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/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/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/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/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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+single+cell+organisms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parametric+studies+of+the+response+decrement+produced+by+mechanical+stimuli+in+the+protozoan,+Stentor+coeruleus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+learning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+specific+habituation+in+a+protozoan.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation:+A+model+phenomenon+for+the+study+of+neuronal+substrates+of+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stentor+coeruleus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macronuclear+genome+of+Stentor+coeruleus+reveals+tiny+introns+in+a+giant+cell.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinase+regulator+Mob1+acts+as+a+patterning+protein+for+Stentor+morphogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+regeneration+in+Stentor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+is+altered+in+neuropsychiatric+disorders-A+comprehensive+review+with+recommendations+for+experimental+design+and+analysis.">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/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/files/ftp_upload/64692/64692fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.01% Poly-ornithine&#160;</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&#34; x 24&#34; x 1/2&#34; (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&#34; x 5&#39;&#39; (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&#160;84oz.in&#160;48mm 4-Lead&#160;</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/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/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/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 ...

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3.0K 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

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.