Paired Whole Cell Recordings in Organotypic Hippocampal Slices

The overall goal of the following experiment is to be able to record electrophysiological from the minimal synaptic circuit in the brain. This is achieved in recordings made from organotypic slice cultures from rat hippocampus grown in plastic culture inserts. These slices make elaborate but appropriate synaptic connections that can be easily recorded after a period of growth in culture.

Making recordings in these slices allows for the isolation of the synaptic activity of the minimal synaptic circuit that made between only two neurons, and thus allows for the study of the most basic properties of neuronal circuitry. This method can be used to probe the function of synaptic micro circuits in the hippocampus, but it can be used for any area of the brain where neighboring neurons are synaptically connected. Visual demonstration of both the recording and dissecting methods are crucial as many of the steps depend on the accurate identification of brain landmarks and neurons, Both before making slices prepare all solutions and the culture plates.

Each larger plate for collecting slices contains seven smaller dishes, each with media and a culture insert during their assembly. Set the inserts into the media at an angle to avoid trapping air. Store these plates in the incubator carefully remove the hippocampus from each half of a P seven rodent brain without damage by navigating the brain using structural landmarks.

First, divide a brain along the midline. Then using fine spatulas, separate the cortex from the midbrain. Peeling open the brain to reveal hippocampus.

Next, cut the fornix with the tip of the spatula and work the spatula underneath the FIA and the hippocampus. Now roll the hippocampus out of the brain and cut it away from the attached tissue. Gently scoop up the hippocampus and put it into ice cold dissection medium.

Proceed with collecting the second hippocampus. Proceed with slicing the two hippo campi. Lay them on the stage of the chopper block lined with two thicknesses of moistens.

Number two, filter paper. Then make consecutive 400 micron transverse slices without lifting any slice. The result is like a sliced loaf of bread.

Consistent thickness and gentle handling are key. Now using a soft white sable, number four artist's paintbrush, gently roll the brush under each sliced hippocampus and lift it off the stage. Once both hippo campi are transferred, cover the dish and swirl it vigorously.

Change directions frequently. The goal is to separate the slices. Now under a dissecting microscope, inspect the slices, discard any that are damaged or smaller than desired.

Also, discard any slices that do not have clearly visible cell layers as this is also an indication of damage. Any slices that did not separate can be turned up on their edge and prodded with dumont forceps to force them apart. First, fill the pipette with medium.

Then gently suck up the slice, keeping it near the opening. Using light pressure. Form a droplet at the tip and wait for the slice to settle into the droplet.

Then touch the droplet to the surface of the tissue culture.Insert. Once the slice is clearly on the insert, withdraw the pipette. If the droplets aren't too large, three slices will fit onto one membrane.Insert.

Once all slices have been transferred, use a pasture pipette to gently aspirate the medium around each slice. Carry out fluid removal for the inserts that were plated. First to allow sufficient time for the slices to settle.

Now cover each small dish, then the large dish, and incubate the assembly. Feed the slices the day after cutting them. Then feed them again on the third day after cutting.

Move them to a 34 degrees Celsius incubator, 5%CO2 after the second feeding, and thereafter feed the cultures twice a week. Organotypic slices are ready to record between one and two weeks after they are cut. At the recording rig, remove an individual slice by completely cutting through the bottom of the culture insert and around the slice with the scalpel.

Remove the cut plastic circle with dumont forceps and place it into the recording chamber at the electrophysiology rig. Use forceps to move the slice to the recording chamber. Now, while keeping the stage, still focus in and scan the surface of the slice under 10 x and then 40 x magnification to locate the CA three cell body layer.

Once the target cells have been identified, raise the objective as far as possible without breaking the fluid meniscus between the objective and the A CSF in the recording chamber. Then place a whole cell recording electrode underneath the objective and focus down to find the tip of the electrode until the electrode makes contact with a target neuron. Be sure to maintain positive pressure on the electrode internal solution to keep the electrode free of debris.

Now lower the electrode and the objective in concert until the electrode is near the surface of the slice. Then further lower the electrode into the tissue and push it gently into the side of the target neuron. Now, remove the positive pressure and apply gentle suction to the internal solution to make a giga ohm seal onto the neuron's membrane.

Once the seal is made, disrupt the membrane under the electrode with pulses of suction or by overcompensating the amplifier's capacitance with one whole cell recording made. Raise the objective again and set up the second electrode to minimize movements that would disrupt the first recording. Keep the amount of positive pressure on the second electrode low.

A reasonably low flow will induce movements at a distance of two to three neurons. Once the second whole cell recording is established, synaptic transmission should be stable for anywhere from one to four hours. Synaptic connectivity is evident by stimulating the pre-synaptic neuron to fire in action potential, and then seeing monos synaptic EPCs in the postsynaptic neuron within five milliseconds.

About one third of the tested ca three cells were monos synaptically coupled to another CA three cell by an active connection. The amplitude of AMPA receptor mediated currents varied between trials within one paired recording and was different in different active connections. EPCs could be less than 10 picoamps or greater than 800 picoamps.

Failure rates varied widely using standard induction methods. Both LTP and LTD could be reliably induced at ca three to CA three synapses, and both forms of plasticity would be maintained for over two hours. The full duration of recording polys synaptic inhibitory events were frequently observed between the CA three parametal neuron pairs.

They were not pharmacologically blocked, however, as they did not significantly interfere with neurosynaptic currents due to the long latencies between these events. After watching this video, you should have a good understanding of how to make organotypic hippocampal slices and how to use them to make dual whole cell recordings from synaptically connected pairs of neurons.