Folia Psychiatrica et Neurologica Japonica, Vol. 30, No. 2, 1976

Regional Distribution of Choline Acetyltransferase and Acetylcholinesterase Activity in Baboon Brain Yutaka Nakamura, M.D.,* Rolf Hassler, M.D., Kiyoshi Kataoka, M.D.,** I. J. Bak, Ph.D.*** and J. S. Kim, M.D. Neurobiologische Abteilung, Max-Planck-lnstitut fur Hirnforschung, Frankfurt/M-Niederrad, West Germany

* National

Musashi Hospital for Mental and Nervous Diseases, Tokyo

* * Department of Physiology, Ehime

University School of Medicine, Matsuyarna

* * * Department of

Neurology, School of Medicine, The Center for the Health Sciences, Los A ngeles, California 90024, USA

INTRODUCTION

Acetylcholine (ACh) has been studied for more than 40 years as a possible transmitter substance in nervous and it is now widely accepted that cholinergic transmission occurs at the neuromuscular junction6 24 and at Renshaw cells in the spinal cord.8 But little is still known about which neuron systems in the brain are actually cholinergic. For studies on this problem less than 1 mg of tissue of a specific area can be used, and two reasons why progress has not been made are that the procedure for biological assay of ACh is tedious and insensitive and, further, tissue ACh is unstable after death. However, the enzymes involved in synthesis and catabolism of ACh, choline acetyltransferase (ChAc) (EC 2.3.1.6) and acetylcholinesterase (AChE) (EC 3.1 .I .7) respectively are very stable, and highly sensitive assay methods for these enzymes have been developed. Furthermore, regional distribu~

-.

Received for publication March 18, 1976.

tion studies have shown parallel distributions of ACh content and the activities of "? :w these enzymes in some regions.:) Therefore, extensive surveys on AChE activity were made using a simple histochemical staining technique to identify cholinergic fibers." However, as pointed out earlier,1fi the distribution of AChE is not always well correlated with that of ACh, and consequently this enzyme activity does not seem to be the best indicator of cholinergic neurons. ChAc seems to be a better marker of cholinergic innervation, because it is well known that ChAc is mainly localized in nerve terminals with Am79h and interruption of the neurons induces a marked reduction in the enzyme activity."' 2(' There have been many investigations on the general distribution of ChAc activity in brain tissue1('l 4 and in restricted regions of the brain.12 However, further information on this matter is still required. In preliminary experiments, we found that certain small nuclei have the same or higher ChAc activity than the putamen, which has previously been

186

Y. Nakarnura et al.

considered to have the highest activity. Therefore, in the present study, we determined the ChAc and AChE activities in more than one hundred regions and subregions of baboon brain. The results in general confirmed previous data and provided data on regions not previously analyzed. The data in this paper provide extensive information on levels of cholinergic innervation in various regions of the central nervous system. MATERIAL AND METHODS Nine young baboons of both sexes weighing 2.5-6 kg were sacrificed by decapitation. The whole brain was quickly removed and the tissue was dissected on a ice-cold glass in a cold room. Samples of 0.2 mg to 30 mg of tissues from various regions were dissected out with small scissors or punched out with a copper tube (internal diameter; 0.8 mm or 2.0 mm). The tissue was weighed on a torsion balance, and then transferred into a small glass vial with a plastic cap and frozen in liquid nitrogen for one hour. Then it was stored at - 80°C until examined. For examination, the frozen brain tissue was thawed and homogenized in a small glass homogenizer with 100 pi of ice-cold 0.25% (viv) Triton X-100 solution. In some cases when more than 5 mg of tissue were used, the volume of homogenate was increased appropriately from 200 pf to 500 pl. The activity of ChAc was assayed essentially by a radiochemical method'" '"I with the following slight modifications. We checked that these modifications did not alterate the results seriously. The sample of 1 to 2 p1 of tissue homogenate was placed in 10 pl of buffered substrate solution in a plastic microtube (Beckman) and incubated at 37°C for 30min. The reaction was terminated by adding 1 pl of 200mM choline (in 3 N trichloracetic acid) in the cold and

the mixture was centrifuged at 10,000 rpm for 4 min in a Mikrofuge (Beckman). Then 5 pl of the resulting supernatant was transferred to another inicrotube containing 10 pl of 5 m M acetyl-CoA (in 0.5 N HCI) and 5 pl of 0.5 N HCI saturated with ammonium Reineckate was added. The mixture was centrifuged and the precipitate was washed with 100 pl of cold acetone and the radioactivity of a sample of 50 pf was counted in normal toluene scintillation solution. The activity of AChE was determined by a spectrophotometric method." The values shown in the tables are means2S.E.M. of values in the numbers of animals shown in parentheses, and ChAc and AChE activities are expressed as mmoles of ACh synthesized/kg wet weight/hr and moles of acetylthiocholine degraded/kg wet weight/hr, respectively. RESULTS 1. Postmortem stability of the enzyme activity Removal of selected tissues takes 120 to 150 min. Therefore the postmortem change in the enzyme activity in baboon brain was first studied. For this we compared the activity in the gyrus rectus at different times after death. Four specimens were removed as soon as possible after isolation of the whole brain. Two of them were rapidly frozen in liquid nitrogen and the other two were stood on ice for 150 min before freezing. The postmortem decrease of ChAc activity thus examined was found to be less than 10%. No further change in ChAc activity was noted when the tissue was stored at -80°C for up to 14 days. The stability of this enzyme in human brain has been reported by other workers.:! Similar results were obtained on glutamic acid decarboxylase activity in brain tissues of the baboon." The ac-

Cholinergic Enzymes Distribution in Baboon Brain

187

tivity of AChE decreased by less than 10% were found in the sensory cortex (area 2, when the tissues were stood on ice for 150 area 3-a and area 3-b), the motor cortex and min and did not change significantly on the visual cortex (area 17). Area 24 had lowstorage at -80°C for 14 days. However, the er activity and area 32 had the lowest ChAc AChE activity of homogenates was less activity of all regions of the cerebral cortex stable than that of whole tissues, and in examined. The values obtained for the cerehomogenates the AChE activity decreased bral cortex were all equivalent to, or far less quicker than the ChAc activity, even in the than 1 mmole ACh synthesized/kg wet frozen state. weight/ hr, which is distinctly lower than the values in other regions of the described data. 2. Cerebral cortex The AChE activities in these cortical subregions were rather similar to each other, but The distribution of ChAc activity in 15 the activity of about 0.1 mole acetylthioregions of the cerebral cortex was studied choline degraded/kg wet weight/hr was and results are shown in Table 1. For exmuch less than the activities in other regions amination, the tissues were dissected out and of the brain. as much white matter as possible was removed by naked eye. The enzyme activity in the cerebral cortex is, in general, much lower than that in the basal ganglia, which are known to have the highest activity in the central nervous system. It is interesting that differences were found in the activities in regions with different functional properties. The premotor areas (area 6-a, area supplementary) and the area recta seem to have rather high activities. Moderate activities

Table 1: Distribution of ChAc and AChE in Different Areas of the Cerebral Cortex of the Baboon Areas Motor cortex 2 3-a 3-b 6 6-a supplementary 8 17 (pole) 17 (convex) recta 39

ChAc 0.74k0.28(3) 0.67k0.10(3) 0.69k0.22(3) 0.76k0.21(3) 0.71 (1) 0.98k0.31(4) 1.0320.13(6) 0.56*0.15 (4) 0.64k0.21(3) 0.66-+0.21(4) 1.08k0.29(4) 0.69zt0.22(4)

AChE 0.09 (2) 0.07 (2) 0.14 (2) 0.15 (2) 0.11 (1) 0.09 (2)' 0.1220.01 (5) 0.06k0.01 (4) 0.09k0.03 (3) 0.12 (2) 0.10~0.01 (4) 0.11 A0.05 (3)

3. Subcortical telencephalic nuclei The activity in the neostriatum (the putamen and the caudate nucleus) was much higher than in other regions and was 10 to 20 times more than those in the cerebral cortex (Table 2). This agrees with earlier reports that the neostriatum has the highest activity not only of ChAc but also of AChE" and the highest ACh content.:"' There was

Table 2: Distribution of ChAc and AChE in Different Subcortical Telencephalic Nuclei of the Baboon Area

ChAc

AChE ~~

Caudate nucleus Fundus caudati 8.83k0.95(8) 1.89k0.13(10) Head 9.5420.77(4) 2.18 (1) Middle 16.5k1.26(4) 3.7020.68(3) Tail 10.0*1.42 (4) 2.28k0.79(2) Putarnen 16.7-Cl.68(4) 2.15k0.11(3) Pallidurn 1.22k0.14(7) 0.40*0.10 (7) interna Pallidurn 1.74zt0.31(5) 0.56k0.10(5) externa Nucleus basalis 8.80rt0.53 (10) 1.01kO.32(9) Claustrurn 1.7720.43 (3) 0.7620.66(3)

Y . Nakamura et al.

188

little difference in the activities of ChAc and AChE in various subregions of the caudate nucleus. The activities were much less in the pallidum, ChAc activity being about one tenth of that in the putamen and AChE activity being about one-third of that in the putamen. The enzyme activities in the internal and external parts of the pallidum were similar. It is interesting that the nucleus basalis, which may be regarded as part of the pallidum had exceptionally high ChAc and AChE activities. The claustrum has activity similar to the pallidum. 4. Rhinencephalon and limbic system As shown in Table 3, the ChAc and AChE activities in the perforated substance were high, being several times higher than those in the cerebral cortex. The activities of both enzymes were also relatively high in the septum, being higher in the dorsal septum than in the ventral septum. Selected regions of the hippocampus, namely CA (1 2) and CA (4+dentate gyrus), and the prepyriform

+

region had lower activities, but these were still higher than those of the cerebral cortex. The entorhinal area had similar ChAc activity to the hippocampus, but lower AChE activity. The amygdaloid nuclei also had quite high ChAc but relatively low AChE activities. A characteristic feature of the distribution patterns of these two enzymes in different amygdaloid nuclei was that ChAc activity was higher in the central amygdaloid nucleus than in the medial and basolateral amygdaloid nuclei, whereas AChE activity differs little from each other. An earlier report described that the central amygdaloid nucleus had practically no AChE activity by a histochemical method.lx 5 . Diencephalon

In regions of the diencephalon, the activities of the cholinergic enzymes were relatively low, being only 2-3 times higher, or the same as those in the cerebral cortex. The enzyme activities in different subregions of the thalamus varied. As summarized in Table

Table 3: Distribution of ChAc and AChE in Different Areas of the Rhinencephalon and the Limbic System of the Baboon Area

ChAc

AChE ~

Substantia perforata Septum dorsalis Septum ventralis Hippocampus CA (1+2) CA (4+gyrus dentatus) Entorhinal area Prepyriform area Area 24 Area 32 Amygdala Medial Basolateral Central

11.3k1.13 (3) 6.20k2.03 (4) 5.50k0.98 (4)

0.57 (1) 1.03 k0.50 (4) 0.85k0.32 (4)

2.28k0.42 1.7 1k0.3 1 2.37k0.35 2.22k0.19 0.32k0.06

(3) (3) (4) (4) (4)

0.21k0.06 (3) 0.19rt0.04 (4)

0.06

(2)

4.49k0.55 (5) 5.22rt0.70 (5) 9.15*1.54 (3)

0.13 (2) 0.28k0.09 (3) 0.05 0.14

(2) (2)

0.16+0.08 (5) 0.23 k0.04 (4) 0.19k0.06 (3)

Cholinergic Enzymes Distribution in Baboon Brain 4, the habenula and the nucleus limitans had relatively high ChAc activity. AChE activity was also high in the latter nucleus but low in the habenula. In nucleus anteromedialis, nucleus medialis fibrosus, nucleus dorsalis

189

superficialis, nucleus intralaminalis and nucleus pulvinaris, ChAc activity was relatively high. On the other hand, the AChE activity was higher in the nucleus limitans, nucleus dorsalis superficialis, nucleus me-

Table 4: Distribution of ChAc and AChE in Different Areas of the Diencephalon of the Baboon Area Habenula Nucleus anteroventralis thalami anteromedialis thalami ventralis anterior thalami ventralis oralis anterior thalami ventralis oralis posterior thalami ventralis posterolateralis thalami ventralis intermedius thalami medialis fibrosus thalami medialis caudalis thalami dorsalis superficialis thalami dorsalis oralis thalami dorsalis intermedius thalami reticularis thalami limitans thalami parafascicularis thalami centromedianus thalami intralaminaris thalami Nucleus pulvinaris Nucleus geniculatum medialis geniculatum lateralis Nucleus subthalamicus Zona incerta Hypothalamus Bed nucleus of stria terminalis lnfundibulum Mammillary body Nucleus hypothalamicus ventromedialis Area preoptica Pineal body Pituitary Posterior lobe Anterior lobe

AChE

ChAc 4.43 20.55 1.6520.34 3.10k1.17 15620.24 1.06+0.10 1.07k0.56 1.52k0.53 1.97k0.48 3.30k0.97 1.34k0.27 3.34k0.59 0.85k0.32 1.4420.13 1.79k0.56 4.52k0.15 1.82k0.3 1 1.74k0.46 2.55k0.98 2.80k0.32 1.34*0.21 2.64k0.73 1.96kO.27 0.79k0.29

(4) (5) (4) (4) (5) (5) (5) (3) (4) (4) (5) (3) (5) (4) (3) (4) (4) (3) (4) (4) (4) (6) (4)

0.46k0.13 0.3 1k0.08 0.57k0.23 0.48k0.16 0.34k0.06 0.34k0.06 0.36k0.03 0.08kO.01 0.71k0.11 0.28k0.01 0.85k0.15 0.18*0.08 0.29*0.07 0.66k0.08 1.80 0.40 0.66k0.39 0.25k0.15 0.49k0.03 0.19k0.04 0.40k0.24 0.50k0.19 0.50k0.05

(3)

(5) (3) (3) (3) (4) (3) (3) (7) (3) (4) (3) (3) (3) (1) (2) (3) (3) (3) (3) (3) (5) (3)

1.42k0.41 (4) 0.84k0.21 (4) 1.51k0.53 (3)

0.64k0.38 (4) 0.31k0.04 (3) 0.23*0.01 (3)

0.57+0.11 (4) 0.91 k0.28 (3)

0.26k0.02 (3) 0.45k0.11 (4) 0.23k0.05 (3)

0.1 1

(2)

0.19

(2)

Y. Nakamura et

190

dialis fibrosus and nucleus centromedianus. The ChAc and AChE activities were both higher in the lateral geniculate nucleus than in the medial geniculate nucleus. The hypothalamic subregions and the subthalamic nucleus had very low ChAc activity like the cerebral cortex. However, the AChE activity in these structures was rather high like those in the thalamic regions. Little enzyme activity was found in the pineal body or the pituitary gland.

6. Midbrain Results are summarized in Table 5 . The ChAc activity in the interpeduncular nucleus was nearly 20 mmoles ACh synthesized/kg wet weight/hr, which is about twice that in the putamen, the region formerly considered to have the highest activity. The AChE activity in this region was also very high, but not so high as in the putamen. High ChAc and AChE activities were found in the superior colliculus and much lower activities in

a/.

the inferior colliculus. The nucleus linearis raphe had moderately high ChAc activity with rather low AChE activity. Three coordination nuclei, the nucleus interstitialis Cajar, the nucleus prestitialis and the nucleus precommisularis also had moderately high activities. The red nucleus, locus caeruleus and the periaquaductal gray had rather low activities. ChAc activity in the substantia nigra was far less than those in other brain nuclei and even less than those in the cerebral cortex. In the subregions of the substantia nigra examined, the activity was highest in the lateral posterior part of the pars compacta and lowest in the pars reticulata. The AChE activity in the substantia nigra was several times higher than those in the cerebral cortex.

7. Cerebellum Table 6 shows results obtained from various regions of the cerebellum. The ChAc activity was very low in all the cerebellar

Table 5 : Distribution of ChAc and AChE in Different Areas of the Midbrain of the Baboon Area Substantia iiigra Pars cornpacta lateralis posterior Pars compacta medialis posterior Pars cornpacta medialis anterior Pars reticulata Nucleus ruber Nucleus interpeduncularis Nucleus interstitialis Cajar prestitialis precommissularis linearis raphe Colliculus superior Colliculus inferior Periaquaductal grey Locus caelureus Tractus retroflexus

ChAc

AChE

1.21+0.34 0.46k0.13 0.71 k0.25 0.34k0.04 2.86k0.97 26.6k1.72 6.93k0.99 3.8520.71 3.72k0.48 6.70+0.15 5.62k1.69 0.63+0.01 3.26k0.60 3.29k0.67

(5)

8.46

(2)

(5) (7) (8) (4) (4) (3) (9) (9) (3) (4) (4) (3) (5)

1.0120.22 0.83*0.18 0.610.15 0.68k0.08 0.33k0.03 2.37k0.08 0.79k0.26 0.41k O . 0 5 0.96k0.23 0.1 1 k0.06 0.9620.10 0.16+0.03 0.24k0.05 0.28k0.08 0.13k0.03

(6) (5) (8) (8) (4) (3) (5) (9) (9) (3) (3) (3) (3) (4) (4)

Cholinergic Enzymes Distribution in Baboon Brain regions studied. The activity in the cerebellar nuclei was higher than in the cerebellar cortical regions examined. On the contrary, the AChE activity was generally rather higher in these cerebellar structures. 8. Medulla oblongata and pons

In the medulla oblongata, the ChAc activity was generally considerably higher in motor nuclei than in sensory nuclei (Table 7). Among the former, the enzyme activity was highest in the oculomotor nucleus, fol-

lowed by the trochlear nucleus. Among the latter, the nucleus gracilis and nucleus cuneatus had rather high ChAc activity. AChE activity in the latter structure has been discussed by earlier authors.:’:’ Among the vestibular nuclei, the nucleus vestibularis descendens and the nucleus Deiters ventralis had higher ChAc activity than the nucleus Deiters dorsalis. The inferior olive nucleus and the pyramis had extremely low ChAc activity. The distribution of AChE activity was somehow different, activity being relatively high in the nucleus hypoglossus, followed by the nucleus cuneatus.

Table 6: Distribution of ChAc and AChE in Different Areas of the Cerebellum of the Baboon Area

ChAc

Culmen Tuber vermis Nodulus Nucleus fastigi interpositus dentatus

AChE

0.20k0.04 (4) 0.88k0.03 (4) 0.25k0.03 (3) 0.38k0.06 (3) 0.35k0.11 (3) 0.38k0.05 (3) 0.99k0.20 (4) 0.25k0.03 (4) 1.95k0.22 (4) 0.37k0.04 (4) 1.4120.25 (4) 0.43k0.04 (4)

191

DISCUSSION To identify the specific cholinergic neuronal systems, a topographical study may be required and accordingly the present study is part of a rough survey of the distribution of the cholinergic mechanism in the central nervous system. Similar studies have been made in the past, but so far available information is still not adequate. The present data

Table 7: Distribution of ChAc and AChE in Different Areas of the Medulla Oblongata and Pons of the Baboon Area Nucleus oculomotorius trochlearis hypoglossus dorsalis vagi solitarius spinalis trigemini Deiters dorsalis Deiters ventralis vestibularis descendens gracilis cuneatus Formatio reticularis oblongata Nucleus olivaris inferior Pyramis

ChAc 8.83 f1.33 (4) 55220.94 (3) 3.11-1-0.81 (4) 3.05k0.77 (6) 2.06k0.73 (5) 3.29k0.70 (3) 0.71k0.21 (5) 1.67-1-0.17 (5) 2.03k0.32 (3) 3.0120.14 (3) 3.21-1-1.05 (3) 5.81k1.82 (4) 0.78k0.04 (3) 0.13*0.01 (4)

AChE 0.73 20.07 0.47k0.11 1.48k0.60 0.53 k0.07 0.3720.18 0.26 0.29k0.06 0.37k0.05 0.47k0.09 0.34 0.8720.06 0.17-1-0.07 0.29k0.01 0.36k0.03

(3) (4) (3) (5) (4) (2) (4) (4)

(5) (2) (3) (3) (3) (3)

192

Y. Nakamura et

in general confirm and extend earlier findings by others on the regional distribution of the activities of enzymes involved in the synthesis and catabolism of ACh." ' ':{ l7 22 28 The results show that the regional distribution patterns of ChAc activity and AChE activity do not entirely coincide. In different hippocampal regions, these two enzymes are distributed almost in parallel.12 However, in the dorsal root there is practically no ChAc activity but high AChE activity.:' ' l' Other examples of this found in the present work are the pallidum and the substantia nigra where ChAc activity is quite low whereas AChE activity is relatively high. Thus, as illustrated in Table 8, the ratios of the activity of ChAc to AChE are very different from region to region. The high values were obtained in amygdala and the interpeduncular nucleus, and the low ratio in the substantia nigra; the values of the former being approximately 20 to 40 times as much as that of the latter. Recently, we found that habenular coagulations induce a marked decrease in ChAc activity but only slight reduction of AChE activity."' These results strongly indicate that validity of AChE activity as a marker enzyme for cholinergic neurons is not always satisfactory. Earlier investigators253:1 traced AChE histochemically in different specific regions of the brain by placing interruption of various neurons. Their extensive studies provide much information on this problem, but still do not seem to identify cholinergic neurons precisely. In this respect, an earlier report should be mentioned that not only cholinergic neurons but also catecholaminergic neurons sometimes give a positive staining reaction for AChE. Furthermore, studies on the subcellular distribution of ChAc clearly indicate that ChAc is located mainly but not exclusively in synaptosomes. The subcellular distribution of AChE is rather different from that of ChAc.' :I4 Therefore loss of ChAc activi-

a/.

Table 8: Ratios of the Activity of ChAc to that of AChE in Different Areas of the Baboon Brain ChAc/ AChE

Area -

- _ _ _ _ _ _ ~ . _ _

Amygdala (central) Interpeduncular nucleus Area 6 Caudate nucleus (Head) Nucleus ventralis anterior thalami Nucleus hypothalamicus ventromedialis Substantia nigra (Pars reticulata)

48.1 11.2 6.45 4.38 3.23 2.19

0.50

-~

ty after neuronal interruption should be one of the most important signs of degeneration of cholinergic synapse. In this work very high ChAc activity was found in the interpeduncular nucleus. As shown in Table 5 , the estimated value was almost twice those in the putamen and the caudate nucleus, which have previously been thought to have the highest ChAc and AChE activities. Using a bioassay technique earlier investigators observed extremely high ChAc activity in this nucleus in the rat, in the order of more than one hundred times that in the striatum."' Therefore, we determined ChAc activity in the interpeduncular nucleus of the rat, the rabbit and the guinea pig, and found that the activities were similar to that in the baboon.'" Cranial motor nuclei, such as the oculomotor nucleus and the trochlear nucleus, have similar ChAc activity to that in the putamen or the caudate nucleus, whereas the cranial sensory nuclei in general have lower cholinergic enzyme activity. It is interesting that in the thalamus the enzyme activities showed some differences among more than 20 subregions. The enzyme activity in the hypothalamus was lower than that in the thalamus. However, our results on the AChE activity in the mamillary body were very different from those reported by othersI3 l e : they reported very high en-

Cholinergic Enzymes Distribution in Baboon Brain

zyme activity in this structure whereas we found low activity. The cerebral and cerebellar cortex also have low enzyme activities as reported by other investigators. This may indicate that cholinergic mechanism is rather sparse in these structures.s Is l4 l7 22 23 28 In conclusion, the present investigation may be a guide and provide a number of hints in quantitative analysis a n d / o r identification of cholinergic neuronal systems in the central nervous system. SUMMARY

( I ) The activities of choline acetyltransferase (ChAc) ( E C 2.3.1.6) and acetylcholinesterase (AChE) ( E C 3.1.1.7) were determined in about one hundred regions and subregions of baboon brain. The activities and distributions of these enzymes were in comparable to those found previously in the brains of other species. (2) ChAc activity was highest in the interpeduncular nucleus, where it was about twice that in the putamen, the region previously thought to be the richest in this enzyme. The caudate nucleus, the substantia perforata, the nucleus basalis, the central part of the amygdala and the oculomotor nucleus also had high activities. The activities in the cerebral and cerebellar cortex were less than one twentieth of that in the interpeduncular nucleus. (3) The distribution of A C h E activity was not entirely in parallel with that of ChAc. REFERENCES Aldridge, W. M. and Johnson, M. K.: Cholinesterase, succinic dehydrogenase, nucleic acid esterase and glutathion reductase in subcellular fractions from rat brain. Biochem J, 73: 270-276, 1959. Bull, G . and Hebb, C. 0.: Choline acetyltransferase activity of human brain tissue during development and at maturity. J

193

Neurochem, 17: 1505-1 5 16, 1970. 3 Burgen, A. S. V. and Chipman, L. M.:Cholinesterases and succinic dehydrogenase in the central nervous system. J Physiol (Lond.), 114: 296-305, 1951. 4 Burgen, A. S. V. and Chipman, L. M.: The localization of cholinesterase in the central nervous system. Quart J exp Physiol, 37: 61-74, 1952. 5 del Castillo, J. and Katz, B.: Localization of active spots with the neuromuscular junction of the frog. J Physiol (Lond.), 1 3 2 630-649, 1956. 6 De Robertis, E., Pellegrino de Iraldi, A., Rodriguez de Lores Arnaiz, G. and Salganicoff, L.: Cholinergic and non-cholinergic nerve endings in rat brain. 1. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J Neurochem 9: 23-35, 1962. 7 De Robertis, E., Rodriguez de Lores Arnaiz, G., Salganicoff, L., Pellegrino de Iraldi, A. and Zieher, L. M.: Isolation of synaptic vesicles and structural organization of the acetylcholine system within nerve endings. J Neurochem 10: 225-235, 1963. 8 Eccles, J. C., Fatt, P. and Koketsu, K.: Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons. J Physiol (Lond.), 1 2 6 524562, 1954. 9 Ellman, G. L., Courtney, K. D., Andres, V. Jr. and Faetherstone, R. M.: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol, 7: 88-95, 1961. 10 Fahn, S. and C8t6, L. J.: Regional distribution of choline acetylase in the brain of the rhesus monkey. Brain Res, 7: 323-325, 1968. 1 1 Feldberg, W.: Present views on the mode of action of acetylcholine in the central nervous system. Physiol Rev, 25: 596-642, 1945. 12 Fonnum, F.: Topographical and subcellular localization of choline acetyltransferase in rat hippocampal region. J Neurochem, 17: 1029-1037, 1970. 13 Goldberg, A. M. and McCaman, R.E.: A quantitative microchemical study of choline acetyltransferase and acetylcholinesterase in the cerebellum of several species. Life Sci, 6: 1493-1500, 1967.

194

Y. Nakamura et ul.

14 Hebb, C. 0. and Silver, A.: Choline acetylase in the central nervous system of man and some other mammals. J Physiol (Lond.), 134: 718-728, 1956. 15 Hebb, C. 0.: Biochemical evidence for the central function of acetylcholine. Physiol Rev, 37: 196-220, 1957. 16 Hebb, C. 0. and Whittaker, V. P.: Intracellular distribution of acetylcholine and choline acetylase. J Physiol (Lond.), 142: 187-196, 1958. 17 Hebb, C. 0. and Ratkovic, D.: Choline acetyltransferase in the evolution of the brain in vertabrates. In Comparative Neurochemistry. Richter, D. (ed.) Pergamon Press, 347-354, 1964. 18 Ishii, T. and Friede, R. L.: A comparative histochemical mapping of the distribution of acetylcholinesterase and nicotineamide adenine dinucleotide diaphorase activities in the human brain. In International Review of Neurobiology. Pfeifer, C. C. and Smythies, J. R. (eds.) Academic Press. vol. 10, 232-275, 1967. 19 Kataoka, K., Nakamura, Y. and Hassler, K.: Habenulo-interpeduncular tract: a possible cholinergic neuron in rat brain. Brain Res, 62: 264-267, 1973. 20 Kataoka, K., Bak, I. J., Hassler, R., Kim, J. S. and Wagner, A,: L-glutamate decarboxylase and choline acetyltransferase activity in the substantia nigra and the striatum after surgical interruption of the strionigral fibers of the baboon. Exptl Brain Res, 19: 217-227, 1974. 21 Kataoka, K., Nakamura, Y., Hassler, R., Bak, I. J. and Kim, J. S.: Activity of Lglutamic acid decarboxylase in different regions of baboon brain. Folia Psychiat Neurol Jap, 29: 361-370, 1975. 22 Koelle, Ci. B.: The histochemical localization of cholinesterases in the central nervous system of the rat. J Comp Neurol, 100: 21 1-235, 1954. 23 Koelle, G. B.: Cytological distributions and physiological functions of cholinesterases. In Handbuch der experimentellen Pharmakologie. Eichler, 0. and Farah, A. (eds.) Springer Verlag. vol. 15, 187-298, 1963. 24 Krnjevic, K. and Miledi, R.: Acetylcholine in mammalian neuromuscular transmission. Nature (Lond.), 182: 805-806, 1958.

25 Lewis, P. R. and Shiite, C. C. D.: The cholinergic limbic system; Projections to hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornical organ and supraoptic crest. Brain, 90: 521-540, 1967. 26 Lewis, P. R., Shiite, C. C. D. and Silver, A.: Confirmation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus. J Physiol (Lond.), 191: 215-224, 1967. 27 Loevi, 0. and Navratil, E.: Ober humarale Obertragbarkeit der Herznervenwirkung. X. Ober das Schicksal des Vagusstoffs. Pfliigers Arch, 214: 678-688, 1926. 28 McCaman, R. E. and Aprison, M. H.: The synthetic and catabolic enzyme systems for acetylcholine and serotonin in several discrete areas of the developing rabbit brain. In Progress in Brain Research. Himwich, W. A. and Himwich, H. E. (eds.) Elsevier vol. 9, 220-233, 1964. 29 McCaman, R. E. and Hunt, J. M.: Microdetermination of choline acetylase in nervous tissue. J Neiirochem, 12: 253-259, 1965. 30 MacIntish, F. C.: The distribution of acetylcholine in the peripheral and central nervous system. J Physiol (Lond.), 99: 436442, 1941. 31 Paton, W. D. M.: Central and synaptic transmission in the nervous system (pharmacological aspect). Ann Rev Physiol, 20: 43 1-470, 1958. 32 Silver, A,: Cholinesterases of the nervous system with special reference to the cerebellum. In International Review of Neurobiology. Pfeiffer, C. C. and Smythies, J. R. (eds.) Academic press. vol. 10, 57-109, 1967. 33 Shute, C . C . D. and Lewis, P. R.: The ascending cholinergic reticular system.: neocortical, olfactory and subcortical projections. Brain, 90: 497-520, 1967. 34 Toschi, G.: A biochemical study of brain microsomes. Exptl Cell Res, 16: 232-255, 1959. 35 Whittaker, V. P.: The application of subcellular fractionation techniques to the study of brain function. In Progress in Biophysics and Molecular Biology. Buttler, J. A. V. and Huxley, H. E. (eds.) Pergamon Press. vol. 15, 41-96, 1965.