Here we present the protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and electrophysiological investigation of retinal geniculate and cortical geniculate synapse function. Retinal ganglion cell axons enter the lateral geniculate nucleus far away from the cortical inputs. This allows the separate stimulations of retinal geniculate and cortical geniculate synapses, which have very different functional properties.
To begin this procedure, prepare two slice chambers, one filled with 100 milliliters of the dissection solution and the other with 100 milliliters of recording solution. Place the two beakers into a water bath at 37 degrees Celsius, and bubble the solutions with carbogen for at least 15 minutes before the dissection. Next fill a plastic beaker with 250 milliliters of near-ice-cold dissection solution, and bubble it with carbogen for at least 15 minutes before use.
Then fill a plastic Petri dish with the cold dissection solution for brain dissection. Place a piece of filter paper in the lid of the Petri dish, and fill it with a small amount of dissection solution. Subsequently cool down the dissection chamber of the vibratome.
Cut the skin of the head from caudal to nasal, and keep it opened with fingers to expose the skull. To obtain the forebrain, first cut the skull along the posterior/anterior midline. Then cut two more times through the coronal suture and lambdoid suture.
Remove the skull between the coronal suture and lambdoid suture to expose the cerebrum. Cut the olfactory bulb, and separate the forebrain from the midbrain with a fine blade. Remove the brain from the skull with a thin spatula, and place it on a filter paper in the lid of the Petri dish.
To preserve the integrity of the sensory and cortical inputs to the dorsal lateral geniculate nucleus in the brain slice, separate the two hemispheres with a parasagittal cut at an angle of three to five degrees. Afterward dry the medial lateral planes of the two hemispheres by placing them on a filter paper. Then glue them onto the cutting stage with an angle of 10 to 25 degrees from the horizontal plane.
Place the stage in the center of the metal buffer tray, and gently pour the rest of the ice-cold dissection solution into the buffer tray. Perform this step carefully, since pouring too hard might remove the pasted brain. Next keep the buffer tray in the ice-filled tray to maintain the low temperature during dissection.
Section 250-micrometer slices with a razor blade at a speed of 0.1 millimeter per second and an amplitude of one millimeter. Transfer the slices to the holding chamber filled with oxygenated dissection solution at 34 degrees Celsius for around 30 minutes, and then allow the slices to recover in the recording solution at 34 degrees Celsius for another 30 minutes. After recovery, remove the slice-holding chamber from the water bath, and keep the slices at room temperature until used in experiments.
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 stimulation pipette on the slice before patching the cell with a recording pipette. To investigate the retinal geniculate synapses, place the stimulating pipette directly on the optic tract where the axon fibers from retinal ganglion cells are bundled. To analyze the cortical geniculate synapses, place the stimulating electrode on the nucleus reticularis thalami, which is rostroventrally adjacent to the dorsal lateral 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 megaohms for analysis. For biocytin labeling, maintain the whole-cell configuration for at least five minutes to allow the diffusion of biocytin into the distal dendrites. After recording, gently remove the pipette from the cell so that the soma is not destroyed.
Prepare a 24-well cell culture plate. Fill each well with 300 microliters of 4%PFA. Take care to not contaminate anything with PFA that will be in contact with the slices to be recorded.
Transfer the slices from the recording chamber to the PFA-containing plate with a rubber pipette, and fix them overnight. The next day, replace the PFA with PBS. Keep the slices in PBS at four degrees Celsius for future processing.
To stain the cells, wash the slices with fresh PBS three times. Block nonspecific binding sites by incubating the slices in the blocking solution for two hours at room temperature on an orbital shaker. Then discard the blocking solution, and incubate the slices with streptavidin Alexa 568 diluted in the blocking solution at four degrees Celsius overnight on an orbital shaker.
The next day, discard the antibody solution, and wash the slices three times with PBS for 10 minutes each at room temperature on an orbital shaker. Then wash the slices with tap water before mounting. Place the slices from one mouse on one glass slide.
Ensure that the stained cells are present on the upper surface of the slices. Absorb the water around the slices with tissues, and then leave the slices to dry for approximately 15 minutes. Subsequently apply two drops of mounting medium on each slice, and mount a cover slip on the slide without producing air bubbles.
Store the labeled slices at four degrees Celsius after viewing them with a confocal microscope. This IR-DIC image shows the soma of a dorsal lateral geniculate nucleus relay neuron with a tip of the patch pipette. Biocytin staining enables us to gain a view of the recorded neuron.
Different from interneurons, which have bipolar morphology, relay neurons have multipolar dendritic arbors containing more than three primary dendrites. Three-dimensional reconstruction of a biocytin-labeled relay neuron was then generated, which shows a radially symmetric dendritic architecture. Preserve the retinal geniculate inputs and the cortical geniculate inputs in the same brain slices are most important in this protocol.
Following this procedure, we can, for example, also investigate the inhibitor inputs from the nucleus reticularis thalami onto dorsal lateral geniculate nucleus neurons. Remember to wear gloves when fixing the brain slices with PFA, as PFA is hazardous.
