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A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Juntendo University Graduate School of Medicine

Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.

Overlapping Projections of Neighboring Direct and Indirect Pathway Neostriatal Neurons to Globus Pallidus External Segment.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

Parkinson's disease (PD) is a neurodegenerative disorder characterized with the progressive loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNc). Quantitative analysis of neuronal loss including neuronal processes, axons and dendrites, would advance the understanding of the pathogenesis of PD. ScaleS, an aqueous tissue clearing method, provides stable tissue preservation while maintaining potent clearing capability, allowing quantitative three-dimensional (3D) imaging of biological tissues. In this chapter, we describe detailed procedures for 3D imaging of brain slice tissues with ScaleS technique. These include brain slice preparation, tissue clarification, chemical and immunohistochemical labeling (ChemScale and AbScale), and observation of labeled tissues using a confocal laser scanning microscope (CLSM).

Application of a Tissue Clearing Method for the Analysis of Dopaminergic Axonal Projections.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaeSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Kenta Yamauchi

Department of Neuroanatomy,
Department of Cell Biology and Neuroscience

Juntendo University Graduate School of Medicine

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Kenta Yamauchi

Department of Neuroanatomy,Department of Cell Biology and Neuroscience

Juntendo University Graduate School of Medicine

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Department of Neuroanatomy,
Department of Cell Biology and Neuroscience