ACTA HISTOCHEMICA ET CYTOCHEMICA
Online ISSN : 1347-5800
Print ISSN : 0044-5991
ISSN-L : 0044-5991
REGULAR ARTICLE
Immunocytochemical Localization of Choline Acetyltransferase in the Microbat Visual Cortex
Gil-Hyun KimHang-Gu KimChang-Jin Jeon
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2018 Volume 51 Issue 5 Pages 153-165

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Abstract

The purpose of the present study was to investigate the organization of choline acetyltransferase (ChAT)-immunoreactive (IR) fibers in the visual cortex of the microbat, using standard immunocytochemistry and confocal microscopy. ChAT-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer III and the lowest density in layer I. However, no ChAT-IR cells were found in the microbat visual cortex. ChAT-IR fibers were classified into two types: small and large varicose fibers. Previously identified sources of cholinergic fibers in the mammalian visual cortex, the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis, all contained strongly labeled ChAT-IR cells in the microbat. The average diameter of ChAT-IR cells in the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis was 16.12 μm, 13.37 μm, and 13.90 μm, respectively. Our double-labeling study with ChAT and gamma-aminobutyric acid (GABA), and triple labeling with ChAT, GABA, and post synaptic density 95 (PSD-95), suggest that some ChAT-IR fibers make contact with GABAergic cells in the microbat visual cortex. Our results should provide a better understanding of the nocturnal bat visual system.

I.  Introduction

Bats are widely distributed around the world and are the second most common mammalian species after rodents. Bats are nocturnal mammals that have the capacity for flight [1, 18, 58, 65]. In general, bats are known to have degenerated eyes and to search for food or hunt primarily by using sophisticated laryngeal echolocation, that is, they acoustically perceive their environment by interpreting returning echoes of emitted sounds [12, 32, 39, 53, 57, 67, 68, 70]. Bats are generally divided into two suborders: Megachiroptera (Megabats, approximately 200 species, 40–220 cm wingspan), also known as fruit bats, which have large eyes and use vision to track wings and food, and Microchiroptera (Microbats, approximately 800 species, 22–135 cm wingspan), which are known to have small eyeballs and somewhat decreased visual acuity. It was discovered, however, that many microbats also depend on visual information for perception, predatism, and self-defense [3, 27, 69, 72, 75, 76]. Several studies have demonstrated the existence of photoreceptors, rod and cone bipolar cells, and subtypes of amacrine and retinal ganglion cells, in microbats [10, 17, 36, 37, 42, 54, 55, 59, 62], suggesting that the microbat might have functional organization for its visual system. The microbat visual cortex has also been used to study the cytoarchitecture of calcium-binding protein-containing neuronal populations [22, 41], and nitric oxide synthase-containing neurons [26].

Acetylcholine (ACh), one of the most important neurotransmitters in the nervous system, has been shown to be involved in various roles, such as learning and memory, arousal and attention, locomotor behavior, and neuronal plasticity [8, 9, 23, 25, 28, 63]. Cholinergic fibers have been found in the visual cortex of many mammals, including mice [43], rats [15, 48, 61], ferrets [30], cats [13, 66], monkeys [29], and humans [50]. However, only cholinergic neurons were found in the rat visual cortex [15, 61]. Cholinergic fibers, with abundant branches and varicosities, are distributed in all layers of the visual cortex. However, there are species differences in the higher density layers of cholinergic fibers in the visual cortex. For example, cats generally have dense cholinergic fibers in layer I, while in humans, cholinergic fibers are particularly intense in layers I, II, and the immediately adjacent rim of layer III. Cholinergic fiber synapses are predominantly symmetrical in the visual cortex, but a small number of asymmetries are observed [13, 61]. Cholinergic fibers are known to form synapses with GABAergic neurons, suggesting that acetylcholine indirectly inhibits visual information through GABAergic neurons [7, 16]. The visual cortex is known to receive cholinergic inputs from cholinergic neurons in the basal forebrain [6, 7, 19, 21, 51, 71, 74]. Among these, the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis are well-known sources of cholinergic fibers in the visual cortex [11, 31, 33, 43, 49, 64, 74].

We previously reported the organization of the cholinergic system in the microbat central visual system, including the retina [59] and superior colliculus [38]. However, the distribution and morphology of cholinergic fibers have not been investigated in the microbat visual cortex. As part of a larger effort in our laboratory to localize cholinergic cells/fibers in the entire bat visual system [38, 59], the primary aim of the present study was to investigate the organization of the cholinergic system in the microbat visual cortex and compare it with other mammals, to understand the extent of species diversity. We also studied sources of cholinergic input into the visual cortex to determine whether the nocturnal microbat has a visual system similar to other predominantly diurnal mammalian species. Finally, we studied whether cholinergic fibers make contact with GABAergic interneurons. Our results contribute to a better understanding of the visual system of the nocturnal microbat.

II.  Materials and Methods

Animal and tissue preparation

For this study, 12 freshly caught adult bats (Rhinolophus ferrumequinum, both sexes: 15–20 g) were used. All bats were captured in a cave in the district of Gimcheon, South Korea. After 5–6 hr to transport and stabilize the bats, they were anesthetized with a mixture of ketamine hydrochloride (30–40 mg/kg) and xylazine (3–6 mg/kg) prior to perfusion. The bats were perfused intracardially with 4% paraformaldehyde and 0.3–0.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.002% calcium chloride. Following a pre-rinse with approximately 30 ml of phosphate-buffered saline (PBS, pH 7.4) over a period of 3–5 min, each bat was perfused with 30–50 ml of fixative for 20–30 min via a syringe needle inserted through the left ventricle and aorta. The animal was decapitated and the head was placed in a fixative for 2–3 hr. The brain was then removed from the skull, stored for 2–3 hr in the same fixative and left overnight in 0.1 M phosphate buffer (pH 7.4) containing 8% sucrose and 0.002% calcium chloride. The brain was then mounted onto a chuck and cut into 50-μm thick coronal sections with a Vibratome 3000 Plus Sectioning System (Vibratome, St. Louis, MO, USA). All animal experiments were approved by the committee of Kyungpook National University (permission NO. 2016-0151). “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) were followed.

Horseradish peroxidase staining

The primary antibody used in this study was goat anti-choline acetyltransferase (ChAT; Millipore, Bedford, MA, USA). Tissues were processed free-floating in small vials at room temperature with gentle agitation. For immunocytochemistry, tissues were incubated in 1% sodium borohydride (NaBH4) for 30 min. Afterwards, these tissues were rinsed for 3 × 10 min in PBS, and incubated in PBS with 4% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 2 hr with 0.5% Triton X-100 added. The tissues were then incubated in the primary antiserum in PBS, with 4% normal serum for 24 hr, with 0.5% Triton X-100 added. The primary antiserum was diluted 1:200. Following three 10-min rinses in PBS, the tissues were incubated in a 1:200 dilution of biotinylated secondary IgG in a blocking solution. The secondary antibody used was bio­tinylated horse anti-goat (Vector Labs.). The tissues were then rinsed for 3 × 10 min in PBS and incubated in a 1:50 dilution of avidin-biotinylated horseradish peroxidase complex (Vector Labs.) in PBS for 2 hr. Then the tissues were rinsed in 0.25 M Tris buffer for 3 × 10 min. Finally, the staining was visualized by reacting with 1,3'-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide in 0.25 M Tris buffer for 3–10 min using a DAB reagent set (Kirkegaard & Perry, Gaithersburg, MD, USA). All tissues were then rinsed in 0.25 M Tris buffer before mounting. As a negative control, some sections were incubated in the same solution without the addition of the primary antibody, and these control tissues showed no ChAT immunoreactivity. Negative control and preabsorption tests for the specificity of this antibody conducted in the central nervous system of the microbat have been described in previous reports [38, 44]. Following the immunocytochemical procedures, the tissues were mounted on Superfrost Plus slides (Fisher, Pittsburgh, PA, USA) and dried overnight in a 37°C oven. The mounted sections were dehydrated, cleared, and coverslipped with Permount (Fisher).

Fluorescence immunohistochemistry

Standard triple-labeling immunohistochemical tech­niques and methods were used, as described previously [56]. The primary antibodies used for triple-labeling im­munofluorescence were goat anti-ChAT (Millipore), mouse anti-GABA (Chemicon International Inc. Temecula, CA, USA), and rabbit anti-PSD-95 (Abcam, Cambridge, UK). Fluorescein-conjugated donkey anti-goat (Millipore), Cy3-conjucated donkey anti-mouse (Millipore), and Alexa fluor 674-conjucated donkey anti-rabbit (Millipore) were used as the secondary antibody. Stained sections were mounted on Superfrost Plus slides and coverslipped with Vectashield mounting medium (Vector Labs.). Immunofluorescence images were obtained using a laser-scanning confocal micro­scope (LSM 700; Carl Zeiss Meditec Inc. Jena, Germany).

Quantitative analysis

ChAT-IR fibers/cells were examined and photographed in the best-labeled sections from each of the three animals with a Zeiss Axioplan microscope, using conventional or differential interference contrast (DIC) optics. In addition, the average diameter/area of these cells were computed using an Axiocam HRc digital camera (Carl Zeiss Meditec Inc.). Analysis was performed using Zeiss AxioVision with Zeiss Plan-Apochromat objectives. To obtain the best image, bright and dark field microscopy was also used to identify the distribution pattern of ChAT-IR fibers, and only ChAT-IR cells containing a nucleus with a nucleolus that was at least faintly visible were included in the analysis. In addition, a standard for the fiber type was set up using the diameter of varicosity (small varicose fiber <0.5 μm, large varicose fiber >0.5 μm) [35, 38]. Images were adjusted with respect to brightness and contrast using Adobe Photoshop CS software (Adobe Systems Inc., San Jose, CA, USA). To quantify the density of IR fibers, we sampled sequential fields (total of nine tissue sections from three animals and three tissue sections from each animal), each 21 μm × 21 μm, across the superficial and deeper layers of the visual cortex. Lines indicating the IR fibers were generated with Adobe Photoshop. In order to quantify the laminar distribution of small and large immunoreactive varicosities (total of nine tissue sections from three animals and three tissue sections from each animal), we used a counting grid in the ocular of the microscope, which subtended 50 μm × 40 μm. We counted the number of varicosities within the grid in each layer. The grid was located in the middle of each layer and oriented perpendicular to the cortical surface.

Synaptic identification

The triple-labeling of ChAT-IR fibers, GABA, and PSD-95 were imaged using a laser-scanning confocal microscope module (Carl Zeiss Meditec Inc.) mounted on a fluorescence microscope. At the contact area of the ChAT fibers and GABA cells, the z-series of ChAT-IR fibers, GABA cell, and PSD-95 images were taken at 0.2 μm interval along the z-axis using a laser-scanning confocal microscope. The images were viewed using an EC Plan-Neofluar 10×, C-Apochromat 40×/1.2 W, and/or 100×/1.2 oil, objectives at 2.5× zoom. We obtained approximately 70–80 confocal images at the presumed synaptic contact. The z-series of the confocal images were reconstructed as three-dimensional (3D) images using the ZEN 2.3 (blue edition) program (Carl Zeiss Meditec Inc.). The 3D images and orthogonal views (xy, xz, and yz axes) were used to find points where the ChAT-IR fibers and PSD-95 made contact with the GABAergic cell.

III.  Results

The distribution of ChAT-IR fibers in the microbat visual cortex

The laminar structure of the microbat visual cortex and the ChAT-immunoreactivity are shown in Figures 1A and 1B, respectively. These images demonstrate that the microbat visual cortex has well-developed cholinergic fibers. ChAT-IR fibers were distributed throughout all layers of the microbat visual cortex. Figures 1C and 1D show highly magnified regions in the square areas of Figure 1B. Figure 1B is dark field figure, captured with a 20× objective, while Figure 1C, including ChAT-IR fibers located within layers I–III, and Figure 1D, showing ChAT-IR fibers located within layers IV–VI, are bright field figures, captured with a 40× objective. A drawing of the ChAT-IR fibers demonstrating their density is shown in Figure 1E. The cholinergic fibers in the microbat visual cortex reveal a plexus of labeled fibers with differential densities in the different layers. Quantitative maps of fiber distribution revealed the density of the ChAT-IR fibers in each layer (Fig. 2). Figure 2 also depicts the laminar distribution pattern of ChAT-IR fibers in the microbat visual cortex analyzed using the image processing program developed at the National Institute of Health. Three different sections taken from each of three different animals (a total of nine sections, each in a different color in Fig. 2C) were analyzed in the present study. Figure 2C shows a density histogram of ChAT-IR fibers in the microbat visual cortex. The frequency of labeled fibers varied in each layer. Thus, 9.95% of the labeled fibers appeared in layer I, 17.06% in layer II, 22.99% in layer III, 18.24% in layer IV, 17.24% in layer V, and 14.51% in layer VI. The highest density of ChAT-IR fibers occurred in layer III, while the lowest density of ChAT-IR fibers was located in layer I (Fig. 2B and 2C).

Fig. 1.

Distribution of ChAT-IR fibers in the microbat visual cortex. (A) The approximate limits of layers are indicated in the thionin-stained section. (B) Low-magnification dark field photomicrograph of ChAT-IR fibers in the microbat visual cortex. (C, D) Medium magnification bright field photomicrograph of ChAT-IR fibers showing the areas marked in (B). Varicose cholinergic fibers generally observed in the microbat visual cortex. (E) Drawing of the ChAT-IR fibers in the microbat visual cortex. Bar = 50 μm.

Fig. 2.

Intensity profile of ChAT-IR fibers in the visual cortex of the microbat. In different coronal sections of the microbat visual cortex, a plot of the intensity of the ChAT-IR fibers characterized by the image processing program Image J developed at the National Institutes of Health. (A) Low-magnification dark field photomicrograph of ChAT-IR fibers in the microbat visual cortex shows well-labeled ChAT-IR fibers. (B) Results of intensity graph analyses from nine coronal sections of the microbat visual cortex are shown. (C) Density histogram of ChAT-IR fibers in the microbat visual cortex. The highest density of ChAT-IR fibers was located in layer III of the microbat visual cortex. Bar = 50 μm.

The morphology of ChAT-IR fibers in the microbat visual cortex

We identified at least two types of ChAT-IR varicose fibers in the microbat visual cortex. The first type of ChAT-IR varicose fibers was small varicose fibers (arrowheads in Fig. 3C and 3D), and the second type was large varicose fibers (arrows in Fig. 3A, 3B, and 3D). Varicose fibers were distributed throughout the rostrocaudal section of the microbat visual cortex and numerous ChAT-IR varicosities were distributed irregularly along the fibers. ChAT-IR fibers in the microbat visual cortex exhibited various orientations, including vertical, horizontal, and oblique orientations. Some ChAT-IR fibers surrounded unlabeled somata in the microbat visual cortex (indicated by asterisks) (Fig. 3E and 3F). The microbat visual cortex does not appear to have cholinergic neurons; no labeled neurons were found in the present study.

Fig. 3.

High-power differential interference contrast (DIC) photomicrographs of ChAT-IR fibers in the microbat visual cortex. (A, B) Arrows indicate large varicosities in the microbat visual cortex. (C) Arrowheads indicate small varicosities in the microbat visual cortex. (D) ChAT-IR fibers with small (arrowheads) and large (arrows) varicosities in the microbat visual cortex. (E, F) Some ChAT-IR fibers surrounded unlabeled cells in the microbat visual cortex (asterisks in E, F). Bar = 10 μm.

Figure 4 and Table 1 show the distributional difference of small and large varicosities in the microbat visual cortex. The distribution of large varicosities in each layer was as follows: 37.44 ± 9.28 (16.72%; mean ± standard deviation [SD]) in layer I, 36.67 ± 6.69 (16.37%; mean ± SD) in layer II, 43.33 ± 3.54 (19.34%; mean ± SD) in layer III, 35.33 ± 4.47 (15.77%; mean ± SD) in layer IV, 33.00 ± 4.87 (14.73%; mean ± SD) in layer V, and 38.22 ± 7.97 (17.06%; mean ± SD) in layer VI. Although the density of the large varicosities was not highly different in each layer, the highest density of large varicosities occurred in layer III. The distribution of small varicosities in each layer was as follows: 154.22 ± 27.97 (15.05%; mean ± SD) in layer I, 166.56 ± 20.70 (16.21%; mean ± SD) in layer II, 187.44 ± 33.29 (18.25%; mean ± SD) in layer III, 172.56 ± 41.80 (16.80%; mean ± SD) in layer IV, 169.56 ± 38.30 (16.50%; mean ± SD) in layer V, and 177.00 ± 44.08 (17.23%; mean ± SD) in layer VI. The highest density of small varicosities also occurred in layer III. Small varicosities were 4.59 times more than large varicosities in the microbat visual cortex.

Fig. 4.

Histogram of the distribution of two types of varicosities in the microbat visual cortex. Although there were not many differences, the layer III contained higher number of both large and small varicosities than other layers. In each layer, there were more small varicosities than large ones. The microbat visual cortex contained 4.59 times more small varicosities than large ones.

Table 1.  Distribution of varicosities at different layers in three visual cortexes
Cortical layers
I II III IV V VI
Small varicosities
Visual cortex 1
Number of varicosities 152 149 185 195 186 184
Density (varicosity/mm2) 76167 74333 92500 97500 92833 92167
Visual cortex 2
Number of varicosities 143 173 169 140 141 143
Density (varicosity/mm2) 71500 86667 84333 70000 70333 71500
Visual cortex 3
Number of varicosities 167 178 209 183 182 204
Density (varicosity/mm2) 83667 88833 104333 91333 91167 101833
Total number (mean ± SD) 154.22 ± 27.97 166.56 ± 20.70 187.44 ± 33.29 172.56 ± 41.80 169.56 ± 38.30 177.00 ± 44.08
Density (mean ± SD) 77111.11 ± 13986.10 83277.80 ± 10350.48 93722.20 ± 16647.30 86277.80 ± 20904.70 84777.80 ± 19149.30 88500.00 ± 22044.00
Percentage 15.01 ± 2.75 16.21 ± 2.03 18.25 ± 0.91 16.80 ± 1.41 16.50 ± 1.64 17.23 ± 2.32
Large varicosities
Visual cortex 1
Number of varicosities 42 33 41 37 34 39
Density (varicosity/mm2) 20833 16667 20667 18500 16833 19500
Visual cortex 2
Number of varicosities 32 37 43 34 31 37
Density (varicosity/mm2) 16167 18667 21833 17333 15667 18667
Visual cortex 3
Number of varicosities 38 39 45 34 34 38
Density (varicosity/mm2) 19167 19667 22500 17167 17000 19167
Total number (mean ± SD) 37.44 ± 9.28 36.67 ± 6.69 43.33 ± 3.54 35.33 ± 4.47 33.00 ± 4.87 38.22 ± 7.97
Density (mean ± SD) 18722.20 ± 4637.56 18333.30 ± 3344.77 21666.70 ± 2236.07 17666.70 ± 2236.07 16500.00 ± 2436.70 19111.10 ± 3982.60
Percentage 16.72 ± 2.02 16.37 ± 1.38 19.35 ± 1.97 15.77 ± 2.69 14.73 ± 2.51 17.06 ± 2.76

SD, standard deviation. * Number of varicosities = mean number from three tissue sections.

The sources of ChAT-IR fibers in the microbat visual cortex

There are several subdivisions of cholinergic neurons in the central nervous system. The predominant groups of cholinergic neurons observed in the brain are found in the striatum, basal forebrain, diencephalon, pons, and cranial nerve nuclei [4, 47, 73, 74]. The nucleus basalis magno­cellularis, the substantia innominata, and the nucleus of the diagonal band are known to project cholinergic fibers into the visual cortex [11, 31, 33, 43, 49, 64, 74].

1)  Nucleus of diagonal band

ChAT-IR neurons were distributed in the horizontal diagonal band of the bat brain (Fig. 5A and 5B) [14, 44, 47]. The labeling of ChAT-IR neurons was intense in various types of neurons in the horizontal diagonal band. The large majority of ChAT-IR neurons were round/oval cells (Fig. 6A), although other cell types were also observed, including pyriform (Fig. 6B) and horizontal cells (Fig. 6C). The average diameter of 86 ChAT-IR neurons in the horizontal diagonal band ranged from 11.8 to 20.21 μm, with a mean of 16.13 μm (SD = 2.00 μm). The area of these cells ranged from 109.31 to 320.8 μm2, with a mean of 207.46 μm2 (SD = 50.05 μm2) (Fig. 7 and Table 2).

Fig. 5.

Photomicrographs showing presumed sources of ChAT-IR fibers in the microbat visual cortex. (A) Low-magnification photomicrograph of coronal sections showing the nucleus of the diagonal band. (B) High-magnification photomicrograph of image in the square marked in (A). (C) Low magnification photomicrograph of a coronal section showing the substantia innominata. (D) High magnification photomicrograph of image in the square marked in (C). (E) Low-magnification photomicrograph of a coronal section showing the nucleus basalis magnocellularis. (F) High-magnification photomicrograph of image in the square marked in (E). LPO, lateral preoptic area; NDB, nucleus of the diagonal band; VP, ventral pallidum; SI, substantia innominata; GP, Globus pallidus; NBM, nucleus basalis magnocellularis. Bar = 200 μm (A, C, E), and 50 μm (B, D, F).

Fig. 6.

High-power differential interference contrast (DIC) photomicrographs of major ChAT-IR cell types in the nucleus of the diagonal band, substantia innominata and the nucleus basalis magnocellularis of microbat. (A) A multipolar round cell with several processes in various directions. (B) A pyriform cell. (C) A horizontal fusiform cell with a horizontal fusiform cell body and horizontally oriented processes. (D) A multipolar round cell. (E) A vertical fusiform cell with a vertical fusiform cell body and vertically oriented processes. (F) A large multipolar stellate cell. (G) A multipolar round cell. (H) A vertical fusiform cell with a vertical fusiform cell body and vertically oriented processes. (I) A horizontal fusiform cell with a horizontal fusiform cell body and horizontally oriented processes. NDB, nucleus of the diagonal band; SI, substantia innominata; NBM, nucleus basalis magnocellularis. Bar = 5 μm.

Fig. 7.

Histogram of ChAT-IR cell area and diameter in the microbat brain. The diameter of 86 ChAT-IR neurons in the horizontal diagonal band ranged from 11.8 to 20.21 μm, with a mean of 16.13 μm (standard deviation, S.D. = 2.00 μm). The area of these cells ranged from 109.31 to 320.8 μm2, with a mean of 207.46 μm2 (S.D. = 50.05 μm2). The average diameter of 98 ChAT-IR neurons in the substantia innominata ranged from 8.33 to 19.83 μm, with a mean of 13.37 μm (S.D. = 2.00 μm). The area of these cells ranged from 54.45 to 308.91 μm2, with a mean of 143.84 μm2 (S.D. = 45 μm2). The diameter of 90 ChAT-IR neurons in the nucleus basalis magnocellularis ranged from 11.77 to 18.08 μm, with a mean of 13.90 μm (S.D. = 1.78 μm). The area of these cells ranged from 108.72 to 252.68 μm2, with a mean of 153.46 μm2 (S.D. = 38.81 μm2). The best-stained cells were measured. NDB, nucleus of the diagonal band; SI, substantia innominata; NBM, nucleus basalis magnocellularis.

Table 2.  Quantitative data of choline acetyltransferase-immunoreactive cells in the microbat nucleus of the diagonal band, substantia innominata, and nucleus basalis magnocellularis
Animal number Number of ChAT-IR cells Diameter ± SD (μm) Area ± SD (μm2)
Nucleus of the diagonal band 1 31 15.08 ± 1.54 180.45 ± 36.06
2 34 16.49 ± 1.92 216.41 ± 48.89
3 21 16.81 ± 2.18 225.53 ± 56.16
Total/Average 86 16.12 ± 2.00 207.46 ± 50.05
Substantia innominata 1 35 12.41 ± 1.67 123.13 ± 33.64
2 35 13.16 ± 1.95 138.84 ± 40.34
3 28 14.54 ± 2.11 169.54 ± 50.41
Total/Average 98 13.37 ± 2.00 143.84 ± 45.00
Nucleus basalis magnocellularis 1 31 13.50 ± 2.02 146.12 ± 43.29
2 31 14.23 ± 1.59 161.19 ± 35.13
3 28 13.95 ± 1.67 153.06 ± 37.15
Total/Average 90 13.90 ± 1.78 153.46 ± 38.81

ChAT-IR, choline acetyltransferase-immunoreactive; SD, standard deviation.

2)  Substantia innominata

ChAT-IR neurons appeared in various shapes in the substantia innominata (Fig. 5C and 5D). The major type of neurons found in the substantia innominata were medium sized round/oval cells (Fig. 6D). However, many vertical fusiform cells (Fig. 6E) and stellate cells (Fig. 6F) were also IR for ChAT. The average diameter of 98 ChAT-IR neurons in the substantia innominata ranged from 8.33 to 19.83 μm, with a mean of 13.37 μm (SD = 2.00 μm). The area of these cells ranged from 54.45 to 308.91 μm2 with a mean of 143.84 μm2 (SD = 45 μm2) (Fig. 7 and Table 2).

3)  Nucleus basalis magnocellularis

ChAT-IR neurons were intense among various types of cells in the nucleus basalis magnocellularis (Fig. 5E and 5F). The large majority of ChAT-IR neurons were round/oval (Fig. 6G), vertical fusiform (Fig. 6H), and horizontal cells (Fig. 6I), although other cell types were also observed, including stellate and pyriform cells. The average diameter of 90 ChAT-IR neurons in the nucleus basalis magnocellularis ranged from 11.77 to 18.08 μm, with a mean of 13.90 μm (SD = 1.78 μm). The area of these cells ranged from 108.72 to 252.68 μm2, with a mean of 153.46 μm2 (SD = 38.81 μm2) (Fig. 7 and Table 2).

The distribution of ChAT-IR fibers and CB-, CR-, PV-, or GABA-IR cells

Among the many calcium-binding proteins (CBP), calbindin-D28K (CB), calretinin (CR), and parvalbumin (PV) are important markers for specific neuronal subpopulations in the central nervous system [2, 5]. CB, CR, and PV are localized in the visual cortex of various mammals [20, 34, 46, 60]. In a previous study, we showed that many round/oval and stellate cells in the microbat visual cortex contained CB, CR, or PV, and many of these cells also contained GABA [41]. In the present study, some ChAT-IR fibers surrounded unlabeled somata in the microbat visual cortex (asterisks, Fig. 3E and 3F). Figure 8 shows that CB-, CR-, PV- or GABA-IR appears in some round/oval (arrowheads) or stellate cells (arrows) in the microbat visual cortex. However, we did not detect any CB-, CR-, PV-, or GABA-IR cells surrounded by ChAT-IR fibers (asterisks).

Fig. 8.

Double labeling of ChAT-IR fibers with CB-, CR-, PV- or GABA-IR cells in the microbat visual cortex. (A) Merged image of fluorescence-reacted ChAT-IR fibers and CB-IR cells. (B) Merged image of fluorescence-reacted ChAT-IR fibers and CR-IR cells. (C) Merged image of fluorescence-reacted ChAT-IR fibers and PV-IR cells. (D) Merged image of fluorescence-reacted ChAT-IR fibers and GABA-IR cells. Some round/oval (arrowheads) cells in B, C, D, or stellate cells (arrows) in A. However, we did not detect CB-, CR-, PV-, or GABA-IR cells surrounded by the ChAT-IR fibers (asterisks). ChAT, Choline acetyltransferase; CB, calbindin-D28K; CR, calretinin; PV, parvalbumin; GABA, gamma-aminobutyric acid. Bar = 20 μm.

Triple-labeling of ChAT-IR fibers, PSD-95 and GABA

In the present study, we performed triple labeling of ChAT-IR fibers, PSD-95, and GABA, in order to identify the synaptic contacts of ChAT-IR fibers with GABA-IR cells. Figure 9 shows the distribution of ChAT-IR fibers (Fig. 9A, green), GABA (Fig. 9B, red), PSD-95 (Fig. 9C, blue), and a merged image (Fig. 9D), in the microbat visual cortex. The crosshair reveals the colocalization of ChAT-IR fibers and PSD-95 with the GABA-IR cells, in an orthogonal projection. The areas marked by white squares in Figures 9A–D are displayed at higher magnification in Figures 9E–P. Figure 9E, 9I, and 9M show merged images of fluorescence ChAT-IR fibers and GABA-IR cells in the xy, xz, and yz planes, respectively. With this method, we identified that ChAT-IR fibers coincided with GABA-IR cells. Figures 9F, 9J, and 9N show merged images of fluorescence ChAT-IR fibers and PSD-95-IR puncta in the xy, xz, and yz planes, respectively. These figures show that ChAT-IR fibers coincided with PSD-95 puncta. Figures 9G, 9K, and 9O show merged images of fluorescence GABA-IR cells and PSD-95 puncta in xy, xz, and yz planes, respectively. These figures demonstrate the coincidence of GABA-IR cells with PSD-95 puncta. Figures 9H, 9L, and 9P show merged images of fluorescence ChAT-IR fibers, PSD-95, and GABA-IR cells. The merged images show that ChAT-IR fibers make contact with GABA-IR cell in the microbat visual cortex.

Fig. 9.

Triple-labeled image of ChAT-IR fibers, GABA and PSD-95 in the microbat visual cortex. (A) Orthogonal view of a confocal z-stack image of the ChAT-IR fibers (green). (B) Orthogonal view of a confocal z-stack image of the GABA-IR cells (red). (C) Orthogonal view of a confocal z-stack image of PSD-95 IR (blue). (D) Orthogonal view of a confocal z-stack image of the merged images. (E–P) High-power images in crosshair from the xy, xz, and yz planes, respectively. (E, I, M) Merged images of fluorescence ChAT-IR fibers and PSD-95 puncta in the xy, xz, and yz planes, respectively. (F, J, N) Merged images of fluorescence ChAT-IR fibers and GABA-IR cell in the xy, xz, and yz planes, respectively. (G, K, O) Merged images of fluorescence GABA-IR cell and PSD-95 puncta in the xy, xz, and yz planes, respectively. (H, L, P) Merged images of fluorescence ChAT-IR fibers, PSD-95, and GABA-IR cell in the xy, xz, and yz planes, respectively. ChAT, Choline acetyltransferase; GABA, gamma-aminobutyric acid. Bar = 10 μm (A–D); 1 μm (E–P).

IV.  Discussion

This study has demonstrated that ChAT-IR fibers are distributed in the microbat visual cortex. However, the distribution pattern of these fibers is rather different from that found in previous studies on the visual cortex in other mammals. We identified at least two types of ChAT-IR varicose fibers in the present study. ChAT-IR cells in the basal forebrain areas that are thought to be the source of ChAT-IR in the visual cortex were also identified. Some of the ChAT-IR fibers contacted GABAergic neurons.

Cholinergic fibers have been found in the visual cortex of numerous mammals, including mice [43], rats [15, 48, 61], ferrets [30], cats [13, 66], monkeys [29], and humans [50]. However, there are species differences in the distribution pattern of cholinergic fibers in the visual cortex among the various animals. For example, dense cholinergic fibers are distributed in layer IV of the mouse visual cortex [43], while dense cholinergic fibers are found in layer IV of male Long-Evans rats’ [15] or in layer V of Sprague-Dawley albino rats’ [61] visual cortex. A low density of cholinergic fibers is found in layer IV of the ferret visual cortex [30], while cholinergic fibers are particularly dense in layer I of the cat visual cortex [13, 66]. In the monkey visual cortex, dense cholinergic fibers are distributed in layers I and IV [29]. In the human visual cortex, cholinergic fibers are particularly intense in layers I, II, and the immediately adjacent rim of layer III [50]. In contrast, in the present study, the highest density of cholinergic fibers was distributed in layer III, while the lowest density of cholinergic fibers was located in layer I in the microbat visual cortex. Thus, the combined results of the previous and the present studies indicate that different animals possess different density of cholinergic fibers among the six layers of the visual cortex. The functional significance of inter-species differences in the density distribution of cholinergic fibers in the visual cortex of mammals remains unknown.

In the present study, ChAT-IR neurons were not found in the microbat visual cortex. This result is in accordance with the majority of previous results from other mammalian visual cortex studies. Thus, no ChAT-IR neurons were found in the visual cortex of mice [43], ferrets [30], cats [13, 66], or humans [50]. In contrast, however, the rat visu­al cortex was found to contain ChAT-IR neurons [15, 48, 61]. ChAT-IR neurons were observed in layers II-VI of the rat visual cortex. Most of these neurons were either round or oval in shape and small in size. The large majority of ChAT-IR neurons were bipolar cells [15, 48, 61]. However, the functional role of cholinergic cells in the rat visual cortex has not yet been described.

Several studies have shown that cholinergic fiber inputs to the neocortex initiate primarily from cholinergic neurons of the basal forebrain [6, 7, 19, 21, 51, 71, 74]. In the forebrain, the nucleus basalis magnocellularis, substantia innominata, and the nucleus of the diagonal band are known to project cholinergic fibers to the visual cortex [11, 31, 33, 43, 49, 64, 74]. In previous studies in bats, ChAT immunoreactivity was found in the basal forebrain areas [14, 44, 47]. In the present study, in the nucleus of the diagonal band, soma diameters varied from 11.8–20.21 μm (mean diameter 16.12 μm) and the predominant type of ChAT-IR cell was round or oval. In the substantia innominata, soma diameters varied from 8.33–19.83 μm (mean diameter 13.37 μm) and the predominant type of ChAT-IR cell was round or oval. In the nucleus basalis magnocellu­laris, soma diameters varied from 9.42–18.08 μm (mean diameter 13.90 μm) and the predominant type of ChAT-IR cell was also round or oval in shape. These results suggest that, although the cholinergic inputs can be confirmed by retrograde tracer injection, cholinergic round/oval cells of various sizes project to the microbat visual cortex, giving rise to different sizes of cholinergic fibers in microbat visual cortex.

In the present study, ChAT-IR varicosities were found throughout the microbat visual cortex. Varicose cholinergic fibers are a common characteristic found in many other mammals. For instance, varicose cholinergic fibers were found in mice [43], rats [15, 48, 61], ferrets [30], cats [13, 66], and humans [50]. In the present study, we found at least two different types of fibers, small and large varicose fibers, in the microbat visual cortex. Two types of ChAT-IR synaptic profiles were observed in the cat superior colliculus using electron microscopy. Small-diameter fibers with few varicosities contained predominantly round synaptic vesicles and formed asymmetric synaptic contacts, while large-diameter fibers with many varicosities also contained round synaptic vesicles and formed symmetric synaptic contacts. These two types of fibers were found only in the superficial layers of the cat superior colliculus and the large fibers were not found in the deeper layers of the superior colliculus [35]. Two types of ChAT-fibers were also found in the microbat superior colliculus [38]. In the microbat superior colliculus, however, the two different types of fibers were found in both superficial and deeper layers. Electron microscopy studies confirmed that the ChAT-IR varicose profiles in the visual cortex contained round synaptic vesicles forming symmetric and asymmetric synaptic complexes in cat [13], and rat [61]. These results suggest that synapses can be made in the varicose fibers of the microbat visual cortex. However, electron microscopy studies will be required to confirm the synaptic profiles of the ChAT-IR fibers in the bat visual cortex.

Previous studies have shown that GABAergic neurons receive more ChAT-IR fiber input than non-GABAergic neurons and most cholinergic boutons formed synapses with dendritic shafts of GABAergic neurons in the cat visual cortex [7]. Beaulieu and Somogyi [7] suggested that “The activation of GABAergic neurons by cholinergic afferents may increase the response specificity of cortical cells during cortical arousal thought to be mediated by the basal forebrain”. The inhibitory effect of cholinergic fibers is thought to be indirectly mediated by activation of GABAergic neurons in the visual cortex [7]. Muscarinic acetylcholine receptors were found in the GABAergic neurons in the cat visual cortex [16]. In the present study, we found that cholinergic fibers make contact with GABAergic neurons in the microbat visual cortex. This suggests that cholinergic fibers in the visual cortex of microbats may also have an indirect inhibitory effect through muscarinic acetylcholine receptors on GABAergic neurons. However, additional physiological and pharmacological studies will be required to identify the functional role of acetylcholine in the visual cortex of microbats.

The cholinergic system is known to play a key role in the central nervous system. Behavioral and physiological regulation including sensory processing, learning, memory, neural plasticity, mood, sleep, arousal, biorhythms, and attention, are regulated by acetylcholine [8, 23, 25, 28, 63]. In the visual cortex, ACh has also been implicated in neural plasticity, attention and learning. For example, ACh plays a role in facilitation of perceptual learning via attention [40] and in regulation of learning to discriminate fine dif­ferences in the temporal stimuli [52]. ACh contributes to experience-dependent synaptic plasticity in visual cortex [25]. In addition, ACh plays a role in regulating the strength of the thalamo-cortical inputs leading to the selection and/or discrimination of visual stimuli [45]. A recent study showed that ACh also participates in improvement of visuospatial perception and processing of visual targets among distractors [24]. Thus, our results show a well-organized cholinergic system in the microbat visual cortex, suggesting that cholinergic fibers in the microbat visual cortex may play a role in synaptic plasticity, attention, and arousal, and improve the signal-to-noise ratio of cortical neurons during visual information processing [24, 25, 40, 45, 52]. However, determining the detailed functional role of cholinergic fibers in the microbat visual cortex requires electrophysiological studies.

In conclusion, we found well-organized ChAT-IR fibers in the microbat visual cortex, with the highest density in layer III. However, ChAT-IR cells were not observed in the microbat visual cortex. The nucleus of the diagonal band, substantia innominata, and nucleus basalis magnocellularis, which are known as sources of cholinergic fibers in the mammalian visual cortex, contained many well-labelled ChAT-IR neurons in the microbat visual cortex. The study also confirmed that some ChAT-IR fibers were in contact with GABAergic neurons. Our study provides important in­formation contributing to better understanding of the visual system of the nocturnal mammal microbat, which has previously been thought to have an atrophied visual system.

V.  Conflicts of Interest

The authors declare that there are no conflicts of interest.

VI.  Acknowledgment

We thank Cactus Communications for proofreading the manuscript. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF2016R1D1A1A09918427).

VII. References
 
© 2018 The Japan Society of Histochemistry and Cytochemistry
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