神经科学

In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain’s curved surface. The protocol enables long-term, high-resolution, and multiscale imaging—from subcellular structures to large neuronal populations—in awake mice over several months.

In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.

The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry.

Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image sensor with a fast sampling rate (500 Hz) detects fluorescent changes in corrective activity. The procedure includes fixing a mouse brain to a stereotaxic apparatus, craniotomy on a large cortical area, VSD staining, and wide-field imaging of cortical activity. The entire procedure can be completed in 5 h (from the administration of anesthesia to the start of cortical VSD imaging).

This protocol comprises the entire process of fluorescent measurement of vesicle recycling using the probe SynaptopHluorin, a pH-dependent GFP variant whose fluorescence increases at the synapse upon vesicle release due to fluorescence quenching in acidic vesicles. This technique provides a genetic tool to monitor synaptic vesicle recycling in real time in cultured hippocampal neurons.

This protocol describes how to visualize neuronal morphology and how to determine neuronal complexity of immature and mature hippocampal neurons in the mouse in vivo including tissue preparation, staining of brain sections and confocal cell analysis.

Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail to fit the requirement of primary rat hippocampal neuronal cultures. It can also be applied to a more general neuronal culture purpose in microfluidics.