<em>In Vivo</em> Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Joseph B. Alzagatiti; Luis Salazar; Heidi Brown; Gabriel Rosas; Lama Adel Jaber; Jaime L. Minaya; Courtney Scaramella; Adam C. Roberts; David  L. Glanzman

doi:10.3791/67301

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Neuroscience

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JoVE Journal Neuroscience

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

体内研究部分束缚斑马鱼幼鱼感觉刺激诱导的神经活动的共聚焦荧光成像

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05:12 min

April 18, 2025

DOI: 10.3791/67301-v

Joseph B. Alzagatiti^1,2, Luis Salazar³, Heidi Brown³, Gabriel Rosas³, Lama Adel Jaber³, Jaime L. Minaya³, Courtney Scaramella⁴, Adam C. Roberts³, David L. Glanzman^2,5,6

¹Department of Molecular, Cellular, and Developmental Biology,University of California, Santa Barbara, ²Department of Integrative Biology and Physiology,University of California, Los Angeles, ³Department of Psychology,California State University at Fullerton, ⁴Department of Neuroscience,University of California, Riverside, ⁵Department of Neurobiology,David Geffen School of Medicine at UCLA, ⁶Integrative Center for Learning and Memory, Brain Research Institute,David Geffen School of Medicine at UCLA

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Overview

This study presents a detailed protocol for examining neural activity in brain regions of transgenic zebrafish expressing GCaMP calcium indicators using confocal microscopy. It aims to investigate dynamic changes in neural activity in response to stimulation, particularly focusing on fluorescence intensity in specific regions.

Key Study Components

Area of Science

Neuroscience
Neuroimaging
Transgenic models

Background

Transgenic zebrafish models allow for real-time imaging of neuronal activity.
GCaMP indicators provide a method for monitoring calcium fluctuations as a proxy for neural activity.
Confocal microscopy offers high spatial resolution to observe fluorescence intensity changes in neurons.
The protocol allows for precise control of imaging conditions to optimize data acquisition.

Purpose of Study

To develop a robust methodology for assessing GCaMP-mediated neural activity.
To characterize the time-lapse imaging responses in specific neuronal clusters of zebrafish.
To investigate the effects of certain stimuli on neural activity dynamics.

Methods Used

Confocal microscopy was used to visualize neuronal activity in vivo.
Transgenic zebrafish larvae aged 2–7 days post fertilization served as the biological model.
Laid out critical steps for acclimatization and imaging settings.
Included post-imaging analysis using Fiji software for assessing fluorescence intensity.

Main Results

The application of aloe isothiocyanate resulted in increased GCaMP fluorescence, indicating heightened neural activity.
Changes in imaging speed affected spatial and temporal resolution of neuronal visualization.
Normalized fluorescence intensity measurements facilitated the generation of neural traces over time.
Neurons in the hindbrain and spinal cord displayed significant activity changes in response to stimulus.

Conclusions

This study establishes a valuable imaging protocol for analyzing neural dynamics in zebrafish.
Findings enhance understanding of the relationship between stimuli and neuronal behavior.
Insights contribute to the broader comprehension of neural mechanisms and plasticity.

Frequently Asked Questions

What advantages does the zebrafish model offer?

The zebrafish model provides a transparent organism with rapid development, allowing for real-time imaging of neural activity in a living system.

How are the larvae prepared for imaging?

Larvae are embedded in low melting point agarose and positioned dorsal side up in embryo medium before imaging.

What types of data can be obtained?

Data includes time-lapse fluorescence imaging revealing changes in calcium dynamics associated with neuronal activity.

How can the method be adapted for other studies?

The protocol can be modified for various stimuli or fluorescent reporters to investigate different aspects of neural function.

What are some limitations of this technique?

Potential limitations include variations in zebrafish genetics and the challenge of capturing very fast neural activity due to resolution constraints.

在这里，我们提出了一个详细的方案，以使用共聚焦显微镜检查表达 GCaMP 钙指示剂的转基因斑马鱼大脑区域的神经活动。

[导师]首先，在胚胎培养基中制备 3% 的低熔点农胶，将农胶加热到可以安全处理斑马鱼而不会造成损坏的温度。在农业冷却和凝固之前，将受精后两到七天的幼虫背面朝上放入玻璃底培养皿中。一旦农脂与幼虫就位凝固，在培养皿中加入五毫升胚胎培养基。使用手术刀，根据需要在幼虫周围切割农作物。将装有斑马鱼的培养皿转移到共聚焦显微镜的载物台上。适应20分钟后，使用明场成像将鱼在室温下40倍水浸物镜下居中。根据荧光分子的质量和表达水平以及组织的深度和光学特性设置共聚焦显微镜采集参数。调整激光功率和主增益设置，以在最佳范围内正确捕获荧光，并防止不必要的 G 营成功相关荧光损失。然后将视野集中在位于脊髓喙部的神经元簇上。以每秒 1.20 帧的帧速率执行视场为 79.86 平方微米的时序扫描，空间分辨率为每像素 0.119 平方微米。根据实验目标调整视场、大小和采集速度。成像开始两分钟后，向培养皿中加入41.67微升芦荟异硫氰酸盐储备液或胚胎培养基，并继续记录约30秒，以通过延时成像观察G Camp 6在感兴趣区域的活性变化。如果记录的输出不是点 TIF 文件格式，请从显微镜软件套件将图像文件导出为点 TIF 文件，以便下游斐济分析。下载斐济神经迹线分析软件后，打开斐济，将点CZI文件导入程序。使用斐济的圆形或手绘工具通过单击相应的图标来突出显示感兴趣的区域或神经元。选择感兴趣区域或 ROI 后，单击斐济工具栏中的分析、工具和 ROI 管理器。单击添加 T 将选定的 ROI 添加到 ROI 管理器窗口。在 ROI 管理器中，单击更多和多度量以分析 ROI。将设置保留为默认值，然后单击确定以生成原始数据。然后将输出另存为 CSV 文件，以便使用 Python 或电子表格进行进一步作。现在，通过平均两分钟预刺激的最后 30 秒，将原始数据归一化，将其表示为神经迹线，并使用给定公式生成归一化荧光强度，并将归一化数据绘制为神经迹线。施用异硫氰酸芦荟导致斑马鱼幼体局部大脑区域内的 G CAMP 6S 荧光增加，表明神经活动增强。在每秒 0.10 帧的慢速捕获速度下，后脑和脊髓中的神经元清晰可见，分辨率高。将捕获速度提高到每秒 0.79 帧会导致空间分辨率略有损失，但时间分辨率有所提高。在每秒 3.16 帧时，神经元显得不那么明显，同时捕捉到了更多的时间动态。

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