老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

Rodent hippocampal slices are frequently used in the study of mammalian synaptic function and plasticity. Here we demonstrate a protocol for obtaining and studying slices from aged rats and mice, which have thicker skulls and tougher connective tissue than younger rats and mice. These features can delay brain extraction and or dissection, and consequently negate or exaggerate real age differences in synaptic function and plasticity.

In addition, aging and amyloid pathology may exacerbate hippocampal damage sustained during the dissection procedure, further complicating any inferences drawn from physiologic assessment. In this demonstration, we discuss steps taken during the extraction and dissection procedures to minimize these problems. First, the brain is rapidly extracted and placed in ice, cold, low calcium, artificial cerebral spinal fluid.

The hippo campi are then gently removed on an ice cold dissection stage, and hippocampal slices are prepared using a vibrato and incubated in oxygenated calcium containing A CSF. Finally, synaptic potentials and currents are evoked in multiple hippocampal sub regions and recorded the obtained results demonstrate the effects of aging and or Alzheimer's like pathology on synaptic function and plasticity. Proper use of this technique has implications for age-related cognitive decline and dementia due to Alzheimer's disease.

This is because synaptic dysfunction is among the earliest biomarkers of these clinical symptoms demonstrating this procedure. Today will be Dr.Chris Norris, the PI of the lab Next to a large sink. Prepare the dissection area for brain removal and hippocampal dissection.

Include a folded paper towel, a number 11 scalpel blade, BB scissors, bone urs, and a hippocampus tool, a specialized dual spatula from fine surgical tool. Also place small surgical scissors, a thin spatula plastic peor pipette watman filter paper, a 100 millimeter glass petri dish filled with ice, a plastic spoon and a beaker of partially thawed calcium free. A CSF covered with param.

Following decapitation of the rat. The brain should be extracted as quickly as possible. Place the head on the paper towel using the scalpel quickly make an incision in the middle of the scalp from the nasal bone to the occipital bone.

Be sure to cut completely through the cutaneous muscle, fully exposing the skull sutures. Hold the head firmly and cut through the occipital, parietal, and frontal plates along the midline suture. Be sure to keep the lower shears firmly against the skull's inner surface and away from the brain.

This is critical to prevent inadvertent gouging. Next slide. The Ron jurors under the left parietal plate and squeeze the jaws together, rolling upward and forward to pull the parietal and occipital plates away.

If necessary, use the Ron jurors to remove the left frontal plate as well. Repeat for the other hemisphere. Once the plates are displaced, use Ron jurors or scissors to gently pull away or cut any dura mater that may be attached to the temporal plates and stretched across the brain's surface.

Now slide the Ron jurors between the brain and the right temporal plate, keeping pressure toward the skull and away from the brain. Squeeze and twist the temporal plate away until a crunch is heard or felt. Repeat for the left side.

Slide the broad spatula ahead of the hippocampal tool between the ventral surface of the brain and the bottom skull plates until it is completely under the brain. Move it laterally from side to side and forward and backwards a few times to sever intact cranial nerves using the hippocampal tool. Scoop the brain out and submerge it in calcium free A CSF cover with Parfum and let it chill for about one minute.

To extract the hippo campi, use a spoon to retrieve the brain from the A CSF and place it on a CSF dampened watman paper on the lid of an ice cold petri dish. Using the scalpel blade, remove the cerebellum and approximately one quarter of the rostral frontal lobes. Now cut through the intra hemispheric fissure to completely separate the two hemispheres.

Place one hemisphere back into the A CSF slushie and stand the other up on the dissecting stage such that the coronal plane of the frontal lobe is facing down the white brainstem and midbrain should be easily distinguished from the pink or grayish overlying cortex. Locate the calli on the midbrain. These will look like two white knobs and will be at the top of the brain.

In this orientation, gently hold the midbrain in place with scissors and slide the spatula between the calli and cortex. Then gently pull the brainstem, midbrain, and thalamus away. Connective tissue or vasculature may need to be snipped with scissors.

Use the sharp edge of the spatula to sever the fornix. A white fiber bundle located at the anterior dorsal portion of the hippocampus. With the scissors gently continue to pull the brainstem, midbrain, and thalamus away without completely severing it from the rest of the brain.

The white fi of fibers that form a shallow hyperbola at the bottom of the hippocampus should now be visible in this orientation. Using the transfer pipette, gently squirt some A CSF into the gap underneath the FIA to help separate the hippocampus from the cortex. Gently slide the spatula into this gap and while holding the brainstem, midbrain and thalamus firmly with the scissors, roll the hippocampus away gently trim any remaining cortex, blood vessels and white matter away from the hippocampus.

Dous with a few milliliters of A CSF. Using the transfer pipette, keep tissue wet and cold with a CSF slushy. Next, remove the other hemisphere and repeat the dissection to section rat hippo Campi.

Fill the reservoir of the vibrato with ice cold, calcium free A CSF such that the cutting stage and blade are completely submerged. Use a scalpel to cut off the rostral and coddle tips of each hippocampus and stand them together on the rostral surfaces with the dentate gyrus of each hippocampus facing one another. Glue the brain tissue onto a mounting block and transfer it to the sectioning stage of the vibrato.

For synaptic physiology experiments, 400 micrometer sections are recommended. Using a wide mouth transfer pipette transfer the slices to a small Petri dish containing ice cold calcium free. A CSF.

Once slicing is complete, slices are transferred to the holding chamber, filled with oxygenated calcium containing A CSF. Gradually bring the chamber from 27 degrees to 32 degrees Celsius to perform basic extracellular recordings. In acute slices, the recording chamber is arranged on the microscope stage, such that pre-war A CSF is gravity fed through a flow regulator and removed by a central vacuum line.

Using a small paint brush, transfer a slice to the recording chamber so that it is submerged in A CSF and resting on the inserted netting. Allow the slice to acclimate for 10 to 15 minutes. While the slice is acclimating, turn on the stimulator or stimulus isolator and dial the output down to zero.

Then position a stimulating electrode over the slice in the stratum radi atom region of ca two near the ca three border. Lower the recording electrode into the ca one stratum radi atom just breaking the surface of the slice. Turn the output on the stimulator up to a moderate level here.

The stimulus isolator is set at about 150 microamps. Slowly lower the stimulating electrode in small intervals until a stimulus artifact is recorded. In ca one, continue to slowly lower both the stimulating and recording electrodes in intervals while acquiring CA one responses.

Until the fiber volley and the excitatory postsynaptic potential amplitudes reach maximal levels using a software acquisition program like clamp X, establish a synaptic strength curve. Then use the software to deliver stimuli at a range of intensities and record the corresponding activity in ca one. The range and number of stimulus intensity levels used should be sufficient to generate a sigmoidal curve when plotted against either FV or EPSP values.

Next to investigate synaptic plasticity, reset the stimulus intensity for each slice so that a one millivolt response is elicited. Begin baseline stimulation at a frequency of 0.033 hertz EPSP. Slope values should be stable for at least 20 minutes prior to the induction of either long-term potentiation LTP or long-term depression LTD.

To record a baseline period, use a clamp X parameter file to stimulate and record every 30 seconds. Next to induce LTP, open a separate parameter file induce LTP using a one second train of 100 hertz stimulation or multiple short bursts of 200 hertz stimulation given every 200 milliseconds for LTD induction. Deliver 900 stimulus pulses at a rate of one hertz.

After the stimulus trains switch back to the baseline parameter file and collect synaptic responses for 60 minutes or more. Compare EPSP slope values obtained before and 60 minutes after high low frequency stimulation to determine the presence of LTP indicated by increase in synaptic strength or LTD indicated by a decrease in synaptic strength. Both processes are widely believed to reflect critical mechanisms for learning and memory.

Here, representative synaptic strength curves from two different hippocampal slices from two A PP PS.One mice are shown. One mouse was treated with a novel anti-inflammatory adeno-associated viral reagent. The other was treated with a controlled viral reagent.

As can be seen when a similar number of presynaptic fibers are activated across treatment conditions. When groups show similar FV amplitudes as indicated on the x axis, the reagent a slice curve is shifted to the left indicating greater EPSP amplitudes. These data demonstrate that basal synaptic strength is greater in the reagent a treated slice shown.

Here are the results from LTP experiments performed on the same slices. Note that EPSP slope values in the reagent a slice are much greater relative to the control slice during the one hour baseline following delivery of 100 hertz stimulation indicating the LTP was present in the reagent a slice but not the control slice. This figure shows what the EPSP wave forms looked like in reagent A and control mouse slices before and 60 minutes after delivery of 100 hertz stimulation.

The increase in the EPSP slope in the post 100 hertz period is very obvious in the reagent a slice indicative of LTP together. The experiments in the figures shown thus far indicate that reagent A increases synaptic strength and ameliorates LTP deficits. In a PP PS one mice similar experiments were performed on hippocampal LACS from aged rats.

Here, the synaptic strength curves from two different hippocampal slices from a rat treated with a novel anti-inflammatory drug and another rat treated with control vehicle reagent are shown. Note that these curves are very similar for the two slices, indicating that drug A has little effect on basal synaptic strength. This figure shows results from LTD experiments performed on the same slices.

Note that typical for aged rats EPSP slope values in the vehicle treated slice are depressed in the one hour baseline following delivery of one her stimulation. However, EPSP slope values in the drug a slice show little to no depression indicating the absence of LTD shown here are the EPSP waveforms from drug A and vehicle rat slices before and 60 minutes after delivery of one hertz stimulation. The decrease in the EPSP slope in the post one hertz period is very obvious in the vehicle slice, but not in the drug a slice together.

The results in these figures show that drug a ameliorates plasticity differences in aged rats blocking LTD induction, but does not affect basal synaptic function. These data show that multiple parameters of synaptic function and plasticity in aged rats and Alzheimer's model mice can be modulated by experimental treatments and highlight the utility of acute hippocampal slices for investigating the nootropic and or neuroprotective potential of experimental new drugs and reagents. While attempting this procedure, it is important to remember to balance your speed and extracting the brain with your precision and gentleness and dissecting, slicing, and handling the hippocampus for electrophysiological measures.