We thank Tatyana Makushok for innumerable discussions about Stentor learning. This work was funded by NSF grant MCB- 2012647 and by NIH grant R35 GM130327, as well as by the I2CELL award from the Foundation Fourmentin-Guilbert.

NOTE: A summary of the habituation experiment workflow is shown in Figure 2.
1. Assembling the habituation device
<ol>
	<li>Hook the motor driver to the motor (see Figure 3).
	<ol>
		<li>Connect the two wires labeled A from the driver board to the blue and red wires on the motor. Connect the two wires labeled B from the driver board to the green and black wires on the motor. 
		NOTE: Looking down on the driver board from above with the motor wires at the top, the four input wires should connect to the motor leads in this order: blue, red, black, and green.</li>
	</ol>
	</li>
	<li>Build the breadboard circuit shown in Figure 4, with special care to connect the LEDs in the correct polarity.</li>
	<li>Connect the Vcc (+5 V) from the driver board to the top rail of the white breadboard and the Gnd from the driver board to the bottom rail of the breadboard.</li>
	<li>Connect the ground of the breadboard to the ground pin of the microcontroller board. Connect the green LED, red LED, switch, and button wires, respectively, to the microcontroller board digital pins 8, 9, 10, and 11.</li>
	<li>Connect the microcontroller board digital pins 2 and 3 to the driver board wires Step and Dir.</li>
	<li>Connect the microcontroller board digital pins 4, 5, 6, and 7 to the driver board wires.
	<ol>
		<li>Connect Pin 4 to MS1, connect Pin 5 to MS2, connect Pin 6 to MS3, and connect Pin 7 to Enable.</li>
	</ol>
	</li>
	<li>Power the driver board with a 12 V power supply. Plug the 12 V supply into the black/green adaptor plug attached by two red wires to the motor driver board. 
	&#8203;NOTE: Do not plug the 12 V supply into the microcontroller board plug.</li>
	<li>Download the control program (https://github.com/WallaceMarshallUCSF/StentorHabituation/blob/main/stentor_habituator_stepper_v7.ino)&#160;onto the microcontroller board.</li>
	<li>Use a USB cable to attach the microcontroller board to a computer, which will also serve as the power source for the microcontroller board.</li>
	<li>Check that user controls are working.
	<ol>
		<li>Confirm that the slide switch turns the automatic mode on and off. In automatic mode, the system will take a step at regular intervals specified by the user (see below).</li>
		<li>Check that the green LED turns on when the automatic mode is on.</li>
		<li>Check that the red LED flashes 1 s before the motor applies a pulse. The red LED is a warning light that indicates when the system is about to deliver a mechanical pulse.</li>
		<li>Test the red button, which triggers a 1/16 micro step every time the button is pushed, regardless of whether the system is in automatic mode.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64692/64692fig03.jpg" /> 
Figure 3: Components of the habituation device. All the labeled electronics are required to assemble the machine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64692/64692fig04.jpg" /> 
Figure 4: Electronics schematic. This is the circuit on the breadboard. The wires connecting to the microcontroller board are numbered as described in the protocol. D1 and D2 are the red and green LEDs, respectively, and are connected to ground through 330 &#937; resistors. The two switches are pulled up with 10 K&#937; resistors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Setting up the habituation experiment
<ol>
	<li>Obtain Stentor.</li>
	<li>Coat a 35 mm plate with 0.01% poly-ornithine solution.
	<ol>
		<li>Add 3 mL of the 0.01% poly-ornithine solution to the plate and leave overnight.</li>
		<li>Wash the plate twice with ultrapure water and once with pasteurized spring water (PSW) (Table of Materials).</li>
	</ol>
	</li>
	<li>Add 3.5 mL of PSW to the 35 mm plate.</li>
	<li>Wash the Stentor in a 6-well plate (Table of Materials).
	<ol>
		<li>Add 3 mL of PSW to the first well and 5 mL of PSW to the second and third wells. Use a P1,000 pipette to add 2 mL of Stentor from a culture dish to the first well of the 6-well plate.</li>
		<li>Identify individual Stentor with a stereo microscope (Table of Materials) and then use a P20 pipette to transfer 100 Stentor from the first well to the second well.</li>
		<li>Identify individual Stentor with a stereo microscope and then use a P20 pipette to transfer 100 Stentor from the second well to the third well.</li>
	</ol>
	</li>
	<li>Use a P200 pipette to transfer 100 Stentor in a total volume of 500 &#181;L from the third well of the 6-well plate into the 35 mm plate such that the final volume in the 35 mm plate is 4 mL.</li>
	<li>Tape a piece (7 cm x 7 cm) of white paper to the metal ruler on the habituation device. Ensure that the left edge of the paper is 2 cm from the end of the ruler closest to the armature.</li>
	<li>Use double-sided tape to adhere the bottom of the 35 mm plate to the center of the 2 in x 2 in paper atop the ruler on the habituation device.</li>
	<li>Leave the 35 mm plate on the habituation device for at least 2 h (this can be extended to overnight) with the lid closed. Throughout this acclimatization period, keep the plate in ambient light conditions that match the experimental light conditions (i.e., do not subject the cells to light/dark fluctuations). Furthermore, ensure that the plate does not experience any mechanical perturbations from accidental jostling.</li>
	<li>Center the USB microscope camera (Table of Materials) directly above the 35 mm plate of Stentor. If necessary, place a prop such as a pipette tip box underneath the universal serial bus (USB) microscope camera to adjust the height. Alternatively, a ring stand can be used to adjust the height.</li>
	<li>Install the Webcam recorder application on a laptop (Table of Materials) and use it to visualize the cells via the microscope input.
	<ol>
		<li>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.</li>
		<li>Adjust the position of the USB microscope camera to maximize the number of cells in the field of view.</li>
	</ol>
	</li>
	<li>Open the microcontroller board serial monitor: select No Line Ending and set it to 9,600 baud.</li>
	<li>Use the l command on the microcontroller board program to lower the armature until it barely touches the ruler. Use the r command to raise the arm if necessary to adjust the exact position. 
	NOTE: If the armature is a significant distance away from the ruler, type in the d command to disable the motor coil current so that the arm can be moved manually toward the ruler. After moving the arm manually, use the e command to enable the motor coil current and keep the arm locked in position. When properly lowered prior to the start of an experiment, the bottom tip of the armature should be 1 cm away from the left edge of the ruler. The armature will deliver the mechanical pulse by hitting the ruler.</li>
	<li>Use the i command to initialize the automatic mode on the habituation device.</li>
	<li>Enter the step size in the command line. Level 5 is the smallest step, and Level 1 is the largest step. Level 4 is the step size used for baseline habituation experiments. 
	NOTE: A Level 5 stimulus results in a downward displacement of the ruler by ~0.5 mm; Level 4 results in downward displacement by ~1 mm; Level 3 results in downward displacement by ~2 mm; Level 2 results in downward displacement by ~3-4 mm; and Level 1 results in downward displacement by ~8 mm. A Level 5 stimulus results in a downward peak force of the armature against the ruler of ~0.122 N; Level 4 results in a downward peak force of ~0.288 N; and Level 3 results in a downward peak force of ~0.557 N. The downward forces generated by Level 1 and Level 2 are more difficult to empirically quantify with a dynamometer due to the significant ruler oscillations that occur after the armature makes contact.</li>
	<li>Enter the time between pulses in minutes. The interval used for baseline habituation experiments is 1 min.</li>
	<li>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.</li>
</ol>
3. Analyzing the experiment video
<ol>
	<li>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 mm plate and extended in an elongated, trumpet-like shape (Figure 5A, Video 1).</li>
	<li>Immediately 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 (Figure 5B, Video 1). 
	NOTE: Contracted cells are easily discernable from elongated cells because Stentor shorten their body length by over 50% within 10 ms during a contraction event3.</li>
	<li>Divide the second count by the first count to determine the fraction of Stentor that contracted in response to the mechanical stimulus.</li>
	<li>Repeat steps 3.1-3.3 for all the mechanical pulses in the experiment video.</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64692/64692fig05.jpg" /> 
Figure 5: Stentor contracting after receiving a mechanical stimulus. (A) The Stentor are in their elongated state and anchored to the bottom of the Petri plate. (B) The Stentor have contracted after receiving a Level 4 mechanical stimulation from the habituation device. The images were taken with a USB microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Video of Stentor contracting. The Stentor receive a Level 4 mechanical stimulus from the habituation device every minute. These cells have not yet habituated, so they contract after receiving the pulse. The cells are in the Petri plate placed atop the habituation device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/Video_1_JoVE.mp4" target="_blank">Please click here to download this Video.</a>

<ol><li>Dussutour, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Learning+in+single+cell+organisms%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Learning in single cell organisms.</a> Biochemical and Biophysical Research Communications. 564, 92-102 (2021).</li>
<li>Sternberg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intelligence%5BTitle%5D)+AND+%22Dialogues+in+Clinical+Neuroscience%22%5BJournal%5D)">Intelligence.</a> Dialogues in Clinical Neuroscience. 14 (1), 19-27 (2012).</li>
<li>Wood, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parametric+studies+of+the+response+decrement+produced+by+mechanical+stimuli+in+the+protozoan%2C+Stentor+coeruleus%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">Parametric studies of the response decrement produced by mechanical stimuli in the protozoan, Stentor coeruleus.</a> Journal of Neurobiology. 1 (3), 345-360 (1969).</li>
<li>Tang, S. K. Y., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+learning%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Cell learning.</a> Current Biology. 28 (20), 1180-1184 (2018).</li>
<li>Wood, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulus+specific+habituation+in+a+protozoan%5BTitle%5D)+AND+%22Physiology+and+Behavior%22%5BJournal%5D)">Stimulus specific habituation in a protozoan.</a> Physiology and Behavior. 11 (3), 349-354 (1973).</li>
<li>Thompson, R. F., Spencer, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Habituation%3A+A+model+phenomenon+for+the+study+of+neuronal+substrates+of+behavior%5BTitle%5D)+AND+%22Psychological+Review%22%5BJournal%5D)">Habituation: A model phenomenon for the study of neuronal substrates of behavior.</a> Psychological Review. 73 (1), 16-43 (1966).</li>
<li>Slabodnick, M. M., Marshall, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stentor+coeruleus%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Stentor coeruleus.</a> Current Biology. 24 (17), 783-784 (2014).</li>
<li>Slabodnick, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+macronuclear+genome+of+Stentor+coeruleus+reveals+tiny+introns+in+a+giant+cell%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The macronuclear genome of Stentor coeruleus reveals tiny introns in a giant cell.</a> Current Biology. 27 (4), 569-575 (2017).</li>
<li>Slabodnick, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+kinase+regulator+Mob1+acts+as+a+patterning+protein+for+Stentor+morphogenesis%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">The kinase regulator Mob1 acts as a patterning protein for Stentor morphogenesis.</a> PLoS Biology. 12 (5), 1001861(2014).</li>
<li>Lin, A., Makushok, T., Diaz, U., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+study+of+regeneration+in+Stentor%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Methods for the study of regeneration in Stentor.</a> Journal of Visualized Experiments. (136), e57759(2018).</li>
<li>McDiarmid, T. A., Bernardos, A. C., Rankin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Habituation+is+altered+in+neuropsychiatric+disorders-A+comprehensive+review+with+recommendations+for+experimental+design+and+analysis%5BTitle%5D)+AND+%22Neuroscience+and+Biobehavioral+Reviews%22%5BJournal%5D)">Habituation is altered in neuropsychiatric disorders-A comprehensive review with recommendations for experimental design and analysis.</a> Neuroscience and Biobehavioral Reviews. 80, 286-305 (2017).</li>
</ol>

The authors have nothing to disclose.

The most critical steps in the protocol relate to ensuring that the Stentor remain in optimal conditions for contractions to occur. The contraction response in the habituation assay requires that Stentors are anchored to a surface using their sticky holdfast since they rarely contract when they are freely swimming. However, the bottom surface of the 35 mm Petri plate used for habituation experiments is not typically conducive for anchoring unless coated with poly-ornithine. Furthermore, the Stentor cannot be exposed to any mechanical perturbation for a minimum of 2 h before the start of the habituation experiment because the Stentor forgetting timescale is 2-6 h3. If Stentor receive mechanical stimulation within 2 h of the habituation experiment start time, there is a possibility that this prior stimulation will induce a slight level of habituation in advance of the experiment, thereby reducing the contraction probability after the habituation device delivers the first mechanical pulse. Finally, during the analysis stage, it is important to only count the number of Stentor that contract after a pulse - rather than any incidental spontaneous contractions that occur prior to the pulse delivery - to obtain an accurate readout of the fraction of cells that contracted in response to the mechanical stimulation.
The protocol can be readily modified to study different types of habituation dynamics by changing the force and frequency of the mechanical pulses delivered by the habituation device. This also provides an opportunity to explore other types of learning, such as sensitization, that might occur in Stentor. The microcontroller board program code itself can also be adjusted to deliver different patterns of mechanical taps to the Stentor.
One potential issue to troubleshoot with this protocol is the low frequency of Stentor anchoring, which could constrain the number of Stentor that can be observed in the habituation experiment. Anchoring frequency is sometimes reduced in Stentor cultures that have not recently been fed or are contaminated. To address this problem, one should wash a fresh batch of Stentor to start a new culture and feed them regularly according to the protocol described in Lin et al.10.
This protocol is limited in that only a single plate of Stentor can be tested at a time, resulting in relatively low-throughput measurements. Furthermore, current software does not allow for the automation of single-cell image analysis. Most data acquired are, therefore, on a population level. Future models of the habituation device and image analysis tools may facilitate high-throughput single-cell experiments.
Habituation in Stentor has been previously studied using methods described by Wood3, but this new protocol allows experiments to be automated. Automation not only allows the researcher to reproducibly deliver mechanical pulses of a specified force and frequency but also facilitates long-term habituation experiments since the device can be left running without supervision for days. Furthermore, using a stepper motor rather than the solenoid employed in Wood&#39;s experiments3 reduces the risk of demagnetization over time and also allows the strength of the stimulus to be varied during the course of a single experiment.
Studying cellular habituation may reveal clinical insights for conditions such as attention-deficit/ hyperactivity disorder (ADHD) and Tourette&#39;s syndrome in which habituation is impaired11. Stentor habituation mechanisms may also unveil new non-synaptic learning paradigms independent of complex cellular circuitry. Finally, insights about single-cell learning could inspire methods for reprogramming cells within multicellular tissues - another potential avenue to fight disease.

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information1, and intelligence is a measure of the computational process that facilitates learning2.
Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus3. Stentor contracts in response to mechanical stimulation3, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability3. The habituated Stentor still contracts after receiving high-force mechanical stimulation4 or photic stimulation5. These observations, which align with Thompson and Spencer&#39;s classic criteria for habituation in animals6, strongly suggest that the original contractile response decrement is due to learning rather than fatigue or ATP depletion. As a free-living cell, Stentor can be studied without much interference from surrounding cells, as would be the case in a multicellular tissue. Several additional features make Stentor a tractable system for studying learning: its large size (1 mm), its quantifiable habituation response3, the ease of injection and micromanipulation7, the fully sequenced genome8, and the availability of RNA interference (RNAi) tools9. Using this model organism to explore cell learning without a brain or nervous system requires a reproducible procedure for stimulating Stentor cells and measuring the response.
Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations (Figure 1). Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64692/64692fig01.jpg" /> 
Figure 1: Habituation experiment setup. The Petri plate containing Stentor is placed atop the flexible metal ruler of the habituation device. The armature of the habituation device then hits the metal ruler at a specified force and frequency, producing a stimulus wave across the field of cells. The USB microscope camera records the responses of the Stentor to the stimulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64692/64692fig02.jpg" /> 
Figure 2: Summary of the habituation experiment workflow. The figure shows the basic steps involved in studying Stentor using the habituation device. The figure was created with BioRender.com. Adapted from &#34;Process Flowchart&#34;, by BioRender.com (2022). Retrieved from&#160;https://app.biorender.com/biorender-templates.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.01% Poly-ornithine&amp;nbsp;</td><td>Millipore Sigma</td><td>P4957</td><td>Used to coat Petri plate</td></tr><tr><td>35-mm Petri plate</td><td>Benz Microscope Optics Center Inc.</td><td>L331</td><td>Contains Stentor during experiments</td></tr><tr><td>6-well plate</td><td>StemCell Technologies</td><td>38016</td><td>Used to wash Stentor</td></tr><tr><td>Aluminum breadboard, 4&quot; x 24&quot; x 1/2&quot; (x1)</td><td>Thorlabs</td><td>MB424</td><td>Used to construct habituation device</td></tr><tr><td>Big easy driver stepper motor driver board (x1)</td><td>Sparkfun</td><td>ROB-12859</td><td>Used to construct habituation device</td></tr><tr><td>Construction rail, 1&quot; x 5&#039;&#039; (x2)</td><td>Newport</td><td>Newport CR-1</td><td>Used to construct habituation device</td></tr><tr><td>Laptop</td><td>Apple Store</td><td>https://www.apple.com/macbook-air-m1/</td><td>Connect laptop to USB microscope to visualize experiments</td></tr><tr><td>Large right-angle bracket (x1)</td><td>Thorlabs</td><td>AP90RL</td><td>Used to construct habituation device</td></tr><tr><td>Microcontroller board</td><td>Arduino</td><td>A000066</td><td>Used to control habituation device</td></tr><tr><td>Nema 17 Stepper Motor Bipolar 59Ncm 2A&amp;nbsp;84oz.in&amp;nbsp;48mm 4-Lead&amp;nbsp;</td><td>Stepperonline.com</td><td>5-17HS19-2004S1</td><td>Used to construct habituation device</td></tr><tr><td>Pasteurized spring water</td><td>Carolina</td><td>132458</td><td>Media for Stentor experiments</td></tr><tr><td>Right-angle bracket (x3)</td><td>Thorlabs</td><td>AP90</td><td>Used to construct habituation device</td></tr><tr><td>Stemi 2000 stereo microscope</td><td>Zeiss</td><td></td><td>Used to visualize Stentor during wash steps</td></tr><tr><td>Stentor coeruleus</td><td>Carolina</td><td>131598</td><td>These are the cells used for habituation experiments</td></tr><tr><td>USB microscope</td><td>Celestron</td><td>44308</td><td>Used to visualize and record experiments</td></tr><tr><td>Webcam recorder</td><td>Apple Store</td><td>https://apps.apple.com/us/app/webcam-recorder/id1508067444?mt=12</td><td>Install this application to take videos of experiments</td></tr></tbody></table>

studying habituation in stentor coeruleus

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

The method described above, using the Level 4 mechanical pulse at a frequency of 1 tap/min, should result in a progressive reduction in the contraction probability of the Stentor within 1 h. This is indicative of habituation (see Figure 6, Video 2).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64692/64692fig06v2.jpg" /> 
Figure 6: Baseline habituation. The contraction probability of Stentor progressively declines over the course of 1 h after receiving Level 4 mechanical pulses at a frequency of 1 tap/min (n = 22-27). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 2. Video of habituated Stentor. The cells receive a Level 4 mechanical stimulus after 1 h of receiving mechanical pulses of the same force at a frequency of 1 tap/min. Most of the cells have habituated to the stimuli during the hour and, thus, do not contract. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/Video_2_JoVE.mp4" target="_blank">Please click here to download this Video.</a>
Altering the force and/or frequency of the mechanical pulse delivery can change the Stentor habituation dynamics. For example, using the Level 2 pulse at a frequency of 1 tap/min precludes habituation over the course of 1 h (see Figure 7). A Level 5 pulse should elicit contractions in few to zero Stentor.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64692/64692fig7v2.jpg" /> 
Figure 7: Lack of habituation within 1 h for stronger forces. The contraction probability of Stentor does not appreciably decline over the course of 1 h after receiving Level 2 mechanical pulses at a frequency of 1 tap/min (n = 7-33). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64692/64692fig07largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Habituation in Stentor coeruleus at JoVE.com

Studying Habituation in Stentor coeruleus

We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

Studying Habituation in Stentor coeruleus

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display ...

studying-habituation-in-stentor-coeruleus

Nanyang Technological University

Research

JoVE Journal

Biology

3.1K Views.  University of California San Francisco. We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations. 

Video: Studying Habituation in Stentor coeruleus

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Behavior

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Neuroscience

A Method for Investigating Change Blindness in Pigeons (Columba Livia)

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. While many laboratory procedures have been developed that produce change blindness in humans, the flicker paradigm has emerged as a particularly effective method. In the flicker paradigm, two visual displays are presented in alternation with one another. If successive displays are separated by a short inter-stimulus interval (ISI), change detection is impaired. The simplicity of the procedure and the clear, performance-based operational definition of change blindness make the flicker paradigm well-suited to comparative research using nonhuman animals. Indeed, a variant has been developed that can be implemented in operant chambers to study change blindness in pigeons. Results indicate that pigeons, like humans, are worse at detecting the location of a change if two consecutive displays are separated in time by a blank ISI. Furthermore, pigeons&#39; change detection is consistent with an active, location-by-location search process that requires selective attention. The flicker task thus has the potential to contribute to investigations of the dynamics of pigeons&#39; selective spatial attention in comparison to humans. It also illustrates that the phenomenon of change blindness is not exclusive to humans&#39; visual perception, but may instead be a general consequence of selective attention. Finally, while the useful aspects of attention are widely appreciated and understood, it is also important to acknowledge that they may be accompanied by specific imperfections such as change blindness, and that these imperfections have consequences across a wide range of contexts.

A Method for Investigating Change Blindness in Pigeons Columba Livia

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. This protocol describes a variation on the flicker paradigm for investigating change blindness that is appropriate and effective for research with pigeons.

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. While many ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Developmental Biology

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Studying Habituation in Stentor coeruleus

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Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

A Method for Investigating Change Blindness in Pigeons (Columba Livia)

Methods for the Study of Regeneration in Stentor

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Long-term Sensory Conflict in Freely Behaving Mice

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking