TECHNICAL NOTE |
Correspondence to: John G. McHaffie, Dept. of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest U., Winston-Salem, NC 27157-1010.
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Summary |
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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
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Introduction |
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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
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.
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Materials and Methods |
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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 (
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;
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) (
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).
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Results |
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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.
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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 (
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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 (
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Discussion |
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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 (
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 (
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 (
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 (
The results of this study are in agreement with previous studies showing that AChE is found in cholinergic and non-cholinergic neurons (
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Acknowledgments |
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Supported by NIH grants NS 22543, EY 06562, and NS 35008.
Received for publication November 12, 1996; accepted December 9, 1996.
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