This procedure aims for the ion aporetic application of pharmacological agents to microcircuits in the brain while simultaneously recording neural activity. First, pull a single barrel glass electrode for single cell recording and bend the tip. Also, pull a multi barrel glass electrode and break its tip to the desired diameter.
Proceed to assemble the two electrodes. Ultimately, the produced electrode is used for extracellular in vivo recordings, where upon demand pharmacological agents can be ontically applied to manipul neural microcircuits. Hi, my name is Dr.Achim Klu, and our lab will show you today how to produce piggyback multi barrel electrodes.
These electrodes can be used to ionophore neural agonists and antagonists into the microenvironment of a neuron, and thus to manipulate neural circuits of a neuron in vivo. Hello, I'm Dr.An Zillow. I'm postdoc in Dr.Ah him Crow lab, and I will be demonstrating the procedure today For a single barrel electrode, use a single barrel glass capillary with filament and pull the tip to a diameter of about one to two micrometers shaft length of 10 to 12 millimeters.
Verify the resistance of the pulled electrode sporadically by filling them with three molar sodium chloride and testing them with a standard O meter in 0.9%saline. Once the electrode is filled with salt solution, the solution dries out quickly, rendering the electrode useless. Therefore, only perform this testing.
Step on some electrodes that will not be used for the piggyback production. To create a full multi barrel electrode, pull the glass to a pipette tip of about 10 micrometers total diameter or less. An electrode with a correctly pulled tip is critical to success.
In addition to being long and thin, the tip must still be firm and broken easily to the desired tip diameter. The tip will be broken to the correct diameter in the next step. So the exact tip size is less critical during the pooling process than the overall shape of the electrode tip, which should be long and relatively thin.
Now, hold the single barrel electrode at about 45 degrees, tip pointing downward and move it through the flame relatively quickly. Aim to have the flame melt the glass at the transition area at about 10 millimeters away from the electrode tip to bend it by about 20 degrees. Place the multi barrel electrode in a bed of modeling clay on a glass slide, and then insert it into the microscope stages.
Slide holder Using the microscope stages XY manipulators, gently move the electrode tip against the plexiglass piece. Observe the breaking of the tip through the microscope oculars. The electrode assembly setup is shown here.
The multi barrel electrode is held on a glass slide on the microscope table. While the single barrel electrode is held by a manual multi axis manipulator that allows a user to lower the single barrel pipette onto the multi barrel pipette with great precision. Lower the single barrel electrode directly into the groove of the multi barrel electrode that is formed by the arrangement of the five barrels with its tip protruding the tip of the multi barrel tip by about five to 10 micrometers.
Glue the shafts of the two electrodes together using cyanoacrylate. Start at the position most distal of the tips and slowly move a toothpick with a glue drop along the electrode shafts towards the electrode tips. Applying glue too close to the electrode tips will clog the tips and render the electrode at least partially non-functional.
Mix a small amount of dental cement. Allow about 15 minutes to dry. Then carefully remove the completed piggyback electrode.
First from the micro manipulator holder, and then detach from the glass slide. Just before using the electrode, prepare to backfill each barrel with its respective drug. Fill the four outer barrels of the five barrel configuration with four drugs of choice and the center barrel with three molar sodium chloride as a balancing barrel.
Then fill the single barrel recording electrode with three molar sodium chloride. Now turn on the ion pheresis pump modules and test all the barrels. The electrode test function of each pump module will help determine if the electrode barrel is functional For deep brain recordings.
A finished five barrel electrode assembled together with a single barrel recording electrode has a long shaft of about seven millimeters. Several synaptic agonists and antagonists are commonly used with iontophoresis blocking glycine inhibition with drugs like the glycine receptor antagonists. Trick nine hydrochloride typically increases firing in neurons.
This sample data measures an auditory neurons discharge rate versus intensity function, and shows responses to sinusoidal sound stimuli of increasing intensity delivered to the animal's ears. Louder sounds resulted in higher spike rates before application of strict nine. The neurons rate intensity function shows low spike rates.
Application of an initial iontophoresis current at 15 nano amps stimulates a slight increase in ejection current. Progressively higher ejection currents block progressively more glycine receptors at the neuron resulting in progressively higher firing rates. At 45 nano amps current, all glycine receptors of that neuron appear to be blocked by strict nine hydrochloride.
Any further increase of the ejection current and releasing even more strict nine did not result in a further change. The neurons discharge rate level function after termination of the iontophoresis, complete recovery of the neural responses back to baseline occurs after about 25 minutes. This recovery process is dependent on the type and amount of drug ejected.
It is important to remember that the ferre drug will diffuse into the vicinity of the recording site and may also block receptors at neighboring neurons. After watching this video, you should have a good understanding of how to combine a recording electrode with a multi barrel drug electrode to obtain low noise deep brain recordings, along with time control delivery of up to four pharmacological agents.
