Sezione tecnica del Patch Clamp per l'analisi di plasticità indotta su apprendimento

Hi, my name is Dai Mitsushima. Here we will show you how to make brain slices in trained animals by comparing patch clamp data in untrained animals. We can analyze learning-induced synaptic plasticity.

Hi, my name is Hiroyuki Kida. In this experiment, we investigated the the neuromechanism of motor running by using Rotor rod test. The rotor rod test is widely used to evaluate the motor cognition of rodents.

The advantage of this method is that, we can change the level of discovery during the test by increasing rotator speed. Rotor rod test. Prior to the motor task, the rotor rod apparatus should be set to accelerating mode from 4 to 40 rpms over 300 seconds.

Rats were made to perform ten attempts for each test. In the time interval between attempts was 30 seconds. During the first training session, rats usually fall off the rod, even at low speeds and sometimes walk in the wrong direction.

After repeating training, however, the rat can walk at a higher speed. By measuring latency on the rod, we can estimate the rat's learning performance of the skill. Here we can see the results of the rotor rod test.

As shown in this figure, two days of training is sufficient to acquire the motor skill. Compared with latency at first trial, post hoc analysis showed significant improvement on the training days final trial. By measuring latency on the rod, we can estimate the rat's learning performance of the skill.

Next, we will show you an inhibitory avoidance test. This test is useful in analyzing contextual learning performance. The inhibitory avoidance apparatus consists of light and dark sides separated by a trap door.

During the training session, rats are placed in the lighted area and given a short time to become acclimated to the environment. Once the door opens, rats are free to enter the darkened area at will. Upon entering the darkened area, the door is closed and rats are given a mild electric shock for two seconds.

After being returned to their cages for 30 minutes upon completion of the trial, rats are placed in the apparatus'lighted area again. Thirty minutes post-shock, the trained rats consistently showed a longer latency before entering the dark area. Here we can see the results of the inhibitory avoidance test.

After the electric shock, the rats learned to avoid the darkened area and stay on the lighted side, which they would not prefer normally. This tendency to avoid the darkened side indicates the acquisition of contextual memories. Post-training brain slices.

Prior to incision, we cool down all scissors, hemostats and beakers with crushed ice. Here you can see our preparation for dissection. Brain dissection should be performed as quickly as possible.

After this deeply anesthetized, the rat is placed in a shallow tray with crushed ice and an incision is made to open the abdominal cavity. After diaphragm incision, a further lateral incision is made. To open the thoracic cavity, the costal cartilage should be clumped-shut using a hemostat.

After exposing the heart, an 18-gauge stainless steel needle is inserted in the posterior part of the left ventricle. The tip of the needle, should be visible through the wall of the aorta. After cutting the right auricle, perfusion is started.

Be sure that both the needle and syringe are first filled with gassed ice-cold dissection buffer. Any air bubbles should be also removed prior to perfusion. First, cut at the posterior of the skull.

Then make a lateral cut, followed by a center cut to expose the brain. Upon dissection, the brain should be placed in bubbling ice-cold buffer for five minutes. Before proceeding with dissection, the filter paper should be wet using ice-cold buffer.

Then, the brain is placed on an ice-cold stainless steel stage. The angle of the cutting stage is crucial for ensuring a correct cutting angle for the brain slice. An incorrect angle could potentially cut the target pyramidal neurons.

After trimming, one drop of superglue should be placed on the stage of the vibratone. To ensure tight adhesion, excess dissection buffer should be removed using a piece of filter paper. Using the vibratone, thin brain slices can be made maintained in a bubbling ice-cold buffer.

Our target area is the primary motor cortex. The target brain area can be trimmed away using Iris scissors. The interface chamber can be made using a plastic food container.

The lid of the interface chamber is necessary to contain the gas. Slices are observed using an infrared-DIC microscope. Here we can see an example of Layer 2/3 neurons in primary motor cortex.

Patch recording pipettes are filled with the appropriate intracellular solution. The solution for the current clamp analysis is different from the voltage clamp analysis. Representative results.

The current clamp technique is useful for analyzing intrinsic cell properties. After Rotor rod training, we were able to obtain the current clamp data from Layer 2/3 neurons in the primary motor cortex. Panel A indicates typical traces induced by current injections.

Panel B indicates the relationship between the injected current and the number of spikes. One-day trained rats induced less spikes, while two-days trained rats induced many more spikes than untrained rats. As can be seen in the lower panels, one-day trained rats exhibited lower resting potential, higher spike threshold, and deeper afterhyperpolarization.

Two-days trained rats exhibited higher resting potential and membrane resistance. The voltage clamp technique is useful for analyzing the learning-induced synaptic plasticity. Here, we can see data from CA1 neurons after contextual training.

We obtained miniature post synaptic currents from CA1 neurons induced by single vesicles of glutamate or GABA. Panels A and B show representative traces of miniature post synaptic current. In the presence of Tetrodotoxin, miniature EPSCs at minus 16 millivolts and miniature IPSCs at zero millivolts were measured sequentially in the same neurons.

Panel C shows two-dimensional plots of the miniature EPSC and miniature IPSC amplitudes in untrained, trained, unpaired and walk-through rats. Panel D shows the plots for the miniature frequencies. As can be seen in the lower panels, contextual trainings significantly strengthened both excitatory and inhibitory synapses promoting diversity of synaptic inputs to the neurons.

In summary, the current-clamp technique is useful for analyzing learning-induced changes in cell property. Also, the voltage-clamp technique is a powerful tool for analyzing learning-induced plasticity at excitatory and inhibitory synapses. The detailed results of these analyses can be found in the following publications.