Three-dimensional structural analysis of GABAergic neurons in the mouse claustrum using tissue-clearing technology

Hiroyuki Hioki, Megumu Takahashi, Tomoyo Kobayashi, Haruhi Mizuma, Kenta Yamauchi, Shinichiro Okamoto, Kazuki Okamoto

Department of Neuroanatomy, Juntendo University Graduate School of Medicine, Tokyo, Japan

Objective: The claustrum is composed of the core and shell subregions. To elucidate how GABAergic neurons are involved in the circuitry in each subregion, we here examined claustral GABAergic neurons for its distribution, neurochemical marker expression, and dendritic and axonal arborizations within the subregions.
Methods: Combination of fluorescence in situ hybridization and immunofluorescence labeling was performed to investigate the distribution of parvalbumin-, somatostatin-, and vasoactive intestinal polypeptide-expressing neurons (PV, SOM, and VIP neurons) in each subregion. We then selectively visualized PV or SOM neurons by injecting recombinant adeno-associated virus (AAV) vectors into the claustrum of PV- or SOM-Cre knock-in mice and optically cleared 1-mm-thick brain slices. After acquisition of three-dimensional image stacks by confocal microscopy, we reconstructed and analyzed their dendrites and axons in each subregion at the single-neuron level.
Results: About 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for PV, SOM, and VIP, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. 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.
Conclusion: The present study showed that claustral GABAergic neurons are homogeneously distributed. Claustral PV and SOM neurons might participate in independent information processing within the local circuits of the core and shell regions.

Keywords: GABAergic; adeno-associated virus; claustrum subregion; single-neuron reconstruction; tissue clearing

Ethical statement: All animal experiments were approved by the Institutional Animal Care and Use Committees of Juntendo University and conducted in accordance with Fundamental Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan (2006). All animal experiments were performed in compliance with ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

Funding statement: This study was supported by KAKENHI (JP21H02592 to H.H.; JP22J15034 to M.T.; JP20K07231 to K.Y.; JP22K20694 to S.O.; JP20K16112 to K.O.) from the Japan Society for the Promotion of Science (JSPS), Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) project from the Japan Agency for Medical Research and Development (AMED) (JP21dm0207112 to H.H.), Moonshot R&D from the Japan Science and Technology Agency (JST) (JPMJMS2024 to H.H.), Fusion Oriented Research for disruptive Science and Technology (FOREST) from JST (JPMJFR204D to H.H.).