Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

The overall goal of this procedure is to generate a gene chimeric preparation of ventral hippocampal accumbens circuit in vitro that allows direct live imaging to identify and analyze both pre and postsynaptic components of the synaptic transmission. This is accomplished by first dissecting out ventral hippocampal tissue strips containing the CA one CIC region. The second step is to cut the ventral hippocampal strips into small thin slices and then plate those micro slices at the center of the cover slip inside the 24 well tissue culture plate overnight.

Next, dissect out the nucleus accumbens from the brain tissues. The final step is to dissociate the nucleus accumbens strips into disperse neurons with trypsin and add the nucleus accumbens neurons to the cover slip plated with ventral hippocampal implants and incubate for five to seven days. Ultimately, presynaptic localization of nicotinic acetylcholine receptors and nicotinic acetylcholine receptor mediated calcium signaling and neurotransmitter release.

Along ventral hippocampal axons can be identified and monitored by spinning disc confocal live cell imaging. The main advantage of this technique is that this micros slices dissociate neuron co culture preparation allows easy recognition of their pre-synaptic exons and post-synaptic targets. In vitro The co-culture preparation recreates the ventral hippocampal to accu circuit in vitro.

This allows direct live imaging of either or both the presynaptic and the postsynaptic compartments. This in vitro synaptic preparation permits high resolution imaging of the spatial and temporal effects of activation of presynaptic receptors on glutamatergic synaptic transmission. As a result, one can obtain information on both pre and post synaptic mechanisms that contribute to glutamatergic synaptic plasticity Before tissue collection.

Pre incubate 12 millimeter poly de lysine, laminated coated glass cover slips in the 24 well tissue culture plates with 500 microliters of culture media at 37 degrees Celsius for at least 30 minutes. Then remove the media before plating the micro slices to collect tissue for culture. Remove the top of the skull to expose the whole brain of each sacrificed pup under a dissecting microscope.

Starting at the juncture where the cerebral cortices apart from each other, cauley, obtain a coronal section containing the posterior cortices, including the ventral hippo.Campi. Next, dissect the ventral hippocampal tissues containing the ca one seum regions out. After that, transfer the tissues to a 35 millimeter culture dish containing cold culture media.

Subsequently cut the tissues further into small thin slices. Plate the slices with a fire polished pasture pipette at the center of the cover. Slip with 50 microliters of media to facilitate the attachment of the x explants.

Then plate the ventral hippocampal micro slices on the cover slip after the X explants have settled onto the cover slip gently add 100 microliters of additional media by the wall so that the level of the media is high enough to completely cover the explan on the periphery, but not those at the center of the cover slip. Then incubate the explan overnight in 5%CO2 at 37 degrees Celsius before the dispersion of the nucleus accumbens neurons. Now obtain the nucleus accumbens tissues from embryonic day 18 to postnatal day one while type mice cut them into small pieces, then transfer them to a 15 milliliter tube.

Treat the tissues with two milliliters of point 25%trypsin for 15 minutes at 37 degrees Celsius. Subsequently, wash the tissues three times with five milliliters of four degrees Celsius washing media and allow the suspension to rest for five minutes in each wash step. Then wash the tissues in five milliliters of four degrees Celsius culture.Media.

Once next, dissociate the cells by gentle ation in two milliliters of culture media with a lightly fire polished pasture pipette. Afterward, transfer the cell suspension to another 15 milliliter tube and centrifuge at 2000 RPM for five minutes. After five minutes, remove the S supernatant and resuspend the cells in culture media at one milliliter per six pups then disperse the cells by gently pipetting the media several times with a fire polished pasture pipette.

At the end, add point 25 milliliters of the dispersed nucleus accumbent cells to each cover slip with the ventral hippocampal explants. Place the culture plates on dampen sterile gauze pads to mechanically stabilize and maintain humidity in order to facilitate synapse formation. Then incubate the plates at 5%CO2 at 37 degrees Celsius.

In this procedure, transfer the cultures to an imaging chamber one at a time. Mount the chamber on the spinning disc confocal microscope. Continuously perfuse the sample with HBS cocktail at one milliliter per minute at room temperature.

Next, stop the profusion and stain the culture with 10 micromolar FM 1 43 in 56 millimolar potassium A CSF for 90 seconds. Then wash the external dye away for 15 minutes with calcium free HBS containing 0.1 millimolar of A-D-V-A-S-E-P seven. After that, de stimulate the culture with 56 millimolar potassium A CSF without FM 1 43 for 120 seconds.

For detaining. Reload the culture with FM 1 43 under the same conditions and wash again with calcium free HBS containing A-D-V-A-S-E-P seven for 15 minutes. Now acquire the FM 1 43 fluorescent images with a spinning disco confocal microscope equipped with a plan apochromatic objective and capture images with a CCD camera.

Collect the baseline FM 1 43 fluorescent images every two seconds for one minute as pre nicotine control. Then apply one micromolar of nicotine by rapid perfusion at two milliliters per minute for one minute before washing the nicotine out with HBS cocktail. Keep capturing the time lapse images before, during, and after nicotine application every two seconds for five minutes.

In this step, rinse the cultures quickly with normal HBS. Then load the cultures with five micromolar flu oh four calcium indicator and 0.02%onic F1 27 in two milliliters of HBS for 30 minutes at 37 degrees Celsius and 5%CO2. After that wash flu oh four solution out with HB S3 times each wash for five minutes.

Return the cultures to the incubator for at least 30 minutes. Then transfer the cultures one at a time to the imaging chamber. Under same conditions as described in FM 1 43 based vesicular fusion procedures.

Collect the baseline flu oh four fluorescent images of axonal projections from the ventral hippocampal micro slices as pre nicotine control. Next, apply one micromolar of nicotine by rapid perfusion at two milliliters per minute for one minute, and then wash the nicotine away with HBS cocktail. Keep capturing the time-lapse images before, during, and after nicotine application every 10 seconds for 30 minutes.

This figure demonstrates directly that the projections from the ventral hippocampal micro slices are glutamatergic and that the contacts are made with dispersed GABAergic. Medium spiny neurons from nucleus accumbens, both alpha seven and non alpha seven nicotinic acetylcholine receptors were found along the ventral hippocampal axons, and specifically at the sites where ventral hippocampal projections contact nucleus accumbens neurons. It should be noted that the dispersed neurons from nucleus accumbens do not express alpha seven nicotinic acetylcholine receptors, which means that the receptor clusters at contacts are strictly presynaptic.

The time-lapse images of flu oh four calcium fluorescence along ventral hippocampal axons were acquired with a 60 x subjective water lens every 10 seconds for 30 minutes with a spinning disc confocal microscope. The acquired images were indicated on pseudocolor scale and played back as a video at two frames per second. This video shows nicotine application induced sustained flu oh four calcium fluorescence activity along live wild-type ventral hippocampal axons.

While attempting this procedure, it is important to remember plate the ventral hippocampal micros in a minimal volume of media and maintain the culture plates on dampen stereo growth paths. These steps are critical for the attachment of the micros, the outgrowths of the projections, and the formation of the synapsis. So this co culture allows one to vary the genotypes of presynaptic input separately from their postsynaptic targets.

In addition, it allows selective expression of exogenous genes in either compartment independently. This is great potential for dissecting pre versus post-synaptic mechanisms of synaptic plasticity. After you watch this video, you should understand how to generate gene chimeric synaptic co cultures that recapitulate the ventral hippocampal to accumbens circuit.

You should then be able to use live imaging techniques to analyze both pre and post-synaptic neurons.