ARTICLE |
Correspondence to: Riitta A. Miettinen, Dept. of Neuroscience and Neurology, University of Kuopio, PO Box 1627, FIN-7021 Kuopio, Finland. E-mail: riitta.miettinen@uku.fi
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Summary |
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This study was undertaken to estimate the total number of cholinergic cells and the percentage of cholinergic cells that contain estrogen receptor- (ER
) in the rat basal forebrain. Double immunostaining for choline acetyltransferase (ChAT) and ER
was carried out on 50-µm-thick free-floating sections. Because routine mounting method causes considerable flattening of the sections, we embedded immunostained sections in Durcupan, an epoxy resin known to cause virtually no shrinkage. When this procedure was used the section thickness was well preserved, individual cells could be clearly identified, and subcellular localization of ER
immunoreactivity was easy to verify. Cell counting in these sections revealed that the rat basal forebrain contains 26,390 ± 1097 (mean ± SEM) cholinergic neurons. This comprises 9674 ± 504 in the medial septumvertical diagonal band of Broca, 9403 ± 484 in the horizontal diagonal band of Broca, and 7312 ± 281 in the nucleus basalis. In these nuclei, 60%, 46%, and 14% of the cholinergic neurons were co-localized with ER
, respectively. We believe that our results are an improvement on existing data because of the better distinction of individual neurons that the Durcupan embedding method brings. (J Histochem Cytochem 50:891902, 2002)
Key Words: stereology, cell counting, Durcupan, resin, embedding, choline actetyltransferase, Alzheimer's disease
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Introduction |
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THE BASAL FOREBRAIN AREA contains a number of cholinergic cell groups that are involved in a variety of physiological and behavioral processes. There is evidence that the gonadal estrogens can directly influence on these neurons in females ( and ß, have been identified and characterized (for review see
(ER
), only few cells contain ERß. However, in these studies, the total numbers of the cholinergic cells containing or not containing ERs were not reported. Therefore, this study was undertaken to obtain these numeric values, which are required for evaluating the effect of estrogens on the survival of the cholinergic neurons in the basal forebrain in future experiments. The importance of these data is highlighted by the fact that one of the most consistent findings in Alzheimer's disease (AD) is the deterioration of the cholinergic system (
Choline acetyltransferase (ChAT), the enzyme responsible for the synthesis of acetylcholine, is located in the cytoplasm, and ER is expressed primarily in the nucleus (see, e.g.,
, one could not be sure whether a certain ER
-immunoreactive (ir) nucleus belongs to the cell that has a ChAT-ir cytoplasm or whether that particular nucleus has been pushed into the proximity of a ChAT-ir cell that does not have an ER
-ir nucleus. Therefore, for this type of investigation it is very important to prepare the material in a way that avoids these artifacts. Therefore, the major objective would be to prevent section shrinkage and flattening.
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In this study we tested and concluded that embedding the samples in an epoxy resin, Durcupan, after immunostaining could be used to maintain section volume during mounting. We illustrate this improvement by presenting the numbers of the cholinergic neurons containing or not containing ER in different basal forebrain nuclei of the rat. This information provides a baseline in understanding the effects of various experimental manipulations aimed at investigating the role of estrogens in forebrain cholinergic dysfunction that is observed in human diseases, most notably in AD.
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Materials and Methods |
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Male 3-month-old Wistar rats (n=4) were used. These rats were housed in one cage in a controlled environment (constant temperature 22 ± 1C; humidity 5060%; lights on 07001900 hr) and had free access to food and water. The experiments were approved by the committee for the welfare of laboratory animals of the University of Kuopio.
The rats were deeply anesthetized with a mixture (0.4 ml/100 g, IP) of sodium barbiturate (Synopharm, concentration in the mixture 9.7 mg/ml), chloral hydrate (Merck, Dormstadt, Germany; 10 mg/ml), magnesium sulfate (Merck; 21.2 mg/ml), propylene glycol (Merck; 40%), and absolute ethanol (10%). Then they were perfused transcardially, consecutively with saline (3 min) and then with 300 ml of fixative (30 min). The fixative contained 4% paraformaldehyde (P001; TAAB Laboratories Equipment, Aldermaston, Berks, UK), 0.05% glutaraldehyde (G002; TAAB Laboratories Equipment) and 0.26% picric acid (623; Merck) in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed from the skull and 50-µm-thick sections were cut on a vibratome (Leica VT 1000 S, Leica Instruments, Wetzlar, Germany) into six series (Fig 2). Sections were stored in a cryoprotectant solution (30% ethylene glycol and 30% glycerol in PB) at -20C until processed.
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To test Durcupan's ability to prevent shrinkage and flattening of the sections, different series of sections were processed as follows: (a) no staining; (b) double immunostaining followed by mounting with Depex; and (c) double immunostaining followed by embedding in Durcupan.
The first series of sections (series a, randomly selected) was washed in PB three times for 10 min. These sections were mounted on slides and immediately covered with a coverslip (to avoid drying). Native section thickness was measured using the set-up described below.
Two series of sections (series b and c) were double-immunostained for ER and ChAT. Sections were first washed six times for 30 min in PB and then in 0.05 M Tris-buffered saline, pH 7.4 (TBS), twice for 20 min. After this, sections were incubated in 10% normal goat serum (NGS; CS-0922, Colorado Serum Company, Denver, CO) containing 0.5% Triton X-100 in TBS for 40 min and in 1% NGS containing 0.5% Triton X-100 in TBS for 15 min. Incubation in rabbit anti-ER
antiserum (1:10, 000, SC-542; Santa Cruz Technology, Santa Cruz, CA) was carried out at 4C for 48 hr. This was followed by incubation in biotinylated anti-rabbit IgG (1:300, BA-1, 000; Vector Laboratories, Burlingame, CA) overnight at 4C and then in avidin-biotinylated horseradish peroxidase complex (1:500, PK-4, 000; Vector Laboratories) for 3 hr at room temperature (RT). The immunoperoxidase reaction was developed using ammonium nickel sulfate (0.2%, 10029; BDH, Poole, UK)-intensified 3,3'-diaminobenzidine (0.015% DAB, D-5637; Sigma, St Louis, MO) as chromogen, giving a blue-to-black granular reaction end product. Then sections were incubated in rat anti-ChAT antiserum (1:10, 770990; Roche, Basel, Switzerland) 48 hours at 4C, followed by an incubation in rabbit anti-rat IgG (1:50, AB-136; Chemicon, Temecula, CA) for 6 hr at RT and finally in rat peroxidase anti-peroxidase complex (1:300, PAP-20; Chemicon) overnight at 4C. The second immunoperoxidase reaction was developed using 0.05% DAB as a chromogen, which gives a homogeneous brown end product. The dilutions of the antisera and all the washing steps (three times for 30 min) that were carried out between the antibody incubations were done in 0.05 M TBS, pH 7.4, containing 1% NGS and 0.5% Triton X-100.
After thorough washing in TBS, the series of sections aimed for routine mounting (series b) were mounted on gelatin-coated slides and dried overnight at 37C. Thereafter, these sections were dehydrated in absolute ethanol, cleared in xylene, and covered with Depex and a coverslip. The third series of sections (series c) was washed in TBS, rinsed with distilled water, dehydrated in a series of ethanol (50%, 70%, 90%, 96% for 5 min in each and in absolute ethanol twice for 5 min) and propylene oxide twice for 5 min. The sections were then immersed in Durcupan (AMC; Fluka, Buchs, Switzerland). The sections were freely floating during the dehydration procedure and immersion in Durcupan. After 3 hr at RT in Durcupan, the sections were transferred onto slides and covered with a coverslip. To ensure that the sections were planar between the coverslip and the objective slide and that excess Durcupan was removed, a glass block (weight 50 g) was placed on the coverslip to slightly press the coverslip. Durcupan was polymerized at 60C for 24 hr.
Controls for Immunostaining
Control stainings for IHC were carried out by omission of primary antibodies. In addition, omission of only one primary antibody in double immunostaining was performed. Both of these procedures resulted in no staining in those structural components that were found to be positive when the corresponding primary antibody was included. The specificity of both ChAT and ER antibodies has been previously characterized showing ability to react with these proteins in rat brain (
Section Thickness Measurement
Section thickness measurements were accomplished with the aid of Stereo Investigator software (MicroBrightField; Colchester, VT). The integrated hardware-software set-up consisted of a PC computer system connected to an ECLIPSE E600 microscope (Nikon; Tokyo, Japan) via 3-chip CCD color video camera (HV-C20; Hitachi, Tokyo, Japan). A motorized stage (three-axis computer-controlled stepping motor system with a 0.1-µm resolution) with a microcator (Heidenhain EXE 610C) attachment (providing a 0.1 µm resolution in the z-axis) was mounted on the microscope. The objective used for the measurements (and later for cell counting) was a CFI Plan Fluor x100 oil immersion objective having the properties NA 1.30 and WD 0.20 mm. For measuring the section thickness, the top of the section was first brought into focus and the depth position (z-axis coordinate) was set to 0. Thereafter the bottom of the section was brought into focus and the z-axis value (i.e., section thickness) was registered. Measurements were made from all sections sampled from each animal used in this study. From each section at least three different sites were measured.
Estimation of Cell Numbers
The number of ChAT-ir cells in the medial septum, vertical and horizontal diagonal band of Broca, and in the nucleus basalis was estimated using the optical fractionator method (
was used to estimate the total number of neurons, where Q- is the number of neurons actually counted in the specimens.
Digital Photography
Low-magnification photomicrographs demonstrating a general view of the distribution of ChAT- and ER-ir cells in different basal forebrain nuclei (Fig 5) were taken with a Nikon Coolpix 990 digital camera attached to the Optiphot-2 Nikon microscope using a Plan x10 objective (NA 0.30). High-magnification photomicrographs with a CFI Plan Fluor x100 oil immersion objective (NA 1.30) demonstrating optical views of Durcupan-embedded and Depex-mounted sections (Fig 3 and Fig 4) were taken using a 3-chip CCD color video camera (HV-C20; Hitachi), which belongs to the set-up for integrated hardware-software package used for cell counting (described above).
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Results |
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Section Thickness Comparison
Measurements of section thickness revealed that (a) unstained sections were 48.8 ± 1.15 µm (mean ± SEM), (b) double-immunostained sections that were mounted with Depex were 12.8 ± 0.1 µm (see also Fig 4), and (c) double immunostained sections embedded in Durcupan were 40.8 ± 0.35 µm (see also Fig 3). The surfaces of the Durcupan-embedded sections were clear and bright and it was easy to focus on the level at which the field of view on the top of the section turned from unfocused exterior into focused interior of the sample and vice versa at the bottom of the section.
General Observations About Durcupan-embedded Material
Macroscopically, sections mounted in Durcupan were darker than the sections in Depex because of Durcupan's intrinsic brownish color. However, at the microscopic level this did not interfere with identification of the cell bodies or the color contrast between different chromogens in the double-immunostained sections. The major advantage of Durcupan embedding was that individual cells could be clearly identified as their own entities at different focal planes (Fig 3). In contrast, the cells in sections mounted with Depex were flattened and the depth resolution was poor. This made it difficult to identify individual cells, especially those that were close to each other (Fig 4).
Microscopic examination revealed that ER-ir (dark blue color) had a punctate or granular appearance (Fig 3 and Fig 4). In the majority of cells, ER
-ir was present in the nucleus, where it formed dense aggregates. However, we found that in some cells, ER
-ir occurred in granules scattered throughout the cytoplasm. ChAT-ir (observed as brown) was located exclusively in the cytoplasm (Fig 3 and Fig 4). The texture of ChAT-ir was more homogenous than ER
-ir, filling up the entire cytoplasm of the cell soma and dendrites. The color difference between ER
-ir and ChAT-ir was clear in all sections and was independent of the mounting method used (Fig 3 and Fig 4). However, individual cells and their different subcellular compartments were easier to distinguish from each other in Durcupan-embedded sections because different focal levels were better monitored inside the thicker sections (Fig 3). Counting on Depex-mounted sections was difficult because it was hard to distinguish individual cells. Therefore, cells were counted only from Durcupan-embedded sections.
Estimation of Cell Numbers
The distribution of the ChAT-ir cells in the basal forebrain was in agreement with previous studies (
The ChAT-containing cells referred to as the Ch4 cell group by Mesulam (
In general, the ER-ir cells of the basal forebrain levels examined followed the distribution of ChAT-ir cells (Fig 5), except that they were more numerous and were present also in high numbers in the areas surrounding the ChAT-ir regions. Very high densities of ER
-ir cells were observed both in the islands of Calleja and the hypothalamic regions. They were also numerous in the lateral septum and preoptic areas. In addition, some scattered ER
-ir cells were detected in the globus pallidus. No ER
-ir cells were observed in the caudate putamen. Because our primary interest was to estimate the incidence of ER
-ir in the ChAT cells, the neurons containing ER
-ir alone were not counted.
The subcellular distribution of ER-ir in the ChAT-ir cells was registered while counting the cell numbers. On the basis of the subcellular distribution three types of subclasses of ChAT cells were made: (a) ChAT-ir neurons without ER
-ir; (b) ChAT-ir neurons that had ER
-ir in the nucleus; and (c) ChAT-ir neurons that had ER
-ir in the cytoplasm (Fig 3). In the last group, ER
-ir typically appeared as scattered granules in the cytoplasm, and these types of cells were clearly distinguishable from those ChAT-ir cells having ER
-ir in the nucleus. A few cells contained both nuclear and cytoplasmic ER
staining. The possible ER
content of each ChAT neuron was always confirmed through microscope oculars.
Estimation of the cell numbers in Durcupan sections revealed that the rat basal forebrain (including MS, VDB, HDB, and NB of both hemispheres) contained 26,390 ± 1097 (mean ± SEM) ChAT-ir cells, 42% of which contained ER-ir (Table 1). In the individual basal forebrain nuclei, the total number of ChAT-ir neurons was 9674 ± 504 (CV=0.10) in the MSVDB, 9403 ± 484 (CV=0.10) in the HDB, and 7312 ± 281 (CV=0.07) in the NB (Table 1). The total number of ChAT-ir neurons containing ER
-ir was 5736 ± 71 in the MSVDB (CV=0.02), 4304 ± 237 in the HDB (CV=0.11), and 1044 ± 140 in the NB (CV=0.26) (Table 1). The number of ChAT-ir neurons containing ER
-ir in the nucleus or the cytoplasm was 3908 ± 200 and 1828 ± 186 in the MSVDB, 2330 ± 55 and 1975 ± 186 in the HDB, and 512 ± 119 and 532 ± 79 in the NB, respectively.
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The rat brains were cut at 50 µm into six series (Fig 2). One series from each animal was randomly selected for processing sections for cell counting. In other words, the sections that were sampled for cell counting were 5 times 50 µm apart from each other. With this sampling system, three to five sections contained the MSVDB, five to seven sections contained the HDB, and seven sections contained the NB (Table 1). With the sampling grid 80 µm x 80 µm and counting frame 35 µm x 35 µm, the number of counted ChAT-ir cells (including all three categories of ChAT-ir cells) was in the MSVDB from 203 to 262, in the HDB from 201 to 253, and in the NB from 155 to 184 (Table 1). Because the ChAT-ir cells in the HDB were more evenly dispersed along different rostrocaudal levels than in other nuclei, it would have been unnecessary to count so many cells to be efficient in cell counting. Therefore, we performed an additional counting using a sampling grid size 160 µm x 160 µm. This resulted in the number of counted cells from 48 to 59. The estimate of total number of ChAT-ir (including all three categories of ChAT-ir) cells was then 9151 ± 200 (CV=0.08). This indicates that for the HDB even such a low number of counted cells would yield a good estimate of the total cell number.
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Discussion |
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In this article we describe a method by which we could prevent shrinkage and flattening of the immunohistochemically stained sections. We found that embedding sections in the epoxy resin Durcupan after immunostaining preserves the three-dimensional structure of the tissue. These sections are excellent for analyses of cell counts for which the optical fractionator method is employed. In addition, the present report is the first to describe estimates of both the total numbers of ChAT-ir neurons and the total numbers of the ChAT-ir neurons that contain ER-ir in the basal forebrain of male Wistar rats.
Embedding of Immunostained Sections in Durcupan Is Superior to Routine Mounting Methods
The development of modern stereological counting methods, especially the optical fractionator, has led to a great improvement in quantitative studies in many respects. First of all, the shrinkage problem, which cannot be handled unequivocally in biological material is basically avoided (see above,
Despite the fact that the optical fractionator method is reported to be unaffected by shrinkage of the tissue or absolute section thickness (
Resin embedding has also been described earlier (
Optically, the Durcupan-embedded sections are very clear, and it is easy to scan on various levels through the section. This facilitates verification of the penetration of immunohistochemicals as well. Because the flatness of Depex-mounted sections it is difficult to determine whether the penetration is uniform throughout the section. If penetration problems go undetected, they will lead to underestimation in the structure counts. Because antibody penetration can be easily verified in Durcupan-embedded sections, this can be considered as a further advantage of the Durcupan embedding method.
Cholinergic Cells in Different Basal Forebrain Nuclei Show Variations in Content of ER
Our study showed that the total number of ChAT-ir cells in the basal forebrain is 26,390 ± 1097, and this is composed of 9674 ± 504 ChAT-ir neurons in the MSVDB, 9403 ± 484 in the HDB, and 7312 ± 281 in the NB. These numbers differ from the previously published studies. However, we believe we have a more accurate representation of the numbers of ChAT-containing cells because of the ability to distinguish individual neurons associated with our method.
Any direct correlation between our studies and others is difficult because of differences in the age, strain, and sex of the rats, possible operations carried out on the animals (e.g., ovariectomy), the antibodies used, the section mounting method, and the definition of the anatomic boundaries for the different nuclei analyzed. However, it should be noted that we find significantly more ChAT-containing cells in our study. For example, the most recent study by
We also investigated the proportion of cholinergic cells that contain ER. In an earlier study,
, using another method. Their findings showed significantly lower percentages (i.e., 41% in the MS, 32% in the VDB, 29% in the HDB, and 4% in the NB) compared to our study. The major causes for these differences can be age, sex, ovariectomy, and strain of the rats, as well as the method used (in situ hybridization using radioactively labeled probes for estrogen receptors combined with immunohistochemistry for ChAT). First of all, it is possible that the level of mRNA and the corresponding protein in the neurons is not directly correlated. However, second, the mounting and detection method can also have a strong effect on the results. Using the embedding method described in here we could clearly distinguish individual neurons and study in different focal planes their possible content for ER
. Furthermore, the subcellular distribution could be clearly identified at its natural location while scanning through the section. However, in autoradiography, the signal is detected in the emulsion, which covers the section. Thus, the "co-localization" is examined by matching the immunohistochemically stained ChAT neurons, which are within the section (35 µm thick in Shughrue's paper), with the silver grains in the emulsion that is developed by the radioactive signal originating from the section and is localized on the top of but not in the section. Therefore, these two factors are spatially non-co-localized in strict sense and do not represent proper co-localization.
The antibody against ER used in the present study is raised against a peptide corresponding to an amino acid sequence at the C-terminus which is believed to be responsible for nuclear localization (
-ir: ER
-ir was located either in the nucleus or in the cytoplasm. At the current stage of knowledge it is difficult to express what is the direct functional importance of this observation. However, we have observed (unpublished observation) in female mice that the receptor location in cholinergic cells changes during the aging, having a decreased preference for nuclear localization at older ages. Because aged females have lower circulating estradiol levels, the cytoplasmically located ER
s may represent receptors that are free of ligand, because ER
s would translocate to the nucleus when estradiol is bound to it. Alternatively, it is also possible that the different distribution reflects the fact that there are functionally different estrogen receptors; nuclear receptors may mediate genomic effects and cytoplasmically located ER
s are involved in the non-genomic activities of estrogens (
s in distinct ChAT-containing neurons can also suggest about different functional status of distinct cholinergic neurons in respect to receptor trafficking.
In conclusion, we have described here critical caveats that can affect quantification and which should therefore be accounted for before material is processed for cell counting. In addition, the present study shows that even though the number of cholinergic cells does not vary greatly among different basal forebrain nuclei, their contents of ER differ from each other. This is likely to have fundamental significance to the proposed action of estrogens in these nuclei.
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Acknowledgments |
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Supported by EVO grant (5510) of the University Hospital of Kuopio, the European Commission (QLK6-CT-1999-02112), the National Technological Agency, and Hormos Medical, Ltd. (40414/00).
We thank Prof Asla Pitkänen for her constructive criticism and thoughtful suggestions on the manuscript, Dr Thomas Dunlop for revising the language, and AnnaLisa Gidlund for excellent technical assistance.
Received for publication February 25, 2002; accepted February 27, 2002.
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