Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

Neural responses give insight to brain activity, but can vary across experiments and individuals. This protocol helps by automating stimulation recording and analysis, and simplifying the programming of different types and patterns of stimuli. The automated technique allows recordings for multiple neurons and organisms in the same microfluidic format in parallel.

This improves the repeatability of recordings and allows the exploration of neuronal variability across populations. Different stimulation patterns are needed to reveal neural phenomena like adaptation and sensory integration. This method is generalizable to other dynamic signals from cells and organoids to organisms and plants.

To begin, turn on the microscope, fill and remove the air bubbles from the reservoir tubing using the priming syringe, then fill the outflow tubing with buffer. Remove the microfluidic device from the vacuum desiccator. Place the device on the microscope and quickly insert the outlet tubing.

Gently push on the outlet syringe to inject fluid into the device until a droplet emerges from an inlet. At this droplet emerged inlet, use a drop-to-drop connection to insert the corresponding fluidic inlet tube, ensuring that liquid drops are present on both the inlet tubing and the device porthole to avoid introducing a bubble. Connect the next inlet tube to the next droplet.

Inject more fluid from the outlet if needed and repeat until all the inlets are filled. Insert a solid blocking pin at unused inlets and the worm loading port. Check the flow on the screen to ensure the flow goes in the direction desired.

Flip valve one on the valve controller and observe stimulus flow flowing into the microfluidic arena. If the flow is imbalanced, adjust the reservoir heights. Transfer the young adult animals onto an unseeded nematode growth medium or NGM agar plate using a wire tip pick, then flood the plate with approximately five milliliters of one XS basal buffer such that the animals can swim.

Draw the worms into a one to three milliliter loading syringe using the attached tubing prefilled with one XS basal buffer. Using a stereoscope, move the tubing in below the liquid surface with one hand to each desired animal and draw it into the tubing using the syringe held in the other hand. Close the outlet outline, remove the worm loading pin, and connect the worm loading syringe to the device using a drop-to-drop connection.

Then gently flow the animals into the arena. Establish buffer flow and allow up to one hour for immobilization by tetramisole. Using the text editor, create a stimulus definition text file named userdefinedacquisitionsettings.

txt containing the stimulation settings for the automated image acquisition. This file defines microscope acquisition parameters such as exposure and excitation timing, trial duration, intervals, and save directory. It also defines the experiment type and stimulation timing settings.

For a single stimulus experiment, use a format with only one repeated stimulation command. For a multi pattern experiment, use a format-with multiple stimulation commands by including a pattern sequence of digits that represents the order of the stimulus patterns. For each pattern, enter a stimulation command on a separate line.

Next, run the microscope control software. Verify that all the fluidic inlets are open, the flow is desired within the arena, and the neurons of interest are in focus within the live window. Then close the live window and run the script multipatternrunscript.

bsh within the software. To analyze the data, run NeuroTracker by sequentially clicking on plugins, then tracking and then NeuroTracker. Select the folder containing the tiff video files to be tracked and select the range of files to be processed.

Next, using the image and adjust menus, open the brightness contrast and threshold control windows. Check the dark background and don't reset range in the threshold window, setting the thresholding method to default and the visualization to red. Then click auto in the brightness contrast window for neuron visibility.

In the brightness contrast window, adjust the minimum and maximum sliders until the neurons are clearly distinguishable. Then adjust the threshold level until all neurons appear as small red spots separate from other objects. Adjust the frame slider to observe the neuron movement and intensity changes, noting any animals to exclude from tracking, such as due to overlap with other animals.

After identifying the neurons for tracking, adjust the threshold level for each animal if needed, such that the red threshold area above the neuron is visible in every frame. Click on the neuron to record its position and threshold level. When all the neurons are selected, press the space bar to begin tracking.

Monitor the tracking process for each animal and make any necessary corrections. If NeuroTracker pauses, it lost the neuron. Adjust the threshold level as needed and re-click the neuron.

If the integration box jumps to another nearby animal or non-neuronal structure, press the space bar to pause. Move the slider back to the first erroneous frame and re-click on the correct neuron location. Run the neurotrackersummarypdf.

m file in MATLAB and select the folder containing the NeuroTracker data text files. Wait for a summary PDF to generate, allowing verification of the tracking process. The animals can be identified by the numbers and the neural responses from each animal and trial and be viewed to assess the population variability.

use the function databrowse. m to explore the neural data grouped by trial number, animal number, stimulation pattern, or by another category. Temporal inhibition phenomenon was tested in a paired pulse experiment which produced eight patterns consisting of two one-second odorant pulses separated by an interval ranging from zero to 20 seconds.

The first one-second odor pulse elicited an equal response magnitude in the diacetyl detecting AWA neurons and responses in the second pulse varied with the inter-stimulus interval. Disinhibition was assessed by the catch trial experiment where adaptation was observed to the diacetyl stimulus, but the presentation of the novel 2-methylpyrazine stimulus did not elicit a strong disinhibition effect. The tested multimodal stimulation revealed that chemical stimulation alone caused adaptation whereas optical stimulation alone did not.

However, the combined multimodal stimulus pattern demonstrated that optogenic responses were susceptible to chemically-induced adaptation. By modifying the microfluidic design to include two arenas, we can compare genetic or other perturbations alongside a matched reference at the same time. Once set up, the system can be modified in many ways.

For example, we have automatically measured chemical dose responses and state-dependent changes to neural activity such as between sleep and awake states.