Optical measurement of neuronal activity in the developing cerebellum of zebrafish using voltage-sensitive dye imaging

Abstract

Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAA receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.

In vivo wide‐field voltage imaging in zebrafish with voltage‐sensitive dye and genetically encoded voltage indicator

“…Although Di-4-ANEPPS is expected to be distributed across the cellular membrane, it is desirable to confirm whether the Di-4-ANEPPS is distributed properly after the staining step. For example, the localization of Di-4-ANEPPS on the cellular membrane can be confirmed using confocal microscopy (Figure 2b,c; Okumura et al, 2018). If there is an ectopic distribution or an aggregated signal, it is better to adjust the concentration of the dye, amount of dye injected, and/or washing process.…”

“…This approach is used to analyze evoked responses. To characterize the responses of the voltage sensors in the zebrafish brain in vivo, we conduct electrical stimulation of the cerebellum and performed voltage imaging simultaneously (Figures 4 and 5a-c;Miyazawa et al, 2018;Okumura et al, 2018).…”

“…Set a bath electrode in the recording chamber, usually at the opposite side of the stimulus electrode (Figure 3a). For quantitative analysis of the responsive area and size in the cerebellum, heatmap analysis is also useful (Figure 4c,d;Miyazawa et al, 2018;Okumura et al, 2018).…”

“…Here, we describe in vivo voltage imaging in zebrafish that we have recently established using VSDs and GEVIs (Miyazawa et al., 2018; Okumura et al., 2018). For both types of voltage indicators, experimental procedures are composed of three steps: sample preparation, image acquisition, and data analysis (Figure 1).…”

Similarities of developmental gene expression changes in the brain between human and experimental animals: rhesus monkey, mouse, Zebrafish, and Drosophila

“…They have advantages in rich genetic resources, their small size, ease of maintenance, and short life cycle, enabling the effective implementation of the diseases of humans [6][7][8][9]. Non-mammal animals, such as Zebrafish [10][11][12][13][14][15][16][17], and Drosophila [18][19][20][21], are also employed as experimental animals because of their technical advantages in maintenance, spatial requirements, fertility, genetic manipulation, and observation. In developmental neuroscience and the related fields using animal models, information about the developmental changes of the gene expression patterns in the brain of experimental animals and their correlation with human transcriptomics are important.…”