An essential experimental control for functional connectivity mapping with optogenetics

To establish functional connectivity between two candidate neurons that might form a circuit element, a common approach is to activate an optogenetic tool such as Chrimson in the candidate pre-synaptic neuron and monitor fluorescence of the calcium-sensitive indicator GCaMP in a candidate post-synaptic neuron. While performing such experiments in Drosophila, we found that low levels of leaky Chrimson expression can lead to strong artifactual GCaMP signals in presumptive postsynaptic neurons even when Chrimson is not intentionally expressed in any particular neurons. Withholding all-trans retinal, the chromophore required as a co-factor for Chrimson response to light, eliminates GCaMP signal but does not provide an experimental control for leaky Chrimson expression. Leaky Chrimson expression appears to be an inherent feature of current Chrimson transgenes, since artifactual connectivity was detected with Chrimson transgenes integrated into multiple genomic locations. While these false-po..., Calcium imaging in the larva
The CNS of larvae at the 3rd instar developmental stage was dissected in saline (135 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1.8 mM Trehalose, 12.6mM Sucrose, 2mM CaCl2) and placed dorsal side down on a lysine-coated coverslip. We used a Hyperscope two-photon scanning microscope (Scientifica, UK) with a MaiTai laser (Spectra Physics, CA, USA). For imaging, we used a two-photon excitation wavelength of 920 nm. When CsChrimson was stimulated with 590nm (e.g., Figure 1b), an amber LED (LCS-0590-03-22, Mightex, Canada) with peak wavelength at 590 nm and a 605/55 bandpass filter (Chroma, VT, US) was used. In Figure 3, CsChrimson was activated with a red LED (LCS-0656-03-22, Mightex, Canada) with peak wavelength at 656 nm and a 650/10 bandpass filter (Chroma, VT, US). To feed the CsChrimson excitation light into the light path, we used a 560LP dichroic (T560LPXRXT-UF1, Chroma, VT, USA). The power of the LED from the objective was measured with a PM100D Power meter with an ..., , # Data from: An essential experimental control for functional connectivity mapping with optogenetics

Dataset DOI: [10.5061/dryad.t4b8gtj77](10.5061/dryad.t4b8gtj77)

## Description of the data and file structure

**Data from: An essential experimental control for functional connectivity mapping with optogenetics**

This repository contains raw data of larval calcium imaging and adult immunohistochemistry files shown in the paper titled 'An essential experimental control for functional connectivity mapping with optogenetics'

### Files and variables

**High level overview**

Folder 'IHC_data.zip' contains both the raw and processed immunohistochemistry data shown in Figure 5.

Folder 'larval_ca_data.zip' contains both the raw and the preprocessed calcium imaging data shown in Figures 1, 3, and 6.

The `leaky_expression_publication` folder (on Zenodo) contains preprocessed data and the scripts used to create Figures 1, 2, 3, 4, and 6 of the manuscript.

**Detailed information about each ...,

为在可能构成环路元件的两个候选神经元间建立功能连接，常规方法是在候选突触前神经元中激活光遗传工具（optogenetic tool）如Chrimson，并在候选突触后神经元中监测钙敏感指示剂GCaMP的荧光信号。在果蝇（Drosophila）中开展此类实验时，我们发现，即便未在特定神经元中刻意表达Chrimson，低水平的渗漏性Chrimson表达仍会在推定的突触后神经元中引发显著的伪影GCaMP信号。

全反式视黄醛（all-trans retinal）是Chrimson响应光信号所需的辅因子发色团，去除该物质虽可消除GCaMP信号，但无法为渗漏性Chrimson表达提供有效的实验对照。渗漏性Chrimson表达似乎是当前Chrimson转基因的固有特征，因为在整合至多个基因组位点的Chrimson转基因中均检测到了伪影连接现象。尽管此类伪影连接……

### 幼虫钙成像
将处于第三龄发育阶段的幼虫中枢神经系统（CNS）在生理盐水中解剖，该生理盐水的配制成分为：135 mM氯化钠、5 mM氯化钾、4 mM氯化镁、1.8 mM海藻糖、12.6 mM蔗糖、2 mM氯化钙，随后将其背侧朝下放置于包被赖氨酸的盖玻片上。本研究使用英国Scientifica公司生产的Hyperscope双光子扫描显微镜，搭配美国加利福尼亚州Spectra Physics公司的MaiTai激光器。成像时采用920 nm的双光子激发波长。当使用590 nm光刺激CsChrimson时（如图1b所示），需使用峰值波长为590 nm的琥珀色发光二极管（LED，型号LCS-0590-03-22，加拿大Mightex公司）及605/55带通滤光片（美国佛蒙特州Chroma公司）。在图3的实验中，则使用峰值波长为656 nm的红色LED（型号LCS-0656-03-22，加拿大Mightex公司）及650/10带通滤光片（美国佛蒙特州Chroma公司）。为将CsChrimson的激发光导入光路，我们使用了560 LP二向色镜（型号T560LPXRXT-UF1，美国佛蒙特州Chroma公司）。物镜输出的LED光功率通过搭载了……的PM100D功率计进行测量。

# 数据来源：用于光遗传学功能连接图谱绘制的必需实验对照
数据集DOI: [10.5061/dryad.t4b8gtj77](10.5061/dryad.t4b8gtj77)

## 数据与文件结构说明
**数据来源：用于光遗传学功能连接图谱绘制的必需实验对照**
本数据集仓库包含发表论文《用于光遗传学功能连接图谱绘制的必需实验对照》中展示的幼虫钙成像原始数据与成虫免疫组织化学文件。

### 文件与变量
**总体概览**
文件夹"IHC_data.zip"包含图5展示的免疫组织化学原始数据与处理后数据。
文件夹"larval_ca_data.zip"包含图1、图3及图6展示的钙成像原始数据与预处理后数据。
Zenodo平台上的`leaky_expression_publication`文件夹包含预处理后的数据，以及用于绘制论文图1、图2、图3、图4和图6的脚本。
**各文件详细信息……