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Coloring and lighting up the brain: new techniques allow investigators to map the human brain
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The inception of the Golg stain in 1887 literally revolutionized the field of neurobiology. This staining technique was further refined and improved by Spanish neurobiologist Santiago Ramon y Cajal which allowed neurobiologists and neuroanatomists to see how individual neurons are connected to neighboring neurons. This technique that is still employed by researchers worldwide today.
Although this technique allows you to visualize the morphology, shape and connectivity of neurons, it does not allow for the in vivo analysis of the morphology and connectivity of individual neurons since neurons are killed during fixation of the brain tissue. It is not known how the Golgi stain randomly stains neurons while others are not. This is a drawback and severe limitation of the technique as it is not possible to study how neighboring neurons connect with each other and how axonal-dendritic connections are generated in a specific subset of neurons.
It took more than 130 years to come up with better techniques that enable researchers to map the human brain and to understand how neurons communicate, interact and relay with each other to form complex electrochemical circuits that respond to certain stimuli.
One very remarkable technique called “Brainbow” mapping has allowed researchers to simultaneously color many neurons in the brain with hundreds of colors by using transgenic strategies that allows for the random, combinatorial expression of green, red, yellow and blue fluorescent proteins in mice. Essentially, this techniques works in a similar fashion to generate colors on a computer screen or television which mix and matches three colors to generate an array of colors in the screen. This technique was published in the journal Nature two years ago by group of researchers from Harvard University which has enabled researchers to:
a. Reconstruct how hundreds of neighboring neurons connect with each other.
b. Allow investigators to map the territory of certain populations of neurons and glia (the support cells of neurons) in vivo.
c. The Brainbow technique allowed researchers to uniquely color and label many individual cells within a population on a large scale basis.
Although this technique is great for reconstructing the connectivity of a particular group of neurons, it is not possible to determine the physiological relevance or implications of a particular neuronal circuit. Enter optogenetics. This novel technique made possible to study the effects of activating a group of neurons using laser light in a group of neurons that express light sensitive proteins.
Indeed, at a press conference at the Society for Neuroscience which concluded last Wednesday at the McCormick center in Chicago, a panel of researchers showed the use of a novel technique that has been christened as “optogenetics”. Optogenetics is a novel neurobiology technique that employs light to switch on or switch off neurons of the central nervous system and has enabled researchers to study specific brain circuits with high precision and with minimal invasive techniques.
How does this work? This technique involves the electroporation of light sensitive genes in neurons or the stereostatic injection of lentiviruses that carry up to three specific light-sensitive proteins (ie., halo-rhodopsin) in a specific population of neurons of the rodent brain. In other words, these light sensitive proteins are inserted into small brain regions or specific neurons of interest. These neurons of interest can transiently express up to three different light sensitive proteins simultaneously in a specific brain region of the mouse. Following the preparatory steps, the following stage of this technique is a “kicker” for most researchers who have fun observing the effects of stimulating certain brain areas on the behavior of mice. In order to do this, a fiber optic device is implanted directly into the area of interest in the brain where the neurons express the light sensitive proteins using microsurgery techniques. By shining different laser light of different wavelengths, different populations of neurons can be stimulated and activated at the same time. In addition, researchers can simultaneously record hundreds of neurons that are activated by light. Moreover, researchers can trace back and reconstruct how all these neurons are connected with each other using complex imaging software similar to the Brainbow technique described above; a task that was considered science fiction just a few years ago.
Here is a synopsis of the findings reported by this distinguished group of researchers:
1. There are currently only two widely employed light sensitive proteins used in the field of optogenetics. Dr. Feng Zhang from Standford University discovered additional light sensitive genes that might be used in the field of optogenetics and developed precise cell targeting techniques that restricts the expression of these proteins to a specific populations of neurons by employing different neuronal specific promoters. More importantly, he discovered a set of novel opsins, some of which were discovered in plants (obviously plants use photosynthesis and are likely candidates for discovering more of this genes), has both excitatory and inhibitory functions in neurons respectively.
2. Dr. Herbert E. Covington from Mount Sinai School of Medicine has employed optogenetic techniques to study depression in mice and has opened the possibility for a novel treatment for depression that does not involve pharmacological intervention. Using optogenetics, he found that stimulating a population of neurons in the medial prefrontal cortex of the brain with laser light alleviated some of the symptoms of depression in mice that were chronically stressed. As further proof of this finding, researchers found that stimulating the prefrontal cortex with laser light restored social behavior and physical activity of depressed mice. In other words, the prefrontal cortex is a brain region that plays an important role in depression and finding ways to stimulating this region may be an important therapeutic alternative. Another remarkable side observation with therapeutic implications is that drugs that block the enzyme histone deacetylase for 10 days infused in the prefrontal cortex by a small pump also alleviated the symptoms of depression.
3. Dr. Garret Stuber form the University of California in San Francisco used optogenetic techniques to determine the specific neuronal populations and circuitry in mice involve in drug addiction and drug seeking behavior. Although it has been known that dopaminergic neurons at certain brain regions are responsible for rewarding and drug-addictive behavior, this is the first time that researchers determined in very fine detail the brain connections that are responsible for these actions, especially given the billion of neurons making connections in the human brain.
By expressing light sensitive channels in glutaminergic (excitatory) neurons of the basal lateral amygdala to the nucleus accumbens pathways in brain of mice, researchers found that activating these neurons with laser light recapitulated drug seeking behavior of mice. Activation of this specific brain circuitry in mice led to increase “nose-poke” behavior in a rodent model of drug addiction. Nose-poking involves the pressing of a lever with the nose of the mice; by completing this action, mice are rewarded with more optical stimulation and this behavior could be carried on for hours! This study shows that blocking the signals from the basal lateral amygdale to the nucleus accumbens can be a therapeutic alternative to block drug-seeking and addictive behavior in human beings.
4. Complex memories can be triggered by a tiny fraction of neurons in the human brain, a concept that is currently hotly debated in the field of memory reconsolidation. Moreover, exactly how the brain retrieves complex memories remains to be elucidated. By using optogenetics, Dr. Michael Hausser’s group at Wolfson Institute for Biomedical Research in London have discovered that reactivation of complex memories may only involve a tiny fraction of brain cells, specifically in the hippocampus.
Mice that expressed light sensitive proteins in the dentate gyrus of the hippocampus underwent classical fear conditioning (either by foot shocks or by employing loud noises). An optic fiber was then implanted into this brain region of mice and laser stimulation of this specific brain region recapitulated fear induced behavior in mice as assessed by freezing behavior. Remarkably, this group of researchers found that triggering a complex memory recalls such as fear occurs in only a small group of neurons in the dentate gyrus since randomly stimulating other populations of cells in the brain did not lead to this behavior.
Concluding remarks-
In the words of Karl Deisseroth, MD, Ph.D. of Standford University, “Although relatively new, optogenetics has already proven to be an extremely powerful tool and there are a hundred billion neurons in the human brain and countless sub-groupings and intersecting populations of different cell types. Using techniques like optogenetics, we can map how, why and when those types of neurons are used. Today's findings bring us one step closer toward unlocking the brain’s mysteries and a better understanding of th origins of disease, behavior, and memory”.
Click on the following link in order to obtain more information:
From the author of the technique, a narrative on the history of optogenetics: http://http://www.scientificamerican.com/article.cfm?id=neural-light-show&page=4
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