With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed. The ultimate goal of optogenetics is the modulation of neuronal circuitries with high temporal and spatial resolution in the behaving animal.

In contrast to pharmacological or electrical manipulations, optogenetics allows for the precise control of specific cell types on neuronal circuitries. Optogenetics is the perfect way to target specific cell types in specially defined region during behavior, and to scrutinize the direct effect of changes in such microcircuits. We will now present a step to step guide how to prepare and plant and conduct optogenetics in a freely moving mice.

For the preparation of the optical implant, place a ceramic ferrule flat side up in a bench vise. Strip the code of a 200 micrometer diameter glass fiber, with a fiber stripping tool and cut two to three centimeters non pieces with a ceramic fiber scribe. Place the piece of glass fiber into the ceramic ferrule then place a drop a superglue at the flat side with an injection cannular.

On the round side of the ceramic ferrule, cut the glass fiber as short as possible and place the pre-implant at a ferrule polishing puck to polish the round side on four different publishing papers. Afterwards, cut the glass fiber on the flat side of the ceramic ferrule, to the length needed for implantation. Use a mouse brain atlas to calculate the length of the implant.

The implant has to end directly above the region of interest. Prior to the implantation, anesthesia is applied. The precise and aesthetic technique is described in the manuscript.

When the mouse reaches a deep state of anesthesia, place it on a heating plate and fix the head in a stereotactic frame. Fix the nose and the teeth in the front and the ears on both sides. Apply an algesia with a cuprofen subcuntaneously into the back of the mouse and apply opaque eye ointment on both eyes to protect them from drying.

Moisten the hair on the scalp with a wet paper towel and then cut it off using scissors. Make sure to remove all the loose hair with a wet paper towel. Use a cotton stick to disinfect the scalp with a tincture containing iodine.

Raise the scalp about the region of interest with a tweezer and cut one centimeter along the midline. To roughen the skull for implantation, apply a small amount of phosphoric acid on the scar and let it take effect for 15 seconds. Remove all acid with a cotton stick and rinse the scar with sodium chloride twice.

For proper injection, calculate the F factor for individual coordinates. Place a glass cannular in the stereoctatic frame and locate it directly above bregma. Now zero the coordinate system and move the glass cannula to Lambda.

Calculate the F factor with the following formula. Bregma minus Lambda divided by 4.2 equals the F factor. Exact coordinates are very important for injection and implantation because otherwise, if the wrong structure is stimulated then the behavioral effects are unspecific.

All mice has minimum deviations in the size of their skull, therefore, the coefficient is absolutely mandatory. Use the adjusted coordinates to find the location on the scar directly above the structure of interest. Use an injection cannula to drill a hole into the skull, by rotating the cannula on the spot.

To take a virus solution into the glass cannula connected to syringe, place a drop of sodium chloride on the skull and a piece of pirophen on top. Place a drop of two microliter virus solution onto the pirophen and lower the tip of the glass cannula into the virus. Apply negative pressure and wait until the virus solution is taken up by the cannula.

Zero to that coordinate when the tip of the virus with cannula is at the level of the skull and slowly lower it into the borehole to the lowest position of the injection side. Apply a small amount of positive pressure with the syringe until the meniscus is lowered. Let the virus spread for two to three minutes before moving the glass cannula upwards to the next position.

Remove the glass cannula very slowly and discard it after the final injection. Now prepare the skull for implantation. Dry the skull with compressed air, apply the primer with this corresponding stick and let it dry for 15 seconds.

Apply the bond with the same stick and cure it for 20 seconds with UV light. Place the implant in the corresponding holder and position the tip of the glass fiber directly above the borehole and lower it carefully. Stop lowering the implant when the remaining valve of superglue touches the skull.

The implantation of optical fibers can also be made for bilateral structures like the hippocampus. This enables slight stimulation in both hemispheres at the same time or allows the stimulation of two different structures. It is also possible to inject the virus at a location different from the optical fiber implant.

If the injection and implantation are being done in different regions, drill all the necessary holes after applying phosphoric acid but before the two component adhesion. Check again if the skull is still completely dry, then apply fluid dental cement around the implant and in the surrounding area then cure for 20 seconds with UV light Apply two more lesser cement in completely fur free and dried skull area. Cure every layer with UV light.

To finish the surgery apply iodine anointment on the whole wound. Release the nose and ear fixation, bring the mouse into a fresh cage and place it under a heating lamp to avoid the loss of body heat. Check it's health status at least once a day and it will post operative and algesia after three days if necessary.

After two weeks of recovery, mice can be used for behavioral experiments. Open the Pulser Software and select the USB com port that light source is plugged into. Choose operation mode three to allow an external software to control the light source.

Choose the correct adjustment for 20 Hertz stimulation with five millisecond light pulse. To locate division to communicate with the pulser, press on start sequence. This status will remain until the experiments are finished.

Now, create a new experiment in EthoVision XT, go to file, choose new from template and select apply a predefined template. Choose live tracking and select the camera with source and confirm the connected Puslar Gen-Eye cam. Choose mouse as the animal which should be recorded.

Select the arena template, open field square and the zone template center, border, corners. Confirm one subject which should be tracked and select center point, nose point and tail base. Confirm the animal color compared to the background is darker and the recommended sample rate to finish the step.

Name the experiment appropriate and choose a location to save. Until you find the arena settings, the camera will automatically open a background image. Adapt the predefined zones to the real arena by using the arrow and the two symbols on it's right.

If some zones are unnecessary, delete them. Press draw scale to calibrate and put a line from one corner of the maze to the other. Enter the length of the real distance in centimeters.

Repeat that for the other axis. To define the trial control settings, prepare the main rule by adjusting the condition time to 20 minutes. The condition for Star Trek should be, when subject is in arena for two seconds, for an automatic status of the tracking when the mouse is in the arena.

To create a sub rule for the light stimulation, go to structures, more and select sub rule. Place it below the main rule and spread out the two boxes. Go to conditions, time and give it a name like light on one, adjust condition is met with after five minutes.

Place the box directly behind the rule begin box of the sub rule. Go to action custom hardware and name it with light on one. Select action to perform as:I'll put one high.

Place the box directly behind the condition box. Repeat the steps to program light off to in light onto. Now go to structures, sub rule reference and check that the reference belongs to the correct sub rule.

Choose start conditions as without delay and start conditions as execute once per start condition. Place the reference box between the extra books one and condition books two of the main rule. Now define the detection settings to show the system what it should track.

Place a test miles into the arena and select automated setup. Choose rodent as animal type and use the mouse cursor to draw a box around the mice in the arena. Confirm the results of care question with yes.

For the open field experiment, bring the experimental mouse to the behavior room right before the experiment to ensure a proper level of anxiety. Couple the mouse be a slit of the light source by pressing it gently to the grid of the cage. Place it into a waiting cage with fresh litter for 10 minutes to get acclimatized to the light cable.

Start acquisition by pressing the stop button and you to vision XT.Transfer the mouse from the waiting cage into the upper left corner of the open field. Leave the visual field of the mouse during the experiment and keep calm. After 20 minutes, when the experiment is finished, remove the mouse from the maze, disconnect the light cable and put it back into its own cage.

By a check visualization, the differences in center time between light off and light on faces can be directly seen. The mouse spends less time in the center when the light is on. For the Barnes maze experiment, bring all experimental mice into the behavior room around one hour before the experiment.

Prepare the Barnes maze by closing all holes except one, and now which an escape box is placed. Connect one mouse in the light source at both implants and place it directly into the middle of the Barnes maze. Press start and need to visit next T and remove the carton box.

Be prepared to stop the trial manually just in case the software does not recognize the whole transition. Take the mouse out of the maze and remove the connection to the light cable. When performing, learning, and memory task, not only the immediate effect of the manipulation is investigated, but also the long-term effect of manipulation during acquisition, consolidation or retrieval.

For data analysis, you find the treatment condition treated or controlled, go to nesting and select trial control state. Choose the state interval from the element action Star Trek to the element action light goes on one. Place the nesting box between the filter box treatment and the corresponding result box.

This defined interval is of one. Repeat the steps for intervals on one of two and on two as also for the control group. Also define the parameters to analyze in the analysis profile.

Choose the dependent variable in zone and select the zone center, the body point, center point, and go on trial statistics. Select frequency, cumulative duration, and let there to see the first. Also add distance move as variable.

With this profile, the data for the time spent in the center, center entries and told to distance move is available. To extract data from each division, go to results and select statistics and charts, press calculate to see the analyzed data. Press export data and select the trial statistics and the location to save.

The exported data is saved as an excel file and with individual values for every mouse. Additionally, go to heat map visualization and press plot heat maps, select trials on the right to see individual heat maps for every mouse and trial. Do a right click on the mouse and export the heat maps as images.

Later, open the excel file the computer and calculate the mean and standard euros SCM for all four trials and every measured conditioning group. To generate graphs and Sigma plot, copy the means and SCM into the correct order from the extra file to Sigma plot. The rows have to contain the data for off one, on one, and the columns contain trial, mean and SCM hertz.

Select all three columns and go to create graphs. Select the bar box and choose ungrouped box with arrow. Label the whole graph, go to home, select the graph box and press export.

Select the destination folder and choose meta file as format. To calculate statistics for obtained data, copy right data from Xcel off one on one, et cetera, and to single columns of Sigma plot. Mark the columns you want to compare and go to analysis, choose T test and press run.

Finally, behavioral data can be shown divided into let off and on trial with additional heat map specialization. In the open field experiment, during light stimulation of Channelrhodopsin2 and pyramidal neurons, the center duration is decreased, which indicates increased anxiety levels. Via immunohistochemistry, the injection site and expression of the virus can be determined, also at the specificity at the special Cre-dependent mouse line can be validated.

One can clearly see that the light stimulation of Channelrhodopsin2 improve mind neurons of this specific cortex region increases anxiety levels, compared to baseline anxiety before that stimulation. This immediate effect of the manipulation on behavior is the reason why optogenetic is used today for so much research questions regarding behavior. This procedure can be used for Optogenetic modulation of any desired cell type or neuronal circuitry in the mouse brain.

By combining this procedure with suitable behavioral tests for your research, a deeper insight into neuronal circuitries involved in behaviors and diseases of interest can be gained.

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