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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

Juntendo University

Parvalbumin-positive (PV+) neurons in the cerebral cortex, mostly corresponding to fast-spiking basket cells, have been implicated in higher-order brain functions and psychiatric disorders. We previously demonstrated that the somatic compartment of PV+ neurons received inhibitory inputs mainly from vasoactive intestinal polypeptide (VIP)+ neurons, whereas inhibitory inputs to the dendritic compartment were derived mostly from PV+ and somatostatin (SOM)+ neurons. However, a substantial number of the axosomatic inputs have remained unidentified. Here we show preferential innervation of the somatic compartment of PV+ neurons by cholecystokinin (CCK)+ neurons in the mouse primary somatosensory cortex. CCK+ neurons, a minor population of GABAergic neurons (3.2%), displayed no colocalization with PV or SOM immunoreactivity but partial overlap with VIP immunoreactivity (27.7%). Confocal laser scanning microscopy observation of CCK+ synaptic inputs to PV+ neurons revealed that CCK+ neurons preferred the somatic compartment to the dendritic compartment of PV+ neurons and provided approximately 33% of the axosomatic inhibitory inputs to PV+ neurons. Additionally, 20.9% and 12.1% of the axosomatic inputs were derived from CCK+/VIP+ and CCK+/VIP-negative (-) neurons, presumably double bouquet and large basket cells, respectively. Furthermore, the densities of the axosomatic inputs from CCK+ and/or VIP+ neurons to PV+ neurons were not significantly different among the cortical layers. The present findings suggest that, by preferentially innervating the cell bodies of PV+ neurons, both CCK+/VIP- basket and CCK+/VIP+ double bouquet cells might efficiently interfere with action potential generation of PV+ neurons, and that the two types of CCK+ neurons might have a large impact on cortical activity through PV+ neuron inhibition.

Preferential inputs from cholecystokinin-positive neurons to the somatic compartment of parvalbumin-expressing neurons in the mouse primary somatosensory cortex.

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.

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.

Shinichiro Okamoto

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

Shinichiro Okamoto

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}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