Journal of Histochemistry and Cytochemistry, Vol. 45, 895-902, Copyright © 1997 by The Histochemical Society, Inc.


TECHNICAL NOTE

A Simple Enzyme Histochemical Method for the Simultaneous Demonstration of Acetylcholinesterase and Monoamine Oxidase in Fixed-Frozen Sections

Daniel D. Dunninga, John G. McHaffieb, and Barry E. Steinb
a Department of Anatomy and Neurobiology, University of California, Irvine, California
b Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina

Correspondence to: John G. McHaffie, Dept. of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest U., Winston-Salem, NC 27157-1010.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We describe an enzyme histochemical technique for the simultaneous demonstration of acetylcholinesterase (AChE) and monoamine oxidase (MAO) (Types A, B, or A+B) in fixed-frozen sections. Several regions in the mesencephalon and brainstem were examined for both somatic and neuropil labeling. The results obtained are equivalent or superior to those obtained using previous methods for the individual localization of these enzymes. The simultaneous visualization of AChE and MAO in the same section allows the relationship of the two enzymes to be easily assessed with brightfield microscopy. (J Histochem Cytochem 45:895-902, 1997)

Key Words: acetylcholinesterase, monoamine oxidase, histocytochemistry, double labeling, locus coeruleus, superior colliculus, dorsal raphe


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Because acetylcholinesterase (AChE) is found in monoaminergic structures, a number of investigators have sought a technique with which to co-localize markers for AChE and monoamines. One such technique, described by Albanese and Butcher 1979 , is based on the glyoxylic acid-induced fluorescence of monoamines originally developed by de la Torre and Surgeon 1976 , coupled with the enzyme histochemical demonstration of AChE. Unfortunately, this technique requires localization in two steps. First, the tissue is processed for glyoxylic acid-induced fluorescence to localize monoaminergic neurons. The fluorescent product of the monoamines is then viewed, after which the tissue is subjected to a second enzyme histochemical reaction to allow visualization of AChE. Therefore, it is not possible to view both reaction products simultaneously in the same section or cell bodies. Development of a technique for simultaneous visualization of histochemical markers would markedly facilitate determination of their relative distributions and would also enable one to directly examine whether or not they are present in the same neuron.

We describe here a comparatively simple enzyme histochemical method with which to co-localize AChE and monoamine oxidase (MAO). Of significant value is the ability of this method to distinguish among serotonergic (MAO-B-positive) and noradrenergic (MAO-A-positive) nuclei and to enable simultaneous viewing of the distribution of AChE and MAO with brightfield microscopy.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Tissue was obtained from adult male Long-Evans hooded rats, Dutch-belted rabbits, ferrets, and mongrel cats, in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. After a lethal injection of sodium pentobarbital (125 mg/kg IP), rats were perfused transcardially with 100 ml room-temperature PBS (pH 7.4) for 2 min, 100 ml cold (3-5C) 1% paraformaldehyde-1% glutaraldehyde fixative in 100 mM sodium phosphate buffer, pH 7.4 (referred to here as phosphate buffer) for 2 min, 400 ml cold fixative at a slower rate for 20 min, and finally with 100 ml cold phosphate-buffered sucrose (15% w/v, pH 7.4) for 2 min. Perfusion volumes were adjusted appropriately for the other species. The brains were immediately removed, blocked, and immersed overnight in phosphate-buffered sucrose with 0.2% paraformaldehyde (pH 7.4) at 3-5C. Serial sections were cut at 50 µm on a freezing microtome, collected in cold phosphate buffer, and processed immediately after cutting while floating freely in compartmentalized staining nets.

Enzyme Histochemistry
To determine the possible effects of one enzyme histochemical procedure on the reaction product of the other, sections processed for either AChE or MAO alone were compared with a third set of sections processed for double labeling. Typically, sections received the following treatment. (a) Two sets of alternate sections were first processed for MAO activity by the nickel-intensified/coupled-peroxidase method (Kitahama et al. 1984 , Kitahama et al. 1986 ) in the same media. (b) One set of MAO-labeled sections was then transferred to the compartmentalized staining net containing the third, unlabeled set of sections. (c) These two sets of sections (one for double labeling and one for AChE only) were then processed for AChE activity by the post-coloring copper ferricyanide version of Koelle's method (Koelle and Friedenwald 1949 ) developed by Tsuji 1974 for electron microscopy. By incubating sections together, the single-labeled sections served as controls for the double-labeled sections. Additional controls were also performed using inhibitors for the various enzymes.

Method for MAO Localization
Tissues processed for total MAO activity (Types A+B) received the following treatment: (a) sections were rinsed three times for 1 min each in phosphate buffer, (b) preincubated for 15 min in 50 mM Tris-HCl buffer at pH 7.6, (c) incubated for 30 min to 9 hr at 22-25C in darkness with agitation at 1-hr intervals (note: sections must not be allowed to overlap or become folded because this significantly reduces contact of the section with the incubation medium), (d) rinsed three times for 1 min each in phosphate buffer, and (e) mounted the following day from phosphate buffer onto chromalum-gelatin-subbed slides and allowed to air-dry overnight. The MAO incubation medium consisted of 0.02% tyramine HCl (Sigma Chemical; St Louis, MO, except where otherwise noted), 0.005% diaminobenzidine-HCl, 0.05% horseradish peroxidase (Type II), 0.065% sodium azide (Aldrich Chemical; Milwaukee, WI), and 0.3% nickel sulfamate (Aldrich) in 50 mM Tris-HCl buffer, pH 7.6. Sections labeled for MAO-A activity received 0.1 µM (1/2500 v/v of a 250-µM stock solution) L(-)-deprenyl HCl (a selective inhibitor of MAO-B; Fowler et al. 1981 ), and those labeled for MAO-B activity received 0.1 µM (1/2500 v/v of a 250-µM stock solution) clorgyline-HCl (a selective inhibitor of MAO-A; Fowler et al. 1981 ) added to the pre-incubation and incubation media. Control sections were treated identically except for the addition of both deprenyl and clorgyline to the preincubation and incubation media. MAO inhibitors were obtained from Research Biochemicals (Natick, MA).

Method for AChE Localization
Tissues processed for AChE activity received the following treatment: (a) sections were rinsed seven times for 1 min each in distilled deionized water, (b) preincubated for 15 min in 100 mM sodium acetate buffer, pH 5.2 (referred to here as acetate buffer) with 20 µM tetraisopropyl pyrophosphoramide (iso-OMPA), (c) incubated for 30 min to 2 hr at 22-25C or 37C with constant agitation, (d) rinsed seven times for 1 min each in distilled deionized water, (e) postincubated for 15 min in coloring solution, (f) rinsed seven times for 1 min each in distilled deionized water, and (g) mounted the following day from water or gelatin-alcohol solution onto chromalum-gelatin-subbed slides and allowed to air-dry overnight. The AChE incubation medium consisted of 4 mM acetylthiocholine iodide (ASChI), 2 mM copper, 10 mM glycine, and 20 µM iso-OMPA (also called tetra[monoisopropyl] pyrophosphortetramide, a selective inhibitor of non-acetyl cholinesterases) (Koelle et al. 1974 ; Aldridge 1953 ; Austin and Berry 1953 ) in acetate buffer. The incubation medium was prepared no more than 30 min before use, usually during the pre-incubation period, by combining a copper/glycine solution (cupric sulfate and glycine free base in 1/4 of the final volume of acetate buffer) and a solution containing the ASChI and iso-OMPA (1/2500 v/v from a 50-mM stock solution) in 3/4 of the final volume of acetate buffer. The incubation medium was then preheated to 37C (22-25C incubations excluded) and the final pH adjusted to 5.2. The postincubation coloring solution consisted of 3% potassium ferricyanide in acetate buffer. Control sections were treated identically except for the addition of 100 µM (1/2500 v/v 250 mM stock solution) 1,5-bis[4-allyldimethylammoniumphenyl]pentan-3-one dibromide [BW284c51 (also called BW297c50), a selective inhibitor of AChE] (Koelle 1955 ; Fulton and Mogey 1954 ; Austin and Berry 1953 ) to the pre-incubation and incubation media. Because non-somatic AChE staining was very intense in some neural regions, it was necessary in some cases to pretreat the animal with 0.6 mg/kg IM di-isopropyl fluorophosphate (DFP, an inhibitor of acetyl and non-acetyl cholinesterases) (Hawkins and Mendel 1947 ) 5.5 hours before sacrifice to reduce non-somatic AChE staining (Butcher 1978 ). Therefore, pretreatment with DFP facilitated visualization of AChE-positive somata that would normally have been obscured by intense neuropil staining. In addition, DFP pretreatment allows those AChE-positive somata to be classified as rapidly synthesizing AChE, a criterion that has been used in the identification of cholinergic somata (Mizukawa et al. 1986 ; Butcher and Woolf 1984 ; Mesulam et al. 1984 ; Satoh et al. 1983 ).

All mounted sections were dehydrated in graded alcohols (15 min each in 70%, 90%, 100%, 100%), cleared in xylene (three times for 30 min), and coverslips applied with DPX mountant (BDH, Poole, UK, imported by Gallard-Schlesinger Industries; Carle Place, NY).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The method was evaluated for its suitability in several experimental applications: (a) the simultaneous visualization of AChE and MAO in neuronal somata, (b) the determination of MAO-A- and MAO-B-positive cell populations paired with the simultaneous demonstration of AChE, and (c) the simultaneous visualization of non-somatal AChE and MAO staining. Appropriate controls were also performed to evaluate the potential effects on one another of the two staining reactions. Because the MAO procedure was always performed before AChE histochemistry, MAO-stained sections were evaluated for the possible loss of staining product due to the subsequent AChE procedures. As shown in Figure 1A and Figure 1B, MAO staining was not affected by subsequent reactions required for visualization of AChE activity. In addition, AChE-stained sections were evaluated for the possible interference in staining by the preceding MAO procedure. As shown in Figure 1D and Figure 1E, AChE staining was not affected by the prior reactions required to visualize MAO. Furthermore, single-labeled control sections treated with MAO (i.e., deprenyl and clorgyline) or cholinesterase inhibitors (i.e., iso-OMPA and BW284c51), as well as the double-labeled control sections treated with all four inhibitors, were completely devoid of staining (Figure 1C). This suggests that the staining for both MAO and AChE activity was specific when performed either individually or in combination.



View larger version (116K):
[in this window]
[in a new window]
 
Figure 1. Absence of crossreactivity in MAO and AChE histochemical reactions. (A) Coronal section through the rat dorsal raphe nucleus single-labeled for MAO-A/B. (B) Section adjacent to that shown in A was double labeled for MAO-A/B (black) and AChE (brown) in the presence of an inhibitor of AChE and an inhibitor of non-acetyl cholinesterases (BW284c51 and iso-OMPA). Although the brown reaction product indicative of AChE is absent, the black reaction product for MAO is unchanged. (C) Section from the same series double labeled for MAO/AChE with block of both MAO-A/B and AChE/non-AChE by all four inhibitors (clorgyline, deprenyl, BW284c51, and iso-OMPA). Note the absence of both the black MAO and the brown AChE reaction products. (D) Coronal section through the rat locus coeruleus single labeled for AChE. (E) Section adjacent to that shown in D was double labeled for AChE and MAO-A/B in the presence of both MAO-A and MAO-B inhibitors. Although the black reaction product indicative of MAO is absent, the brown reaction product indicative of AChE is unchanged. Bars: A-C = 250 µm; D,E = 100 µm.

Double Labeling
Simultaneous visualization of AChE and MAO labeling of somata and neuropil (in the same section), using the double-labeling protocols outlined in Materials and Methods, is shown in Figure 2. Because the copper ferrocyanide reaction product of similar AChE procedures has been shown to have peroxidase-like activity (Tago et al. 1986 ), there was the possibility that the copper ferricyanide reaction product could cause nonspecific staining if it was performed before the MAO procedure. Therefore, the MAO procedure was always performed first in the double-labeling experiments. The low-power photomicrograph of a section through the dorsolateral aspect of the pontine tegmentum of the rat shown in Figure 2A reveals the distribution of AChE- and MAO-A-positive somata within the locus coeruleus and the adjacent mesencephalic nucleus of the trigeminal nerve. The presence of somatal profiles containing a reddish-brown, homogeneously distributed precipitate indicates AChE-labeled neurons, whereas the black granular deposits reveal the presence of MAO-A neurons. Both the color and the nature of the reaction products produced by this double-labeling technique allow each marker to be simultaneously co-localized within individual neurons. A prominent delineation is also evident between those neurons double labeled for AChE and MAO-A in the locus coeruleus and those that are single labeled for AChE in the mesencephalic nucleus of the trigeminal nerve. The region outlined in Figure 2A, which is shown at higher magnification in Figure 2A', shows that double-labeled somata can be distinguished within the same section.



View larger version (113K):
[in this window]
[in a new window]
 
Figure 2. Simultaneous visualization of AChE and MAO labeling in various regions of the rat CNS. (A) Low-power photomicrograph of locus coeruleus (LC) and mesencephalic nucleus of the trigeminal nerve (Mes 5). This section has been reacted for AChE and MAO-A activity. The region outlined by the box is shown at higher magnification in A'. (B) Low-power photomicrograph of the section adjacent to that shown in A. This section has been reacted for AChE and MAO-B activity. Note the absence of black MAO-B reaction product in the locus coeruleus (LC) compared to the abundance of MAO-A positive somata seen in section A. (C) Low power photomicrograph of dorsal raphe (DR) and paradorsal raphe nucleus. This section was reacted for AChE and MAO-A activity. (D) Low-power photomicrograph of a section adjacent to that shown in C. This section was reacted for AChE and MAO-B activity. Compare the presence of the black reaction product, indicating MAO-B-positive neurons here, with the absence of MAO-A labeling in the dorsal raphe of section C. The region outlined by the box is shown at higher magnification in D'. (A') High-power photomicrograph of the region outlined in A shows that the two reaction products (AChE and MAO-A) are present in locus coeruleus somata (left), but only the AChE reaction product is present in Mes 5 (right). Virtually all neurons in LC appeared to be double labeled. Incubation times (A,B,A') were 30 min at 22C for MAO-A followed by 2 hr at 22C for AChE. This animal received an injection of DFP before sacrifice to reduce non-somatic AChE staining. (D') High-power photomicrograph of the region outlined in D shows single-labeled AChE somata (closed arrows) and double-labeled AChE/MAO-B somata (open/closed arrows) in paradorsal raphe nucleus. (E) The simultaneous visualization of non-somatal AChE and MAO labeling in the superior colliculus (SC). The section was reacted for total MAO (A+B) and AChE without DFP pretreatment. Note the complementary clusters of AChE (closed arrowheads) and MAO (open arrowheads) labeling in the intermediate layers. In some regions, the two labels were overlapping (open/closed arrowheads). Incubation times were 4.5 hr at 24C for MAO followed by 70 min at 37C for AChE. Bars: A-D = 100 µm; A'-D' = 25 µm; E = 250 µm.

An adjacent section of the dorsolateral pontine tegmentum, shown in Figure 2B, illustrates the absence of MAO-B (i.e., no black, granular reaction product) in locus coeruleus sections double labeled for AChE and MAO-B. However, the distribution of reddish-brown AChE-positive somata in locus coeruleus is similar to the MAO-A/AChE distribution seen in Figure 2A. Note that several black MAO-B-positive somata can be seen to the left of the locus coeruleus. Such somata were usually in close approximation to blood vessels.

Simultaneous visualization of AChE and MAO labeling was also observed in other brainstem loci (Figure 2C and Figure 2D) in tissue obtained from the same animal and reacted at the same time as that shown in Figure 2A and Figure 2B. Low-power photomicrographs of the region around the dorsal raphe and paradorsal raphe nucleus reacted for AChE and MAO-B activity (Figure 2D) reveal many somatal profiles containing both the reddish-brown AChE precipitate and the black granular deposits of MAO-B. The area outlined in Figure 2D, which is shown at higher magnification in Figure 2D', illustrates double- and single-labeled somata within the same section.

In addition to the somatal staining described above, it was also feasible with the present technique to simultaneously visualize neuropil labeling. In the deep layers of the superior colliculus, where afferents, efferents, and various neurochemical markers are organized as a mosaic of discontinuous patchy domains (Graybiel and Illing 1994 ; Illing and Graybiel 1984 , Illing and Graybiel 1994 ; Huerta and Harting 1984 ), regions containing dense neuropil labeling for AChE and MAO were readily apparent (Figure 2E). In this case, the MAO visualized was for total MAO (Types A+B), and AChE staining was performed without DFP pretreatment. Some domains positive for AChE (closed arrowheads) and MAO (open arrowheads) were complementary, whereas others appeared to be overlapping (open/closed arrowheads). Observations of this material at higher magnifications (not shown) suggest that it is possible to resolve individual axonal and/or dendritic profiles.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The results obtained here using a single-step double-labeling method revealed that many neurons in locus coeruleus that reacted positively for MAO-A also stained positively for AChE. These results are consistent with those previously obtained with the two-step double-labeling localization process (Albanese and Butcher 1979 ; de la Torre and Surgeon 1976 ) and with previous studies of the distribution of AChE by enzyme histochemical techniques (Caffe 1994 ; Ennis and Shipley 1992 ; Mizukawa et al. 1986 ; Paxinos and Watson 1986 ; Butcher and Woolf 1984 ; Mesulam et al. 1984 ; Satoh et al. 1983 ; Albanese and Butcher 1979 ; Lewis and Schon 1975 ), and of MAO-A by enzyme histochemical (Kitahama et al. 1986 , Kitahama et al. 1994 ; Konradi et al. 1989 ; Willoughby et al. 1988 ; Arai et al. 1986 ) and immunohistochemical (Kitahama et al. 1994 ; Konradi et al. 1988 ; Thorpe et al. 1987 ; Westlund et al. 1985 , Westlund et al. 1988 , Westlund et al. 1993 ) techniques. The distinct color and nature of the reaction products produced by the present double-labeling technique allow both markers to be simultaneously co-localized within individual neurons. To our knowledge, only one other study has been done using similar techniques to co-localize AChE and MAO. However, this study (see Nakamura et al. 1993 ) did not produce the clarity of staining possible with the present technique.

The clarity and specificity of simultaneous AChE/MAO labeling are approximately equivalent to those seen when each of these procedures was used independently. Consequently, despite the extensive use of heavy metals and phosphates in the MAO procedure and the observation that these significantly impair AChE staining (van Ooteghem and Shipley 1984 ), no degradation of AChE labeling was observed. Although it is not immediately evident why the expected interference in AChE labeling was not apparent, one possibility is that the metals are rendered inactive by binding to the reaction products in the MAO procedure and that a sufficient amount of phosphate is removed by the washes to eliminate any inhibitory effects they might have on the subsequent AChE procedure. Regardless of the specific factors that allow the combined AChE/MAO-labeling paradigm to work, it has proved to be an extremely effective means for examining the distribution of these enzymes simultaneously in the neuropil and among individual somata. Furthermore, the results indicate that the method is applicable in different structures and species.

There are several obvious advantages of the technique presented. One is its usefulness in discriminating between MAO-A and MAO-B. As noted earlier, this ability can be utilized to distinguish noradrenergic cell populations that exhibit enhanced MAO-A activity from serotonergic cell populations that exhibit enhanced MAO-B activity. In addition, with regard to neuropil labeling, the ability to localize each of the MAO subtypes may prove useful for distinguishing putative monoaminergic terminal from non-terminal labeling. Because MAO-A is believed to be neuronal in origin (Westlund et al. 1985 , Westlund et al. 1993 ; Student and Edwards 1977 ), whereas MAO-B is more likely extraneuronal (e.g., glial) (Konradi et al. 1989 ; Westlund et al. 1985 ; Levitt et al. 1982 ; Student and Edwards 1977 ) except in serotonergic nuclei, the present method could be used to distinguish between these elements in the neuropil. Moreover, because both AChE and MAO reaction products are electron-dense, they can be viewed by electron microscopy (Maeda et al. 1987 ; Tsuji and Fournier 1984 ; Tsuji 1974 ). Another advantage of the method is its ease of use. It does not require animal decapitation or fresh-frozen sections, and the reaction products are non-labile and can be viewed simultaneously with brightfield microscopy. Because of the low cost and ease of use of the method, it is well suited to the routine processing of archival materials and/or large-scale comparative studies.

A negative aspect of this technique for co-localization is that the staining products for the two enzymes may obscure each other. This can sometimes lead to the inability to unequivocally identify double-labeled somata. Immunohistochemical methods tend to give more discrete localization and are therefore more likely to give unequivocal identification of double-labeled somata. Because antibodies for AChE, MAO-A, and MAO-B have recently become available from a variety of sources (e.g., Accurate Chemical & Scientific, Westbury, NY, and Chemicon International, Temecula, CA), immunohistochemical double labeling should be possible. However, enzyme-labeled immunohistochemical techniques suffer from the same diffusion artifact problem that enzyme histochemical techniques do, and immunofluorescent techniques would still be required to achieve unequivocal identification of double-labeled somata. The need for specialized equipment to view double-labeled immunofluorescence and the increased expense of immunohistochemical reagents needed to achieve this level of accuracy for identification of double-labeled somata must therefore be considered. Another shortcoming of the present method is that when DFP pretreatment is not possible (e.g., human postmortem specimens), visualization of AChE-positive somata may be obscured by non-somatal staining. Under these circumstances, immunohistochemical (Mesulam et al. 1984 ) or alternative enzymatic methods (Kujat et al. 1993 ; Schatz et al. 1992 ; Kugler 1987 ; Tago et al. 1986 ; Hedreen et al. 1985 ) for AChE staining may be preferable when coupled with other methods of MAO staining (e.g., Nakos and Gossrau 1993 ; Gossrau and Richter 1992 ).

The results of this study are in agreement with previous studies showing that AChE is found in cholinergic and non-cholinergic neurons (Mizukawa et al. 1986 ; Butcher and Woolf 1984 ; Mesulam et al. 1984 ; Eckenstain and Sofroniew 1983 ; Satoh et al. 1983 ). In recent years, AChE has been associated with non-cholinergic functions within the central nervous system, e.g., degradation of amides (Checler et al. 1994 ), neuronal differentiation and development (Coleman and Taylor 1996 ; Jones et al. 1995 ; Layer and Willbold 1994 , Layer and Willbold 1995 ), and interactions with excitatory amino acid (Appleyard 1994 ) and monoaminergic (Abo et al. 1992 ; Ennis and Shipley 1992 ) neurotransmitters. The relationship of non-cholinergic AChE to monoamines has been best studied in the dopaminergic neurons of the substantia nigra (Greenfield 1991 , Greenfield 1995 ), and the method described here may prove particularly helpful in examining such functions in noradrenergic and serotonergic nuclei.


  Acknowledgments

Supported by NIH grants NS 22543, EY 06562, and NS 35008.

Received for publication November 12, 1996; accepted December 9, 1996.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Abo V, Viera L, Dajas F (1992) Different functional pools of acetylcholinesterase induce changes in rat locus coeruleus noradrenaline metabolism. Neurosci Lett 141:111-114[Medline]

Albanese A, Butcher LL (1979) Locus coeruleus somata contain both acetylcholinesterase and norepinephrine: direct histochemical demonstration on the same tissue section. Neurosci Lett 14:101-104[Medline]

Aldridge WN (1953) The differentiation of true and pseudo cholinesterase by organophosphorus compounds. Biochem J 53:62-67[Medline]

Appleyard ME (1994) Non-cholinergic functions of acetylcholinesterase. Biochem Soc Trans 22:749-755[Medline]

Arai R, Kimura H, Maeda T (1986) Topographic atlas of monoamine oxidase-containing neurons in the rat brain studied by an improved histochemical method. Neuroscience 19:905-925[Medline]

Austin L, Berry WK (1953) Two selective inhibitors of cholinesterase. Biochem J 54:695-700

Butcher LL (1978) Recent advances in histochemical techniques for the study of central cholinergic mechanisms. In Jenden DJ, ed. Advances In Behavioral Biology. Vol 24. Cholinergic Mechanisms and Psychopharmacology. New York, Plenum Press, 93-124

Butcher LL, Woolf NJ (1984) Histochemical distribution of acetylcholinesterase in the central nervous system: clues to the localization of cholinergic neurons. In Bjorklund A, Hökfelt T, Kuhar MJ, eds. Handbook of Chemical Neuroanatomy. Vol 3. Classical Transmitters and Transmitter Receptors in the CNS. Part II. New York, Academic Press, 1-50

Caffe AR (1994) Light microscopic distribution of some cholinergic markers in the rat and rabbit locus coeruleus and the nucleus angularis grisea periventricularis of the domestic pig (Sus scrofa): a correlative electron microscopic investigation of cholinergic receptor proteins in the rabbit. Microsc Res Tech 29:186-199[Medline]

Checler F, Grassi J, Vincent JP (1994) Cholinesterases display genuine arylacylamidase activity but are totally devoid of intrinsic peptidase activities. J Neurochem 62:756-763[Medline]

Coleman BA, Taylor P (1996) Regulation of acetylcholinesterase expression during neuronal differentiation. J Biol Chem 271:4410-4416[Abstract/Free Full Text]

de la Torre JC, Surgeon JW (1976) A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method. Histochemistry 49:81-93[Medline]

Eckenstain F, Sofroniew MV (1983) Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase. J Neurosci 3:2286-2291[Abstract]

Ennis M, Shipley MT (1992) Tonic activation of locus coeruleus neurons by systemic or intracoerulear microinjection of an irreversible acetylcholinesterase inhibitor: increased discharge rate and induction of c-fos. Exp Neurol 118:164-177[Medline]

Fowler CJ, Oreland L, Callingham BA (1981) The acetylenic monoamine oxidase inhibitors clorgyline, deprenyl, pargyline and J-508: their properties and applications. J Pharm Pharmacol 33:341-347[Medline]

Fulton MP, Mogey GA (1954) Some selective inhibitors of true cholinesterase. Br J Pharmacol 9:138-144[Medline]

Gossrau R, Richter W (1992) Is it still adequate to study the nervous system using methods of catalytic enzyme histochemistry? Acta Histochem 52(suppl):S39-S49

Graybiel AM, Illing RB (1994) Enkephalin-positive and acetylcholinesterase-positive patch systems in the superior colliculus have matching distributions but distinct developmental histories. J Comp Neurol 340:297-310[Medline]

Greenfield SA (1995) A non-cholinergic function for acetylcholinesterase. In Quinn DM, Balasubramanian AS, Doctor BP, Bhupendra P, Taylor P, eds. Enzymes of the Cholinesterase Family. New York, Plenum Press, 415-421

Greenfield SA (1991) A non-cholinergic action of acetylcholinesterase (AChE) in the brain: from neuronal secretion to the generation of movement. Mol Cell Neurobiol 11:55-77

Hawkins RD, Mendel B (1947) Selective inhibition of pseudo cholinesterase by diisopropyl fluorophosphonate. Br J Pharmacol 2:173-180

Hedreen JC, Bacon SJ, Price DL (1985) A modified histochemical technique to visualize acetylcholinesterase-containing axons. J Histochem Cytochem 33:134-140[Abstract]

Huerta MF, Harting JK (1984) The mammalian superior colliculus: Studies of its morphology and connections. In Vanegas H, ed. Comparative Neurology of the Optic Tectum. New York, Plenum Press, 687-773

Illing RB, Graybiel AM (1994) Pattern formation in the developing superior colliculus: ontogeny of the periodic architecture in the intermediate layers. J Comp Neurol 340:311-327[Medline]

Illing RB, Graybiel AM (1984) Complementary and non-matching afferent compartments in the cat's superior colliculus: innervation of the acetylcholinesterase-poor domain of the intermediate gray layer. Neuroscience 18:373-394

Jones SA, Holmes C, Budd TC, Greenfield SA (1995) The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain. Cell Tissue Res 279:323-330[Medline]

Kitahama K, Arai R, Maeda T, Jouvet M (1986) Demonstration of monoamine oxidase type B in serotonergic and type A in noradrenergic neurons in the cat dorsal pontine tegmentum by an improved histochemical technique. Neurosci Lett 71:19-24[Medline]

Kitahama K, Maeda T, Denney RM, Jouvet M (1994) Monoamine oxidase: distribution in the cat brain studied by enzyme- and immunohistochemistry: recent progress. Prog Neurobiol 42:53-78[Medline]

Kitahama K, Sakai K, Tago H, Kimura H, Maeda T, Jouvet M (1984) Monoamine oxidase-containing neurons in the cat hypothalamus: distribution and ascending projection to the cerebral cortex. Brain Res 324:155-159[Medline]

Koelle GB (1955) The histochemical identification of cholinesterase in cholinergic, adrenergic and sensory neurons. J Pharmacol Exp Ther 114:167-184

Koelle GB, Davis R, Dilberto EJ, Koelle WA (1974) Selective, near-total, irreversible inactivation of peripheral pseudocholinesterase and acetylcholinesterase in cats in vivo. Biochem Pharmacol 23:175-188[Medline]

Koelle GB, Friedenwald JS (1949) A histochemical method for localizing cholinesterase activity. Proc Soc Exp Biol 70:617-622

Konradi C, Kornhuber J, Froelich L, Fritz J, Heinsen H, Beckman H, Schulz E, Riederer P (1989) Demonstration of monoamine oxidase A and B in the human brainstem by a histochemical technique. Neuroscience 33:383-400[Medline]

Konradi C, Svoma E, Jellinger K, Riederer P, Denney R, Thibault J (1988) Topographic immunocytochemical mapping of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase in human post mortem brain stem. Neuroscience 26:791-802[Medline]

Kugler P (1987) Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase. Histochemistry 86:531-532[Medline]

Kujat R, Rose C, Wrobel K-H (1993) The innervation of the bovine ductus deferens: comparison of a modified acetylcholinesterase-reaction with immunoreactivities of choline acetyltransferase and panneuronal markers. Histochemistry 99:231-239[Medline]

Layer PG, Willbold E (1995) Novel functions of cholinesterases in development, physiology and disease. Prog Histochem Cytochem 29:1-94[Medline]

Layer PG, Willbold E (1994) Cholinesterases in avian neurogenesis. Int Rev Cytol 151:139-181[Medline]

Levitt P, Pintar JE, Breakefield XO (1982) Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons. Proc Natl Acad Sci USA 79:6385-6389[Abstract]

Lewis PR, Schon FEG (1975) The localization of acetylcholinesterase in the locus coeruleus of the normal rat and after 6-hydroxydopamine treatment. J Anat 120:373-385[Medline]

Maeda T, Imai H, Arai R, Tago H, Nagai T, Sakumoto T, Kitahama K, Onteniente B, Kimura H (1987) An improved coupled peroxidatic oxidation method of MAO histochemistry for neuroanatomical research at light and electron microscopic levels. Cell Mol Biol 33:1-11[Medline]

Mesulam M-M, Mufson EJ, Levey AI, Wainer BH (1984) Atlas of cholinergic neurons in the forebrain and upper brainstem of the Macaque based on monoclonal choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry. Neuroscience 12:669-686[Medline]

Mizukawa K, McGeer PL, Tago H, Peng JH, McGeer EG, Kimura H (1986) The cholinergic system of the human hindbrain studied by choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry. Brain Res 379:39-55[Medline]

Nakamura S, Akiguchi I, Kimura J (1993) A subpopulation of mouse striatal cholinergic neurons show monoamine oxidase activity. Neurosci Lett 161:1441-1144

Nakos G, Gossrau R (1993) Light microscopic visualization of monoamine oxidase using a cerium method. Acta Histochem 95:203-219[Medline]

Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates. 2nd ed. New York, Academic Press

Satoh K, Armstrong DM, Fibiger HC (1983) A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry. Brain Res Bull 11:693-720[Medline]

Schatz CR, Geula C, Morecraft RJ, Mesulam M-M (1992) A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue. J Histochem Cytochem 40:431-434[Abstract/Free Full Text]

Student AK, Edwards DJ (1977) Subcellular localization of types A and B monoamine oxidase in rat brain. Biochem Pharmacol 26:2337-2342[Medline]

Tago H, Kimura H, Maeda T (1986) Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure. J Histochem Cytochem 34:1431-1438[Abstract]

Thorpe LW, Westlund KN, Kochersperger LM, Abell CW, Denney RM (1987) Immunocytochemical localization of monoamine oxidases A and B in human peripheral tissues and brain. J Histochem Cytochem 35:23-32[Abstract]

Tsuji S (1974) On the chemical basis of thiocholine methods of demonstration of acetylcholinesterase activities. Histochemistry 42:99-110[Medline]

Tsuji S, Fournier M (1984) Ultrastructural localization of acetylcholinesterase activity by means of the electron dense precipitate derived from Koelle's cuprous thiocholine iodide by treatment with phosphomolybdic acid and osmium tetroxide. Histochemistry 80:19-21[Medline]

van Ooteghem SA, Shipley MT (1984) Factors affecting the sensitivity and consistency of the Koelle-Friedenwald histochemical method for localization of acetylcholinesterase. Brain Res Bull 12:543-553[Medline]

Westlund KN, Denney RM, Kochersperger LM, Rose RM, Abell CW (1985) Distinct monoamine oxidase A and B populations in primate brain. Science 230:181-183[Medline]

Westlund KN, Denney RM, Rose RM, Abell CW (1988) Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem. Neuroscience 25:439-456[Medline]

Westlund KN, Krakower TJ, Kwan S-W, Abell CW (1993) Intracellular distribution of monoamine oxidase A in selected regions of rat and monkey brain and spinal cord. Brain Res 612:221-230[Medline]

Willoughby J, Glover V, Sandler M (1988) Histochemical localization of monoamine oxidase A and B in rat brain. J Neural Transmission 74:29-42





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Dunning, D. D.
Articles by Stein, B. E.
Articles citing this Article
PubMed
PubMed Citation
Articles by Dunning, D. D.
Articles by Stein, B. E.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]