortical development. Of all cortical tissue man's area 17 (striate cortex) had the lowest concentration of enzyme (Tables 2, 3). In the same species area 4 synthesized more, about double that synthesized by area 17; TABLE 2. Choline acetylase activity as,ug ACh/g/hr in cerebral cortex of mammals. Figures in brackets are results obtained from tissues extracted hr after death (see Methods) Cortical areas I. A 'N Ammon's Species horn Man * (290) (580) - ( ) (100) Pig (990) Cat Sheep Dog (1300) Rabbit * * Guinea-pig 4000* 4000* 3750t 5000 *Includes some cortical tissue from adjacent areas. tareas 28 and 51 pooled. TABLE 3. Choline acetylase activity expressed as tg ACh/g/hr of fresh samples of human cerebral cortex and subjacent white matter. Figures in brackets are from post-mortem samples of brain which are included for comparison Subjacent Mixed grey Cortex white tissue and white tissue Occipital lobe including striate (Area 17 and peristriate areas only, 100) Occipital lobe excluding striate area Temporal lobe (260) Frontal lobe (280) 46 PHYSIO. CXXXIV

5 CATHERINE 0. HEBB AND ANN SILVER while in the rhinencephalon the values were again higher. Next to man was the pig, synthesizing about 4 times as much ACh in areas 4, 17 and 28. Then came, with successively higher cortical values, the cat, sheep, dog, guinea-pig and rabbit. The largest species differences were shown by areas of the neocortex, e.g. 4 and 17, and the remainder of the occipital cortex in man and rabbit. The amount of ACh synthesized by these areas in the rabbit was 20 times that synthesized by the same areas in man. The corresponding difference for areas of the rhinencephalon or allocortex (area 28 and Ammon's horn) was that in the rabbit these tissues synthesized 4-6 times the amount which they synthesized in man. TABLE 4. Choline acetylase activity as mg ACh/g/hr in the basal ganglia, thalamus and centres of mid- and hind-brain. Figures in brackets as in Table 2 Species Guinea- Region of brain Man Pig Sheep Dog Cat Rabbit pig Caudate nucleus ( ) (15) (13.5) Amygdaloid nucleus (2-6) Thalamus (0 325) Lateral geniculate (0 23) Medial geniculate (020) Hypothalamus (0-17) _ Mammillary body (0 09) Superior colliculus (0.12) Inferior colliculus Cerebellar cortex *075 0* ( ) 02-0*3 0(.04 Cerebellar peduncles J Basal ganglia (Table 4). Only the caudate and amygdaloid nuclei have been examined systematically. The highest concentration of choline acetylase within the central nervous system of each species was found in the head and body of the caudate nucleus. The values tail off, however, since the concentration of choline acetylase in the amygdaloid nucleus, although high, is always 20-50% less than the concentration in the caudate. The lentiform (tested only in man) had about half the activity of the caudate. With the exception of the cat, in which the activity was slightly lower, the rate of synthesis of ACh by the caudate nucleus in man, pig, sheep, dog, rabbit and guinea-pig was surprisingly constant, between 10 and 15 mg/g/hr. The potential rate of synthesis by the basal ganglia of man is such that together they might conceivably produce between 40 and 50 mg of ACh/hr in one brain. A rough calculation on an average rate of synthesis of 200,g/g dried brain/hr suggests that the potential output of all of the rest of the human brain would be of the same order. Thalamus. Here the concentration of enzyme was found to be considerably lower than in the basal ganglia, but nevertheless on the average some 4-6 times higher than the concentrations reported by Feldberg & Vogt (1948). The values for the lateral and medial geniculates,were near the average for the whole thalamus. The values for the hypothalamus were less.

6 CHOLINE ACETYLASE IN CENTRAL NERVOUS SYSTEM 723 Cerebellum. The concentration of enzymes was low in all species examined. Our results showing low but variable concentrations of enzyme in the cerebellar cortex, higher values in the cerebellar peduncles and an over-all low average for the whole organ agreed well with those of Feldberg & Vogt. Species variations were slight, no more than might be expected from associated variations in the amount of connective and vascular tissue present. The enzyme concentration of the pons was found to be about double that of the cerebellar peduncles. No other parts of the hind-brain were tested. Choline acetylase activity in the optic nerves, dorsal spinal roots and ventral spinal roots expressed as,ug ACh/g/hr Dorsal spinal r~ I, Ventral spinal Species Optic nerve roots ganglia roots Rabbit Ox 0 0-6,800 Dog ,750-12,000 Cat ,800-10,000 Man (0) TABLE 5. TABLE 6. Choline acetylase activity in the retina and superior colliculus or optic lobes of some mammals and two birds. Values are expressed as mg ACh/g/hr Species Retina Superior colliculus Domestic fowl S812* Pigeon * Sheep Guinea-pig Pig Rabbit Ox Horse Dog Cat Man 0-12 * Optic lobe. Dorsal spinal roots. Here, as already indicated, little or no enzyme was found (Table 5); usually there appeared to be none at all. Dorsal root ganglia from the dog which might have been slightly contaminated from adjacent ventral spinal nerves did synthesize small but significant amounts of ACh. By contrast, extracts of the ventral spinal roots synthesized on the average only slightly less than the caudate nucleus. The optic nerve which was examined in five species synthesized little or no ACh. Retina and superior colliculi. These were both examined in six mammals. The results are summarized in Table 6. Of all parts of the mammalian nervous system the retina exhibited the largest-about 40-fold-species variation. The two carnivores, the cat and dog, had the lowest concentration of choline acetylase and the sheep had the highest, while in descending order of concentration, values for the retinas of the pig, guinea-pig, rabbit and horse, were between these two extremes. As in earlier experiments (Hebb, 1955), no 46-2

7 724 CATHERINE 0. HEBB AND ANN SILVER evidence was found for the presence of significant quantities of choline acetylase in the optic nerves of mammals. In the superior colliculus, however, moderate concentrations of choline acetylase were present. It is of interest that the concentration in this centre showed a positive correlation with the retinal concentration in different species. For purposes of comparison we have included in Table 6 data for the retina and optic lobes (homologous with the superior colliculi) of the hen and pigeon (Hebb, 1955). In these, as well as in the mammals, there was evidence to show that, in all species save the dog and cat, the concentration of enzyme in the mid-brain visual centre although at a lower level varied from one species to another parallel with the variations in the retinal concentration. The range of values for the superior colliculus was not so wide as the range for the retina. TABLE 7. Choline acetylase activity as,ug ACh/g/hr in peripheral division of olfactory brain. Figures in brackets as in Table 2 Tissues Species Olfactory bulb Olfactory tract Olfactory trigone Man (570) (250) (4,500) Pig 790-1,300 1,400-2,000 9,750-10,800 (790) (2,000) Sheep 1,200-1,400 1,700-3,000 9,000-9,400 Dog 1,150-1,600 2,400-3,700 7,500-9,000 (1,450) (3,750) Cat 2,200 3,600 Rabbit 1,200-1,750 3,800 10,000 Guinea-pig 1,000-1,800 The olfactory pathway was the only other system studied (see Table 7). In all species the olfactory bulbs synthesized moderate amounts of ACh. Man's olfactory bulb, synthesizing 570,ug ACh/g/hr was one-quarter to one-half as active as the olfactory bulbs of other species. The human olfactory tract had in turn less enzyme than the bulb, possibly because of the admixture of a larger proportion of connective tissue. The olfactory tract of other species contained more choline acetylase than did the bulbs; and there was evidence that the more central parts of the tract in the dog had more choline acetylase than had the peripheral parts. In the olfactory trigone (the surface layer of tissue at the roots of the olfactory tracts) the concentration of enzyme was very high in all species including man. DISCUSSION In this survey of the neuroanatomy of choline acetylase the results obtained go far to substantiate the most important conclusion reached earlier by Feldberg & Vogt (1948). Although compared with their method ours is a more sensitive means of estimating choline acetylase and with it we have observed a much wider range of values of choline acetylase concentrations, our results confirm theirs in showing that choline acetylase is present only in a proportion

8 CHOLINE ACETYLASE IN CENTRAL NERVOUS SYSTEM 725 of neurones and is virtually absent from the axons of two important groups of sensory neurones, the dorsal spinal and the optic nerves. Our conclusion that these two types of sensory nerves contain no choline acetylase is based on the failure to find any evidence of ACh synthesis in a large number of tests. As the tables indicate, however, we have occasionally observed a small rate of ACh production by optic nerves and dorsal roots which did not exceed 25,ug/g/hr. We have considered that these were not significant, in part because they were not reproducible at will being observed in only about 25% of our tests, in part because slight contamination with other more active tissue could not be wholly and certainly excluded as their cause, and finally because they fell so far below the rates observed in cholinergic nerves. The question then arises what meaning can be attached to the claim by other workers that the sensory nerves do synthesize ACh. Cohen (1956) finds that the dorsal roots of the ox synthesize slightly less than 50,g/g/hr in a system which enables the ventral roots to synthesize about 7 mg/g/hr or more. On testing the same tissues of the same species we have found with our system a similar rate of synthesis by the ventral roots but little or no synthesis by the dorsal roots. We think the differences in results may be due to two separate causes. One factor may easily be contamination of the inactive dorsal root tissue with dorsal root ganglia and/or attached ventral root tissue. Another cause of disagreement, and this we think is the most important, is the method of identifying the ester in the incubate. In our experiments we have preferred to check doubtful results by assay on the guinea-pig ileum. We find too that in tests on both guinea-pig ileum and frog rectus it is important that the effect of sensitizing substances in the incubate and the alkali-lability of the active principle should be assessed independently. So long as the spread of values for choline acetylase was represented by so low a ratio as 42:1, while the equivalent ratio for true cholinesterase was about 10 times greater, it was justifiable to suspect that the method of sampling choline acetylase was insensitive. Now, however, this criticism disappears and the evidence is, if anything, more strongly in favour of a discrete distribution of choline acetylase limited to certain types of neurone. No other explanation can adequately account for all the observations. Among those who have taken a different view, Nachmansohn and his coworkers (Nachmansohn & Wilson, 1951) have proposed that both choline acetylase and true cholinesterase are present in all conducting tissues. Nevertheless, they have not yet been able to find any explanation in keeping with this view that would account for the large variation in both enzymes which are observed in mammalian tissues. Nachmansohn in a recent publication has remarked, apropos of cholinesterase in the central nervous system, 'We don't know anything about the meaning of the variations in concentrations of

9 CATHERINE 0. HEBB AND ANN SILVER acetylcholinesterase' (Waelsch, 1955). On the other hand, once it is accepted that there exist neurones of two kinds, one which contains choline acetylase and one which does not, both individual and species variations can be simply explained. The variations in true cholinesterase concentration can be largely accounted for in the same way. It has long been recognized, however, that this enzyme, although probably present in all neurones which contain choline acetylase, is found in other tissues as well. These may include some neurones and very probably the glial tissue in certain parts of the nervous system. The distribution of the two enzymes is not therefore strictly parallel, and in the case of the cerebellum, in which true cholinesterase is relatively concentrated and choline acetylase only sparsely distributed, the discrepancy is large. Our results in the optic system also support Feldberg & Vogt's (1948) suggestion that in some nervous pathways cholinergic alternate with noncholinergic neurones. The large species variations in retinal concentration of choline acetylase are possibly related to a number of differences in the organization of its neural elements. From histochemical evidence (Francis, 1953; Koelle, Wolfand, Friedenwald & Allen, 1952; Hebb, Silver, Swan & Walsh, 1953) it appears that both bipolar and amacrine cells are cholinergic. If so, it is in these cells that the choline acetylase will be found and one might expect its concentration to vary according to both the numbers of amacrine and bipolar cells and the richness of their endings in synaptic relation to the ganglion cells. On these grounds the high concentration of choline acetylase in the bird retina is readily explained. It is not so easy, however, to account for the low concentration of enzyme in the cat retina which contains numerous small amacrine cells. Possibly some amacrine cells are not cholinergic or they may have a less diffuse type of ending. Associated with the large variations in the retinal concentration of choline acetylase are similar variations in the enzyme of the superior colliculus. These two centres are connected by the non-oholinergic third-order neurones which arise from the optic ganglion cells and form the optic nerve and tract. Accordingly, the choline acetylase of the superior colliculus (or optic lobe ofthe birds) is determined by the number of cholinergic neurones which have their cell bodies in and originate from the superior colliculus. The evidence of a quantitative relation between the numbers of cholinergic neurones in the retina and the superior colliculus in different species therefore reinforces the idea that this pathway is composed of alternating cholinergic and noncholinergic neurones. The observations made on the cerebral cortical concentration of choline acetylase of any one species have more meaning when they are considered in relation to corresponding values obtained for other species. In the rabbit and guinea-pig in which there has been the least evolutionary development of the

10 CHOLINE ACETYLASE IN CENTRAL NERVOUS SYSTEM 727 cortex there is scarcely any variation from one part to another. In all other species, however, there are well-marked differences which though quantitatively dissimilar follow the same general pattern. Thus in all these species area 17 has the lowest concentration of enzyme; area 4 has a consistently higher concentration but the areas with the highest concentration are found in certain parts of the allocortex or rhinencephalon. It is also noticeable that it is in the neocortical areas that the enzyme is most affected during the enlargement of the cortex; the change in the rhinencephalon is consistently much less. The olfactory bulb which can be regarded as part of the allocortex-it has a similar neural pattern-is the least changed. It appears then that the great evolutionary development of the neocortex which reaches its maximum in man has been due largely to the multiplication of neurones which are not cholinergic. Because ofthe diminished concentration of cholinergic neurones in the more highly developed brains it has been suggested that they represent a more primitive type of cell (Feldberg, 1945). Our results might also be interpreted in this way, but we feel that on the basis of our present knowledge it is an oversimplification to discuss cholinergic neurones in these terms. 'Cholinergic' or 'non-cholinergic' are descriptions, each of which can apply to many morphological types of neurones including those that have different kinds of axons, myelinated and non-myelinated, fast-conducting and slow-conducting. Apart from the fact that one secretes ACh and the other does not there are no special characters distinguishing the two types of nerve from which it could be decided that one is more primitive than the other. We lack also precise knowledge of the comparative distribution of cholinergic neurones, and until we have this information the question of how primitive is the acetylcholine-releasing nerve cell must be left undecided. SUMMARY 1. The distribution of choline acetylase which has been measured in the nervous system of man and a number of other mammals including the dog and rabbit has been found to vary widely in each species from values approaching zero in certain sensory nerves to very high values in the caudate nucleus and ventral spinal roots, each of which synthesize between 10 and 15 mg ACh/g/hr. 2. The caudate nucleus with a very high concentration and the cerebellar cortex with a very low concentration of enzyme are two tissues which show little or no species variation; while the retina exhibits the largest species variation which has been encountered. 3. Species variations in the cerebral cortical concentration of enzyme are found to be negatively correlated with the degree of cortical development. 4. The evidence supports the hypothesis that choline acetylase is present only in a proportion of the neurones which make up the central nervous system of mammals.

11 728 CATHERINE 0. HEBB AND ANN SILVER We are indebted to Mr G. Bull, Miss Miriam Leonard and Miss Mary Lawes for assistance in the biochemical preparations and the bio-assays in this research. We also wish to make acknowledgment to Dr Marthe Vogt for advice on the neuroanatomy of the dog's brain and to Mr Wylie McKissock and his assistant Miss Joan Hooker, for enabling us to obtain fresh samples of human brain tissue, and to Miss Mary Lawes for preparing neuroanatomical diagrams of each dissection. REFERENCES BURGEN, A. S. V. & CHIPMAN, L. M. (1951). Cholinesterase and succinic dehydrogenase in the central nervous system of the dog. J. Phy8iol. 114, COHEN, M. (1956). Concentration of choline acetylase in conducting tissue. Arch. Biochem. Biophys. 60, FELDBERG, W. (1945). Present views on the mode of action of acetylcholine in the central nervous system. Physiol. Rev. 25, FELDBERG, W. & MANN, T. (1946). Properties and distribution of the enzyme systems which synthesize acetylcholine in nervous tissue. J. Physiol. 104, FELDBERG, W. & VOGT, M. (1948). Acetylcholine synthesis in different regions of the central nervous system. J. Physiol. 107, FRANCIs, C. M. (1953). Cholinesterase in the retina. J. Physiol. 120, HEBB, C. 0. (1955). Choline acetylase in the mammalian and avian sensory systems. Quart J. exp. Physiol. 40, HEBB, C. O., SILVER, A., SWANN, A. A. B. & WALSH, E. G. (1953). A histochemical study of cholinesterases of rabbit retina and optic nerve. Quart. J. exp. Physiol. 38, HEBB, C. 0. & SMALLMAN, B. N. (1956). Intracellular distribution of choline acetylase. J. Physiol. 134, HEBB, C. 0. & WAITES, G. M. H. (1956). Choline acetylase in antero- and retro-grade degeneration of a cholinergic nerve. J. Physiol. 132, KAPPERS, C. U. A., HUBER, G. C. & CROSBY, E. C. (1936). The comparative Anatomy of the Nervous System of Vertebrates including man, vol. II. New York: The Macmillan Co. KLEMPIN. (1921). tber die Architektonick der Grosshirnrinde des Hundes. J. Psychol. Neurol., Lpz., 26, KOELLE, G. B., WOLFAND, L., FRIEDENWALD, J. S. & ALLEN, R. A. (1952). Localization of specific cholinesterase in ocular tissues of the cat. Amer. J. Ophthal. 35, $4. NACHMANSOHN, D. & WILSON, I. B. (1951). The enzymic hydrolysis and synthesis of acetylcholine. Advanc. Enzymol. 12, WAELSCH, H. (1955). Biochemistry of the Developing Nervous System, p New York: Academic Press Inc. WILKIE, J. (1937). The Dissection and Study of the Sheep's Brain, as an Introduction to the Study of the Human Brain. London: Oxford University Press. WrNKLER, C. & POTTER, A. (1911). An Anatomical Guide to Experimental Researches on the Rabbit's Brain. Amsterdam: Versluys. WINKLER, C. & POTTER, A. (1914). An Anatomical Guide to Experimental Researches on the Cat's Brain. Amsterdam: Versluys. ZETLER, G. & SCHLOSSER, L. (1955). fber die Verteilung von Substanz P und Cholinacetylase im Gehirn. Arch. exp. Path. Pharmak. 224,