Sodium channel cluster, βIV-spectrin and ankyrinG positive “hot spots” on dendritic segments of parvalbumin-containing neurons and some other neurons in the mouse and rat main olfactory bulbs
Introduction
Axon initial segments (AISs) and nodes of Ranvier are considered as the sites for action potential (AP) or spike generation, which are also known to be highly enriched in voltage-gated sodium channels and some cytoskeletal/scaffolding molecules such as ankyrinG, βIV-spectrin as well as multiprotein complexes of cell adhesion molecules (Hedstrom and Rasband, 2006). In our previous study (Kosaka and Kosaka, 2008a) we analyzed the structural features of parvalbumin (PV) positive intrinsic neurons in the mouse main olfactory bulb (MOB). In that study we revealed that most PV positive neurons in the external plexiform layer (EPL) did not have typical axons but they had some particular segments displaying some of characteristic chemical markers of conventional AISs, βIV-spectrin (Berghs et al., 2000, Komada and Soriano, 2002, Uemoto et al., 2007) and sodium channel clusters, on their dendritic processes. Then several issues related with βIV-spectrin/sodium channel cluster positive dendritic segments of the PV positive EPL neurons were raised; among them (1) whether or not some proteins suggested to play some central roles in organizing the molecule assembly at the AISs and nodes of Ranvier such as ankyrinG (Kordeli et al., 1995, Zhou et al., 1998, Jenkins and Bennett, 2001, Hedstrom and Rasband, 2006, Pan et al., 2006, Yang et al., 2007) and phospho-IκBα (Schultz et al., 2006, Politi et al., 2008, Sanchez-Ponce et al., 2008) are also concentrated at these βIV-spectrin/sodium channel cluster positive dendritic segments of the PV positive EPL neurons, (2) whether such particular dendritic patches display some ultrastructural features common to the AISs and/or nodes of Ranvier (Peters et al., 1991), (3) whether such particular dendritic patches distribute throughout the dendritic trees or are limited to some parts of the dendritic trees, (4) whether or not these sites are specific to PV positive EPL neurons and (5) whether or not similar βIV-spectrin/sodium channel cluster positive segments are localized on the PV positive EPL neurons in the rat MOB.
In the present study we addressed these five issues. We revealed that ankyrinG was concentrated on the βIV-spectrin positive segments of the dendritic processes of PV positive EPL neurons. These dendritic patches also displayed the ultrastructural features characteristic to the AISs and nodes of Ranvier, membrane undercoating. These results revealed that the particular dendritic segments of the PV positive EPL neurons displayed characteristics of the AISs and/or nodes of Ranvier and further suggested that these particular sites play the functional roles as the AP generation sites, “hot spots”. Our quantitative analyses showed the variety of the numbers, sizes and locations of these dendritic “hot spots” on dendritic trees of the PV positive EPL neurons. Furthermore we encountered neurons other than PV positive EPL neurons expressing similar dendritic “hot spots”, that is, some granule cells and nitric oxide synthase (NOS) positive periglomerular cells. Thus sodium channel cluster/βIV-spectrin/ankyrinG positive dendritic segments are not necessarily specific to the PV positive EPL neurons. We also confirmed that multiple patch-like sodium channel cluster/βIV-spectrin/ankyrinG positive segments were present on the dendritic processes of most PV positive neurons in the EPL in the rat MOB. Thus they resembled those in the mouse MOB.
Section snippets
Tissue preparations
All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996) and the institutional guidance for animal welfare (the Guidelines for Animal Experiment in Graduate School of Medical Sciences, Kyushu University) and have been approved by the Committee of the Ethics on Animal Experiment in Graduate School of Medical Sciences, Kyushu University. All efforts were made to minimize the number of
Immunofluorescent multiple labeling
The sections fixed with fixatives PFA4, PFA2 and PFA1 were used for confocal laser scanning microscopic (CLSM) examinations and those fixed for PGA were used for combined CLSM and electron microscopic (EM) examinations. The sections for CLSM were incubated overnight with 1% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 and 0.05% sodium azide at room temperature. Then, they were incubated for 10 days at 20 °C in mixtures of primary antibodies raised in different species (Table 1
Combined CLSM and EM
For combined CLSM and EM examinations we used the sections fixed with the fixative PGA. After cryoprotection in 0.1 M PB containing 30% sucrose and freeze–thaw procedure using liquid N2, the sections were treated with 1% sodium borohydride in PBS for 30 min before immunocytochemical procedures (Kosaka et al., 1986). The sections were preincubated overnight with 1% BSA in PBS containing 0.05% sodium azide at 4 °C, and then incubated in a mixture of rabbit anti-PV antibody (1:5000) and chicken
Analysis and figure preparation
The confocal image stacks were transferred to a personal computer, analyzed using the image analysis software NIH ImageJ 1.33 (NIMH Bethesda, MD) and subsequently processed by Adobe Photoshop CS2 image-editing software (Adobe Systems, San Jose, CA).
The immunostainings for ankyrinG and pan sodium channels were occasionally so noisy that we applied the median filter to reduce the noise.
To reveal the three-dimensional locations and sizes of dendritic patches immunopositive for βIV-spectrin,
βIV-spectrin, sodium channels and ankyrinG are localized in register at particular dendritic segments of the PV positive EPL neurons in the mouse MOB
Both the antibody against pan sodium channels and antibody against ankyrinG were mouse monoclonal antibodies. As we could not use these two monoclonal antibodies simultaneously for the multiple fluorescent immunostaining, we compared their localizations with that of βIV-spectrin in the present study. In addition, as explained in the Experimental procedures, we compared the phospho-IκBα positive elements with those positive for βIV-spectrin but did not compare them directly with those positive
Discussion
In the present study we showed that the PV positive EPL neurons in the mouse and rat MOBs and some other interneurons in the mouse MOB had some particular dendritic portions resembling the AISs and nodes of Ranvier in their molecular assembly and ultrastructural features. AnkyrinG and βIV-spectrin have been generally regarded as the specific markers for AISs and nodes of Ranvier (Berghs et al., 2000, Komada and Soriano, 2002) and been considered to play some essential roles in clustering
Acknowledgements
The authors are grateful to Drs. Emson, Heizmann, and Nagatsu for providing the primary antibodies, to Dr. Fukuda for his help in preparing samples for the combined CLSM-EM analysis, and to Drs. Ohmori and Yamada for suggestions on the functional aspects of the channel distributions. The authors would like to thank Ms. Chie Goto and Ms. Kazuyo Sawai for their technical and secretarial assistance. This work was supported by Grants-in Aid for Scientific Research on Priority areas (Elucidation of
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