Elsevier

Neuroscience Letters

Volume 738, 1 November 2020, 135386
Neuroscience Letters

Research article
Variations in GABA immunoreactivity among granule cells of the mouse olfactory bulb, as revealed by high-voltage electron microscopy

https://doi.org/10.1016/j.neulet.2020.135386Get rights and content

Highlights

  • Here we report the variation of GABA immunoreactivity among interneurons in mice.

  • We quantitatively analyzed the variation of immunoreactivity in interneurons.

  • The unique feature of this study is the use of high-voltage electron microscopy.

  • GABA immunoreactive intensity could be a novel method of cell classification.

Abstract

Odor information is processed in the olfactory bulb (OB), which is organized into olfactory inputs, interneurons, projection neurons, and centrifugal inputs, and these various structures regulate olfactory information processing. Similar to other brain regions, the OB structures include many types of interneurons, including γ-aminobutyric acid (GABA)ergic interneurons. Many interneurons are granule cells that are found in the granule cell layer (GCL), which is a deep layer of the OB. Interestingly, these interneurons exhibit variations in GABA immunoreactivity, and previous studies have observed differing intensities among morphologically and chemically similar neuronal populations. However, the numbers and distribution patterns of cells that show variations in GABA immunoreactivity are unknown. Therefore, we observed and quantitatively analyzed this diversity in the GCL of the mouse OB using immunogold, high-voltage electron microscopy, combined with light microscopy. Consequently, our results clearly show variations in the GABA immunoreactivity among GCL interneurons, which suggested heterogeneity in the amount of GABA present in each interneuron and reflected the possibility that different amounts of neuroactive substances may be associated with different functions for the various GABAergic interneuron groups. Variations in GABA immunoreactivity could be a novel criterion for classifying interneuron subpopulations.

Introduction

The olfactory bulb (OB) is the primary center of the olfactory system, where odor information is integrated and processed [15]. The mouse OB exhibits a distinct layered structure, defined by cellular distribution, comprised of the glomerular layer (GL), external plexiform layer, mitral cell layer, internal plexiform layer, and granule cell layer (GCL) [24]. The olfactory receptor neurons express odorant receptors, receive odor information, and project their axons to the olfactory glomeruli in the GL, providing sensory inputs to the OB. Then, the information is transmitted to higher brain centers, via projection mitral/tufted neurons, as output from the OB. In the OB, the transduction of information is regulated by both interneurons and afferent neurons [24]. Notably, more interneurons are present in the GL and GCL than in the other layers, and these neurons have been chemically and morphologically defined in previous studies [1,13,14,17]. In the GCL, 98%–99% of interneurons are granule cells [21,23], whereas the remaining 1%–2% of interneurons are short-axon cells [6,22,24]. These cell groups can be clearly distinguished using morphological analysis. For example, the cell body diameters of granule cells and short-axon cells are 6–8 μm and 10–20 μm, respectively. Previous studies have demonstrated at least five morphologically distinct subpopulations of granule cells in the rodent OB [9,[12], [13], [14],18,24]. The periglomerular cells found in the GL contain many subpopulations, which are characterized by the expression of different molecular markers. However, the interneurons in the GCL are less molecularly diverse. GABAergic interneurons represent the major population of granule cells in the GCL [19,20]. At the moment, GABA is the only transmitter known to be used by granule cells [19,20]. Furthermore, a morphological and physiological study showed that the short-axon cells in the GCL are also GABAergic neurons [6]. Interestingly, in our preliminarily studies, differences in the intensity of GABA immunoreactivity were observed among interneurons in the GCL. However, the numbers and distribution patterns of cells with varying levels of GABA immunoreactivity are not known. Hence, we aimed to quantitatively reveal this diversity.

In this study, we quantitatively analyzed variations in the GABA immunoreactivity across all types of GABAergic interneurons in the GCL of the mouse OB using immunogold, high-voltage electron microscopy (HVEM), combined with fluorescence, laser-scanning, light microscopy (LSM).

Section snippets

Animals

Eleven male C57BL/6 J mice (8–16 weeks old, 24–30 g), obtained from Japan SLC (Shizuoka, Japan), were used in this study. Mice were maintained in standard laboratory cages on a 12 -h light/dark cycle at a constant temperature (24 ± 1 °C). Food and water were available ad libitum. All animal experiments performed in the present study were approved by the Animal Research Committee of Kawasaki Medical School (approval# 19-012) and were conducted according to the “Guide for Care and Use of

Results

The immunohistochemical analysis showed the presence of GABA-immunoreactive interneurons in the OB (Fig. 1A). Variations in GABA immunoreactivity were observed among the interneurons in all layers of the mouse OB (Fig. 1A). In the GCL, high-magnification light microscopy (LM) demonstrated that several cells were closely aligned horizontally and formed “islet” structures [24] that contained between a few and 100 cells (Fig. 1B). Within the same islet, GABA immunoreactivity varied, with intense (

Discussion

In this study, we used LM to examine the diversity of GABA immunoreactivity among interneurons in the GCL of the mouse OB and quantitatively measured the immunoreactive intensity. Moreover, HVEM with gold particle labeling was used to accurately measure the variations in GABA immunoreactivity. Consequently, we were able to examine the variations in GABA immunoreactivity among GCL cells.

LSM showed continuous variations in GABA immunoreactivity, which prevented the cells from being grouped by

Conclusions

This study clearly showed GABA immunoreactivity variations among OB interneurons, using immunogold HVEM and LSM analyses. This diversity could represent a novel criterion for the classification of interneuron subpopulations in the OB. Furthermore, classifications according to GABA immunoreactivity variations may be applied to interneurons in other brain regions. In future studies, whether this diversity is correlated with interneuron subpopulations that have previously been characterized by

CRediT authorship contribution statement

Keita Satoh: Formal analysis, Data curation, Writing - original draft, Writing - review & editing. Emi Kiyokage: Validation, Writing - review & editing. Satoshi Ichikawa: Formal analysis, Data curation. Kazunori Toida: Project administration, Conceptualization, Methodology, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

We thank Dr. Kanako Inoue and Prof. Hidehiro Yasuda, Director of Research Center for Ultra-High Voltage Electron Microscopy, of Osaka University for electron microscopic observation and Dr. Sawa Horie and Ms. Rie Ohmori of the Department of Anatomy and Mr. Nobuyuki Matsuda and Ms. Kazuko Yamane of the Central Research Institute of Kawasaki Medical School for their technical assistance. We thank Lisa Kreiner, PhD, and Lisa Giles, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/ac

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