Abstract:

We have been studying the neural network structures to unravel the grand design of the central nervous system. We have shown that GABAergic neurons in the neocortex form specific network structures, indicating rules in synaptic connections. Our work also involves developing imaging techniques to facilitate morphological analysis. To improve the gene-transduction efficiency and label neuronal cells clearly, we have developed an adeno-associated virus (AAV) vector expression system using the Tet-Off system [1]. We have also designed a multi-color fluorescent signal amplification system, enhancing signals by 10 to 30 times more than the indirect method and 60 to 180 times more than the direct method [2]. Furthermore, we have developed a tissue clearing technique that preserves ultrastructures and have established a multiscale imaging pipeline that zooms in from the whole brain to the synaptic scale [3,4].

In this lecture, I will give an overview of these imaging techniques, hoping that they will be useful for your research work. Moreover, I will briefly describe the 3D structure of GABAergic neurons in the mouse claustrum using these imaging techniques [5]. The mouse claustrum has two subregions: the core and shell subregions. By using AAV vectors, we labeled single parvalbumin-expressing (PV) or somatostatin-expressing (SOM) GABAergic neurons in the core or shell subregions and analyzed their neurites in 3D with tissue clearing technology. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. These efficient 3D structural analyses benefit from advances in tissue clearing technology, and I’d like to discuss their usefulness and future prospects.

References:

1. Sohn J, Takahashi M, Okamoto S, Ishida Y, Furuta T, *Hioki H. A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System. PLoS One,12, e0169611, 2017.

2. Yamauchi K, Okamoto S, Ishida Y, Konno K, Hoshino K, Furuta T, Takahashi M, Koike M, Isa K, Watanabe M, Isa T, *Hioki H. Fluorochromized Tyramide-Glucose Oxidase as a

multiplex fluorescent tyramide signal amplification system for histochemical analysis. Sci Rep, 12, 14807, 2022.

3. Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, Kaneko T, Akagi T, Saito T, Saido T, *Miyawaki A. (2015) ScaleS: an optical clearing palette for biological imaging. Nat Neurosci, 18, 1518-1529, 2015.

4. #Furuta T, #Yamauchi K, Okamoto S, Takahashi M, Kakuta S, Ishida Y, Takenaka A, Yoshida A, Uchiyama Y, Koike M, Isa K, Isa T, *Hioki H. Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF. iScience, 25, 103601, 2022.

5. Takahashi M, Kobayashi T, Mizuma H, Yamauchi K, Okamoto S, Okamoto K, Ishida Y, Koike M, Watanabe M, Isa T, *Hioki H. Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum. Neurosci Res, 190, 92-106, 2023