An Electrophysiological Study of Retinogeniculate and Corticogeniculate Synapses in Mouse Brain Slices
Transkript
In this procedure, pull the recording pipettes using borosilicate glass capillaries and a filament puller. Next, pull the stimulating pipettes using the same protocol but break the tip slightly after pulling to increase the diameter. Subsequently, fill the recording pipettes with intracellular solution, and biocytin and fill the stimulating pipettes with recording solution.
Then, place the slices in the recording chamber, and continuously perfuse them with oxygenated recording solution at room temperature. Visualize the slices with an upright microscope equipped with IR-DIC video microscopy. Check all the slices and select the ones that display intact optic tracts. Place the stimulating pipette on the slice before patching the cell with the recording pipette.
To investigate the retinogeniculate synapses, place the stimulating pipette directly on the optic tract where the axon fibers from retinal ganglion cells are bundled. To analyze the corticogeniculate synapses, place the stimulating electrode on the nucleus reticularis thalami, which is rostroventrally adjacent to the dorsolateral geniculate nucleus. Once the recording pipette is immersed in the recording solution, apply a five-millivolt step to monitor the pipette resistance.
Set the holding potential to zero millivolt, and cancel the offset potential so that the holding current is zero picoampere. Approach the cell with a recording pipette while applying positive pressure. When the pipette is in direct contact with the cell membrane, release the positive pressure, and set the holding potential to minus 70 millivolts.
Then, apply slight negative pressure to allow the cell membrane to attach to the glass pipette such that a gigaohm seal forms. Compensate the pipette capacitance and open the cell by applying negative pressure pulses. To investigate the synaptic function, apply 0.1-millisecond current pulses via the stimulation pipette. Monitor the series resistance continuously by applying a five-millivolt step.
The series resistance can be estimated by dividing five millivolts by the peak amplitude of the evoked current. Use only the cells with a series resistance smaller than 20 mega ohms for analysis.
