The overall goal of this study was to develop a telemetric, bidirectional device to perform multichannel neural recordings and stimulation in freely behaving rats. This new device can help answer key questions in the behavioral neuroscience field such as the causal relationships between brain activation patterns and behavior. In order to validate the telemetric system, we choose the inferior colliculus as a target neurostructure, because an overt behavioral response can be elicited by it's electrical stimulation.
It's widely know that the electrical stimulation of the inferior colliculus elicits a conditioned fear-like behavioral responses in rats, such as alertness, freezing, and escape behavior. We assumed that to be able to elicit such a clear and unequivocal behavior would provide a real challenge to our telemetric system. The integrated telemetric system consists of a head stage, battery, receiver, and a microelectrode unit to be implanted during stereotaxic surgery.
Shown here is a completely integrated, wireless system developed by us, which consists of recording single electrode, tetrode, stimulation electrode, electrode fiber connection board, flexible connection cables, ground wire, connector board, male or female connector for TWS, TWS microelectrode unit connected to the pre-amplifier, and the holder for attaching to a stereotaxic frame. To begin this procedure, fix and horizontally align the animal's head in the stereotactic apparatus using ear bars and upper incisor bar. Next, shave the surgical field, using surgical clippers or scissors and sterilize the scalp with povidone iodine.
Place a heating pad under the animal to prevent hypothermia and treat its eye with dexpanthenol to prevent them from drying. Then inject Xylocaine in the center of the surgical site. Afterward, make a small incision with the scalpel to expose the skull.
Separate the skin gently and remove residual tissue. Next, carefully clean the skull using hydrogen peroxide coated cotton buds. Subsequently, drill four smalls in the skull for fixation of stainless steel screws.
Following that, drill a hole approximately seven millimeters in the skull above the target area for fixation of the electrode unit. Connect the microelectrode unit and electrode holder to the pre-amplifier which is attached to the stereotactic micromanipulator. Absorb any blood with cotton buds.
Then, vertically introduce the microelectrode unit until the tips reach the target area. Position the ground cable along the stainless steel screws and under the skin. After that, carefully adjust the electrode with the micromanipulator until it reaches a zone of active neurons in the the target structure.
In the meantime, monitor the spike activity and detect neural activity with the signal to noise ratio suited for spike sorting. Next fix the microelectrode unit to the skull with ultraviolet glue. Cover the screws and the ground cable with acrylic resin.
After that, disconnect the microelectrode unit from the electrode holder. Cover the contact plate and small cables with acrylic resin. Carefully remove the brad from the stereotactic frame and disconnect the pre-amplifier from the microelectrode unit.
Then connect the cap protection on the microelectrode unit implanted and disconnect it only during the experimental procedures. Monitor the animal welfare and fill a score sheet everyday up to the end of the experiments. Keep the rats individually in the home cage for at least one day after surgery to allow recovering and then house the rats in pairs.
Single cages should be covered with high acrylic lids. Avoid conventional lids made of metal grid, since implants can get stuck and become damaged or unstable over time. Open the TWS software and connect the transceiver to the computer's USB port.
The transceiver can be placed within five meters above the animal. Once no metal barrier is introduced between transceiver and the animal head stage, the TWS is applicable to a broad set of behavioral tasks. Connect the battery to the top of the head stage.
Make sure that a green light comes on in the head stage while the battery is connected to it. Then connect the head stage directly to the implanted microelectrode. As exemplary behavioral tests, we use the open field for the measurement of general behavioral activity and the elevated plus maze, a standard test to assess anxiety like behavior in rats.
For the open field assay place the rat into the center of the open field. Allow the rat to explore the apparatus for at least five minutes. Push the connect button to connect the host computer with the telemetric system.
The status should change from offline to online. Select the stimulus parameter setting menu. Select the stimulation generator parameters according to you stimulation experiment.
For example, pulse amplitude one, pulse amplitude two, pulse frequency, and train frequency. If all stimulation parameters are set to the required values move the mouse to Stimulus Start and push the Stimulus Start button. Move the mouse pointer to the Graph Start Display button.
Move the mouse pointer to the Graph View menu to select the graphs to display the stimulation signal. For the elevated plus maze assay, the plus maze used in these experiments is made of gray acrylic and consists of two open arms and two closed arms that extend from a central platform elevated 50 centimeters above the floor. Place the rate into the center plus maze facing toward an open arm.
Allow it to freely explore the apparatus for five minutes. Push the connect button to connect the host computer with the telemetric system. The status should change from offline to online.
The communication strength and remaining battery capacity is displayed by the green bar in the upper left corner. Push the configuration menu to see the graph options. Select the file path menu to enter the file name and path where the recorded data should be stored.
Press save to save the file name and path. Push start the display to start the display of the detected signals of the four recording channels. Pay attention to the fact that the recorded signals are not yet stored.
Select the gain of the integrated TWS head stage amplifier. Start the audio monitor by pushing the green arrow and select a signal threshold with the slider to reduce the background noise and acoustically display the recorded spikes. Push the red record button in the recording menu.
When pushing the recording button, the recorded signal is stored on the hard disk. The recorded signal is displayed continuously on the right side. Shown here is the recording performance of the TWS with a single bipolar recording electrode implanted in the inferior colliculus.
Afterward, disconnect the head stage from the implanted microelectrode unit and connect the cap protection. Following that, return the rat to its home cage. Extracellular multi-unit activity was successively recorded in the inferior colliculus from the same implanted electrode using TWS and a conventional tethered recording system.
This figure shows representative raw data recorded using both systems while the animal was freely moving in an open field. Here direct comparison of the signals suggests similar spike wave forms and noise levels. Here we presented our wireless recording and stimulation system for electrophysiological and behavioral studies in freely moving animals.
Our approach has several advantages over existing ones. Firstly, this system uses a single portable head stage. It keep it with a pre-amplifier and a rechargeable battery.
The head stage is light and compact, weighing 12 grams including battery. Secondly, it was well tolerated by the animals and for that it can be used to replace the tethered system since it does not restrict the animal's mobility. Thirdly, through simultaneous bidirectional recording and stimulation of neural activity, this system provides a sophisticated tool to assess the causal relationships between specific brain activation patterns and behavior.
Finally, we have developed a chronically implantable microelectrode unit that can be easily implanted during a conventional stereotaxic surgery. After watching this video you should have a good understanding of how to implant the microelectrode unit and perform electrical stimulation and extracellular neural recordings. The telemetric system is a useful tool to investigate the brain stimulation mechanisms.
The telemetric system is an important tool that can be combined with a broad set of behavior tests in order to investigate how electrical activity in defined neural circuits generates certain forms of behavior, a fundamental quest to neuroscience.
