Brainbow
Encyclopedia
Brainbow is a term used to describe the process by which individual neurons in the brain
can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein
in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of connectomics
, or the study of neural connections in the brain.
The technique was originally developed in the Spring of 2007 by a team led by Jeff W. Lichtman and Joshua R. Sanes, both professors of Molecular & Cellular Biology in the Department of Neurobiology at Harvard Medical School
. Their demonstration of the technique in mice first appeared in the November 1, 2007 issue of the journal Nature. The original technique has recently been adapted for use with other model organisms including Drosophila melanogaster
and Caenorhabditis elegans
.
While earlier labeling techniques allowed for the mapping of only a few neurons, this new method allows more than 100 differently mapped neurons to be simultaneously and differentially illuminated in this manner. The resulting images can be quite striking and have in fact won awards in science photography competitions.
in 2007. This particular group of scientists was led by professors Jeff W. Lichtman and Joshua R. Sanes, both of whom specialize in Molecular and Cellular Biology and are highly renowned for their work. The team constructed Brainbow using a two-step process: first, a specific genetic
construct was generated that could be recombined in multiple arrangements to produce one of either three or four colors based on the particular fluorescent proteins (XFPs) being implemented. Next, multiple copies of the same transgenic construct were inserted into the genome
of the target species, resulting in the random expression of different XFP ratios and subsequently causing different cells to exhibit a variety of colorful hues.
Brainbow was originally created as an improvement over more traditional neuroimaging
techniques, such as Golgi staining and dye injection, both of which presented severe limitations to researchers in their ability to visualize the intricate architecture of neural circuitry in the brain
. While older techniques were only able to stain cells with a constricted range of colors, often utilizing bi- and tri-color transgenic mice to unveil limited information in regards to neuronal structures, Brainbow is much more flexible in that it has the capacity to fluorescently label individual neurons with up to approximately 100 different hues so that scientists can identify and even differentiate between dendritic and axonal processes. By revealing such detailed information about neuronal connectivity and patterns, sometimes even in vivo, scientists are often able to infer information regarding neuronal interactions and their subsequent impact upon behavior and function. Thus, Brainbow filled the void that previous neuroimaging methods were unable to fulfill.
With the recent advent of Brainbow in neuroscience
, researchers are now able to construct specific maps of neural circuits and better investigate how these relate to various mental activities and their connected behaviors (ie. Brainbow reveals information about the interconnections between neurons and their subsequent interactions that affect overall brain functionality). As a further extrapolation of this method, Brainbow can therefore also be used to study both neurological and psychological disorders by analyzing differences in neural maps.
, in which the protein Cre recombinase
drives inversion
or excision of DNA
between loxP sites. The original Brainbow method includes both Brainbow-1 and Brainbow-2, which utilize different forms of cre/lox recombination. Brainbow-1 uses DNA construct
s with different fluorescent protein genes (XFPs) separated by mutant and canonical forms of loxP. This creates a set of mutually exclusive excision possibilities, since cre mediated recombination
occurs only between identical loxP sites. After recombination occurs, the fluorescent protein that is left directly after the promoter is uniquely expressed. Thus, a construct with four XFPs separated by three different loxP sites, three excision events, and the original construct can produce four different fluorescent proteins.
Brainbow-2 uses Cre excision and inversion to allow multiple expression possibilities in a given construct. In one DNA segment with two oppositely oriented XFPs, Cre will induce a random inversion event that leaves one fluorescent protein in the proper orientation for expression. If two of these invertible sequences are aligned, three different inversion events are possible. When excision events are also considered, one of four fluorescent proteins will be expressed for a given combination of Cre excisions and inversions. For both Brainbow-1 and-2, the expression of a given XFP is a stochastic, or random, event.
Brainbow is implemented in vivo
by crossing two transgenic
organism strains: one that expresses the Cre protein and another that has been transfected with several versions of a loxP/XFP construct. Using multiple copies of the transgene
allows the XFPs to combine in a way that can give one of approximately 100 different colors. Thus, each neuron is labeled with a different hue based on its given combinatorial and stochastic expression of fluorescent proteins.
In order to elucidate differential XFP expression patterns into a visible form, brain slices are imaged with confocal microscopy
. When exposed to a photon
with its particular excitation wavelength, each fluorophore
emits a signal that is collected into a red, green, or blue channel, and the resultant light combination is analyzed with data analysis software. Superimposition of differentially colored neurons allows visual disentanglement of complicated neural circuits.
Brainbow has predominantly been tested in mice to date; however, the basic technique described above has also been modified for use in more recent studies since the advent of the original method introduced in 2007.
has 4,000,000 neurons and is more similar to a human brain than both drosophila
and other commonly used organisms to model this technique, such as C. elegans. Mice were the first organisms in which the Brainbow method of neuroimaging was successfully employed. Livet et al. (2007) developed two versions of Brainbow mice using Brainbow-1 and Brainbow-2, which are described above. In using these methods to create a complete map and track the axons of a mouse muscle, it is necessary to collect tens of thousands of images and compile them into stacks to create a complete schematic. It is then possible to trace each motor axon and its synaptic contacts to construct a complete connectome
of the muscle.
More examples of neurons examined using the Brainbow technique in transgenic mice are located in the motor nerve innervating ear muscles, axon tracts in the brainstem, and the hippocampal dentate gyrus
.
Ultimately, this technique provides the ability to efficaciously map the neuronal circuitry in Drosophila so that researchers are able to uncover more information about the brain structure of this invertebrate and how it relates to its ensuing behavior.
In addition, due to the random nature in the expression of the fluorescent proteins, scientists are unable to precisely control the labeling of neural circuitry, which may result in the poor identification of specific neurons.
The use of brainbow in mammalian populations is also hampered by the incredible diversity of neurons of the central nervous system
. The sheer density of neurons coupled with the presence of long tracts of axons make viewing larger regions of the CNS with high resolution difficult. Brainbow is most useful when examining single cell resolution against the background of a complex multicellular environment. However, due to the resolution limits of optical microscopy, conclusive identification of synaptic connections between neurons is not easily accomplished. This issue is somewhat avoided by the use of synaptic markers to supplement the use of optical microscopy in viewing synaptic connections.
Fluorescence
Cre-Lox Recombination
Brain
The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...
can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of connectomics
Connectomics
Connectomics is a high-throughput application of neural imaging and histological techniques in order to increase the speed, efficiency, and resolution of maps of the multitude of neural connections in a nervous system...
, or the study of neural connections in the brain.
The technique was originally developed in the Spring of 2007 by a team led by Jeff W. Lichtman and Joshua R. Sanes, both professors of Molecular & Cellular Biology in the Department of Neurobiology at Harvard Medical School
Harvard Medical School
Harvard Medical School is the graduate medical school of Harvard University. It is located in the Longwood Medical Area of the Mission Hill neighborhood of Boston, Massachusetts....
. Their demonstration of the technique in mice first appeared in the November 1, 2007 issue of the journal Nature. The original technique has recently been adapted for use with other model organisms including Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
and Caenorhabditis elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
.
While earlier labeling techniques allowed for the mapping of only a few neurons, this new method allows more than 100 differently mapped neurons to be simultaneously and differentially illuminated in this manner. The resulting images can be quite striking and have in fact won awards in science photography competitions.
History & Development
The Brainbow neuroimaging technique was initially developed by a team of researchers in the Department of Neurobiology at Harvard Medical SchoolHarvard Medical School
Harvard Medical School is the graduate medical school of Harvard University. It is located in the Longwood Medical Area of the Mission Hill neighborhood of Boston, Massachusetts....
in 2007. This particular group of scientists was led by professors Jeff W. Lichtman and Joshua R. Sanes, both of whom specialize in Molecular and Cellular Biology and are highly renowned for their work. The team constructed Brainbow using a two-step process: first, a specific genetic
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
construct was generated that could be recombined in multiple arrangements to produce one of either three or four colors based on the particular fluorescent proteins (XFPs) being implemented. Next, multiple copies of the same transgenic construct were inserted into the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of the target species, resulting in the random expression of different XFP ratios and subsequently causing different cells to exhibit a variety of colorful hues.
Brainbow was originally created as an improvement over more traditional neuroimaging
Neuroimaging
Neuroimaging includes the use of various techniques to either directly or indirectly image the structure, function/pharmacology of the brain...
techniques, such as Golgi staining and dye injection, both of which presented severe limitations to researchers in their ability to visualize the intricate architecture of neural circuitry in the brain
Brain
The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...
. While older techniques were only able to stain cells with a constricted range of colors, often utilizing bi- and tri-color transgenic mice to unveil limited information in regards to neuronal structures, Brainbow is much more flexible in that it has the capacity to fluorescently label individual neurons with up to approximately 100 different hues so that scientists can identify and even differentiate between dendritic and axonal processes. By revealing such detailed information about neuronal connectivity and patterns, sometimes even in vivo, scientists are often able to infer information regarding neuronal interactions and their subsequent impact upon behavior and function. Thus, Brainbow filled the void that previous neuroimaging methods were unable to fulfill.
With the recent advent of Brainbow in neuroscience
Neuroscience
Neuroscience is the scientific study of the nervous system. Traditionally, neuroscience has been seen as a branch of biology. However, it is currently an interdisciplinary science that collaborates with other fields such as chemistry, computer science, engineering, linguistics, mathematics,...
, researchers are now able to construct specific maps of neural circuits and better investigate how these relate to various mental activities and their connected behaviors (ie. Brainbow reveals information about the interconnections between neurons and their subsequent interactions that affect overall brain functionality). As a further extrapolation of this method, Brainbow can therefore also be used to study both neurological and psychological disorders by analyzing differences in neural maps.
Methods
Brainbow techniques rely on the Cre-Lox recombinationCre-Lox recombination
Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice . Subsequently, the laboratory of Dr...
, in which the protein Cre recombinase
Cre recombinase
Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...
drives inversion
Chromosomal inversion
An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself. Inversions are of two types: paracentric and pericentric.Paracentric inversions do not include the...
or excision of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
between loxP sites. The original Brainbow method includes both Brainbow-1 and Brainbow-2, which utilize different forms of cre/lox recombination. Brainbow-1 uses DNA construct
DNA construct
A DNA construct is an artificially constructed segment of nucleic acid that is going to be "transplanted" into a target tissue or cell...
s with different fluorescent protein genes (XFPs) separated by mutant and canonical forms of loxP. This creates a set of mutually exclusive excision possibilities, since cre mediated recombination
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
occurs only between identical loxP sites. After recombination occurs, the fluorescent protein that is left directly after the promoter is uniquely expressed. Thus, a construct with four XFPs separated by three different loxP sites, three excision events, and the original construct can produce four different fluorescent proteins.
Brainbow-2 uses Cre excision and inversion to allow multiple expression possibilities in a given construct. In one DNA segment with two oppositely oriented XFPs, Cre will induce a random inversion event that leaves one fluorescent protein in the proper orientation for expression. If two of these invertible sequences are aligned, three different inversion events are possible. When excision events are also considered, one of four fluorescent proteins will be expressed for a given combination of Cre excisions and inversions. For both Brainbow-1 and-2, the expression of a given XFP is a stochastic, or random, event.
Brainbow is implemented in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
by crossing two transgenic
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
organism strains: one that expresses the Cre protein and another that has been transfected with several versions of a loxP/XFP construct. Using multiple copies of the transgene
Transgene
A transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another....
allows the XFPs to combine in a way that can give one of approximately 100 different colors. Thus, each neuron is labeled with a different hue based on its given combinatorial and stochastic expression of fluorescent proteins.
In order to elucidate differential XFP expression patterns into a visible form, brain slices are imaged with confocal microscopy
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
. When exposed to a photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...
with its particular excitation wavelength, each fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
emits a signal that is collected into a red, green, or blue channel, and the resultant light combination is analyzed with data analysis software. Superimposition of differentially colored neurons allows visual disentanglement of complicated neural circuits.
Brainbow has predominantly been tested in mice to date; however, the basic technique described above has also been modified for use in more recent studies since the advent of the original method introduced in 2007.
Mice
The mouse brainBrain
The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...
has 4,000,000 neurons and is more similar to a human brain than both drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
and other commonly used organisms to model this technique, such as C. elegans. Mice were the first organisms in which the Brainbow method of neuroimaging was successfully employed. Livet et al. (2007) developed two versions of Brainbow mice using Brainbow-1 and Brainbow-2, which are described above. In using these methods to create a complete map and track the axons of a mouse muscle, it is necessary to collect tens of thousands of images and compile them into stacks to create a complete schematic. It is then possible to trace each motor axon and its synaptic contacts to construct a complete connectome
Connectome
A connectome is a comprehensive map of neural connections in the brain.The production and study of connectomes, known as connectomics, may range in scale from a detailed map of the full set of neurons and synapses within part or all of the nervous system of an organism to a macro scale description...
of the muscle.
More examples of neurons examined using the Brainbow technique in transgenic mice are located in the motor nerve innervating ear muscles, axon tracts in the brainstem, and the hippocampal dentate gyrus
Dentate gyrus
The dentate gyrus is part of the hippocampal formation. It is thought to contribute to new memories as well as other functional roles. It is notable as being one of a select few brain structures currently known to have high rates of neurogenesis in adult rats, .The dentate gyrus cells receive...
.
Drosophila
The complexity of the Drosophila brain, consisting of about 100,000 neurons, makes it an excellent candidate for implementing neurophysiology and neuroscience techniques like Brainbow. In fact, Stefanie Hampel et al. (2011) combined Brainbow in conjunction with genetic targeting tools to identify individual neurons within the Drosophila brain and various neuronal lineages. One of the genetic targeting tools was a (UAS)-GAL4 binary expression system that controls the expression of UAS-Brainbow and targets the expression to small groups of neurons. Utilizing ‘Flip Out’ methods increased the cellular resolution of the reporter construct. The expression of fluorescent proteins, as with the original Brainbow, depended on Cre recombination corresponding with matched lox sites. Hampel et al. (2011) also developed their own variation of Brainbow (dBrainbow), based on antibody labeling of epitopes rather than endogenous fluorescence. Two copies of their construct yield six bright, separable colors. This, along with simplifications in color assignment, enabled them to observe the trajectories of each neuron over long distances. Specifically, they traced motor neurons from the antennal lobe to neuromuscular junctions, allowing them to identify the specific muscle targets of individual neurons.Ultimately, this technique provides the ability to efficaciously map the neuronal circuitry in Drosophila so that researchers are able to uncover more information about the brain structure of this invertebrate and how it relates to its ensuing behavior.
Limitations
Just as any technique, Brainbow has a number of limitations that stem from the methods required to perform it. For example, the process of breeding at least two strains of transgenic animals from embryonic stem cells is both time consuming and complex. Even if two transgenic species are successfully created, not all of their offspring will show the recombination. Thus, this requires extensive planning prior to performing an experiment.In addition, due to the random nature in the expression of the fluorescent proteins, scientists are unable to precisely control the labeling of neural circuitry, which may result in the poor identification of specific neurons.
The use of brainbow in mammalian populations is also hampered by the incredible diversity of neurons of the central nervous system
Central nervous system
The central nervous system is the part of the nervous system that integrates the information that it receives from, and coordinates the activity of, all parts of the bodies of bilaterian animals—that is, all multicellular animals except sponges and radially symmetric animals such as jellyfish...
. The sheer density of neurons coupled with the presence of long tracts of axons make viewing larger regions of the CNS with high resolution difficult. Brainbow is most useful when examining single cell resolution against the background of a complex multicellular environment. However, due to the resolution limits of optical microscopy, conclusive identification of synaptic connections between neurons is not easily accomplished. This issue is somewhat avoided by the use of synaptic markers to supplement the use of optical microscopy in viewing synaptic connections.
See also
GFPGreen fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
Fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
Cre-Lox Recombination
External links
- Podcast on NPR's Science Friday
- "Brainbow" A cool use of GFP