Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABA_A Receptors

The aim of this procedure is to set up co cultures between embryonic, medium spining neurons and HEC 2 9 3 cells stably expressing GABA A receptors to study the molecular mechanisms that coate GABAergic synapse formation in vitro. This is accomplished by first dissecting the stri AUM from E 16 to 17 rats or mice embryos. In the second step, the stri AAL cells are plated onto cover slips.

In a 24 well plate next HEC 2 9 3 cells stably expressing GABA A receptors are transfected with an M cherry fluorescent indicator. In the final step, the transfected cells are co cultured with the medium spiny neurons at 14 days in vitro for 24 hours before fixation. Ultimately, the formation of the synapses between the two cell subsets can be visualized by immunofluorescence and confocal microscopy.

The implications of this technique extend towards the therapy of many neurological disorders as GABAergic synapses coordinate the activity of neuronal cells and are responsible for many fundamental structural and functional elements of neuronal circuits in the central nervous system. Before starting the experiment incubate overnight 13 millimeter polyol lysine coated cover slips. In a 24 well plate, a 37 degrees Celsius and 5%carbon dioxide with humidity the following day aspirate the excess polyol lysine and wash the cover slips with two ten second and two five minute long washes in sterile water.

Then incubate the cover slips overnight in laminin under the same conditions on the day of the experiment. First place a pregnant mouse in the supine position onto an ethanol disinfected dissection pad. Then pinch the skin with tweezers and cut around the abdomen through the skin muscle and peritoneum to reveal the internal organs and the uterus.

Next, extract the embryonic day 16 to 17 embryos from the uterus and place them in a Petri dish containing chilled PBS. Transfer the embryos into the laminar flow hood to capitate the heads into a new Petri dish with chilled HBSS and then use curved and straight forceps to dissect out each of the brains. Collect the brains in a new Petri dish containing chilled HBSS.

And then for each brain, separate the two cerebral hemispheres and carefully remove the meninges. Next, cut along the line of the hippocampus and peel back the cortex to reveal the striatum. A sated white structure at the anterior of the hemisphere dissect out the stratum very carefully to avoid including tissue from the corset and place into a new Petri dish.

Containing chilled HBSS Cut the ator into very small one to two millimeter pieces using the curved forceps. Then use a fire polished pasta pipette to transfer the tissue into a sterile 15 milliliter centrifuge tube. Reduce the volume of tissue suspension to a total of one milliliter and pipette the solution a further four to six times bio polish the tip of the pipette to approximately 30%of its original diameter to tri rate the solution of further four times when the solution appears homogenous built to the cell slurry through a 100 micrometer nylon strainer into a sterile 50 milliliter centrifuge tube.

Then after counting plate seven times 10 to the power of four cells into 500 microliters of neuronal culture, medium power well into a 24 well tissue culture plate. Agitate the well's left to right and then incubate the cells for 14 days as described in the accompanying text protocol. Aspirate the cell culture medium and wash each well twice briefly with PBS to detach the cells from the six well plate.

Add tripsin EDTA solution to each well and incubate a 37 degrees Celsius and 5%carbon dioxide with humidity for five minutes to stop the reaction and quench the in add one milliliter of cell culture medium to each well and transfer the contents to a 15 milliliter centrifuge tube and centrifuge the cells at 440 times G for five minutes At room temperature, remove the supinate and fully resuspend the pellet in 500 microliters of neuronal culture. Medium by pipetting up and down gently after counting seed, three times 10 to the four cells per well in the 24 well tissue culture plate containing the neurons and agitate the plate to disperse the cells. Incubate the co cultures at 37 degrees Celsius in a humidified 5%carbon dioxide atmosphere for 24 hours.

In these images, cell surface expression of the alpha one, beta two, and gamma two GABA AR subunits or the alpha one, beta three and gamma two GABA AR subunits was observed with a high degree of colocalization on the HC 2 93 cells.Here. Analysis of the cell to cell contacts between the HC 2 93 cells and the neuronal cells at 14 days in vitro indicates that GAD 65 positive GABAergic Axon terminals form only sporadic contacts with the control HEC 2 9 3 cells compared to the numerous synapse like contacts formed between HEC 2 93 cells expressing the alpha one, beta two gamma two gabaa receptors. In these experiments, only the vesicles actively engaged in the neurotransmitter release were labeled with the antis synap toin antibody indeed view.

If any of the contacts between the control HEC 2 9 3 cells and the medium spining neuron terminals were active. This is illustrated by the lack of colocalization between the presynaptic GAD 65 synaptic tein fluorescence and the mCherry fluorescence within the HEC 2 93 cells. In contrast, many active contacts were formed between the mediums binding neuron terminals and the alpha one, beta two gamma two gabaa receptor expressing HEC 2 93 cells as revealed by a high degree of colocalization between the GAD 65 synap Taman and the M cherry fluorescence expressed specifically in the HEC 2 93 cells.

In these final images, HEC 2 93 cells stably expressing a different subtype of GABA AR were co cultured with medium spiny neurons. Analysis demonstrates numerous synapse like contacts formed between synapse in one positive GABAergic acts on terminals and alpha one B two three gamma two expressing HEC 2 9 3 cells after 24 hours. In co-culture, This technique has the potential to pave way for researchers in the field of molecular neuroscience to explore the role of various synaptic adhesion molecules involved in the formation of GABAergic synapses.