The overall goal of this experiment is to study the activity dependent nucleo cytoplasmic shuttling of proteins using acute hippocampal slices where neuronal connectivity and function are well preserved To achieve this goal, the hippocampus of an adult male rat is first isolated and acute transverse slices are prepared as a second step. The hippocampal slices are transferred to the recording chamber and the late form of LTP is induced in the CA one stratum radi atom. Next, the potentiated and control slices are snapped frozen, and the stimulated ca one regions are dissected in order to isolate the nuclear enriched fraction for further immuno blott Analysis.
Results based on western blotting analysis show differences in nuclear phosphoprotein levels between the potentiated and control slices 30 minutes after the induction of LTP. Generally, individuals new to this method will struggle because of two reasons. First, the low amount of tissue sample, and second, the critical timeframe that is recurred for lysis of the samples in hypotonic lysis buffer, allowing release of the nuclei Visual demonstration of this method is critical as the hippocampus life preparation and isolation of the C one region require fast and very accurate dissections, but there are certain tricks.
To make it easier one would need to follow both written and visualized instructions for preparation of the nuclear enriched fraction from the C one area of the hippocampus. Lysis Demonstration of procedure will be performed by pinon Postal Laboratory. She will show how to prepare acute hippocampus lysis and induce LTP.
After the dissection of C one region through Berra g student from our lab will show you how to isolate nuclear and destruction. Begin this procedure by isolating the rat brain and immerse it in the pre carbonated ice cold gaze solution. Next, remove the cerebellum and part of the enteral cortex.
Separate the cortical hemispheres with a mid sagittal cut. Afterward, make a 50 to 70 degree cut along the dorsal edge of each hemisphere. Then place each hemisphere down on its medial surface glue each hemisphere with the freshly cut surface on the slicing platform of the vibrato.
Subsequently cover the platform with the pre carbogen ated ice cold gaze solution. Cut 350 micrometer slices containing the hippocampal formation subular and enteral cortices from the anterior to posterior side with the vibram. After that, transfer the slices to a U-shaped and submerged type incubator and incubate them for at least two hours at 32 degrees Celsius with carbogen A CSF.
Now transfer a hippocampal slice to the submerged type recording chamber mounted under a microscope. Perfused the slice with carbonated A CSF at six milliliters per minute for at least 30 minutes at 32 degrees Celsius. After 30 minutes, fill the glass capillary micro electrodes with a CSF.
Place a micro electrode in the CA one Schaefer collateral fibers for stimulation, and another one in the ca one stratum ready atom for field EPSPs recording with a distance of 300 micrometers apart. Then evoke the field EP SSPs by delivering three to four volts by phasic rectangular current pulses to the Schafer collateral fibers. Perform the maximum stimulation test by measuring the input output relationship and define the stimulation strength as 40%of the maximum field EPSPs slope value.
Next, record the baseline for at least 15 minutes by measuring the responses to test stimuli every minute throughout the experiment. To induce late LTP, apply high frequency tein to increase the evoked field EPSPs in ca one region. Apply five micromolar by COE to the A CSF two minutes before tetin and wash out immediately after the last tetanus.
Stop the recording. Two minutes or 30 minutes after late LTP induction afterward, remove the electrodes and quickly transfer the slice onto a pre chilled metal platform placed on dry ice. Then collect each slice in a 1.5 milliliter eend orph tube and store at negative 80 degrees Celsius.
In this step, take the frozen slices out of the negative 80 degrees Celsius and keep them on ice. Add 0.5 milliliters of fresh cold TBS buffer containing protease and phosphatase inhibitors into the tube. Incubate them for two to three minutes before transferring to a stereo microscope.
Next, dissect the CA one stratum parama dole region of the hippocampus by holding the slice with one needle and cutting the CA one area with the other. Afterward, collect the dissected ca one regions from five slices per group in a new one point milliliter tube containing 50 microliters of lysis buffer. Then homogenize the collected tissues by carefully pipetting up and down with a 200 microliter pipette.
Incubate the lysate on ice for five to seven minutes to allow the cells to swell. After that, take two microliters of the lysate and drop it on a microscope slide. Visualize the swelling of the cells under a bright field microscope.
The nuclei appear as round intact structures with proper swelling. Now, collect 20 microliters of sample in a fresh 1.5 milliliter tube as the homogenate fraction. Add eight microliters of denaturing four XSDS sample buffer.
Then centrifuge the remaining lysate at 1100 RCF for one minute. After one minute, carefully collect the supernatant from the top and transfer it to a new tube. Subsequently, add 20 microliters of four x sample buffer.
Then resuspend the pellet in 60 microliters of hypotonic buffer and add 20 microliters of four x sample buffer. This fraction is referred to as a nuclear enriched fraction. Store the homogenate, cytosolic and nuclear enriched fractions at negative 20 degrees Celsius or negative 80 degrees Celsius.
For later immuno blotting in this procedure thaw the samples and boil them for five minutes at 95 degrees Celsius before measuring the protein concentration by AM mito black test, or BCA test load, an equal amount of protein samples from the homogenate, cytoplasmic and nuclear enriched fractions. On an SDS page, gel samples from the control and LTP slices should be placed on the same gel for direct comparison in the Western blot. This figure shows an immuno blot for the homogenate, cytosolic and nuclear enriched fractions of ca one lysate probed with cytosolic and nuclear markers.
This is an immuno blot analysis of PJS 180 levels in the homogenate two minutes and 30 minutes after induction of tein, and this is an immuno blood analysis of PJS 180 levels in the nuclear enriched fraction. Two minutes and 30 minutes after ization phospho Jacob levels remained unaltered in total CA one protein homogenates and in the nuclear enriched fraction two minutes after ization. But a significant increase was found in p Jacob, S 180 Immunoreactivity 30 minutes after the induction of LTP.
While attempting this procedure, it's important to remember to use an appropriate volume of hypotonic lysis valve. Also, the critical timeframe for lysis of the samples needs to be standardized. In particular, when this method is applied for isolation of nuclear and fraction from rat or mouse brain tissue samples other than hippocampus Following this procedure, other methods like complementary immune staining with antibody against candidate proteins imported to the nucleus after induction of OTP or signaling molecules like active forms of kinase or phosphatase, can be helpful for verifying those results.
Pharmacological treatment can be performed to answer additional questions. For example, what signaling cascades are involved in translocation of synaptic nuclear protein meers, or how they influence a nuclear function. In addition, one could study the role of synap nuclear messenger proteins in disease involving hippocampus.
For example, Alzheimer's disease.
