The overall goal of this pressure injection procedure is to reliably manipulate the direct vicinity of the recording site pharmacologically. This method can help answer key questions in the field of cognitive neuroscience, such as showing the specific contribution of neurotransmitters to the top-down modulation of feedforward cortical information processing. The main advantage of this technnique is that we are able to reliably manipulate the direct surrounding of the recording electrode for a defined time period, with a wide selection of agonists and antagonists.
The modulation of feedforward cortical information processing is functionally important for many cognitive processes, including the top-down modulation of sensory information processing by attention. The rhesus monkey is a well suited animal model to investigate the involvement of neurotransmitter systems in such modulations. While the animal's performing a cognitive task, single cell recordings combined with local and temporary neuropharmacological manipulation are conducted.
The system described here can provide stable measurement of single neuron activity with and without pharmacological injections in the direct vicinity of the recording site in an awake, behaving, monkey. To begin this procedure, clean the guide tubes of the recording system by dipping the cleaning wires in sterile silicon oil and feeding them through the individual guide tubes several times. Subsequently, insert the quartz glass micropipette into a guide tube of the recording system.
After fixing the micropipette in the recording system, attach the sterile tube to the metal pin of the micropipette. Take care not to break the micropipette by using two sterile tweezers to apply equal pressure on the pin and tube. Then, use superglue to seal the junction between the tube and the micropipette.
Wait at least three hours for the glue to harden before filling the micropipette with liquid. After the micropipette insertion, insert the microelectrodes into other positions of the recording system. Before each recording, collect the tube of the substance to be injected and allow it to reach room temperature if refrigerated.
Next, apply sterile silicon oil to the guide tube gap and the tips of electrodes and micropipette in order to lubricate the system for smooth movement. Then, check the tips of the electrodes and micropipette using a microscope to ensure they are intact. Align the electrodes and the micropipette so that they extend out of the guide tubes at the same length.
To position the electrodes and the micropipette at zero position, drive them into the guide tubes until they are no longer visible. Subsequently, set the depth of the electrodes and the micropipette to zero in the software. Next, fill a sterile syringe with sterile saline and insert the needle into the tube taking care not to pierce the wall.
Drive the micropipette out of the guide tube for visual control of the substance flow. Then, flush at least two milliliters of sterile saline through the tube and micropipette to ensure no air remains in the syringe or in the tube. Do not apply too much pressure to the plunger of the syringe and ensure the junction between the tube and micropipette is sealed.
After that, fill a new sterile syringe with the solution to be injected. Exchange it with the saline syringe barrel and make sure that no air is transferred into the system. In order to completely remove saline from the tube, flush the system with 250 microliters of the solution to be injected.
Now, using the motor control software, retract the electrodes and micropipette into the guide tubes to a depth of at least 500 micrometers. Then, lower the guide tube ring to the bottom of the guide tubes to maintain their fixed relative position. Clean the base of the system with ethanol, in particular where it will touch the monkey's recording chamber.
Then, close the recording system by replacing the front cover and tightening the screws. In this procedure, set the x-y position of the recording system to define the point at which the guide tubes reach the dura, within the chronically implanted recording chamber. Make sure the guide tubes are retracted completely.
Next, bring the recording system into position and place the syringe in the microinjection pump. Slide the movable part of the pump until it is firmly in place behind the plunger of the syringe. If a drop of substance is visible at the tip of the guide tubes, carefully remove it using a sterile cotton bud.
Then, securely mount the recording system on the monkey's recording chamber. Slowly lower the guide tubes into the recording chamber until it reaches the dura. Next, drive the electrodes using the motor control software.
Check the impedance of the electrodes regularly at different depths. After the electrodes have successfully penetrated the dura, advance the micropipette. Drive the electrodes and the micropipette to the depth of the area of interest.
Then, slowly drive the electrodes and micropipette until an electrode is close enough to record the activity of a single unit with good signal to noise ratio, and keep in mind to position the recording electrode and the micropipette at the same depth, ensuring minimum distance between them. During an injection block, inject a predefined amount of the substance at regular intervals, by defining the injection volume using the step function. Then, press the injection button every minute according to the clock of the recording software.
Record the time and the trial during which the substance is injected, the depth of the electrodes and micropipette, as well as the amount of ejected substance. Follow the injection block with the recovery block, in which no substance is injected. Monitor and maintain the recording quality of the selected single units until the end of the recovery block.
After recording, retract the electrodes and micropipette into the guide tubes, and then manually retract the guide tubes. Next, remove the recording system from the recording chamber of the monkey. Then, release the syringe from the injection pump and transfer the system to the preparation area for cleaning.
We used this system to investigate pharmacological effects on neuronal activity in different attentional states. This figure shows the effect of scopolamine on the average firing rate of the sample neuron in each of the three attentional conditions. The neurons firing rate for the two spatial attention conditions, as well as for the sensory condition, dropped shortly after the first injection of the injection block and during the recovery block, increased, after a delay to the same level as before the injection.
This figure shows a control recording from a second sample neuron in which saline was injected using the same protocol as for the scopolamine injection. During the injection block, no change in the neuron's firing rate was observed compared to the control block. Here, we have illustrated in detail how to perform reliable, pre-sized injections, and high-quality single cell recordings with an off the shelf pressure injection system.
We hope that this method inspires other scientists to investigate neuromodulatory contributions to the dynamics of neuronal activity.
