The overall goal of the following experiment is to probe changes in network function of the brain areas affected by an experimental model of mild traumatic brain injury Experiments begin by administering a lateral fluid percussion injury to a mouse, which mimics both the pathology and symptomology of human mild traumatic brain injury. As a second step, a hippocampal dependent behavior, contextual conditioned fear response is used to assess brain injury induced cognitive impairment. Next, extracellular and intracellular recordings along with voltage sensitive dyes are used to determine the regionally specific changes in synaptic balance between excitatory and inhibitory synaptic transmission In the region of interest, the hippocampus, the results show subregion specific changes in net synaptic efficacy in the hippocampus based on differences between the control and brain injured mice across the three recording techniques described.
The main advantage of this approach is the ability to discern the effects of both the pathological state and the potential therapeutics on multiple physiological scales, allowing a more coherent picture of this circuit deficits. This method can help answer key questions about traumatic brain injury, such as specifically which neurons are most susceptible to injury, and therefore making the largest contribution to hippocampal dysfunction after injury. The implications of our approach extend toward the treatment of traumatic brain injury as it allows us to delineate first the underlying mechanisms, and then develop potential therapies to reinstate a droid physiology in areas that are damaged by brain injury.
In this procedure, anesthetize the mouse using a mixture of ketamine and xylazine given intraperitoneal. Next, perform a C craniectomy over the right parietal area using a tine with three millimeters outer diameter. Then secure the lure lock needle hub over the C craniectomy using cyanoacrylate and dental acrylic.
24 hours later anesthetize the mouse using isof fluorine via inhalation Once normal breathing resumes, but before the mouse has become sensitive to stimulation, deliver a 20 millisecond pulse of saline to the skull via the fluid percussion injury device. Remove the hub immediately after injury. Subsequently re anesthetize the mouse using isof fluorine and suture the scalp.
Handle the mouse on two consecutive days prior to conditioned fear response training. On the day of training, place the mouse in the conditioning chamber for three minutes before administering a 1.5 minute amp floor shock for two seconds. Then leave the mouse in the chamber for an additional 30 seconds before removal.
After a delay period of 24 hours, return the mouse to the conditioning chamber for five minutes, then assess freezing at five second intervals seven days after injury. Prepare one liter of artificial cerebral spinal fluid and 250 milliliters of sucrose cutting solution. Ensure that all instruments and solutions used during brain slice preparation are ice cold with ice.
Anesthetize the mouse using isof fluorine quickly and gently. Remove the brain from the mouse and place in sucrose. Next, trim the brain.
Place the freshly cut coddle surface of the brain on a drop of superglue on the Vibram stage in front of an agar block. Cut 350 micron thick coronal slices. You should be able to get four or five slices with the intact hippocampal circuit.
After that incubate slices for at least one hour at 37 degrees Celsius. Now prepare some two to five mega ohm bo silicate glass electrodes. Using the vertical puller, transfer a slice to the chamber, then place an electrode onto the electrode holder for extracellular field potential recording.
Position the stimulating electrode above the axonal tract on the slice, such as the perent path or Shafer collaterals. Then continue lowering the recording electrode into the slice. Next, deliver a single electrical pulse to evoke the field extracellular postsynaptic potential.
Continue stimulation varying the depth of the recording electrode between stimuli when the maximal response is achieved. This is an indication that the electrodes are at the same. Z level analysis typically consists of measurements, comparing the amplitude of the fiber volley in both brain injured and sham operated mice to evaluate pre-synaptic activation, as well as comparing the slope of the F-E-P-S-P to evaluate post-synaptic activation for patch clamp recording again, transfer a slice to the chamber and lower the recording electrode.
Approach a cell with positive pressure on the electrode to ensure that the recording electrode does not get clogged as it moves down through the tissue. When the electrode gently touches the cell, apply negative pressure in order to create a giga seal between the electrode and the plasma membrane. Next, apply short bursts of negative pressure in order to rupture the plasma membrane to achieve whole cell configuration.
Eliminate the capacitance transient using the amplifier and compensate for series resistance to ensure accurate measurements. Then quantify and compare the rate and size of spontaneous synaptic currents in both brain injured mice and sham operated controls. In this step, prepare the dye stock by mixing one milligram of dye three onap DHQ in 50 microliters of ethanol.
Dispense two microliter aliquots into foil wrapped tubes, and store at negative 20 degrees Celsius. Make the working dye solution daily in one to 200 dilution in A CSF. Then incubate a slice on the A CSF moistened filter paper and stain with 90 microliters of dye for 16 minutes.
After that, rinse it with and place it in the interface recording chamber. Place the stimulating and recording electrodes as described in the extracellular field. Potential recording.
Adjust the light stimulation intensity until the response is centered in the middle of the camera. Range images are typically acquired at a rate of 500 to 1000 frames per second. Trigger the shutter for light stimulation 200 milliseconds before the electrical stimulation to allow initial rapid photo bleaching to stabilize.
Pausing 10 seconds in between trials. Alternate the acquisition trials between electrical stimulation and non-electrical stimulation to allow later subtraction of non-electrical stimulation background. Then record the resting light intensity prior to opening the shutter.
Each VSD trial can be analyzed as a movie of fluorescence readings. That is, each trial is a three dimensional matrix of data with values in X and Y spatial dimensions across time. Record 10 to 12 VSD trials in each test condition and calculate the intra pixel pre illumination normalized fluorescence change according to the accompanying manuscript.
This figure shows an example of an F-E-P-S-P recording in area CA one. The first downward deflection is the stimulus artifact followed by the presynaptic fiber volley, and then the F-E-P-S-P. Here are the input output curves depicting a decrease in net synaptic efficacy in CA one following fluid percussion injury.
And here are the input output curves depicting an increase in net synaptic efficacy in dentate gyrus following FPI. Here is an example of a spontaneous excitatory post-synaptic current from a CA one parametal cell shown. Here is an example of the action potential train from a CA one parametal cell and a fast spiking ca one inter neuron.
This is a Lucifer yellow filled ca one inhibitory inter neuron without dendritic spines, and here is a Lucifer yellow filled ca one parametal neuron with dendritic spines. These figures show a mouse coronal hippocampal slice. The yellow to red pixels represent the excitatory activity in area ca one 14 milliseconds after the afferent shafer collateral stimulation.
Red indicates more depolarization while yellow indicates a lesser but still significant depolarization. The blue pixels represent the sites of inhibitory activity in area ca 1 56 milliseconds after the same Afferent Shafer collateral stimulation while darker blue indicates more hyperpolarization. After watching this video, you should have a good understanding of how to combine multiple ex vivo recording techniques to address a clinically relevant pathological condition involving circuit dysfunction.
