This protocol is one of the first demonstrations of the direct application of electrical noise to individual neurons in tissue slices. In order to observe neuronal responses to electrical stimuli. The main advantage of this technique is, that we're able to assess a specific set of stimulus parameters on individual neurons.
This technique allows for the optimization of stimulus parameters to target particular, functional neuronal types. This has implications for the development of better therapeutics for balance disfunction. Before beginning the brainstem extraction, Equilibry s-ACSF with CARPOGEN and cool the solution at minus 80 degrees Celsius for 25 minutes until an ice slurry has formed.
After the solution has chilled, use a number 22 rounded razor blade to make a sagittal skin incision in the skull of a three to five week old C-57 black six mouse and use the pointed end of a pair of standard pattern scissors to make a small incision, starting at the lambda, along the sagittal suture. Using a pair of shallow bend Pierson von Jours, carefully reflect away the repaired parietal and exoccipital bones, and bathe the brain in ice cold s-ACSF. Then, isolate the brain stem from the forebrain and its boney encasing using a number 11 straight razor blade to cut down the parietal exoccipital sulcus and at the caudal negula.
Mount the isolated brain stem ventral and down on perviously cut trapezoidal polystyrene block, and use a piece of tissue paper to remove any excess fluid from around the dissected tissue. Use cyanoacrylate glue to fix the polystyrene block to the cutting stage with the attached brainstem rostral and down. Use an advanced speed of 0.16 millimeters per second and a vibration amplitude of three millimeters to obtain 200 micrometer transverse slices of the MVN.
Then use a plastic trimmed pipette to transfer the tissue slices on to a filter paper disk incarbinginated ACSF at 25 degrees Celsius for at least 30 minutes. For whole-cell patch clamp electrophysiology, first pull micro pipettes with a final resistance that will range from three to five mega Oms when filled with a potassium-based internal solution and placed in the bath. To obtain whole-cell patch clamp recordings from individual neurons in the MVN, transfer a single tissue slice from the incubation chamber to the recording chamber of a standard electrophysiological set up, and use a nylon thread on a U shaped weight to secure the slice.
Continuously perfuse the recording chamber with carbonginated ACSF at 25 degrees Celsius at a flow rate of three milliliters per minute and fill a micro pipette with internal solution. Apply a small amount of positive pressure using a pipette to push debris away from the pipette tip. Using a lower power objective, locate the MVN before switching to a high power to locate individual neurons within the MVN.
Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip, and use the micro manipulator to move the pipette toward the selected neuron. A small dimple should form on the neuronal membrane. Release the positive pressure, and apply a small amount of negative pressure.
Once a one giga Om seal has been achieved, apply gentle short and sharp negative pressure to the pipette holder through the suction port to rupture the membrane and to create a whole-cell configuration. Then obtain whole-cell current clamp recordings according to standard protocols. To apply stochastic and sinusoidal noise to individual medial vestibular nucleus neurons, set the range of amplitudes from three to 24 pico amps to determine the neuronal threshold and firing rate and group the higher and lower stimulus intensities to determine the sensory threshold.
Next, calculate the average firing rate over the 10 second period, during which the depolarizing current step will be injected for each individual current level. Use the average firing rate values to generate a firing rate versus current plot. Then perform a linear regression analysis to determine the gradient of the line of best fit to determine the neuronal gain.
Neither sinusoidal nor stochastic noise changes basil firing rates of MVN neurons compared to control no noise recordings. In this experiment, the selected noise level of 6 pico amps is sub threshold, as it can be observed that the average firing rate begins to increase from the experimental threshold of 12 pico amps. This threshold was determined objectively by grouping the stimulus levels above and below the experimental threshold.
The neuronal gain was evaluated by subjecting the neurons to a suite of depolarizing current steps from zero to 50 pico amps in 10 pico amp increments with and without noise. Indeed, sinusoidal and stochastic noise applied at sub threshold amplitudes of six pico amps can alter the gain of MVN neurons. The established stimulus parameters can be applied to the nerve innovating the individual neurons to provide a more naturalistic stimuli.
