BrainResearchBulletin,Vol. 24, pp. 113-118. © PergamonPress plc, 1990. Printed in the U.S.A.
0361-9230/90 $3.00 + .00
A Double-Labeling Method for AChE and Fluorescent Retrograde Tracers M A T T H E W E N N I S , * t M I C H A E L T. S H I P L E Y t I A N D M I C H A E L M. B E H B E H A N I *
*Department of Physiology and Biophysics (ML 576) and #Department of Anatomy and Cell Biology (ML 521) Division of Neurobiology University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267 R e c e i v e d 1 S e p t e m b e r 1989
ENNIS, M., M. T. SHIPLEY AND M. M. BEHBEHANI. A double-labelingmethodfor AChEandfluorescent retrogradetracers. BRAIN RES BULL 24(1) 113-118, 1990.--Staining for the degradative enzyme acetylcholinesterase (ACHE) is an important tool in studying central cholinergic/cholinoceptive systems. AChE staining has also been useful in identifying the projections of AChE-containing neurons and codistribution of AChE with other neurotransmitters. The intensity and opacity of conventional AChE histochemical reaction products, however, pose problems for such double-labeling studies. Here, we have successfully combined a modified version (37) of the Koelle-Friedenwald AChE reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). By omitting the final intensification steps of the Koelle-Friedenwald reaction, a translucent, light-stable reaction product is created. Viewed under darkfield illumination, this precipitate is of similar intensity and sensitivity to that produced by conventional AChE histochemical processing. Prior administration of an AChE-inhibitor yields preferential staining of AChE-positive neuronal somata. This nonintensified darkfield AChE (NIDA) histochemical method was compatible with visualization of retrogradely transported FG in AChE-positive neurons, allowing unambiguous identification of the projections of AChE-containing neurons. Acetylcholine Fluoro-Gold
Acetylcholinesterase (ACHE) Double labeling Locus coeruleus Diagonal band
HISTOCHEMICAL visualization of acetylcholinesterase (ACHE) is an important, widely used technique in the study of the CNS. This staining method has been used to identify putative cholinergic/cholinoceptive neurons and the distribution of AChE-positive fibers and terminals (7). In combination with Nissl staining, localization of AChE is very useful in architectonic studies of nuclear subdivisions and cortical lamination (30,37). AChE staining has also been used to study development, plasticity and neurodegenerative changes in the CNS (8, 14-16, 32, 41). AChE staining has been combined with retrograde tracing to examine the projections of putative cholinergic and cholinoceptive neurons (3, 5, 9, 17, 18, 21, 23, 25, 26, 29, 42-46), and in double-labeling studies to assess colocalization and codistribution of this marker with other putative neurotransmitters (2, 9, 12, 20, 27, 39) and receptors (4, 15, 27). However, there are potential problems of interpretation in such combined labeling studies, primarily because the tissue processing used in the histochemical visualization of AChE, and the intensity of the opacity of the AChE reaction product, are not optimal for visualization of retrograde tracers or neurochemicai markers. Here, we have combined a modified version (37) of the Koelle-Friedenwald (19) AChE histochemical reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). The modified AChE histochemical reaction yields a translucent, light-stable reaction
Histochemistry
Fluorescent tracers
product. Viewed under darkfield illumination, this precipitate is of similar intensity to that produced by the standard AChE histochemical process. This nonintensified darkfield AChE (NIDA) histochemical method is compatible with visualization of retrogradely transported FG in the same section, thus allowing identification of the projections of AChE-positive neurons. METHOD Rats were anesthetized with chloral hydrate (400 mg/kg, IP) and placed in a stereotaxic instrument. Holes were drilled in the skull over the olfactory bulb (6.7-7.7 mm rostral to bregma, 1.1 mm lateral to midline) and injections of 15 to 50 nl of Fluoro-Gold (FG, 1% solution in distilled water) were made with glass micropipettes (40-100 ixm tip diameter) connected to hydraulic injection system (35). Several injections of FG were placed in the olfactory bulb (1-4 mm ventral to brain surface) at different rostrocaudal sites. The olfactory bulb was selected as a model system for these experiments as this structure receives inputs from neurons that contain both choline acetyltransferase (CHAT) and AChE (presumed cholinergic neurons) from the horizontal limb of the diagonal band [HDB; (11, 33, 47)], as well as inputs from CHAT-negative, AChE-positive neurons in the locus coeruleus [LC, (2)]. The pharmacohistochemicai method of Butcher (6) was
~Requests for reprints should be addressed to M. T. Shipley, Department of Anatomy and Cell Biology, Division of Neurobiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521.
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used to preferentially limit AChE staining to cell bodies. To irreversibly inhibit AChE, animals were injected intramuscularly with a 77.7 mg/kg of soman (pinacolylmethylphosphonofluoridate) 4-7 days after FG injections. Two hours after soman injections, animals were deeply anesthetized and perfused transcardially with 0.9% saline containing 0.2% dimethyl sulfoxide until venous return was clear. This was followed by one liter of one of the following 3 fixatives: (1) 4% paraformaldehyde in 0.1 M phosphate buffer containing 2% sucrose; (2) 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer containing 2% sucrose, pH 7.4; (3) 2% paraformaldehyde and 0.7% glutaraldehyde in 0.1 M phosphate buffer containing 2% sucrose; all fixatives were at room temperature. Finally, the animal was perfused with one liter of 10% sucrose in 0.1 M phosphate buffer (pH 7.4; 4°C). The brain was removed and stored overnight in 20% sucrose in 0.1 M phosphate buffer (pH 7.4, 4°C). Frozen, 30--40 ~xm thick sections were cut with a freezing microtome and alternately placed into 3 trays containing 0.1 M phosphate buffer. For tissue perfused with fixatives containing glutaraldehyde, sections were agitated for 5-7 min at room temperature with 1% sodium borohydride in 0.1 M potassium phosphate buffer (pH 8.0) to reduce glutaraldehyde-induced autofluorescence. For each brain, sections in the first tray were immediately rinsed (2 × 1 rain) with distilled water and mounted on gelatinized slides (FG-Only seties). Sections in the second tray were processed according to the standard silver-intensifiedKoelle-Friedenwald AChE reaction (19) as previously described [Standard-AChE series; (40)]. Sections in the third tray underwent a modified version (37) of the KoelleFriedenwald reaction consisting of an incubation step only (nonintensified darkfield AChE, NIDA), i.e., the sulfide and silver steps were omitted. All sections were dehydrated through graded alcohols and xylene, and were coverslipped with DPX. RESULTS
The Standard-AChE reaction produced an opaque, dark brown reaction product. Staining was discrete and primarily localized to cell bodies due to the pretreatment with soman. However, even with the relatively high level of soman used in these experiments (0.7 LD50), some animals still had pockets of residual ACHEstained neuropil in areas which have high levels of AChE (e.g., striatum, olfactory tubercle). When present, this residual neuropil staining partially obscured visualization of AChE-positive neurons in the HDB and LC. The nonintensified darkfield AChE (NIDA) histochemical reaction produced a light-stable, white to light brown, translucent reaction product. Under brighffield illumination, the intensity of this reaction product is of no practical value for visualizing ACHE. However, as shown in Fig. 1A and 2A, when viewed under darkfield illumination the NIDA reaction yielded sensitive and discrete AChE staining. Robust AChE staining of cell bodies in HDB and LC was obtained with all three of the fixatives tested. Consistent with previous reports (10, 24, 36, 38), inspection of the FG-Only series revealed that FG injections into the olfactory bulb retrogradely labeled substantial numbers of neurons in the HDB, (Fig. 1), anterior olfactory nucleus, primary olfactory cortex, nucleus of the lateral olfactory tract, LC (Fig. 2), and the median and dorsal raphe. With all three fixatives tested, satisfactory retrograde labeling was observed in the FG-Only series. There were no apparent differences in numbers or intensity of retrogradely labeled neurons across fixatives, however, possible small differences of this type were not investigated with quantitative methods. FG-labeled neurons were also readily observed in NIDA-stained sections. In cases of optimal inhibition of AChE neuropil staining by soman, no obvious differences in patterns or
numbers of retrogradely labeled neurons were noted between the FG-Only and NIDA series. Thus, the modified AChE reaction does not appear to compromise the visualization of retrograde labeling with FG. As illustrated in Figs. 1 and 2, by merely switching from darkfield to ultraviolet (UV) illumination, it was possible to clearly identify individual neurons that contained FG, ACHE, or both labels. There appeared to be little or no interference between the two lables as FG was visualized in neurons intensely stained with AChE and vice versa. However, in cases of incomplete AChE inhibition, extrasomatic pockets of AChE-stained neuropil obscured visualization of FG. This problem may be mitigated with higher levels of AChE inhibitors but there is always some animal to animal variability in AChE inhibition using the same dose of soman (M. T. Shipley, unpublished observations). In contrast to the results above, very few FG-labeled neurons were observed in the AChE-Standard series. While some FGlabeled neurons were present, only very few also contained ACHE, and these contained only very weak deposits of the enzyme. Thus, the Standard-AChE reaction is not compatible with combined visualization of FG in the same tissue. DISCUSSION
The present report describes a simple, double-labeling method for AChE and retrogradely transported fluorescent tracers. Our results suggest that fixation and tissue processing parameters for localization of AChE and FG are compatible and, therefore, do not cause appreciable loss of sensitivity for either label. In addition, both labels are easily visualized in the same section, robust in intensity, and there is little or no optical interference of one label by another. The present technique obviates many of the time consuming steps, procedural difficulties, and problems of interpretation associated with combined AChE-retrograde tracing studies. For example, conventional AChE staining combined with retrograde tracing often involve laborious procedures, such as first processing, coverslipping and photographing tissue specimens for retrogradely transported tracers, then decoverslipping and processing for ACHE, followed by further microscopic analysis (5, 9, 20, 42--46). Other studies have attempted to simultaneously demonstrate AChE and retrograde tracers in the same tissue (17, 18, 21, 25, 26, 29). However, combined processing for AChE and one of the most widely used retrograde tracers, horseradish peroxidase (HRP) or related lectin/toxin-HRP conjugates, is problematic. For example, the intense, opaque brown/black AChE reaction product often occludes or masks identification of the opaque, granular brown/ black (or brown/blue, when the chromogen TMB is used) HRP reaction product in the same cell, and vice versa, thus making it difficult to ambiguously differentiate doubly labeled neurons. In addition to the problem of distinguishing AChE and HRP staining on the basis of subtle tinctorial differences, the fixation and histochemical parameters necessary for demonstration of AChE and retrogradely transported HRP have been reported to produce a loss in sensitivity of one or both labels, often leading to falsenegatives, and also to higher than usual levels of artifactual reaction product (21,25). While it has been reported that AChE and retrogradely transported HRP may be localized within different cytoplasmic organelles, double-labeling studies relying on such differential intrasomatic localization patterns require electron microscopic techniques (17,18). Conventional histochemical staining for AChE has also been combined with demonstration of retrogradely transported fluorescent tracers in the same section (1, 3, 23, 39), however, in the present study visualization of the retrogradely transported FG in the Standard-AChE series was obscured by the nearly opaque
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FIG. 1. ACHE- and FG-positive neurons in the nucleus of the horizontal limb of the diagonal band (HDB) 4 days following injections of FG into the olfactory bulb. (A) Photomicrograph (darkfield illumination) of a coronal section showing AChE-positive neurons in HDB. (B) Photomicrograph (UV illumination) of the same section showing neurons retrogradely labeled with FG. Arrows in (A) and (B) indicate neurons that contain both AChE and FG. Orientation: midline is to the left, dorsal is at the top.
AChE reaction product. By contrast, the NIDA reaction yields a nearly translucent AChE reaction product which is entirely compatible with visualization of retrogradely transported FG in the same tissue section. In addition, the present results indicate that the optimal fixative for visualization of FG (31,34) is also compatible with NIDA histochemistry, thus there is nearly optimal sensitivity for both labels. Although we have not attempted to visualize other fluorescent retrograde tracers (e.g., fluorescent-
labeled latex microspheres, True Blue, Fast Blue) it seems very likely that these tracers should work as well as FG as the major success of the present method is due to the elimination of the optical interference by the chromagens commonly used in the AChE histochemical reactions. The utility of AChE as a marker for cholinergic cell bodies and fibers has long been debated. While it has been suggested that only neurons that stain "intensely" for AChE after administration of
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FIG. 2. ACHE- and FG-positive neurons in the LC 7 days following injections of FG into the olfactory bulb. (A) Photomicrograph (darkfield illumination) of a coronal section showing AChE-positive neurons in LC. (B) UV illumination of the same section shows LC neurons retrogradely labeled with FG. Arrows indicate neurons containing both FG and ACHE. Abbreviation: IV, fourth ventricle. AChE inhibitors are cholinergic [for review see (7)], conflicting results have been published (11,13). For example, in rodent cerebral cortex the synthetic enzyme ChAT did not colocalize in all AChE-containing neurons (22). In a subsequent study, however, all ChAT-positive neurons in the caudate-putamen and basal forebrain were reported to contain AChE and vice versa; neurons containing only AChE were found in several areas (11). The significance of neurons containing AChE and not ChAT is still not clear, but it has been suggested that such AChE-positive, CHATnegative neurons in the olfactory bulb (28) and LC (2), for example, may be targeted by cholinergic inputs, and hence, cholinoceptive. If such potential pitfalls in the interpretation of AChE staining are recognized and if other indices of cholinergic function (i.e., ChAT staining; biochemical assays of ACh, AChE and CHAT) are taken into consideration, then AChE staining is still a key method in studying central cholinergic/cholinoceptive systems. AChE staining has been useful in studies of abnormalities of cholinergic
systems in senile dementia of the Alzheimer's type (8,32) and in studies of neurotrophic effects of nerve growth factor on central cholinergic neurons (14-16). The method described here offers a simple, direct way to analyze the projections of AChE-containing neurons. Future studies may reveal other features (i.e., efferent projections, morphology, neurotransmitter identity, neurotransmitter colocalization) that further characterize subpopulations of AChE-positive neurons. In this regard, it is noteworthy that with 4% paraformaldehyde fixation, our NIDA histochemical reaction should be compatible with immunocytochemistry. Thus, it may be possible to double label for AChE and other neuroactive/neurotrophic molecules. ACKNOWLEDGEMENTS We thank Beata Frydel, Grace Kenney and Libang Fu for technical assistance. Supported by NINDS 23348, 20643; NS24698, NS22053; U.S. Army Medical Research and Development Command DAMD 17-86C-6005, and HL08097.
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A Double-Labeling Method for AChE and Fluorescent Retrograde Tracers M A T T H E W E N N I S , * t M I C H A E L T. S H I P L E Y t I A N D M I C H A E L M. B E H B E H A N I *
*Department of Physiology and Biophysics (ML 576) and #Department of Anatomy and Cell Biology (ML 521) Division of Neurobiology University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267 R e c e i v e d 1 S e p t e m b e r 1989
ENNIS, M., M. T. SHIPLEY AND M. M. BEHBEHANI. A double-labelingmethodfor AChEandfluorescent retrogradetracers. BRAIN RES BULL 24(1) 113-118, 1990.--Staining for the degradative enzyme acetylcholinesterase (ACHE) is an important tool in studying central cholinergic/cholinoceptive systems. AChE staining has also been useful in identifying the projections of AChE-containing neurons and codistribution of AChE with other neurotransmitters. The intensity and opacity of conventional AChE histochemical reaction products, however, pose problems for such double-labeling studies. Here, we have successfully combined a modified version (37) of the Koelle-Friedenwald AChE reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). By omitting the final intensification steps of the Koelle-Friedenwald reaction, a translucent, light-stable reaction product is created. Viewed under darkfield illumination, this precipitate is of similar intensity and sensitivity to that produced by conventional AChE histochemical processing. Prior administration of an AChE-inhibitor yields preferential staining of AChE-positive neuronal somata. This nonintensified darkfield AChE (NIDA) histochemical method was compatible with visualization of retrogradely transported FG in AChE-positive neurons, allowing unambiguous identification of the projections of AChE-containing neurons. Acetylcholine Fluoro-Gold
Acetylcholinesterase (ACHE) Double labeling Locus coeruleus Diagonal band
HISTOCHEMICAL visualization of acetylcholinesterase (ACHE) is an important, widely used technique in the study of the CNS. This staining method has been used to identify putative cholinergic/cholinoceptive neurons and the distribution of AChE-positive fibers and terminals (7). In combination with Nissl staining, localization of AChE is very useful in architectonic studies of nuclear subdivisions and cortical lamination (30,37). AChE staining has also been used to study development, plasticity and neurodegenerative changes in the CNS (8, 14-16, 32, 41). AChE staining has been combined with retrograde tracing to examine the projections of putative cholinergic and cholinoceptive neurons (3, 5, 9, 17, 18, 21, 23, 25, 26, 29, 42-46), and in double-labeling studies to assess colocalization and codistribution of this marker with other putative neurotransmitters (2, 9, 12, 20, 27, 39) and receptors (4, 15, 27). However, there are potential problems of interpretation in such combined labeling studies, primarily because the tissue processing used in the histochemical visualization of AChE, and the intensity of the opacity of the AChE reaction product, are not optimal for visualization of retrograde tracers or neurochemicai markers. Here, we have combined a modified version (37) of the Koelle-Friedenwald (19) AChE histochemical reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). The modified AChE histochemical reaction yields a translucent, light-stable reaction
Histochemistry
Fluorescent tracers
product. Viewed under darkfield illumination, this precipitate is of similar intensity to that produced by the standard AChE histochemical process. This nonintensified darkfield AChE (NIDA) histochemical method is compatible with visualization of retrogradely transported FG in the same section, thus allowing identification of the projections of AChE-positive neurons. METHOD Rats were anesthetized with chloral hydrate (400 mg/kg, IP) and placed in a stereotaxic instrument. Holes were drilled in the skull over the olfactory bulb (6.7-7.7 mm rostral to bregma, 1.1 mm lateral to midline) and injections of 15 to 50 nl of Fluoro-Gold (FG, 1% solution in distilled water) were made with glass micropipettes (40-100 ixm tip diameter) connected to hydraulic injection system (35). Several injections of FG were placed in the olfactory bulb (1-4 mm ventral to brain surface) at different rostrocaudal sites. The olfactory bulb was selected as a model system for these experiments as this structure receives inputs from neurons that contain both choline acetyltransferase (CHAT) and AChE (presumed cholinergic neurons) from the horizontal limb of the diagonal band [HDB; (11, 33, 47)], as well as inputs from CHAT-negative, AChE-positive neurons in the locus coeruleus [LC, (2)]. The pharmacohistochemicai method of Butcher (6) was
~Requests for reprints should be addressed to M. T. Shipley, Department of Anatomy and Cell Biology, Division of Neurobiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521.
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used to preferentially limit AChE staining to cell bodies. To irreversibly inhibit AChE, animals were injected intramuscularly with a 77.7 mg/kg of soman (pinacolylmethylphosphonofluoridate) 4-7 days after FG injections. Two hours after soman injections, animals were deeply anesthetized and perfused transcardially with 0.9% saline containing 0.2% dimethyl sulfoxide until venous return was clear. This was followed by one liter of one of the following 3 fixatives: (1) 4% paraformaldehyde in 0.1 M phosphate buffer containing 2% sucrose; (2) 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer containing 2% sucrose, pH 7.4; (3) 2% paraformaldehyde and 0.7% glutaraldehyde in 0.1 M phosphate buffer containing 2% sucrose; all fixatives were at room temperature. Finally, the animal was perfused with one liter of 10% sucrose in 0.1 M phosphate buffer (pH 7.4; 4°C). The brain was removed and stored overnight in 20% sucrose in 0.1 M phosphate buffer (pH 7.4, 4°C). Frozen, 30--40 ~xm thick sections were cut with a freezing microtome and alternately placed into 3 trays containing 0.1 M phosphate buffer. For tissue perfused with fixatives containing glutaraldehyde, sections were agitated for 5-7 min at room temperature with 1% sodium borohydride in 0.1 M potassium phosphate buffer (pH 8.0) to reduce glutaraldehyde-induced autofluorescence. For each brain, sections in the first tray were immediately rinsed (2 × 1 rain) with distilled water and mounted on gelatinized slides (FG-Only seties). Sections in the second tray were processed according to the standard silver-intensifiedKoelle-Friedenwald AChE reaction (19) as previously described [Standard-AChE series; (40)]. Sections in the third tray underwent a modified version (37) of the KoelleFriedenwald reaction consisting of an incubation step only (nonintensified darkfield AChE, NIDA), i.e., the sulfide and silver steps were omitted. All sections were dehydrated through graded alcohols and xylene, and were coverslipped with DPX. RESULTS
The Standard-AChE reaction produced an opaque, dark brown reaction product. Staining was discrete and primarily localized to cell bodies due to the pretreatment with soman. However, even with the relatively high level of soman used in these experiments (0.7 LD50), some animals still had pockets of residual ACHEstained neuropil in areas which have high levels of AChE (e.g., striatum, olfactory tubercle). When present, this residual neuropil staining partially obscured visualization of AChE-positive neurons in the HDB and LC. The nonintensified darkfield AChE (NIDA) histochemical reaction produced a light-stable, white to light brown, translucent reaction product. Under brighffield illumination, the intensity of this reaction product is of no practical value for visualizing ACHE. However, as shown in Fig. 1A and 2A, when viewed under darkfield illumination the NIDA reaction yielded sensitive and discrete AChE staining. Robust AChE staining of cell bodies in HDB and LC was obtained with all three of the fixatives tested. Consistent with previous reports (10, 24, 36, 38), inspection of the FG-Only series revealed that FG injections into the olfactory bulb retrogradely labeled substantial numbers of neurons in the HDB, (Fig. 1), anterior olfactory nucleus, primary olfactory cortex, nucleus of the lateral olfactory tract, LC (Fig. 2), and the median and dorsal raphe. With all three fixatives tested, satisfactory retrograde labeling was observed in the FG-Only series. There were no apparent differences in numbers or intensity of retrogradely labeled neurons across fixatives, however, possible small differences of this type were not investigated with quantitative methods. FG-labeled neurons were also readily observed in NIDA-stained sections. In cases of optimal inhibition of AChE neuropil staining by soman, no obvious differences in patterns or
numbers of retrogradely labeled neurons were noted between the FG-Only and NIDA series. Thus, the modified AChE reaction does not appear to compromise the visualization of retrograde labeling with FG. As illustrated in Figs. 1 and 2, by merely switching from darkfield to ultraviolet (UV) illumination, it was possible to clearly identify individual neurons that contained FG, ACHE, or both labels. There appeared to be little or no interference between the two lables as FG was visualized in neurons intensely stained with AChE and vice versa. However, in cases of incomplete AChE inhibition, extrasomatic pockets of AChE-stained neuropil obscured visualization of FG. This problem may be mitigated with higher levels of AChE inhibitors but there is always some animal to animal variability in AChE inhibition using the same dose of soman (M. T. Shipley, unpublished observations). In contrast to the results above, very few FG-labeled neurons were observed in the AChE-Standard series. While some FGlabeled neurons were present, only very few also contained ACHE, and these contained only very weak deposits of the enzyme. Thus, the Standard-AChE reaction is not compatible with combined visualization of FG in the same tissue. DISCUSSION
The present report describes a simple, double-labeling method for AChE and retrogradely transported fluorescent tracers. Our results suggest that fixation and tissue processing parameters for localization of AChE and FG are compatible and, therefore, do not cause appreciable loss of sensitivity for either label. In addition, both labels are easily visualized in the same section, robust in intensity, and there is little or no optical interference of one label by another. The present technique obviates many of the time consuming steps, procedural difficulties, and problems of interpretation associated with combined AChE-retrograde tracing studies. For example, conventional AChE staining combined with retrograde tracing often involve laborious procedures, such as first processing, coverslipping and photographing tissue specimens for retrogradely transported tracers, then decoverslipping and processing for ACHE, followed by further microscopic analysis (5, 9, 20, 42--46). Other studies have attempted to simultaneously demonstrate AChE and retrograde tracers in the same tissue (17, 18, 21, 25, 26, 29). However, combined processing for AChE and one of the most widely used retrograde tracers, horseradish peroxidase (HRP) or related lectin/toxin-HRP conjugates, is problematic. For example, the intense, opaque brown/black AChE reaction product often occludes or masks identification of the opaque, granular brown/ black (or brown/blue, when the chromogen TMB is used) HRP reaction product in the same cell, and vice versa, thus making it difficult to ambiguously differentiate doubly labeled neurons. In addition to the problem of distinguishing AChE and HRP staining on the basis of subtle tinctorial differences, the fixation and histochemical parameters necessary for demonstration of AChE and retrogradely transported HRP have been reported to produce a loss in sensitivity of one or both labels, often leading to falsenegatives, and also to higher than usual levels of artifactual reaction product (21,25). While it has been reported that AChE and retrogradely transported HRP may be localized within different cytoplasmic organelles, double-labeling studies relying on such differential intrasomatic localization patterns require electron microscopic techniques (17,18). Conventional histochemical staining for AChE has also been combined with demonstration of retrogradely transported fluorescent tracers in the same section (1, 3, 23, 39), however, in the present study visualization of the retrogradely transported FG in the Standard-AChE series was obscured by the nearly opaque
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FIG. 1. ACHE- and FG-positive neurons in the nucleus of the horizontal limb of the diagonal band (HDB) 4 days following injections of FG into the olfactory bulb. (A) Photomicrograph (darkfield illumination) of a coronal section showing AChE-positive neurons in HDB. (B) Photomicrograph (UV illumination) of the same section showing neurons retrogradely labeled with FG. Arrows in (A) and (B) indicate neurons that contain both AChE and FG. Orientation: midline is to the left, dorsal is at the top.
AChE reaction product. By contrast, the NIDA reaction yields a nearly translucent AChE reaction product which is entirely compatible with visualization of retrogradely transported FG in the same tissue section. In addition, the present results indicate that the optimal fixative for visualization of FG (31,34) is also compatible with NIDA histochemistry, thus there is nearly optimal sensitivity for both labels. Although we have not attempted to visualize other fluorescent retrograde tracers (e.g., fluorescent-
labeled latex microspheres, True Blue, Fast Blue) it seems very likely that these tracers should work as well as FG as the major success of the present method is due to the elimination of the optical interference by the chromagens commonly used in the AChE histochemical reactions. The utility of AChE as a marker for cholinergic cell bodies and fibers has long been debated. While it has been suggested that only neurons that stain "intensely" for AChE after administration of
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FIG. 2. ACHE- and FG-positive neurons in the LC 7 days following injections of FG into the olfactory bulb. (A) Photomicrograph (darkfield illumination) of a coronal section showing AChE-positive neurons in LC. (B) UV illumination of the same section shows LC neurons retrogradely labeled with FG. Arrows indicate neurons containing both FG and ACHE. Abbreviation: IV, fourth ventricle. AChE inhibitors are cholinergic [for review see (7)], conflicting results have been published (11,13). For example, in rodent cerebral cortex the synthetic enzyme ChAT did not colocalize in all AChE-containing neurons (22). In a subsequent study, however, all ChAT-positive neurons in the caudate-putamen and basal forebrain were reported to contain AChE and vice versa; neurons containing only AChE were found in several areas (11). The significance of neurons containing AChE and not ChAT is still not clear, but it has been suggested that such AChE-positive, CHATnegative neurons in the olfactory bulb (28) and LC (2), for example, may be targeted by cholinergic inputs, and hence, cholinoceptive. If such potential pitfalls in the interpretation of AChE staining are recognized and if other indices of cholinergic function (i.e., ChAT staining; biochemical assays of ACh, AChE and CHAT) are taken into consideration, then AChE staining is still a key method in studying central cholinergic/cholinoceptive systems. AChE staining has been useful in studies of abnormalities of cholinergic
systems in senile dementia of the Alzheimer's type (8,32) and in studies of neurotrophic effects of nerve growth factor on central cholinergic neurons (14-16). The method described here offers a simple, direct way to analyze the projections of AChE-containing neurons. Future studies may reveal other features (i.e., efferent projections, morphology, neurotransmitter identity, neurotransmitter colocalization) that further characterize subpopulations of AChE-positive neurons. In this regard, it is noteworthy that with 4% paraformaldehyde fixation, our NIDA histochemical reaction should be compatible with immunocytochemistry. Thus, it may be possible to double label for AChE and other neuroactive/neurotrophic molecules. ACKNOWLEDGEMENTS We thank Beata Frydel, Grace Kenney and Libang Fu for technical assistance. Supported by NINDS 23348, 20643; NS24698, NS22053; U.S. Army Medical Research and Development Command DAMD 17-86C-6005, and HL08097.
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