Recordings of Neural Circuit Activation in Freely Behaving Animals

For recordings of neural and muscular field potentials. Electronic recordings from a pair of bath electrodes are synchronized with high-speed video recordings and displayed on a laptop monitor. A juvenile crayfish is placed in a small aquarium and allowed to acclimatize for five minutes.

The animal is touched with a handheld probe by the experimenter after the recordings have been started and the resulting behavioral and physiological responses are measured and subsequently analyzed. Hi, I'm Dr.Y Savaha from the Department of Psychology at the University of Maryland College Park. Today we will show you a procedure to record non-invasively neural and muscular field potentials in freely behaving animals.

We use this procedure in our laboratory to identify neural circuits that are activated during escape responses in juvenile crayfish. So let's get started. To begin constructing the recording electrodes nor 0.5 to one millimeter of insulation is stripped off one end of an insulated copper wire.

This process is repeated on a second identical wire to create a pair of recording electrodes using non-toxic glue. The recording electrodes are attached to the inside of a thin walled glass recording chamber. The recording electrodes are positioned opposite each other in the center of both short sides of the chamber.

Next, the ground wire is made by stripping two to three centimeters of insulation of one end and sold. During this end to a longer piece of wire. The ground electrode is positioned on one of the long sides of the recording chamber, perpendicular to the recording electrodes.

The recording chamber should be 8.5 centimeters long by two centimeters wide by five centimeters high. Next, a stimulating probe is made of a glass perpet equipped with a pair of fine wire electrodes. The electrode tips are exposed to produce an electronic signal when the animal is touched by the probe.

This allows for precise measurement of the time of stimulation. The bath recording electrodes the probe and the ground wire are connected to an extracellular amplifier. So first, I'm connecting the bath electrodes to the amplifier.

Next, I'm connecting the probe to the amplifier. And finally, I'm grounding the bass and the probe through the ground of the amplifier. Outputs from the amplifier are connected to a breakout box and data acquisition board using BNC cables.

The chamber is filled with gravel to provide traction for the animal. The chamber is now filled with deionized water. Optimal results are obtained when water of high resistance, approximately 18 mega ohms is used with construction of the recording electrodes, ground wire, and stimulation probe completed.

We can see how to conduct experimental recordings before beginning the experiment. A high speed video camera is positioned perpendicular to the recording chamber to provide a side view using a gooseneck illuminator. The brightness inside the recording chamber is adjusted using a breakout box and data acquisition board.

Videography is synchronized with electronic recordings. When the experimenter is ready to begin recording, an external hand switch trigger starts to synchronize video and data acquisition. To begin the experiment, a single animal is introduced into the chamber and allowed to acclimatize for five minutes.

Once the animal is acclimatized, synchronized video and data acquisition are initiated by the external hand switch trigger. The videos recorded at a frame rate of 1000 frames per second, and electronic field potentials are recorded at a frequency of 25 kilohertz. And so we are looking here.

Each frame here is one millisecond long and again, the upper trace is the the neuro muscular response of the animal. The lower trace is the probe. Heres the deflection in the probe, which indicates this is when the animal was hit.

And then here you have the signal from the animal. The neural signal here preceding the large deflection, which is the muscles and the muscles contract Using the stimulation probe escape tail flips are elicited by single taps of different intensities to the head or abdomen. This process is repeated to look at tail flips during different behavioral contexts.

Finally, using photon motion tool software, the data obtained during the recording session is analyzed to determine the nature of the animal's movement resulting from each particular activated neural circuit. Here is a look at a series of single high-speed video frames and corresponding electric field recordings for an escape tail flip in response to tactile stimulus delivered to the head or tail of a juvenile crayfish. In this trace, a strong tactile stimulus to the head evoked a tail flip controlled by the medial giant circuit.

The recorded spike of the giant neuron and the large phasic deflection that follows enables non ambiguous identification of the tail flip as mediated by giant neuron activity. The backward movement shown in the video traces determines the identity of the activated neural circuit. Here is a tail flip mediated by the lateral giant circuit.

After a strong tactile stimulus was applied to the tail upward and forward motion seen in the video traces. Together with a synchronized electronic trace displaying the giant spike and the large phasic initial deflection determines the identity of the activated neural circuit. This video demonstrates a tail flip controlled by non-GI circuitry.

A more gradual tactile stimulus was delivered to the thorax of the animal. While the movement captured on video does not allow unambiguous identification of the activated circuit, the electronic recording lacks a giant spike and consists of much smaller deflections identifying the activated circuit. Here one can see latency measurements For all three types of escape tail flips.

Time between probe contact and physiological response was measured for seven animals. Giant mediated tail flips are elicited significantly faster than non-GI tail flips. We've just shown you how to record neural and muscular field potentials generated by juvenile gray fish when they escape from a tactile stimulus.

When doing this procedure, it's important to adjust the protocol to your research question. For example, you may or may not need high speed video and you may or may not need to synchronize your video with your bass recordings. When testing a new species for the first time, you need an independent measurement to verify the recordings from the bass electrodes in crayfish.

This can be done by temporarily implanting a pair of electrodes around the ventral nerve court, and by comparing the recordings from the implanted electrodes to the recordings from the bath electrodes, it's also important to adjust chamber size. The recording chamber should be as small as possible, but it should not restrain the animal in its natural behavior. So that's it.

Thank you for watching and good luck with your experiments.