Onderzoek van Synaptic Tagging / Capture en Cross-capture met Acute hippocampus Plakjes van Knaagdieren

The overall goal of this procedure is to study long-term plasticity and its associative processes such as synaptic tagging, capture, and cross tagging in the CA one parametal neurons using Q hippocampal slices from rodents. This is accomplished by first dissecting the brain and quickly isolating the hippocampus into cold oxygenated artificial cerebra spinal fluid. The second step is to prepare acute hippocampal slices using a manual tissue slicer and incubate them in an interface chamber.

Next two stimulating electrodes and two recording electrodes are placed in the CA one region to record field EPSP and population spike responses from two synaptic inputs. The final step is to induce a transient form of plasticity in once synaptic input and a persistent form of plasticity in another input within a specific time window. Ultimately, field EPSP responses from both inputs are recorded for extended durations to investigate synaptic tagging capture and cross capture mechanisms.

Synaptic tagging and capture provides a conceptual basis for how short-term memory transforms to long-term memory within a particular timeframe. Visual demonstration of this method is critical as the experimental steps are difficult to learn because they involve stimulation and recording from multiple synaptic inputs. Demonstrating the procedure will be Mahesh Shira, machete and maima Sharma graduate students from my laboratory.

To begin this procedure, prepare A CSF and bubble it with 95%oxygen and 5%carbon dioxide. Fill the bottom of the interface chamber with distilled water to about 70%of its capacity. Then turn on the temperature controller preset to 32 degrees Celsius.

Next, wash the upper chamber for 10 to 15 minutes by running distilled water through the inflow tubing at a high flow rate before switching to A CSF at a flow rate of one milliliter per minute. Then place a net in the upper chamber to provide a resting surface for the slices. Start carbogen the lower chamber immerse the inflow tubing in the A CSF that is being continuously carbogen.

Allow 20 minutes for the A CSF to be saturated with carbogen and for it to fill the upper chamber. In this step, mount a razor blade onto the tissue chopper and ensure that the cutting edge is evenly aligned. Test by chopping a small filter paper to ensure that the blade is firmly secured.

Then set the sliding vernier micrometer to its starting position. Now obtain the head of a euthanized rat and using an iris scissors, make a cut through the posterior skull to remove the brainstem. Then make a small incision along the right side of the skull and a longer incision on the left.

Carefully remove the skull with a bone UR starting from the left side and going to the right side of the skull. To reveal the cortex and the thin layer of dura covering it carefully remove the dura with a thin spatula and then remove the frontal plates with the bone. Ro.Subsequently, remove the remaining dura specifically in the junction between the cortex and cerebellum with the flat end of a spatula, and avoid damaging the brain by maintaining the pressure upward.

Using the spatula, gently scoop the brain into a Petri dish on an aluminum cooling block filled with cold and carbonated A CSF to isolate the hippocampus using a number 11 scalpel, make a straight cut to remove the cerebellum and another cut to remove one quarter of the anterior portion of the brain. Then make a shallow sagittal cut along the midline. Starting from the midline.

Carefully remove the cortex with the sickle scaler to reveal the dorsal hippocampus. After that, remove the layer of cortex above the hippocampus. Make a small cut to the hippocampal commissure.

Gently remove the hippocampus with the sickle scaler, starting from the dorsal end using rolling motions. Subsequently, remove any cortex and connective tissues around the isolated hippocampus using the curved end of the sickle scaler. To slice the hippocampal tissue, place a piece of a CSF soaked 30 millimeter watman filter paper on the slicing stage of the manual slicer.

Transfer the hippocampal tissue onto the filter paper. Using the flat end of the sickle scalor, move the filter paper to align the hippocampus at a proper orientation in relation to the blade of the slicer, so that the hippocampus is sliced at an angle of about 70 degrees to the fia. Next, blot the excess solution surrounding the hippocampal tissue.

With a folded filter paper, slice the hippocampus, transversely, and discard tissue from the extreme end of the hippocampus where the slice morphology is not clear. Then slice the remaining tissue into 400 micron thick slices by adjusting the vernier micrometer after every round of slicing. After each cut, transfer the slice gently from the blade using a brush with soft bristles to a small beaker filled with cold carbonated A CSF.

Then transfer the slices gently onto the net in the slice chamber using a clean plastic past pipette. With a broad tip, carefully adjust the position of the slices in a manner that facilitates electrode location and recording check to make sure that the slices are sufficiently surrounded by a layer of A CSF, but not completely submerged. After that, cover the chamber and incubate the slices for two to three hours in synaptic tagging and capture experiments.

Under the microscope position the two stimulating electrodes in the stratum radi atom of the CA one region to stimulate the Schaffer collateral fibers and when recording electrode in the apical dendritic region of ca one midway between the stimulating electrodes. To record F-E-P-S-P responses, place another recording electrode in the stratum parid. DO layer for recording population spike.

When all the electrodes touch the slice, deliver a test stimulation to ensure a proper field EPSP signal can be obtained in both the inputs. Once a proper F-E-P-S-P signal is obtained, carefully lower the electrodes about 200 microns further using the fine movement knobs of the manipulators. Then allow 20 minutes for the slice to recover.

After that, determine the input output relation by measuring the slope of field EPSP upon a range of current intensities from 20 microamps to 100 microamps. Then for each input, set the stimulation intensity that evokes 40%of the maximum field EPSP slope as the constant stimuli throughout the experiment. After 15 to 20 minutes, start recording the baseline record a stable baseline of at least 30 to 60 minutes before L-T-P-L-T-D induction in one input, induce an early LTP using a weak tetin protocol consisting of a single high frequency stimulation.

While the other input continues to record the baseline 30 minutes after the early LTP induction, induce a late LTP in input S two, using a strong tetin protocol involving repeated high frequency stimulation with an intertrain interval of 10 minutes. Then record the field EPSP responses for extended to durations to reveal the transformation of early LTP in input S one to late LTP by the synaptic tagging and capture. Similarly, in a cross capture experiment, first induce an early LTP and one input followed 30 minutes later by late LTD induction in another input, using a strong low frequency stimulation protocol consisting of 900 bursts over a 15 minute duration.

Then record the field EPSP responses for extended durations to reveal the transformation of early LTP in input S one to late LTP by the cross capture. In this representation, two stimulating electrodes are positioned in the stratum radi atom of the CA one region to stimulate two independent but overlapping synaptic inputs. Onto CA one parametal neurons two extracellular recording electrodes.

One to record F-E-P-S-P from the apical dendritic compartment. And another to record somatic population spike from the parametal cell bodies are located in the stratum radi atom and stratum para respectively. This figure shows the week before strong paradigm to study synaptic tagging and capture.

Weak tetin is applied to S one for inducing early LTP, followed by strong tetin of S two at 30 minutes to induce late LTP, and this figure shows the week before strong paradigm to study cross tagging. Early LTP is induced by weak tetin in S one, followed by the induction of late LTD in S two using a strong low frequency stimulation protocol. After 30 minutes in S one, the early LTP is transformed to late LTP lasting six hours, showing cross tagging and capture.

Once mastered, this technique of dissection and slice preparation can be performed very quickly within three to five minutes. Thus, the viability of the slices can be preserved for long-term recordings. Using this method, one can also test the effects of different pharmacological agents on the synaptic tagging and capture processes.

After watching this video, you should have a good understanding of how to conduct long-term functional plasticity experiments and the processes of synaptic tagging and capture.