A subscription to JoVE is required to view this content. Sign in or start your free trial.
We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.
Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.
Calcium imaging techniques have allowed the noninvasive recording of in vivo neural dynamics in real time using fluorescence microscopy and genetically encoded calcium indicators expressed in target cells1,2,3. These sensors typically use a green fluorescent protein (GFP), such as the GFP-calmodulin-M13 peptide (GCaMP) family, to increase the fluorescence intensity upon neuronal activation and elevated intracellular calcium levels. Calcium imaging has been especially powerful in the nematode C. elegans for examining how neurons and neural circuits function in living, behaving animals4,5,6,7,8,9,10, as their transparent nature means no surgical process is required for optical access, and cell-specific gene promoters target expression to the cells of interest. These techniques often make use of microfluidic devices, which provide precisely controlled environments to study biological, chemical, and physical phenomena at a small physical scale11,12. Microfluidic devices abound for measuring neural activity, with new designs continually under development, and they are readily fabricated in the research lab. However, many designs trap a single animal at a time, limiting the experimental throughput7,9,13. Neural responses often vary substantially across animals due to differences in prior experience, internal states such as stress or hunger, or stochastic factors such as gene expression levels. These differences establish a need for methods that can simultaneously stimulate and observe many animals and extract information from individuals4.
In addition, certain neuromodulatory phenomena become apparent only under specific stimulation conditions, such as temporal inhibition14, which refers to the brief suppression of responses when stimulation occurs in rapid succession. Electrophysiological systems can drive neural activity across a broad stimulus space for this purpose, modulating, for example, the electrical pulse current, voltage, frequency, waveform, duty cycle, and timing of periodic stimulus trains. Indirect stimulation by naturally detected stimuli or optogenetic systems would benefit from a similar breadth of control mechanisms. Currently, many natural stimuli are presented in a simple "on-off" manner, such as odor presentation and removal, using commercial systems that have been slow to add flexibility. However, inexpensive microcontrollers can now automate the delivery of several types of stimuli in a manner that is customizable to the researchers' needs. Combined with microfluidics, these systems have achieved the goal of increased experimental throughput and flexibility, allowing neural responses to a variety of precise stimuli to be measured simultaneously in many animals4,6. Multimodal stimulation can be used to further interrogate the neuronal circuitry, such as by monitoring changes in neural excitability when consistently stimulating before, during, and after an orthogonal perturbation such as drug exposure4. The benefits of inexpensive, open microscopy systems are clear for advancing scientific research, yet in practice, the need for part sourcing, construction, and performance validation can impede the adoption of these techniques.
This protocol aims to alleviate some of these technical challenges. Whereas previous protocols have focused on microfluidic device use and basic stimulation9,15,17, we describe here the construction and use of a flexible, automated, multimodal stimulus delivery system for neural imaging in C. elegans or other small organisms utilizing previously described microfluidic devices4. The open-source system is programmed via simple text files to define the experiments, and the NeuroTracker data analysis program semi-automatically extracts the neural activity data from the microscope videos. We demonstrate this system with examples of assessing temporal inhibition, disinhibition, and stimulus crosstalk using the chemosensory neuron AWA, which depolarizes in response to different food odors or in response to light when expressing optogenetic light-sensitive ion channels5,6.
1. Neural imaging equipment
NOTE: See Lawler and Albrecht15 for detailed instructions on building the imaging and stimulation system, which controls the microscope illumination timing, image acquisition, and stimulus delivery (Figure 1). An inexpensive Arduino Nano stimulus controller actuates the fluidic valves through digital signals to a valve controller and controls the optogenetic illumination through analog voltage signals to an LED controller. Other stimuli, such as vibration motors and thermal heaters, can be controlled using digital or analog signals. The stimulus controller synchronizes the stimulation and image recording via camera signals, as specified by the open-source Micro-Manager microscope control software (µManager)16. See the Table of Materials for details related to all the materials, reagents, equipment, and organisms used in this protocol.
2. Microfluidic device fabrication
NOTE: See Lagoy et al.17 for detailed information about obtaining or fabricating the master molds and the production, use, and cleaning of the microfluidic devices. These steps are summarized below.
3. Animal preparation
4. Solution preparation
5. Microfluidic device preparation
NOTE: See Reilly et al.9 for a video protocol showing the reservoir generation, device setup, and the loading of the animals. See also Lagoy et al.17 for a written protocol including many helpful tips.
6. Animal loading
NOTE: See Lagoy et al.17.
7. Automated stimulation and neuronal recording
8. Data analysis using NeuroTracker
NOTE: NeuroTracker4,18,19 is an ImageJ/FIJI20 software plugin for tracking the fluorescence intensity of multiple neurons and animals, even as they move during trials. This plugin saves data as text files with each neuron's position and background-corrected fluorescence intensity (F). The fluorescence data are normalized to the baseline fluorescence (F0), for example, the average of several seconds prior to stimulation, as ΔF/F0 = (F -F0)/F0, which can be averaged across populations.
9. Data exploration and visualization
NOTE: The MATLAB analysis script is used for data processing and visualization and to generate summary PDFs for each analysis.
We present several examples of stimulus patterns that assess different neural phenomena, including temporal inhibition, adaptation, and disinhibition. Temporal inhibition is the momentary suppression of a neural response to a second stimulus presentation occurring shortly after the initial presentation14. To test this phenomenon, in a paired-pulse experiment, eight patterns consisting of two 1 s odorant pulses separated by an interval ranging from 0 s to 20 s were presented (
In this protocol, we describe an open-access microscopy system for the assessment of neural activity phenomena using the temporally precise delivery of different stimulus patterns. The microfluidic platform delivers repeatable stimuli while keeping tens of animals in the microscope field of view. Few commercial microscopy software packages allow for the easy programming of various stimulus timing patterns, and those that do often require the manual entry of each pattern or proprietary file formats. In contrast, experimen...
The authors have no conflicts of interest to disclose.
We thank Fox Avery for testing these protocols and reviewing the manuscript and Eric Hall for programming assistance. Funding for the methods presented herein was provided in part by the National Science Foundation 1724026 (D.R.A.).
Name | Company | Catalog Number | Comments |
Bacterial strains | |||
E. coli (OP50) | Caenorhabditis Genetics Center (CGC) | Cat# OP50 | |
Experimental models: Organisms/strains | |||
C. elegans strains expressing GCaMP (and optionally, Chrimson) in desired neurons | Caenorhabditis Genetics Center (CGC) or corresponding authors of published work | NZ1091, for example | |
Chemicals, Treatments, and Worm Preparation Supplies | |||
2,3-Butanedione | Sigma-Aldrich | Cat# B85307 | diacetyl, example chemical stimulus |
Calcium chloride, CaCl2 | Sigma-Aldrich | Cat# C3881 | |
Fluorescein, Sodium salt | Sigma-Aldrich | Cat# F6377 | |
Glass water repellant | Rain-X | Cat #800002250 | glass hydrophobic treatment (single-use) |
Magnesium chloride, MgCl2 | Sigma-Aldrich | Cat# M2393 | |
Nematode Growth Medium (NGM) agar | Genesee | Cat #: 20-273NGM | |
Petri dishes (60 mm) | Tritech | Cat #T3305 | |
Poly(dimethyl siloxane) (PDMS): Sylgard 184 | Dow Chemical | Cat# 1673921 | |
Potassium phosphate monobasic | Sigma-Aldrich | Cat# P5655 | |
Potassium phosphate dibasic | Sigma-Aldrich | Cat# P8281 | |
Sodium chloride, NaCl | Sigma-Aldrich | Cat# S7653 | |
(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (TFOCS) | Gelest | CAS# 78560-45-9 | glass hydrophobic treatment (durable) |
Software and algorithms | |||
Arduino IDE | Arduino | https://www.arduino.cc/en/software | |
ImageJ | NIH | https://imagej.nih.gov/ij/ | |
MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
Micro-manager | Micro-manager | https://micro-manager.org/ | |
Microscope control software | Albrecht Lab | https://github.com/albrechtLab/MicroscopeControl | |
Neurotracker data analysis software | Albrecht Lab | https://github.com/albrechtLab/Neurotracker | |
Automated Microscope and Stimulation System | |||
Axio Observer.A1 inverted microscope set up for epifluorescence (GFP filter cubes, 5× objective or similar) | Zeiss | Cat #491237-0012-000 | |
Excelitas X-cite XYLIS LED illuminator | Excelitas | Cat #XYLIS | |
Orca Flash 4.0 Digital sCMOS camera | Hamamatsu | Cat #C11440-22CU | |
Arduino nano | Arduino | Cat #A000005 | |
3-way Miniature Diapragm Isolation Valve (LQX12) | Parker | Cat #LQX12-3W24FF48-000 | Valve 1: Control |
2-way normally-closed (NC) Pinch Valve | Bio-Chem Valve Inc | Cat #075P2-S432 | Valve 2: Outflow |
3-way Pinch Valve | NResearch | Cat #161P091 | Valve 3: Stimulus selection |
Optogenetic stimulation LED and controller (615 nm) | Mightex | Cat #PLS-0625-030-S and #SLA-1200-2 | |
ValveLink 8.2 digital/manual valve controller | AutoMate Scientific | Cat #01-18 | |
Wires and connectors | various | See Fig. 2 of Cell STARS Protocol (Lawler, 2021) | |
Microfluidic Device Preparation | |||
Dremel variable speed rotary cutter 4000 | Dremel | Cat #F0134000AB | Set speed to 5k RPM for cutting glass |
Dremel drill press rotary tool workstation | Dremel | Cat #220-01 | |
Diamond drill bit | Dremel | Cat #7134 | |
Glass slide, 1 mm thick | VWR | Cat #75799-268 | |
Glass scribe (Diamond scriber) | Ted Pella | Cat #54468 | |
Luer 3-way stopcock | Cole-Parmer | Cat #EW-30600-07 | |
Luer 23 G blunt needle | VWR | Cat #89134-100 | |
Microfluidic device | Corresponing author or fabricate from CAD files associated with this article | N/A | |
Microfluidic device clamp | Warner Instruments (or machine shop) | P-2 | |
Microfluidic tubing, 0.02″ ID | Cole-Parmer | Cat #EW-06419-01 | |
Tube 19 G, 0.5″ | New England Small Tube | Cat #NE-1027-12 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved