When a particular protein function is modified by gene manipulation, coordination of irritative failure and motor running impairment is considered a necessary condition for causal relationship between LTD and motor learning. Here we demonstrate the use of multiple protocols to assess LTD which can be induced via compensatory mechanisms in gene-manipulated animals. Before harvesting the brain, chill and oxygenate two 50 milliliter beakers of ACSF on ice.
When the solution temperature reaches below four degrees celsius, add 50 microliters of one millimolar tetrodotoxin to one of the ice cold beakers. To harvest the brain, hold the head and use ophthalmological scissors to cut the superficial skin along the midline. Manually retract the skin to widely expose the skull surface and use the scissors to cut along the skull horizontally from the major spinocerebellar hole to just above the eyes and ears before cutting the skull along a line above both eyes.
Use a scalpel to cut the brain at the middle of the cerebrum and isolate the caudal part of the brain, including the cerebellum, from the skull. Immerse the sample in the ice cold beaker of ACSF and adjust the bubble tubing so that it does not stir the brain block in the beaker. After at least seven minutes, use a spatula to pick up the brain block and use a piece of filter paper to absorb any excess ACSF.
Mount the tissue ventral side down onto a two by two centimeter piece of agar with an appropriate medical adhesive. Use a blade to cut out the right hemisphere of the brain tissue as parallel to the dendritic plane of the Purkinje cells as possible. Cut and remove the other side of the hemisphere and cut the brain between the superior and inferior colliculi.
Cut off the spinal cord and glue the right side of the trimmed cerebellum with the agar block onto the pre-chilled specimen tray. Then, tilting the specimen tray, pour ACSF onto the sample to fix the tissue and to wash away any excess glue. To slice the brain sample, orient the specimen such that the dorsal side of the cerebellum is at the front and pour enough of the ice cold cutting ACSF supplemented with tetrodotoxin to completely immerse the cerebellum.
Place a gas tube into the cutting solution and initiate bubbling with an oxygen-carbon dioxide gas mixture. Using fine tweezers and magnifiers, remove the arachnoid mater and excise the cerebellar peduncle. After removing the brainstem and agar block, rotate the tray 180 degrees so that the dorsal surface of the cerebellum faces the razor blade of the vibratome and adjust the first cutting location.
Set the vibratome amplitude to 5.5 and frequency to 85 Hertz, the speed to three to four, and the slice thickness to 300 micrometers. As the cerebellar slices are obtained, use a nylon net to transfer the sections to an acrylic incubator in a 26 degree celsius water bath and completely immerse the samples in fresh oxygenated ACSF for at least one hour. For whole cell patch clamp reporting, dissolve one micromolar picrotoxin in ACSF by ultasonication for three minutes before perfusing a 30 degree celsius recording chamber with the toxin solution at a two milliliters per minute flow rate.
After a few minutes, transfer a cerebellar slice to the recording chamber and fix the tissue with a platinum weight and nylon threads. Then fill a stimulating electrode with fresh ACSF. To stimulate the parallel fibers, place the stimulating electrode on the surface of the molecular layer about 50 micrometers from the Purkinje cell layer.
For stimulation of the climbing fibers, place the stimulating electrode at the bottom of the Purkinje cell layer and use a microloader to fill a recording electrode with eight microliters of 0.45 micrometer filtered potassium or cesium based internal solution. Apply a weak positive pressure to the recording electrode before immersing the electrode into the ACSF. The electrode resistance should be two to four megaohms and the liquid junctional potential should be corrected.
Approach the healthy bright cell body of a Purkinje cell with the recording electrode and push the surface of the Purkinje cell slightly. Then stop applying the positive pressure and apply negative pressure until a gigaohm seal is formed. Then use negative pressure to establish a whole cell configuration, maintaining the membrane potential at minus 70 millivolts and applying a minus two millivolt 100 millisecond pulses at 0.1 hertz, continuous monitoring of the input resistance, series resistance, and input capacitance.
For long term depression induction, stimulate the molecular layer with a 0.1 millisecond pulse and apply a double pulse stimulus to identify the parallel fiber excitatory post-synaptic currents. A paired pulse facilitation and gradual increase in amplitude relative to the increase in stimulation intensity should be observed. To record the test response, apply a single 0.1 hertz pulse and adjust the intensity of the stimulus so that the evoked amplitude is around 200 picoamps.
Stimulate the climbing fibers at the bottom of the Purkinje cell layer and to identify the parallel fiber excitatory post-synaptic currents elicited by the climbing fiber activation, apply a double pulse stimulus. A paired pulse depression should be observed in an all or none manner in correlation with the increase in stimulation intensity. In this representative experiment, a conjunction of one parallel fiber stimulation and one climbing fiber stimulation under current clamp conditions were used for the slice preparation.
The shape of the complex spike elicited by the conjunctive stimulation was similar to that elicited by the climbing fiber stimulation alone with the first steep spikelet followed by two to three spikelets. A similarly shaped complex spike was observed when one parallel fiber stimulation was followed 50 milliseconds later by a conjunctive second parallel and climbing fiber stimulation. In this test under voltage clamp conditions using a cesium-based internal solution, a parallel fiber stimulation was followed 50 milliseconds later by a concomitant application of a second parallel fiber stimulation and somatic depolarization.
An inward current was elicited upon somatic depolarization from minus 70 to zero millivolts and the tail current was also evoked after repolarization. Finally, five parallel fiber stimuli at 100 hertz were given simultaneously with the somatic depolarization under voltage clamp conditions. Again, a repetitive generation of inward currents was elicited during depolarization and the tail current was induced after the repolarization.
To assess the relationship between cerebellar LTD and motor learning in gene-manipulated animals, multiple protocols should be used to induce LTD under cozy physiological conditions. If cerebellar LTD is visualized in gene-manipulated animals after motor learning, the causal relationship between them can be more directly examined.
