We thank Sandra N. Manosalvas-Kjono, Purim Ladpli, Farah Memon, Divya Gopisetty, and Veronica Sanchez for support in device design and generation of mutant animals. This research was supported by NIH grants R01EB006745 (to BLP), R01NS092099 (to MBG), K99NS089942 (to MK), F31NS100318 (to ALN) and received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement No. 715243 to MK).

<ol>
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The authors have nothing to disclose.

This protocol demonstrates a method for delivering precise mechanical stimulation to the skin of a roundworm trapped in a microfluidic chip. It is intended to facilitate the integration of physical stimuli for answering biological questions and aims to streamline mechanobiology research in biological labs. This method extends previous assays to assess the function of mechanosensory neurons in C. elegans. Previous quantitative and semi-quantitative techniques measured forces1,34 and behavior35, but were difficult to integrate with high-resolution imaging of neuronal activity. We thus believe that the use of this chip extends current applications and its performance is optimized for stimulation of neurons required for gentle touch. We optimized the deflection of the actuator to reach 5 &#181;m, which is a strong enough stimulus to reach nearly maximal neuronal responses. With slight variations, we believe that this design could be used to stimulate harsh touch receptors, such as PVD nociceptors that innervate the body surface36.
Modifications and Troubleshooting 
If no clean room is available for SU-8 lithography, a benchtop clean box or SU-8 soft-lithography stations equipped with UV lamps, programmable hotplates, and spin coaters can be used. A low cost alternative for production of microfluidic devices that avoids the use of clean room facilities can also be followed26.
In the design, we implemented an existing trapping technique, which immobilizes a single animal in a microfluidic channel. The trap design is adapted from a device that is used to study olfactory sensation in the worm36, but it is not the only viable option to suppress worm movement. Other strategies have been reported to hold worms in microfluidic devices: CO2, electrophoretic, and compressive immobilization11,37,38 have been used, and could be integrated into a modified design.
We delivered pressure to the actuation channels using a commercially-available, piezoelectric pressure pump which can control and deliver up to 800 kPa from an external pressure source. We connected the pump to compressed air in the research building, which is capable of delivering ~450 kPa. If no source of compressed air is available, a tank of compressed, inert gas (e.g., N2) could be used. This would have the added benefit of delivering higher pressures and generating large deformations, but would also require high-pressure fittings to connect to the pressure controller to the chip. Fabricating the chip from softer PDMS is alternative way to increase pressure-induced deformations.
In this experiment, the outlet of the pressure pump is connected to the PDMS chip through 1-mm receiving openings (e.g., punch holes) plugged with adhesive-free and reversible interconnectors consisting of a press-fit 20 G metal-tube inserted in a PE tubing. These have been shown to resist pressures up to 700 kPa39. Care must be taken during repeated insertion, however, as the PDMS around the holes tends to tear and thus limit the performance or make the chip unusable. For this reason, the metal tubing was inserted once and left plugged into the chip, without removing and inserting it repeatedly.
Critical Steps 
Stimulation is delivered by a pressurized actuation channel, which indents the skin. The resulting deformation and mechanoreceptor neuron activation can be imaged with high resolution on an optical fluorescence microscope and/or confocal set-up. Several factors can hamper the detection of the GCaMP responses. First, C. elegans express a blue-light receptor30 in many neurons, including TRNs21,23, which activates cells after illumination with blue (488 nm) light. Thus, imaging C. elegans using the genetically encoded calcium sensor GCaMP6s will activate its neurons upon illumination, obscuring calcium transients activated by mechanical stimulation23. Working in the lite-1 mutant background or using red-shifted calcium activity sensors avoids this confound. Second, mechanically-induced calcium transient might not be detected if the stimulus profile does not match the sensitivity of the mechanoreceptors. We failed to detect activation for high-amplitude steps and ramps, but evoked extremely high signals after stimulation with moderate dynamics stimuli (e.g., 200 -kPa buzz). Finally, TRNs habituate to a repeated stimuli delivered with short (&#60;60 s) interstimulus intervals. Thus, it is important to design stimulation sequences that minimize habituation except in cases where habituation is being studied.
We recommend filtering all solutions, as debris and dust in the buffers can easily clog the small trapping channel. Further, animals must be age-synchronized and size-matched to the chip&#39;s design; animals smaller than young adults will slide through the device or fail to be restrained and larger animals cannot enter the trapping channels and are in danger of bursting and/or clogging the chip. In case of clogging, application of excessive pressure to the worm or gravity flow inlet may clean the channels, but could also lead to failure of the fitting.
As mentioned in the results section, several factors can lead to a failure in the analysis program. If problems are encountered in the compiling the code, verifying that the most recent Fiji software is running and that Fiji is using the newest Java compiler is recommended. If the program is running but there are unexpected results please make sure that the recorded movie has a 50-frame buffer before the actual mechanical stimulation is performed to ensure a proper estimation of the baseline and background fluorescence. Further, the field of view of the green, activity-dependent (GCaMP6s) and the red, activity-independent (tagRFP) channel are assumed to be projected onto the upper and lower half of the camera chip, respectively. If the optical beam splitter projects the images onto the left and right half of the sensor, turn the camera 90&#176; to obtain the desired orientation during the recording or rotate the image stacks in a pre-processing step. Furthermore, the program identifies the neuron by its fluorescence and will fail if the signal-to-noise ratio or contrast is too low. In this case, changing the excitation intensity and/or exposure time may solve the problem. The program will also stop if the neuron leaves the field of view during the recording, in which case the neuron should be positioned in the center of the field of view before starting a new acquisition.
Limitations of the Technique 
The present design enables stimulation of the body wall mechanoreceptor neurons like the PVD nociceptors and the TRNs, but not mechanoreceptors that innervate the worm&#39;s head region, such as FLP and ASH. It is limited to pressures &#60;700 kPa. For larger pressures, different tube fittings need to be employed. Since the pressure supply here did not exceed 500 kPa, the maximum pressure possible to apply was limited to 450 kPa. The calcium transients observed here mark the upper boundary of the expected signal. Another limitation for the design of our actuators is their aspect ratio. In principle, thinner diaphragms would enable larger deformations, but structures with high aspect ratio are difficult to fabricate reliably. If larger deformations are essential, wider actuators or parallel deformations from both sides might be useful, as presented by Cho et al.29
Although we can monitor diaphragm deformation, it is difficult to measure the forces applied during pneumatic stimulation of the actuation channels. The principle limitation is that the contact area of the actuator is not well-defined. This is because the actuator itself is elastic and deforming during actuation, which changes the contact radius with increasing deflection. With these caveats in mind, we can estimate that a 5 &#181;m indentation (around 300 kPa actuation pressure in an empty chip) is predicted to apply about 3.8 &#181;N to the worm according to the worm stiffness estimates of Petzold et al.34 Since the force dependence of the neuronal current varies in worms with different stiffnesses1,34, however, monitoring indentation as a measure of stimulus intensity instead of force is recommended.
We hope that the dissemination of this protocol will help users take advantage of microfluidics for mechanobiology research using C. elegans as a model system, and thus accelerate the advancement of science on this important subject in the field.

The sense of touch provides animals with crucial information about their environment. Depending on the applied force, touch is perceived as innocuous, pleasurable, or painful. The tissue deformation during touch is detected by specialized mechanoreceptor cells embedded in the skin that express receptor proteins, most commonly ion channels. The steps linking force perception to ion channel activation during touch and pain are not fully understood. Even less is known about how the skin tissue filters mechanical deformation and whether mechanoreceptors detect changes in strain or stress1,2,3. This gap in understanding arises, in part, from a lack of suitable tools to apply precise mechanical stimulations to the surface of the skin of a living animal while observing the responses at the cellular level. Whereas atomic force microscopy has been used extensively to apply and measure forces in isolated cells4,5 and also to activate Piezo1 receptors in living cells6, similar experiments using living animals, especially C. elegans, have been notoriously challenging due to the intrinsic mobility of the subject. This challenge is traditionally circumvented by using veterinary- or surgical-grade cyanoacrylate glue to immobilize individual animals on agar pads1,7,8,9. This approach has been productive, but has limitations related to the skill required for immobilization by gluing and the soft agar surface on mechanical compliance. A microfluidics strategy is a complimentary alternative that avoids some of the complications linked to gluing.
The nematode C. elegans is a genetic model organism with a completely mapped nervous system that, due to the animal&#39;s size, is a good fit for microfluidics technology. Microfluidics-based devices offer the advantage that the otherwise extremely mobile animals can be restrained while performing high-resolution imaging and delivery of relevant neuro-modulatory stimuli. With the help of microfluidic technologies, living animals can be immobilized without harm10,11, enabling monitoring of behavioral activity over the entire lifetime12,13 and high-resolution imaging of neuronal activity14,15,16,17. Further, many mechanoreceptor neurons needed for the sense of touch and pain can be characterized on their physiological1,8, mechanical4,18,19, and molecular level20,21,22.
C. elegans senses gentle mechanical stimuli to its body wall using six TRNs, three of which innervate the animal&#39;s anterior (ALML/R and AVM) and three of which innervate the animal&#39;s posterior (PLML/R and PVM). The ion channel molecules needed for transducing an applied force into a biochemical signal have been extensively studied in its TRNs8. This article presents a microfluidic platform23 that enables researchers to apply precise mechanical forces to the skin of an immobilized C. elegans roundworm, while reading out the deformation of its internal tissues by optical imaging. In addition to presenting well-defined mechanical stimuli, calcium transients can be recorded in mechanoreceptor neurons with subcellular resolution and correlated with morphological and anatomical features. The device consists of a central trapping channel that holds a single animal and presents its skin next to six pneumatic actuation channels (Figure 1 and Figure 2). The six channels are positioned along the trapping channel to deliver mechanical stimuli to each of the worm&#39;s six TRNs. These channels are separated from the trapping chamber by thin PDMS diaphragms, which can be driven by an external air pressure source (Figure 1). We calibrated the deflection with respect to pressure and provide the measurements in this article. Each actuator can be addressed individually and used to stimulate a mechanoreceptor of choice. The pressure is delivered using a piezo-driven pressure pump but any alternative device can be used. We show that the pressure protocol can be used to activate TRNs in vivo and demonstrate operating devices suitable for delivering mechanical stimuli to adult C. elegans, loading adult animals into devices, performing calcium imaging experiments, and analyzing the results. Device fabrication consists of two main steps: 1) photolithography to make a mold from SU-8; and 2) molding PDMS to make a device. For the sake of brevity and clarity, readers are referred to previously published articles and protocols24,25 for instructions on how to produce the molds and devices.

<table>
	<tbody>
		<tr>
			<td>Chrome mask</td>
			<td>Compugraphics (http://www.compugraphics-photomasks.com/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Mitani-Micronics (http://www.mitani-micro.co.jp/en/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Kuroda-Electric (http://www.kuroda-electric.eu/</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon wafer (B-test)</td>
			<td>Stanford Nanofabrication Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2002</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Laurell Technologies</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposure timer</td>
			<td>Optical Associates, Inc</td>
			<td>OAI 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination controller</td>
			<td>Optical Associates, Inc</td>
			<td>2105C2</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A426F-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethylsilane (TCMS)</td>
			<td>Sigma-Aldrich</td>
			<td>92361-500ML</td>
			<td>Caution: TCMS is toxic and water-reactive</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS prepolymer</td>
		</tr>
		<tr>
			<td>Biopsy punch, 1 mm</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Plasma Asher</td>
			<td>Branson/IPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW</td>
			<td>New England Small Tube Corporation</td>
			<td>NE-1300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene syringe filter, 0.22 &#956;m</td>
			<td>Thermo Scientific</td>
			<td>725-2520</td>
			<td>to filter all solution, small particles would clog the chip</td>
		</tr>
		<tr>
			<td>Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID</td>
			<td>Solomon Scientific</td>
			<td>BPE-T50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>for worm trapping and release</td>
		</tr>
		<tr>
			<td>Syringe, 20 ml</td>
			<td>BD Scientific</td>
			<td>309661</td>
			<td>for gravity-based flow</td>
		</tr>
		<tr>
			<td>Gilson Minipuls 3, Peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td>to suck solutions and worms out of the chip</td>
		</tr>
		<tr>
			<td>Microfluidic flow controller, equipped with 0&#8211;800 kPa pressure channel</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td>pressure delivery</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T52</td>
			<td>connection from house air to pressure pump</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T51</td>
			<td>connect pressure pump to small tubng</td>
		</tr>
		<tr>
			<td>Push-to-Connect Tube Fitting for Air</td>
			<td>McMaster-Carr</td>
			<td>5111K468</td>
			<td>metric - imperial converter</td>
		</tr>
		<tr>
			<td>Straight Connector for 6 mm &#215; 1/4&#8243; Tube OD</td>
			<td>McMaster-Carr</td>
			<td>5779 K258</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI 4000 B microscopy system</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>63&#215;/1.32 NA HCX PL APO oil objective</td>
			<td>Leica</td>
			<td>506081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu Orca-Flash 4.0LT digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C11440-42U</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumencor Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td>With cyan and green/yellow light source</td>
		</tr>
		<tr>
			<td>Excitation beam splitter</td>
			<td>Chroma</td>
			<td>59022bs</td>
			<td>in the microscope</td>
		</tr>
		<tr>
			<td>Hamamatsu W-view Gemini Image splitting optics</td>
			<td>Hamamatsu</td>
			<td>A12801-01</td>
			<td>to split green and red emission and project them on different areas on the camera chip</td>
		</tr>
		<tr>
			<td>Emission beam splitter</td>
			<td>Chroma</td>
			<td>T570lpxr</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters GCamp6s</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters mCherry</td>
			<td>Chroma</td>
			<td>ET632/60m</td>
			<td>in the image splitter</td>
		</tr>
	</tbody>
</table>

using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

SU-8 Lithography and Chip Bonding 
The lithography protocol and PDMS molding follow standard procedures. Details can be found elsewhere23,24,25,26. The PDMS should peel off the wafer without problems after curing. If the SU-8 features rip off during PDMS peeling, either the SU-8 adhesion layer or the silanization was insufficient. If plasma-activation of the glass coverslips and the PDMS chips is not sufficient, weak bonding can result in leakage out of the trapping channel or pneumatic actuators resulting in performance degradation and absence of stimulation. If fluid leakage is evident, the chip is unusable.
Actuator Performance 
All of the PDMS actuators and the tubing connecting the chip to the air reservoir must be properly sealed to ensure proper pneumatic actuation of the diaphragms. Repeated insertion and removal of the PE tubing (plumbing) will damage PDMS holes and lead to a decrease in pressure. Such a loss of pressure will limit actuator deflection and the force applied to the skin of the immobilized animal. Deflection of the diaphragm can be measured using an empty chip (no animal present) by performing several actuation cycles with the desired pressure and comparing the quantitative values with theoretical predictions. The deflection of the diaphragm was not measured in presence of an animal in the trapping channel, because the membrane and worm boundary are difficult to visualize. This makes precision calibration with a trapped animal not possible. Table 1 lists the expected deflection as a function of pressure. These values were calculated using elastic plate theory and details can be found in Nekimken et al.23 This relationship is expected to vary with differences in diaphragm thickness generated during chip casting and other factors such as the integrity of the bonding between the chip and the glass and seals connecting the chip to the pressure sources. Here, values were reproducible with slight variation not exceeding 1 &#181;m at 450 kPa of pressure. We have characterized the performance of the diaphragms for all three different stimulus profiles and refer the interested reader to Nekimken et al.23 In summary, there are several factors to consider if the values deviate from Table 1: 1) a leak in the tubing or tubing connectors, 2) the PDMS has a different elasticity, which can result from an alternative ratio of base polymer and curing agent, or 3) the PDMS has aged and continued to crosslink over time. Therefore, use of chips which have been prepared within a month of their application is recommended.
Trapping Performance 
After inserting the worms into the inlet of the chip, a gentle pressure with the syringe should place one single animal inside the trapping channel and present its skin alongside the six actuators (Figure 1). Importantly, the nose of the animal does not necessarily need to protrude into the buffer reservoir. Instead, it is more important to position the animal such that the neurite is adjacent to the nearest actuator and its cell body is within the field of view of the microscope. To achieve optimal neuronal activation, the distance between the actuator and cell body should be adjusted such that the actuator lies anterior to the cell body of interest. In our experience, for neurons stimulated posterior to the cell body, no activation will take place and calcium transients will be absent. Proper immobilization can be achieved with young adult or one-day old hermaphrodites (devices optimized for these two ages are available on the same mask). Smaller animals will slip into the collection reservoir or move laterally inside the trapping channel. Animals that are too large will be squeezed into the channel, which might result in premature activation of its TRNs and subsequent failure to detect mechanoreceptor transients. Also, removal of large worms is challenging, leading to death of the animal and/or clogging of the device. With this design, the PLM neuron usually cannot be immobilized completely, so it is free to move laterally and vertically. Although we have successfully activated PLM neurons, recording calcium transients in these neurons is difficult due to vertical movement of the tail. Another trapping design has been used to record from PLM33.
Imaging of Calcium Transients and Analysis 
Once in place, the animals can be stimulated using one of the six actuators. The six actuators are positioned to deliver mechanical stimuli to each of the six TRNs. A microfluidic flow controller connected to a 450-kPa in-house pressure source was used to deliver to the animal a stimulus protocol consisting of a 2-s 275 kPa step, a 2-s 0&#8211;275 kPa ramp, and a 2-s buzz (a 75 kPa, 10 Hz sine superimposed with a 275 kPa step), each separated by a 10-s waiting period. The simultaneously recorded image sequences of the calcium transients were analyzed in Fiji, using a custom-written software available on our GitHub account (see above and&#160;https://github.com/HFehlauer/Poking-Analyzer). We found that pressure ramps and pressure steps activated TRNs only if the stimuli achieved pressures above 400 kPa. By contrast, a robust activation of all TRNs stimulated using a sinusoidal, 10-Hz buzz at pressures &#60; 275 kPa was observed (Figure 4). In general, TRNs responded better to a brief sinusoidal stimulus (called &#39;buzz&#39;), in agreement with previous reports using different methodologies1,9. The stimulation with a buzz is highly efficient, leading to ~ 90% of activation in all neurons and trials tested (Figure 4). Surprisingly, there was no strong correlation observed on whether the stimulus was applied to the dorsal or the ventral side, and was independent of the distance between the cell body and the stimulus position on the neurite. Future studies will have to investigate whether the delay of the maximum response correlates with the stimulus distance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56530/56530fig1.jpg" /> 
Figure 1: Overview of the chip design. (A) Schematic layout of the photomask and the resulting chip with all connectors indicated. There are six actuation fingers, three on each side of the central trapping channel. Scale bar = 3 mm. (B) Photograph of the microfluidic device with PDMS bonded to the coverslip and connected to tubing in the worm inlet and the worm outlets. Only one of the actuation fingers is connected to the pressure source, while the other five are not occupied. Parts of the figure have been reproduced from reference23 under creative common licenses. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig1large.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/56530/56530fig2.jpg" /> 
Figure 2: Close-up of the central trapping channel and trapping procedure. (A) Schematic drawing of the trapping channel. The pillars at the worm inlet side help to orient the worm and facilitate loading of the animals. The actuation fingers can be pressurized and indent a thin diaphragm into the trapped specimen. Scale bar = 200 &#181;m. (B) Representative video frame of a worm being trapped in the horizontal channel. The channel is designed such that the worm comes to lay across the six pneumatic actuators. Tail is to the left, head is to the right. Location of individual touch receptor neurons (TRNs) are indicated by arrows and labeled in the figure. Scale bar = 100 &#181;m (in all panels). Parts of the figure have been reproduced from reference23 under creative common licenses. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56530/56530fig3.jpg" /> 
Figure 3: Overview of the provided Fiji plugin to analyze calcium traces acquired with this protocol. The software starts a graphical user interface. The first tab (&#34;Open video&#34;) of the user interface shows the button to open a video, it displays the path and the first frame of the opened video. The second tab (&#34;Define ROIs&#34;) includes the button to define a touch receptor neuron, a checkbox for activating the button &#34;Define Actuator&#34; and the button &#34;Define Actuator&#34;. In the lower part, the first frame of the video is displayed. In the upper part of the third tab (&#34;Analysis&#34;), adjustable parameters for the image analysis are displayed. The middle part shows the &#34;Start Analysis&#34; button. In the lower part, a progress bar shows the progress of the current analysis. In the fourth tab, &#34;Results&#34;, the result is displayed as a graph of the relative fluorescence intensity over time. In the lower part, there is a button to save the result table. In the fifth tab, &#34;Help&#34;, a help text is displayed showing the requirements for the analysis software and instruction on how to use it. Figure adapted from reference23 under creative common licenses. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig3large.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/56530/56530fig4.jpg" /> 
Figure 4: Individual neurons respond to mechanical stimuli. (A) Representative fluorescence micrograph of GCamP6s labeled AVM touch receptor neuron before and after mechanical stimulation with a buzz. Scale bar = 10 &#181;m. Color scale = 1,500-3,500 gray values. (B) Stimulus protocol including 2 s diaphragm excitation representing a 275 kPa step, a 275 kPa ramp, and a sine (75 kPa; 10 Hz) superimposed with a 275 kPa step (buzz). (C) Normalized GCamP6s intensity trace (mean &#177; SEM as shaded area) recorded from AVM stimulated with the profile shown in B of wild type animals (green, n = 14) and animals mutant in the mechanoreceptor channel subunit mec-4 (blue, n = 10), that is known to facilitate responses to mechanical stress. This indicates that the transients are induced by the applied mechanical stimuli. Parts of the figure have been reproduced from reference23 under creative common licenses. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig4large.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>Pressure (kPa)</td>
			<td>Mean Deflection (&#181;m)</td>
			<td>SD</td>
			<td>Expected Deflection (&#181;m)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>-0.01</td>
			<td>0.01</td>
			<td>0</td>
		</tr>
		<tr>
			<td>50</td>
			<td>0.77</td>
			<td>0.26</td>
			<td>0.607143</td>
		</tr>
		<tr>
			<td>100</td>
			<td>1.57</td>
			<td>0.58</td>
			<td>1.21429</td>
		</tr>
		<tr>
			<td>150</td>
			<td>2.32</td>
			<td>0.65</td>
			<td>1.82143</td>
		</tr>
		<tr>
			<td>200</td>
			<td>3.13</td>
			<td>0.74</td>
			<td>2.42857</td>
		</tr>
		<tr>
			<td>250</td>
			<td>3.86</td>
			<td>0.79</td>
			<td>3.03571</td>
		</tr>
		<tr>
			<td>300</td>
			<td>4.61</td>
			<td>0.79</td>
			<td>3.64286</td>
		</tr>
		<tr>
			<td>350</td>
			<td>5.3</td>
			<td>0.82</td>
			<td>4.25</td>
		</tr>
		<tr>
			<td>400</td>
			<td>5.92</td>
			<td>0.82</td>
			<td>4.85714</td>
		</tr>
		<tr>
			<td>450</td>
			<td>6.43</td>
			<td>0.75</td>
			<td>5.46429</td>
		</tr>
	</tbody>
</table>
Table 1: Expected experimental deflection values for applied pressures ranging from 0&#8211;450 kPa and their theoretical predictions. Data derived for a step stimulus. Mean &#177; SD. These data were acquired with an empty trapping channel.

Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

using-microfluidics-device-for-mechanical-stimulation-high-resolution

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.7K Views.  Stanford University. New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Video: Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...