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The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.
Microfluidic devices provide increased access to precisely controlled environments, wherein animals, such as the nematode C. elegans, can be experimentally manipulated1. These studies include behavioral assays, calcium imaging studies, or even screenings for specific phenotypes, resulting in more exact measurements of experimental outcomes1,2,3,4,5,6. Microfluidics provide small-scale liquid conditions through which detailed experiments can be run while utilizing minimal amounts of reagents. There is a constant production of new microfluidic device designs, and the use of each varies, from arenas that allow for the natural sinusoidal motion of C. elegans in behavioral assays and neural imaging studies, to trap devices used in neural imaging and olfactory studies, to devices that allow for high-throughput phenotypic analysis in genetic screens4,5,6,7. Following the fabrication of a master mold, microfluidic devices are inexpensive to construct—given the reusability of the master—and easy to use, allowing for rapid data generation via high-throughput studies. The fabrication of devices using polymers such as polydimethylsiloxane (PDMS) allows for the creation of new devices within hours.
Calcium imaging studies use genetically encoded calcium indicators (GECIs) expressed in target cells to measure the neural dynamics of those cells in real time8,9,10,11. The transparent nature of C. elegans allows for the recording of the fluorescent levels of these proteins in live animals. Traditionally, GECIs rely on the green fluorescent protein (GFP)-based sensor GFP-Calmodulin-M13 Peptide (GCaMP), although more recent studies have adapted these sensors to allow for better signal-to-noise ratios and red-shifted excitation profiles. Following the development of GCaMP3, proteins with these specifications have varied, including sensors such as GCaMP6s and GCaMP6f (slow and fast fluorescence off-rates, respectively), as well as RFP-Calmodulin-M13 Peptide (RCaMP), which has a red-shifted activation profile. The combination of these GECIs with C. elegans cell-specific gene promoter sequences can target cells of interest, particularly sensory neurons12,13,14,15,16.
While the ease of C. elegans use in microfluidic studies is apparent, almost all studies have focused on hermaphrodites. Despite males only accounting for 0.01–0.02% of the wild type population, invaluable findings can arise from their characterization. While the physical connectome of the hermaphrodite nervous system has been fully mapped for decades17, the male connectome remains incomplete, especially in the head region of the animal18. The use of calcium imaging in males will help to generate an understanding of the male nervous system and the differences that arise between the two sexes. The smaller size of C. elegans adult males prevents effective and reliable trapping in the loading ports of traditional olfactory devices designed for larger hermaphrodites. To address this, a modified version of the Chronis Olfactory Chip19 was developed with a narrower loading port, a lower channel height, and turns in the worm loading port (which rotate the animal), allowing for the visualization of bilateral left/right neuronal pairs. This design permits: (1) the effective trapping of young adult males, (2) a more reliable orientation of the animal for the visualization of both members of bilateral paired neurons, and (3) the precise imaging of neural activity in male neurons.
Increasingly, studies show that C. elegans males respond differently than hermaphrodites to a variety of ascarosides (ascr), or nematode pheromones20,21,22,23,24. Therefore, developing an understanding of the neural dynamics and representations within the male connectome has become even more pertinent. Male C. elegans contain 87 sex-specific neurons not present in the hermaphrodite25,26, altering the connectome in as-yet undetermined ways. Being able to image these unique neural dynamics will allow us to better understand sex-specific responses and neural representations.
This protocol describes the use of a male-adapted olfactory chip for the neural imaging of male C. elegans chemosensation. The nociceptive neuron ASH responds reliably to 1 M glycerol in males, consistent with previous hermaphroditic studies27. Exposure to ascarosides may elicit responses that are variable from animal to animal, requiring a larger number of animals to be tested. The response of the male-specific CEM neurons has previously been shown, through both electrophysiology and calcium imaging studies, to respond variably to ascaroside #323.
NOTE: See reference1.
NOTE: Silicon master molds were fabricated using standard photolithographic techniques for patterning SU-8 photoresist on a silicon master1,7. Photomasks for wafer patterning were printed at 25,000 dpi. The male-adapted device features a Chronis Olfactory Chip design19 with a change in the worm loading port, adapting a design obtained from M. Zimmer (personal correspondence, 2016). A turn is included to control the rotation of the animals. The width of the worm loading port channel is narrowed to 50 μm. All channels are 32 μm tall. Once a silicon master mold is available to the user, the user can follow the subsequent protocol, as described previously1.
NOTE: See reference23.
NOTE: See refefence1.
An example of the overall device setup can be seen in Figure 1A–B. Figure 1A depicts the proper reservoir construction and setup. Figure 1B shows the connections of the reservoirs to the microfluidic device. Figure 1C depicts a microfluidic device with individual ports labeled for clarity.
The design of the male-adapted microfluidic device contains a curve in the loading port, but flow dynamics are identical to the device designed by Chronis et al.19(Figure 2A–2C). The flow of buffers can be controlled by altering which flow control valve is open (Figure 2A–2B). The measurements of the device as fabricated vary from the designed file. The measurements provided in Figure 2C are “as fabricated” measurements.
After loading male C. elegans into the male-adapted olfactory device, their placement and orientation, as well as channel flow dynamics, can be verified via both bright-field and fluorescent imaging (Figure 2D–2E). The exposure of worms expressing GCaMP3 in the nociceptive neuron, ASH, to 1 M glycerol results in visible changes in fluorescence within the ASH neuron, indicative of neural activity (Figure 2F). Subtle changes in fluorescence may not be visible by eye, but software can be used to quantify these changes. The free ImageJ software can be used to analyze and quantify the fluorescent intensity of ASH neurons upon exposure to 1 M glycerol over time (Figure 2F). This is similar to what is observed in hermaphrodites27 and, due to the robustness of the ASH neuronal response to glycerol, this is observed in all animals tested.
A small amount of axial or rotational movement is expected in unparalyzed animals, often necessitating a neuron-tracking algorithm during video analysis (Figure 3A). The addition of a paralytic in the buffers (e.g., 1 mM tetramisole) nearly eliminates this effect, although some animals (~10%) still move during the trials. This can be circumvented by: (a) using older males, which are more efficiently trapped; (b) decreasing the width or thickness of the worm loading port even further; or (c) either increasing the concentration of the paralytic used or using another paralytic. This will also ensure that there is not too much of the head past the end of the trap and exposed to the odor channel. If a trial with worm movement occurs, the area of analysis can be moved and reread from the new neuronal location, starting at the frame after which the movement occurs (Figure 3B). Manual reconstruction of the neural traces by the user is required in this instance. Scripts that analyze the fluorescent changes within the neuron and that follow the neuron’s center as it moves can also be written19.
Male C. elegans sense attractive biogenic pheromones called ascarosides via the four sex-specific CEM neurons23. When calcium transients are observed in males, the responses are variable in shape, sign, and magnitude between both neurons and animals (Figure 4A–B). However, male response to pheromones is not as reliably observed as calcium transients in many animals (Figure 4C). This is not discouraging, as most ascarosides do not elicit calcium transients upon sensation13,14,15.
The male-adapted olfactory chip incorporates a turn into a narrower loading port, which allows for more control of the orientation and for the efficient trapping of male C. elegans. This allows for the visualization of both the left and right members of neuronal bilateral pairs, without the need for z-stacking. This curve leads to an orientation away from vertical 100% of the time in worms where only one bilateral pair is targeted with a fluorescent marker, such as ASH (Figure 2D–E)29,30. However, in neuronal classes with four radially symmetric neurons, such as CEM, all four neurons are visible only one-third of the time. Another third of worms tested have only three of the four neurons visible, and for the remaining third, the only distinguishable difference is between the dorsal and ventral cell bodies, not the left-right asymmetry (data not shown). The narrower port is combined with a lower channel height to prevent worm fluctuation across the z-axis. This design allows for the imaging of males in future studies, which, when combined with the constantly increasing knowledge of the male connectome25,31, will allow for a better understanding of sex-specific neural function.
The analysis performed in this protocol uses the free software ImageJ to measure changes in fluorescence in the neuron of interest. With the current design, 1 mM tetramisole in the buffer effectively paralyzes the worms and prevents movement of the neurons being imaged. If movement is not preventable, or if the user wishes to avoid the use of a paralytic, more complex tracking scripts must be written that track the neurons as they move7. However, in this protocol, male worms only move when they were too small to be effectively constrained by the loading port and when presented with an extremely aversive stimulus, such as glycerol. Even in these instances, the movement is brief and does not require large amounts of tracking adjustments—setting an ROI around the new neuron location alleviates the incorrect fluorescent readouts (Figure 2).
A limitation of single-worm, trap-based imaging is that only one worm can be imaged at a time1. Another limitation of these traps is that worms can get stuck within the device, causing devices to be clogged and “used up” after imaging only a few worms. However, the quick turnaround time for the fabrication of new devices from a master mold alleviates this downside. Extended blue-light illumination has also been shown to induce photodamage in C. elegans32,33. The relatively short experimental time frame of this protocol (30 s) allows for imaging without measurable photobleaching. However, to avoid photobleaching and photodamage in longer experiments, the light source can be pulsed7. For example, during each 100-ms exposure, the light can be pulsed for 10 ms. This has been shown to eliminate increased body autofluorescence over time7.
In order to properly test males for their responses to ascarosides, larval-stage 4 (L4) males must first be isolated from hermaphrodites, for at least 5 h, in order to achieve a near-naïve response to the pheromones23. Isolation for less than this length of time may cause animals to fail to respond to the ascarosides. However, this isolation is not necessary when testing non-ascaroside cues, such as glycerol. For the sake of consistency, however, animals were always isolated at least 5 h prior to calcium imaging. In some neurons, such as the CEM, not every stimulation will elicit a neuronal response, and each CEM that does responds does so to generate a certain “code” of neural representation. This phenomenon in CEM has been observed via electrophysiology studies, as well as with calcium imaging studies like the ones described here23. Thus, measurable calcium transients in CEM neurons in every ascaroside-exposed animal are not guaranteed23. In fact, many of the ascarosides investigated to date do not elicit measurable calcium transients13,15,34,35. The successful elicitation of measurable transients was observed during only one of three pulses of pheromone in two of the five animals exposed to the ascaroside of interest (Figure 3). This matches the rate of success previously observed in other labs23. This variability is a limitation when studying pheromone response and is not due to the male-based focus of this protocol.
When investigating calcium transients elicited in response to ascarosides, one should not dismiss a lack of consistent response without further investigation. This can be tested through experiments such as electrophysiology studies to confirm the variable response within a neuronal class. For neurons, such as ASH, that respond reliably, a lack of consistent response could be indicative of larger experimental problems, such as errors in stimulus control. The peak intensity of the responses can also be investigated if variability is expected in the response. The traces can be plotted with the standard deviation or standard error (as in Figure 2F). If the standard deviation is small, the traces can be plotted and analyzed as such. If there is noticeable amount of variation leading to moderate standard deviations, the data can be plotted the same, with accompanying heatmaps sorted by response “type” to show the response-by-response variation. If there is significant variation (Figure 3), wherein the peaks cannot be distributed in a Gaussian manner, responses can be categorized into response “types” (e.g., depolarizing, hyperpolarizing, or non-polarizing)23. Responses that fall into a certain “type” can be plotted and analyzed together. Similarly, heatmaps should accompany this analysis as well.
Moving forward, this device can be adapted to allow for the imaging of larval-staged nematodes by narrowing the loading port even further. Further narrowing of the end of the loading port will allow for the constraint of the animal to allow for imaging of just the cilia of the sensory neurons, as opposed to the cell body. While other devices are designed for the more commonly studied hermaphrodite, this adapted olfactory chip allows for the imaging of neural activity in male neural circuits. As the connectome of the male is still being elucidated, being able to measure neural dynamics in sex-specific networks is critical to fully understanding neuronal signaling. Differences between hermaphroditic and male responses can now be tested and measured using this device.
We would like to thank Manuel Zimmer for providing us with the initial design file that was adapted for use with males; Frank Schroeder for the synthesis and supply of ascr#3; Ross Lagoy for the insight and assistance with imaging and analysis; and Laura Aurilio for the master fabrication and who, alongside Christopher Chute, contributed to the review of this manuscript. Funding for this work was provided under the National Institutes of Health grant 1R01DC016058-01 (J.S.), the National Science Foundation grant CBET 1605679 (D.R.A.), and the Burroughs Wellcome Career Award at the Scientific Interface (D.R.A.).
The video component of this article can be found at https://www.jove.com/video/56026/
The authors have nothing to disclose.