Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

The overall goal of the following experiment is to perform in vitro dynamic clamp in cortical neurons using the freely available software toolbox, LCG. This is achieved by preparing slices of rat somatosensory cortex for patch clamp recordings from the parametal cells. As a second step, a series of automated electrophysiological protocols is applied to the recorded cell, which provides a standard characterization of the cell that will be useful for subsequent analyses and comparisons across cell types.

Next, the injection of excitatory and inhibitory synaptic events is simulated. In order to recreate the high conductant state experienced by the neocortical neurons in vivo, the results show how background synaptic injection modulates the gain of the cortical cells. The main advantage of this technique over existing methods is the possibility of embedding experimental recordings into more complicated workflows by using, for instance, the result of one experimental protocol as the input to another protocol.

This method can help standardize electro physiological protocols, accelerate data analysis and paved the way for data sharing across laboratories. Though this method was originally developed for wholesale patch CLA recordings in vitro, it can also be conveniently used for other experimental preparation and protocols, such as in vivo patch, CLA recordings, or acceler recordings with micro to arrays the race. After extracting the rat brain discard the cerebellum and separate the two hemispheres along the midline using a scalpel, remove excess fluid from one of the hemispheres and glue it on an inclined platform with a drop of super glue.

Then quickly add a few drops of a CSF over the brain and transfer it to the Vibram chamber. Remove the first 2.5 to three millimeters of the brain tissue with a blade. Afterward, adjust the slicing speed and frequency in order to limit the damage to the slice surface.

Next, set the thickness to 300 micrometers and begin slicing. Once the blade has gone past the cortex, use a razor blade or a bent needle to make a cut above the hippocampus and at the edges of the cortical area of interest. After that, transfer the slices to a multi-well incubation chamber at 36 degrees Celsius.

In this procedure, place a slice in the recording chamber with the 40 x magnification lens. Search for healthy cells in layer five and 600 to 1000 micrometers. Below the brain surface, these cells usually have lower contrast, a smooth appearance, and are not swollen.

Once a healthy cell is found, load one third of the micro pipette with intracellular solution. Then place it in the head stage in the pre-configured Linux operating system. Launch a command.

Shell add its prompt. Type this command to ensure that the data acquisition board is not driving the amplifier. Then apply 30 to 50 millibars of positive pressure by pressing the piston of the syringe, which is connected to the pipette holder.

Next, place the pipette approximately 100 micrometers above the slice and move the pipette towards the target cell using the approach mode of the microm manipulator. After that, adjust the pipette offset on the electrophysiology amplifier and output a 10 millivolt Test pulse in voltage clamp mode. Using the command LCG SEAL test to monitor the pipette resistance, then reduce the pressure to 10 to 30 millibars by withdrawing the piston of the syringe.

Gently approach the cell and check for the formation of a dimple by observing the image on the video camera monitor. At the same time, monitor an increase in resistance evoked by the test pulse by watching the current waveform displayed on the monitor. When there is an increase in pipette resistance and the formation of a dimple on the cell, immediately release the pressure and apply gentle negative pressure to the pipette to help the seal formation.

In the meantime, gradually decrease the holding potential to negative 70 millivolts. Apply gentle suction to rupture the cell membrane and establish the whole cell configuration. Then characterize the cell in terms of its electrical properties.

Using the command LCGE code. Here is the average action potential of a patched layer five parametal neuron with a threshold of negative 50.5 millivolts. This figure shows the measurement of passive responses to the hyperpolarizing currents.

The identification of basic active properties of the cell reveals that the cell is a regular spiking neuron, and there is minimal adaptation to inject simulated excitatory, post-synaptic potentials change to the directory where the next experiment will be saved by typing this command at the command prompt of the shell. Instead of acting on conventional hardware controls of the amplifier. To apply bridge balancing and capacitance compensation estimate the electrode kernel needed to perform the active electrode compensation by issuing this command.

The full kernel is the sum of two components, a fast electrode kernel, and a slower membrane kernel that gives rise to the exponential tail observed in the full kernel. The former constitutes a non-parametric model of the pipette Upon current injection to separate membrane and electrode kernels, the LCG kernel command will prompt the user for the number of points that compose the fast electrode kernel. Next, select a number so that the electrode kernel covers the end of the exponential decay tail.

Then perform the dynamic clamp experiment using this command. Finally, visualize the results. Here is the recreation of in vivo like activity using dynamic clamp.

Red and blue traces represent the simulation of excitatory and inhibitory synapses respectively. And here are the voltage traces recorded from an L five parametal neuron, which was subjected to a bombardment of excitatory or inhibitory post-synaptic currents. The corresponding excitatory, inhibitory and total current injected into the cell are shown in the chart.

The raster plot of the spikes generated across 20 trials shows that the neuron can be extremely reliable and precise in response to the same input. After watching this video, you should have a good understanding of how to perform dynamic lamp experiments using the freely available software toolbox.LCG. This procedure can be extended to other closed loop protocols to investigate a wide variety of subjects ranging from neural excitability to complex network interactions.