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Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

Posted by Aliyah Weinstein on Mar 5, 2019 8:08:52 AM

Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This type of cell labeling technique involves the introduction of a transgene construct containing fluorescent proteins (XFP) of different colors to label an organ or entire organism. Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire one of a variety of colors when a combination of XFP are expressed. Each cell remains the same color for its entire lifetime and daughter cells retain the same color, allowing for the fate mapping of cell populations over time. The ability to track single cell dynamics at the organism level has been made possible by tools that allow cells to become persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow, now exist for a variety of species, cell types, and research applications.

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Topics: Fluorescent Proteins, Cell Tracing, Neuroscience

Tetbow: Bright Multicolor Labeling for Neuronal Tracing

Posted by Guest Blogger on Jan 24, 2019 9:24:20 AM

This post was contributed by Richi Sakaguchi from Kyoto University, and Marcus N. Leiwe and Takeshi Imai from Kyushu University.

Stochastic multicolor labeling is a powerful solution for discriminating between neurons for light microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP system to express one of the three fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity will result in a combinatorial expression of these three genes with different copy numbers, producing dozens of color hues (Livet et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level.

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Topics: Fluorescent Proteins, Cell Tracing

Using AAV for neuronal tracing

Posted by Klaus Wanisch on Aug 9, 2018 9:04:52 AM

Background on neuronal tracing

A key aspect to understanding the brain’s function is knowing its architecture, in particular the connections between different brain regions. For example, communication between the hippocampus and the prefrontal cortex brain regions is involved in the formation of episodic memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional flow of information between different parts of the brain is mediated via individual neurons. Neurons are composed of a cell body, with dendrites receiving incoming information, and a projecting axon sending information onwards to other neuronal cells. Synapses at the terminals of axons form connections to dendrites of proximal neuronal cells. In the specific example of episodic memory, a subset of hippocampal neurons projects axons directly to the prefrontal cortex, but also indirectly via synapses to neurons in other brain regions. Further, the connections between regions are often reciprocal, forming a neuronal loop which is activated and strengthened during memory formation and memory retrieval.

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Topics: Viral Vectors, Cell Tracing, Other Viral Vector Tools

Rabies and Neuronal Tracing

Posted by Leila Haery on May 29, 2018 9:51:06 AM

Why study neural connectivity?

One of the early lessons many of us learned in biology is that the body’s architecture and plumbing are important. We started with learning the head is connected to the neck. Shortly after, we learned about organs and the jobs they perform. This became foundational later on when we studied biological processes, like how our stem cells are housed in specific locations and give rise to progenitors during growth and development or that blood flows through the heart and lungs and oxygenates the body. However, in neuroscience, this architecture is frequently still an open question. The connections between neurons are what define how the brain operates, and thus, are a major part of the answer to many biological questions about the brain. To address this, molecular tools to map neuronal connectivity are widely used in neuroscience. In this post, I’ll describe how rabies virus (RABV) can be used in the brain to visualize how neurons are connected.

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Topics: Viral Vectors, Cell Tracing

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