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Morphology of acetylcholinesterase-containing neurons in primary cultures of dissociated rat cerebral cortex
392
Brain Reseurch, 361 ( 19851 392- 395 Elsevier
BRE 21252
Morphology of acetylcholinesterase-containing neurons in primary cultures of dissociated rat c e r e b ~ cortex W. ERIC THOMAS Department of Physiology, Meharry Medical College, Nashville, TN 37208 (U. S. A. )
(Accepted August 6th, 1985) Key words: cerebral cortex - - tissue culture - - acetylcholinesterase - - cholinesterase histochemistry - neuronal morphology - - bipolar cell
Acetylcholinesterase-containing neurons were investigated in primary cultures of cerebral cortex. Neuronal cholinesterase staining was essentially totally attributable to acetylcholinesterase based on its pattern of sensitivity to pharmacological inhibitors. The mean percentage of stained neurons in the cultures was 2.17. Stained neurons of all morphologies were detected; however, the majority of the cells possessed bipolar morphology. The stained bipolar neurons were not a homogeneous morphological population.
The question of the existence of cholinergic neurons intrinsic to the cerebral cortex has long been an unresolved one. A variety of different techniques, including biochemical analysis of cholinergic components following cortical undercutting4,7.9, histochemical staining for cholinesterase t0,11,tS, and immunohistochemical localization of choline acetyltransferase (CHAT, E C 2.3.1.6) 14, have been employed to investigate this question; however, none has provided a definitive answer, A n alternative approach involves the implementation of primary tissue culture techniques. The presence of afferent processes, some fractions of which are known to be cholinergic6.1tA2,15, in cortex from extrinsic neurons represents a major complicating factor in most in vivo studies. This complication is eliminated in tissue culture and the neurochemistry of endogenous cortical cells can be specifically investigated, in addition tissue culture offers the advantages of control over the size of the cell population and increased accessibility of individual neurons. The synthesis of acetylcholine in primary cultures of cerebral cortex has recently been reported L6. These cultures exhibited high affinity uptake of radiolabelled choline and converted a fraction of the choline to acetylcholine. This acetylcholine synthesis
was shown to occur via the enzyme CHAT. Thus the cortical cultures appear to contain a population of cholinergic neurons. The present report describes initial efforts to establish the identity of these cells. Also the relative proportion of cholinergic neurons in the cultures was investigated. Cortical cultures were established and maintained as previously described 16. Briefly, 15-day-old embryos were removed from timed-pregnant rats using aseptic surgical conditions. Cerebral cortical tissue was selectively dissected from the embryos and dissociated by trituration. Dissociated cells were plated in 35 mm tissue culture dishes (5 × 104 cells per dish). Cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C Under these conditions, cortical neurons display mature features by 10-14 davs in vitro. All cultures used in this study were 14-36 days in vitro. Cultures were stained for cholinesterase enzyme according to the method of KoellO 0 as described by Dells et al. 1. In this procedure, formaldehyde-fixed cultures are treated with dimethyl sulfoxide. Thiocholine formed by the action of the enzyme on the substrate acetylthiocholine in the staining solution complexes "~,ith copper and is precipitated with sodium sulfide. In some cases, cholinesterase staining was intensified by a postincuba-
Correspondence: W.E. Thomas, Department of Physiology, Meharry Medical College, Nashville, TN 37208, U.S.A.
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
393 TABLE I
Pharmacological sensitivity of cholinesterase staining Cortical cultures were fixed and stained as described in the text. The indicated inhibitors were included in the staining solution. Stained neurons were determined by direct counting using light microscopy. All values are the total stained neurons per culture under each condition. The N.D. denotes none detected.
Plating no.
No inhibitor
I
113 +
II
187 172 293 199 434
III IV V VI
+ + + + +
23 48 33 51 45 82
Ethopropazine
Iso-O M PA
124 + 10 159 + 25 -
tion in 1% silver nitrate. When esterase inhibitors were used, these compounds were included in the staining solution. Inhibitors were used at the following concentrations: neostigmine 0.1 mM; ethopropazine 0.2 mM; tetraisopropylpyrophosphoramide (IsoOMPA) 1.0 mM; and 1,5-bis-[4-allyldimethyl-ammoniumphenyl]pentan-3-one dibromide (BW284C51) 0.1 mM. Stained cultures were examined under light microscopy using a Zeiss Standard 16 microscope. Cholinesterase reaction product was observed throughout the cortical cultures. Very light staining was detectable in non-neuronal cells, usually confined to the nucleus. Other cells, identified as neurons on the basis of morphological criteria, exhibited more intense staining. Reaction product was present throughout the somata and at least the proximal portion of processes. While this neuronal staining was variable in intensity between different cells, through optimization of the staining reaction time a clearer
TABLE II
Percentage of neurons containing AChE The total stained neurons per culture was determined in cultures from several different platings. The total neurons in the cultures was then determined to evaluate the percentage of cells containing ACHE.
Plating no.
Total neurons per culture
Neurons stained for A ChE
% Neurons stained for AChE
I II III IV V VI
14,622 6448 10,562 13,146 13,156 8488
555 113 187 248 297 133
3.80 1.75 1.77 1.89 2.26 1.57
+ + + + + +
2410 792 753 878 1026 1131
+ 102 + 59 + 48 + 94 -+ 86 + 22
Neostigmine
BW284C51
-
2 + 0
-
164 340 179 472
1+ 0 N.D. N.D. N.D.
N.D. N.D. N.D. N.D. N.D.
+ + + +
10 32 61 104
distinction between stained and unstained neurons was achieved. This allowed the quantitation of stained neurons by direct counting. All neuronal counts are expressed as the mean of 3 or more determinations + S.D. The number of stained neurons per culture in all experiments ranged from 113 + 23 to 555 + 102 (see Tables I and II). In an initial series of experiments, the nature of the activity producing the reaction product was investigated. This was done by using various pharmacological inhibitors. The results of experiments on cultures from six different platings are presented in Table I. The general cholinesterase inhibitor neostigmine abolished virtually all the neuronal staining. This confirmed that the reaction product was produced by a cholinesterase enzyme. The pseudo-cholinesterase inhibitors, ethopropazine and IsoOMPA, were without effect on the mean number of stained neurons per culture. The specific acetylcholinesterase (ACHE, EC 3. !. 1.7) inhibitor BW284C51 always reduced the mean number of stained neurons to a non-detectable level. Thus, on the basis of these pharmacological properties, all or most of the neuronal staining was attributable to ACHE. To determine the percentage of AChE-containing neurons, stained neurons were counted; then, the total neurons were counted in cresyl violet-stained sister cultures. The results of such experiments from six different platings are shown in Table II. The overall mean percentage of AChE-containing neurons was 2.17. Finally, the morphology of the stained cells was evaluated. Entire cultures were scanned and all neurons stained enough to reveal their detailed morphology were classified. Four morphological classes were designated - - pyramidal, stellate, bipolar and bi-
394
A t
..
-
i!ii
Fig. 1. Stained neurons of different morphologies. A-D: various subtypes of AChE-containing bipolar cells. E and F: examples of stained pyramidal and stellate neurons, respectively. Scale bar = 20 u m
tufted (according to the classification scheme of Feldman and PetersS). The accumulative results for the morphological distribution of stained neurons in 34 different cultures are shown in Fig. 1. Pyramidal cells constituted 10.3% of the stained neurons, stellate 11.1%, bipolar 66.9% and bitufted 11.7%. Thus, the majority of the A C h E - s t a i n e d neurons exhibited bipolar morphology; however, stained neurons of all morphologies were observed. Typical stained neurons with p y r a m i d a l and stellate (multipolar) morphology are shown in Fig. 2E and F, respectively. While no systematic subclassification of stained bipolar cells was a t t e m p t e d , variability in aspects of the detailed m o r p h o l o g y of these cells, particularly soma size and shape, was observed (Fig. 2 A - D ) . Some stained bipolars possessed round or oval somata, oth-
ers had distinctively m o r e elongated somata. The smallest cells averaged 5 - 1 0 u m in somal diameter. The largest appearing cells averaged 15-25 ~tm in diameter. 2OOO
o o Z
tO00
E 0
Pyramidal
Stellate Morphological
Biaolar Clal~es
Bitufted
Fig. 2. Histogram showing the morphological distribution of AChE,stained neurons. Morphologically identifiable stained neurons from 34 different cultures (a total of 2708 cells) were classified.
395 These findings provide further support for the presence of cholinergic cells in the cortical cultures. The existence of such cells suggests that there are cholinergic neurons intrinsic to the cerebral cortex in vivo. This contention is also s u p p o r t e d by several recent immunohistochemical studies indicating the presence of ChAT-positive neurons in cortex 3,8,17. The percentage of A C h E - c o n t a i n i n g neurons detected in the present study was a p p r o x i m a t e l y 2. This must be viewed as an u p p e r limit considering the d e m o n strated lack of total specificity of A C h E as a m a r k e r for cholinergic cells 2. H o w e v e r , this finding is in general agreement with o t h e r studies which depict potential cholinergic neurons as a m i n o r c o m p o n e n t of the total cortical neuronal population1.3.13. In the present study, stained neurons of all morphologies were observed; however, the m a j o r i t y of the ACHE-
1 Dells, J.R., Zhu, C.-H. and Dichter, M.A., Coexistence of acetylcholinesterase and somatostatin-immunoreactivity in neurons cultured from rat cerebrum, Science, 223 (1984) 61-63. 2 Eckenstein, F. and Sofroniew, M.V., Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetyicholinesterase, J. Neurosci., 3 (1983) 2286-2291. 3 Eckenstein, F. and Thoenen, H., Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase, Neurosci. Lett., 36 (1983) 211-215. 4 Emson, P.C. and Lindvall, O., Distribution~of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30. 5 Feldman, M.L. and Peters, A., The forms of non-pyramidal neurons in the visual cortex of the rat, J. Comp. Neurol., 179 (1978) 761-794. 6 Fibiger, H.C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 7 Hebb, C.O., Krnjevic, K. and Silver, A., Effect of undercutting on the acetylcholinesterase and choline acetyltransferase activity in the cat's cerebral cortex, Nature (London), 198 (1963) 692. 8 Houser, C.R., Crawford, G.D., Barber, R.P., Salvaterra, P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an iammunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Research, 266 (1983) 97-119.
containing neurons exhibited bipolar morphology. The identification of bipolar cells is also consistent with ChAT immunohistochemical studies 3.8,17. However, these studies vary in the detection of cells with o t h e r morphologies. This may be attributable to the relatively low n u m b e r of these cells. In conclusion, A C h E - c o n t a i n i n g neurons were detected in primary cultures of cerebral cortex. While a m a j o r i t y of these cells were bipolar, stained bipolar neurons did not exhibit h o m o g e n e o u s morphology. The expert technical assistance of Ms. W . D . McC a d d e n is appreciated. G r a t i t u d e is also expressed to Ms. R. M o o r e for assistance in the p r e p a r a t i o n of the manuscript. This work was s u p p o r t e d by M B R S G r a n t RR-08037 and N S F G r a n t R11-8313599.
9 Johnston, M.V., McKinney, M. and Coyle, J.T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. 10 Koelle, G.B., The histochemical localization of cholinesterases in the central nervous system of the rat, J. Comp. Neurol., 100 (1954) 211-236. 11 Krnjevic, K. and Silver, A., A histochemical study of cholinergic fibres in the cerebral cortex, J. Anat., 99 (1965) 711-759. 12 Lehmann, J., Nagy, J.l., Atmadja, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 13 Mesulam, M.-M. and Dichter, M., Concurrent acetylcholinesterase staining and ~,-aminobutyric acid uptake of cortical neurons in culture, J. Histochem. Cytochem., 29 (1981) 306-308. 14 Rossier, J., On the mapping of the cholinergic neurons by immunohistochemistry, Neurochem. Int., 6 (1984) 183184. 15 Shute, C.C.D. and Lewis, P.R., The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections, Brain, 90 (1967) 497-522. 16 Thomas, W.E., Synthesis of acetylcholine and 7-aminobutyric acid by dissociated cerebral cortical cells in vitro, Brain Research, 332 (1985) 79-89. 17 Wainer, B.H., Levey, A.I., Mufson, E.J. and Mesulam, M.-M., Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase, Neurochem. Int., 6 (1984) 163-182.
Brain Reseurch, 361 ( 19851 392- 395 Elsevier
BRE 21252
Morphology of acetylcholinesterase-containing neurons in primary cultures of dissociated rat c e r e b ~ cortex W. ERIC THOMAS Department of Physiology, Meharry Medical College, Nashville, TN 37208 (U. S. A. )
(Accepted August 6th, 1985) Key words: cerebral cortex - - tissue culture - - acetylcholinesterase - - cholinesterase histochemistry - neuronal morphology - - bipolar cell
Acetylcholinesterase-containing neurons were investigated in primary cultures of cerebral cortex. Neuronal cholinesterase staining was essentially totally attributable to acetylcholinesterase based on its pattern of sensitivity to pharmacological inhibitors. The mean percentage of stained neurons in the cultures was 2.17. Stained neurons of all morphologies were detected; however, the majority of the cells possessed bipolar morphology. The stained bipolar neurons were not a homogeneous morphological population.
The question of the existence of cholinergic neurons intrinsic to the cerebral cortex has long been an unresolved one. A variety of different techniques, including biochemical analysis of cholinergic components following cortical undercutting4,7.9, histochemical staining for cholinesterase t0,11,tS, and immunohistochemical localization of choline acetyltransferase (CHAT, E C 2.3.1.6) 14, have been employed to investigate this question; however, none has provided a definitive answer, A n alternative approach involves the implementation of primary tissue culture techniques. The presence of afferent processes, some fractions of which are known to be cholinergic6.1tA2,15, in cortex from extrinsic neurons represents a major complicating factor in most in vivo studies. This complication is eliminated in tissue culture and the neurochemistry of endogenous cortical cells can be specifically investigated, in addition tissue culture offers the advantages of control over the size of the cell population and increased accessibility of individual neurons. The synthesis of acetylcholine in primary cultures of cerebral cortex has recently been reported L6. These cultures exhibited high affinity uptake of radiolabelled choline and converted a fraction of the choline to acetylcholine. This acetylcholine synthesis
was shown to occur via the enzyme CHAT. Thus the cortical cultures appear to contain a population of cholinergic neurons. The present report describes initial efforts to establish the identity of these cells. Also the relative proportion of cholinergic neurons in the cultures was investigated. Cortical cultures were established and maintained as previously described 16. Briefly, 15-day-old embryos were removed from timed-pregnant rats using aseptic surgical conditions. Cerebral cortical tissue was selectively dissected from the embryos and dissociated by trituration. Dissociated cells were plated in 35 mm tissue culture dishes (5 × 104 cells per dish). Cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C Under these conditions, cortical neurons display mature features by 10-14 davs in vitro. All cultures used in this study were 14-36 days in vitro. Cultures were stained for cholinesterase enzyme according to the method of KoellO 0 as described by Dells et al. 1. In this procedure, formaldehyde-fixed cultures are treated with dimethyl sulfoxide. Thiocholine formed by the action of the enzyme on the substrate acetylthiocholine in the staining solution complexes "~,ith copper and is precipitated with sodium sulfide. In some cases, cholinesterase staining was intensified by a postincuba-
Correspondence: W.E. Thomas, Department of Physiology, Meharry Medical College, Nashville, TN 37208, U.S.A.
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
393 TABLE I
Pharmacological sensitivity of cholinesterase staining Cortical cultures were fixed and stained as described in the text. The indicated inhibitors were included in the staining solution. Stained neurons were determined by direct counting using light microscopy. All values are the total stained neurons per culture under each condition. The N.D. denotes none detected.
Plating no.
No inhibitor
I
113 +
II
187 172 293 199 434
III IV V VI
+ + + + +
23 48 33 51 45 82
Ethopropazine
Iso-O M PA
124 + 10 159 + 25 -
tion in 1% silver nitrate. When esterase inhibitors were used, these compounds were included in the staining solution. Inhibitors were used at the following concentrations: neostigmine 0.1 mM; ethopropazine 0.2 mM; tetraisopropylpyrophosphoramide (IsoOMPA) 1.0 mM; and 1,5-bis-[4-allyldimethyl-ammoniumphenyl]pentan-3-one dibromide (BW284C51) 0.1 mM. Stained cultures were examined under light microscopy using a Zeiss Standard 16 microscope. Cholinesterase reaction product was observed throughout the cortical cultures. Very light staining was detectable in non-neuronal cells, usually confined to the nucleus. Other cells, identified as neurons on the basis of morphological criteria, exhibited more intense staining. Reaction product was present throughout the somata and at least the proximal portion of processes. While this neuronal staining was variable in intensity between different cells, through optimization of the staining reaction time a clearer
TABLE II
Percentage of neurons containing AChE The total stained neurons per culture was determined in cultures from several different platings. The total neurons in the cultures was then determined to evaluate the percentage of cells containing ACHE.
Plating no.
Total neurons per culture
Neurons stained for A ChE
% Neurons stained for AChE
I II III IV V VI
14,622 6448 10,562 13,146 13,156 8488
555 113 187 248 297 133
3.80 1.75 1.77 1.89 2.26 1.57
+ + + + + +
2410 792 753 878 1026 1131
+ 102 + 59 + 48 + 94 -+ 86 + 22
Neostigmine
BW284C51
-
2 + 0
-
164 340 179 472
1+ 0 N.D. N.D. N.D.
N.D. N.D. N.D. N.D. N.D.
+ + + +
10 32 61 104
distinction between stained and unstained neurons was achieved. This allowed the quantitation of stained neurons by direct counting. All neuronal counts are expressed as the mean of 3 or more determinations + S.D. The number of stained neurons per culture in all experiments ranged from 113 + 23 to 555 + 102 (see Tables I and II). In an initial series of experiments, the nature of the activity producing the reaction product was investigated. This was done by using various pharmacological inhibitors. The results of experiments on cultures from six different platings are presented in Table I. The general cholinesterase inhibitor neostigmine abolished virtually all the neuronal staining. This confirmed that the reaction product was produced by a cholinesterase enzyme. The pseudo-cholinesterase inhibitors, ethopropazine and IsoOMPA, were without effect on the mean number of stained neurons per culture. The specific acetylcholinesterase (ACHE, EC 3. !. 1.7) inhibitor BW284C51 always reduced the mean number of stained neurons to a non-detectable level. Thus, on the basis of these pharmacological properties, all or most of the neuronal staining was attributable to ACHE. To determine the percentage of AChE-containing neurons, stained neurons were counted; then, the total neurons were counted in cresyl violet-stained sister cultures. The results of such experiments from six different platings are shown in Table II. The overall mean percentage of AChE-containing neurons was 2.17. Finally, the morphology of the stained cells was evaluated. Entire cultures were scanned and all neurons stained enough to reveal their detailed morphology were classified. Four morphological classes were designated - - pyramidal, stellate, bipolar and bi-
394
A t
..
-
i!ii
Fig. 1. Stained neurons of different morphologies. A-D: various subtypes of AChE-containing bipolar cells. E and F: examples of stained pyramidal and stellate neurons, respectively. Scale bar = 20 u m
tufted (according to the classification scheme of Feldman and PetersS). The accumulative results for the morphological distribution of stained neurons in 34 different cultures are shown in Fig. 1. Pyramidal cells constituted 10.3% of the stained neurons, stellate 11.1%, bipolar 66.9% and bitufted 11.7%. Thus, the majority of the A C h E - s t a i n e d neurons exhibited bipolar morphology; however, stained neurons of all morphologies were observed. Typical stained neurons with p y r a m i d a l and stellate (multipolar) morphology are shown in Fig. 2E and F, respectively. While no systematic subclassification of stained bipolar cells was a t t e m p t e d , variability in aspects of the detailed m o r p h o l o g y of these cells, particularly soma size and shape, was observed (Fig. 2 A - D ) . Some stained bipolars possessed round or oval somata, oth-
ers had distinctively m o r e elongated somata. The smallest cells averaged 5 - 1 0 u m in somal diameter. The largest appearing cells averaged 15-25 ~tm in diameter. 2OOO
o o Z
tO00
E 0
Pyramidal
Stellate Morphological
Biaolar Clal~es
Bitufted
Fig. 2. Histogram showing the morphological distribution of AChE,stained neurons. Morphologically identifiable stained neurons from 34 different cultures (a total of 2708 cells) were classified.
395 These findings provide further support for the presence of cholinergic cells in the cortical cultures. The existence of such cells suggests that there are cholinergic neurons intrinsic to the cerebral cortex in vivo. This contention is also s u p p o r t e d by several recent immunohistochemical studies indicating the presence of ChAT-positive neurons in cortex 3,8,17. The percentage of A C h E - c o n t a i n i n g neurons detected in the present study was a p p r o x i m a t e l y 2. This must be viewed as an u p p e r limit considering the d e m o n strated lack of total specificity of A C h E as a m a r k e r for cholinergic cells 2. H o w e v e r , this finding is in general agreement with o t h e r studies which depict potential cholinergic neurons as a m i n o r c o m p o n e n t of the total cortical neuronal population1.3.13. In the present study, stained neurons of all morphologies were observed; however, the m a j o r i t y of the ACHE-
1 Dells, J.R., Zhu, C.-H. and Dichter, M.A., Coexistence of acetylcholinesterase and somatostatin-immunoreactivity in neurons cultured from rat cerebrum, Science, 223 (1984) 61-63. 2 Eckenstein, F. and Sofroniew, M.V., Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetyicholinesterase, J. Neurosci., 3 (1983) 2286-2291. 3 Eckenstein, F. and Thoenen, H., Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase, Neurosci. Lett., 36 (1983) 211-215. 4 Emson, P.C. and Lindvall, O., Distribution~of putative neurotransmitters in the neocortex, Neuroscience, 4 (1979) 1-30. 5 Feldman, M.L. and Peters, A., The forms of non-pyramidal neurons in the visual cortex of the rat, J. Comp. Neurol., 179 (1978) 761-794. 6 Fibiger, H.C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 7 Hebb, C.O., Krnjevic, K. and Silver, A., Effect of undercutting on the acetylcholinesterase and choline acetyltransferase activity in the cat's cerebral cortex, Nature (London), 198 (1963) 692. 8 Houser, C.R., Crawford, G.D., Barber, R.P., Salvaterra, P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an iammunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Research, 266 (1983) 97-119.
containing neurons exhibited bipolar morphology. The identification of bipolar cells is also consistent with ChAT immunohistochemical studies 3.8,17. However, these studies vary in the detection of cells with o t h e r morphologies. This may be attributable to the relatively low n u m b e r of these cells. In conclusion, A C h E - c o n t a i n i n g neurons were detected in primary cultures of cerebral cortex. While a m a j o r i t y of these cells were bipolar, stained bipolar neurons did not exhibit h o m o g e n e o u s morphology. The expert technical assistance of Ms. W . D . McC a d d e n is appreciated. G r a t i t u d e is also expressed to Ms. R. M o o r e for assistance in the p r e p a r a t i o n of the manuscript. This work was s u p p o r t e d by M B R S G r a n t RR-08037 and N S F G r a n t R11-8313599.
9 Johnston, M.V., McKinney, M. and Coyle, J.T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. 10 Koelle, G.B., The histochemical localization of cholinesterases in the central nervous system of the rat, J. Comp. Neurol., 100 (1954) 211-236. 11 Krnjevic, K. and Silver, A., A histochemical study of cholinergic fibres in the cerebral cortex, J. Anat., 99 (1965) 711-759. 12 Lehmann, J., Nagy, J.l., Atmadja, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 13 Mesulam, M.-M. and Dichter, M., Concurrent acetylcholinesterase staining and ~,-aminobutyric acid uptake of cortical neurons in culture, J. Histochem. Cytochem., 29 (1981) 306-308. 14 Rossier, J., On the mapping of the cholinergic neurons by immunohistochemistry, Neurochem. Int., 6 (1984) 183184. 15 Shute, C.C.D. and Lewis, P.R., The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections, Brain, 90 (1967) 497-522. 16 Thomas, W.E., Synthesis of acetylcholine and 7-aminobutyric acid by dissociated cerebral cortical cells in vitro, Brain Research, 332 (1985) 79-89. 17 Wainer, B.H., Levey, A.I., Mufson, E.J. and Mesulam, M.-M., Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase, Neurochem. Int., 6 (1984) 163-182.