Studying habituation at the level of a single cell will help characterize learning paradigms that are independent of complex neural circuitry thus helping us understand the origins of intelligence. This technique allows the force and frequency of mechanical stimulation delivered to cells to be varied under automatic computer control thus greatly increasing the diversity of input sequences. Using these methods to study cellular habituation will help us learn more about conditions like ADHD and Tourette's Syndrome in which habituation is impaired.
To begin, hook the motor driver to the motor by connecting the two wires labeled A from the driver board to the blue and red wires on the motor. Then connect the two wires labeled B from the driver board to the green and black wires on the motor. After building the breadboard circuit with special care to connect the LEDs in the correct polarity, connect the VCC from the driver board to the top rail of the white breadboard.
And the ground from the driver board to the bottom rail of the breadboard. Next, connect the ground of the breadboard to the ground pin of the microcontroller board. Then connect the green LED, red LED, switch and button wires, respectively to the microcontroller board digital pins 8, 9, 10, and 11.
Connect the microcontroller board digital pins two and three to the driver board wires, step and direction. Then connect pin four to MS1, pin five to MS2, PIN six to MS3, and pin seven to enable. To power the driver board, plug the 12 volt supply into the black green adapter plug, attached by two red wires to the motor driver board.
Download the control program onto the micro controller board. Use a USB cable to attach the microcontroller board to a computer, which will also serve as the power source for the microcontroller board. After obtaining Stentor, coat a 35 millimeter plate by adding three milliliters of the 0.01%polyornithine solution to the plate and leave overnight.
Wash the plate twice with ultrapure water and once with pasteurized spring water. Then add 3.5 milliliters of pasteurized spring water to the 35 millimeter plate. Add three milliliters of pasteurized spring water to the first well and five milliliters to the second and third wells.
Using a P1000 pipette, add two milliliters of Stentor from a culture dish to the first well of the six well plate. Identify individual Stentor with a stereo microscope and then use a P20 pipette to transfer 100 Stentor from the first well to the second well. Similarly, after identifying individual Stentor with a stereo microscope as demonstrated previously, transfer 100 Stentor from the second well to the third well using a P20 pipette.
Then using a P200 pipette, transfer 100 Stentor in a total volume of 500 microliters, from the third well of the six well plate into the 35 millimeter plate such that the final volume is four milliliters. Tape a piece of white paper to the metal ruler on the habituation device, ensuring that the left edge of the paper is two centimeters from the end of the ruler closest to the armature. Using double-sided tape, adhere the bottom of the 35 millimeter plate to the center of the two by two inches paper atop the ruler on the habituation device.
Leave the 35 millimeter plate on the habituation device for at least two hours with the lid closed. Center the USB microscope camera directly above the 35 millimeter plate of Stentor. To install the webcam recorder application, open the webcam recorder app and select the USB microscope from the dropdown menu.
Adjust the focus on the USB microscope camera so that the cells are clearly in view and the camera position to maximize the number of cells in the field of view. After opening the microcontroller board serial monitor, select no line ending"and set it to 9, 600 baud. Use the L command on the microcontroller board program to lower the armature until it barely touches the ruler and the R command to raise the arm if necessary to adjust the exact position.
Use the I command to initialize the automatic mode on the habituation device. Enter the steps size and time between pulses in minutes in the command line. Start taking a video using the webcam recorder app by pressing the red record button.
Then flip the switch on the habituation apparatus to begin the experiment with the first automated mechanical pulse delivery. Immediately before the first mechanical pulse appears on the video, pause and count the number of Stentor that are both anchored to the bottom of the 35 millimeter plate and extended in an elongated trumpet-like shape. Similarly, after the first pulse, count the number of Stentor that are both anchored to the bottom of the plate and contracted into a ball-like shape.
Divide the second count by the first count to determine the fraction of Stentor that contracted in response to the mechanical stimulus, while repeating the procedure for all the mechanical pulses in the experiment. The contraction probability of the Stentor was monitored and the results demonstrated that it progressively declines over the course of one hour. After receiving level four mechanical pulses at a frequency of one tap per minute, indicating habituation.
Altering the force or frequency of the mechanical pulse delivery can change the Stentor habituation dynamics. While using the level two pulse set of frequency of one tap per minute, precludes habituation over the course of one hour. We can study different types of habituation dynamics by altering the force and frequency of mechanical stimulation.
This is an opportunity to explore different types of learning, such as sensitization. Quantitative insights about single cell learning, gleaned from our methods, could inspire other avenues for reprogramming cells within multicellular tissues. Another potential way to fight disease.
