Knowledge Sharing: Optogenetic Technology

Optogenetics, also known as optical stimulation plus genetic engineering, is a technique for precisely controlling cell behavior in the brains of living animals through optical and genetic techniques. Due to its high temporal and spatial specificity, optogenetic techniques are widely used in the field of neuroscience.

In 2010, optogenetics technology was awarded to Nature Methods Annual Life Science Technology. In 2010, it was considered by Science to be one of the breakthroughs in the past decade. The optogenetics technology was once again named by Nature Methods as one of the eight most noteworthy technologies of 2016. In the future, optogenetic techniques will contribute more to the treatment of diseases in the neurological and psychiatric fields and to the study of tissue functions beyond neuroscience.

one. Before optogenetics

 

       In 1979, Francis Crick suggested that there is an urgent need to develop a control technique in the field of neuroscience to manipulate certain cells in the brain without changing other conditions. Because the electrical stimulation signal can't accurately locate the cells, and the chemical drugs have a slow onset of action and cannot be precisely controlled, Crick considers whether light control technology can be used. Although microbiologists have long discovered that they can express visible light-gated proteins, no one has ever linked them at the time.

Figure 1. Main research techniques in the field of neuroscience before the emergence of optogenetics

two. Optogenetics

1. The development of optogenetics

      

       In August 2005, an article published by Karl Deisseroth Lab brought a long-awaited new technology, optogenetics, to scientists in the field of neuroscience. The implementation of this technology is based on the discovery of a one-component control tool, the light-sensitive protein.

Figure 2. The history of optogenetics

2. Principle of optogenetics

      

       In neuronal cells, action potentials occur after depolarization of the cell membrane. Conversely, hyperpolarization of the cell membrane inhibits the appearance of a peak potential. By expressing an exogenous light-sensitive protein-encoding gene that changes the membrane potential in a neuronal cell, the peak-potential switch can be controlled by a light-controlled operation.

Figure 3. Resting potential and action potential of neurons

        The first of these microbial-encoded opsin neuron cell switches is ChR2 (channelrhodopsin-2). As a non-selective cation channel protein, ChR2 expressed in neurons immediately depolarizes neurons under blue light irradiation, inducing action potentials.

However, scientists don't always want to activate neuronal cells. There is a test strain called NpHR, halorhodopsin, which is a chloride ion pump. When the neuron cells are irradiated with yellow light, super-chemical reactions occur to suppress the formation of action potentials.

Figure 4. Photogenetic tools for regulating membrane potential - photochannel proteins

(Erika Pastrana, 2011)

3. Auxiliary techniques and basic steps required for optogenetics

The range of optogenetic techniques is extensive. It mainly includes the following.

Figure 5. Photogenetics and its assistive technologies

    In optogenetic manipulation, cells express a specific gene encoding a light-sensitive protein and then use light to alter the behavior of the cell. The basic steps of optogenetics to control cell function are as follows:

Figure 6. Basic steps in optogenetics to control cell function

      Among them, the method of transmitting the genetic information of the exogenous light-sensitive protein to the target cell by virus infection is the most convenient and quick. Thanks to the specific tissue tropism of adeno-associated virus (AAV) to the brain, AAV has become an important tool in optogenetic research .

4. The advantages of optogenetics technology - high spatial and temporal resolution

      The optogenetics technology fundamentally solves the problem of how to precisely regulate cell behavior. This is based on its advantage of having a high degree of spatiotemporal specificity. The optogenetics technology can control the precision in milliseconds (ms) in time, and the spatial precision can reach a single cell level, which is unmatched by electrical signal stimulation and chemical drugs.

Figure 7. High temporal resolution of optogenetics

Third, ViGene provides you with a wide selection of AAV photochannel protein carriers

       Specific promoters and specific AAV serotypes will provide more precise spatial localization for your photosensitivity experiments. ViGene offers you 24 promoters, 7 AAV serotypes and 5 light-sensitive channel proteins, over 840 vector combinations. In addition, you can customize the photo-sensitive vector plasmid according to your needs.

A lot of choices make your personalized experiment needs!

       Table 1. AAV serotypes provided by ViGene

Table 2. Promoter types provided by ViGene

Table 3. Photosensitive channel protein types provided by ViGene

Table 4: ViGene partial optogenetic spot

Attachment: Literature related to optogenetics

1. Application of activated photochannel protein

   

   In 2015, Dheeraj Pelluru et al. published an article in the European Journal of Neuroscience entitled Optogenetic stimulation of astrocytes in the posterior hypothalamus increases sleep at night in C57BL/6J mice, which was found to stimulate the hypothalamus during the active phase of the sleep-wake cycle. Astrocytes cause sleep, revealing the important role of astrocytes in the regulation of sleep-wake.

Figure 1. ChR2-EYFP was specifically expressed in astrocytes by AAV5 . (Dheeraj Pelluru, 2015)

Figure 2. Effect of different light stimulation frequencies on astrocytes to sleep. In the first 6 hours after turning off the lights,

Using optogenetic techniques to activate astrocytes using different frequencies of blue light (473 nm, 10 ms), respectively

Record the time of arousal, non-rapid eye movement sleep, and rapid eye movement sleep. It was found that a star expressing ChR2 was stimulated using 10 Hz.

Glial cells can significantly increase the time of non-rapid eye movement sleep and rapid eye movement sleep, and reduce awakening accordingly

between. (Dheeraj Pelluru, 2015)

Figure 3. Effect of different light stimulation times on astrocytes to sleep. Starting at the 12th hour after turning off the lights,

Activation of astrocytes using luminescence using different frequencies of blue light (473 nm, 10 ms) using optogenetic techniques for 1 min,

Pause for 4 minutes for 6 hours to record the time of arousal, non-rapid eye movement sleep and rapid eye movement sleep. Found different time

The stimulation between the awakening time and the non-rapid eye movement sleep time has a significant effect and peaks in the 15th hour after the light is turned off .

(Dheeraj Pelluru, 2015)

2. Application of inhibitory photochannel proteins

 

In 2013, Michael T. Stefanik et al. published an article in Front Behav Neurosci entitled Optogenetic dissection of basolateral amygdala projections during cue-induced reinstatement of cocaine seeking, which was discovered by inhibiting amygdala neurons and projecting to the central nucleus of the nucleus accumbens. The prefrontal cortex can reduce the possibility of recovery of cocaine in rats.

Figure 4. Specific expression of ArchT in the lateral nucleus of the amygdaloid nucleus by AAV2. It was found that inhibition of neuronal activity by light can reduce the search for cocaine.

The possibility of behavioral recovery. (A) Inhibition of amygdala neurons by 561 nm illumination can significantly reduce the number of active compression bars in rats; (B) DAB staining shows

Overexpression of ArchT-GFP in amygdala neurons; (C) inhibition of amygdala neurons by light to reduce the effect of seeking cocaine, significantly superior

In the absence of light, and close to the effect of subtractive administration; (D) illumination of AAV2-infected amygdala neurons with GFP only

No impact. (Michael T. Stefanik, 2013)

Figure 5. By inhibiting the projection of amygdala neurons to neurons in the central nucleus of the nucleus accumbens, the potential for recovery from cocaine behavior can be reduced.

(A) Inhibition of central nucleus neurons in the nucleus accumbens by 561 nm illumination can significantly reduce the number of active compression rods in rats; (B) DAB staining is shown in

Central nucleus accumbens region ArchT-GFP showed high expression; (C) by light irradiation inhibiting central nucleus accumbens reduce cocaine seeking obtained

Effect, the effect is not significantly better than the acceptable light, and close to the reduction effect obtained by the administration. (Michael T. Stefanik, 2013)

Figure 6. By inhibiting the projection of amygdala neurons on prefrontal neurons, the potential for recovery from cocaine behavior can be reduced.

(A) Prefrontal neurons by inhibiting 561nm light, can significantly reduce the number of active lever in rats; (B) DAB staining display

Prefrontal region ArchT-GFP showed high expression; (C) decreased prefrontal area sought to be obtained by light irradiation inhibiting cocaine

The effect is significantly better than the effect of not receiving light , and is close to the effect obtained by subtractive administration.

(Michael T. Stefanik, 2013)

Figure 7. Schematic diagram of the location of the fiber optic terminal in the experiment. The number indicates the number of millimeters (mm) from the coronal section of the front chimney.

(A) AAV injection of amygdala , located in the fiber end position of the amygdala; (B) AAV injection of amygdala, located in the voltazone

The fiber end position of the nuclear central core; (C) the position of the fiber end position of the prefrontal lobe after AAV injection of the amygdala.

(Michael T. Stefanik, 2013)

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