Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

The overall goal of this method is to study dynamic morphological changes of axon branching and synapse formation in the thalamocortical projection. This method can help answer key questions in the field of neurobiology, such as new neural circuit formation. The main advantage of this technique is simultaneous observation of axon growth and the synapse formation in individual living cells.

This method can provide insight into the relationship between axon branching and synapse formation in the thalamocortical projections. It can also be applied to other neural circuits, such as intrinsic connection in the cerebral cortex. To begin this procedure, sterilize all the surgical instruments with 70%ethanol for 10 minutes or longer.

Next, dissect the whole brain quickly from a postnatal day-two rat under ice-cold anesthesia. Then, place the brain into a 100-millimeter petri dish containing 40 milliliters of cold Hank's Balanced Salt Solution. Under a binocular microscope, remove the pia matter carefully with fine forceps, and cut the primary visual and somatosensory cortex into 300 to 400-micron thick slices using microsurgical scissors.

After that, transfer the cortical slices onto the membrane filter of the culture insert using a disposable plastic pipette. Adjust the positions of the slices near the center of the culture insert with the disposable plastic pipette, and remove excess medium from the insert. Maintain the slices in the serum-containing culture medium at 37 degrees Celsius, and culture them alone for one day before placing a thalamic block from an E15 embryo next to it.

Adjust the level of the medium slightly below the surface of the slice, so that the slices can receive sufficient supply of both the gas and the medium. This is crucial for the viability of the slices. In this step, make the glass capillaries with an electrode puller.

Then break the tips of the glass capillaries for plasmid injection. For the electrode pipettes, grind and polish the tips of the glass capillaries with sandpaper. And then fire polish them to achieve an inner diameter of 50 to 200 microns.

Next, attach the electrode pipette and the ejection pipette to the manipulators. Transfer five microliters of plasmid solution to a petri dish. Then draw the solution into the electrode pipette with capillary action.

Insert the 0.2-millimeter diameter electrode into the electrode pipette. After connecting the ejection pipette to a one-milliliter syringe with a plastic tube, transfer five microliters of plasmid solution to a petri dish again. Then slowly draw the solution into the ejection pipette using the syringe.

Following that, transfer the coculture on the electroporation stage and insert the one-millimeter diameter electrode into the culture medium. Subsequently, place the ejection pipette on the cultured thalamic block. Push the syringe manually after the tip touches the surface of the thalamic block.

Place the electrode pipette on the thalamic block immediately after the retraction of the ejection pipette. Now, deliver the electrical pulses. Monitor the amplitude and the number of trains on the oscilloscope.

Afterward, retract the electrode pipette and return the dish to the incubator. To observe the sample, place the culture dish on the stage of an upright confocal microscope equipped with two filter sets for EGFP and DsRed. Then take images of 10 to 25 optical sections, at one to seven-micron step sizes, with a 20x long working distance objective lens.

Select individually-distinguishable labeled axons, which express both DsRed and Syp-EGFP. Shown here is an organotypic coculture of thalamic and cortical slices after 12 DIV. Here is a magnified image of the boxed region.

And here is a DsRed-labeled thalamic cell extending an axon into the cortical slice, and forming branches in upper layers. These images show the DsRed-labeled thalamocortical axon and Syp-EGFP puncta. Discrete accumulation of Syp-EGFP was observed along a single thalamocortical axon.

DsRed plus Syp-EGFP plasmids were introduced into cultured thalamic cells at one DIV using electroporation. This axon was imaged daily over four days. Axon branching and synapse formation were dynamic, with addition and elimination.

Following these procedures, electroporation with interesting genes together with Syp-EGFP and DsRed can be performed in order to investigate the role of the gene in axon branching and synapse formations. This technique paves the way for researchers in the field of the neurobiology to explore the mechanisms of dynamic changes of neural circuits.