High-throughput optical quantification of mechanosensory habituation reveals neurons encoding memory in Caenorhabditis elegans

Abstract

A major goal of neuroscience studies is to identify the neurons and molecules responsible for memory. Mechanosensory habituation in Caenorhabditis elegans is a simple form of learning and memory, in which a circuit of several sensory neurons and interneurons governs behavior. However, despite the usefulness of this paradigm, there are hardly any systems for rapid and accurate behavioral genetic analysis. Here, we developed a multiplexed optical system to genetically analyze C. elegans mechanosensory habituation, and identified two interneurons involved in memory formation. The system automatically trains large populations of animals and simultaneously quantifies the behaviors of various strains by optically discriminating between transgenic and nontransgenic animals. Biochemical and cell-specific behavioral analyses indicated that phosphorylation of cyclic AMP response element-binding protein (CREB), a factor known to regulate memory allocation, was facilitated during training and this phosphorylation in AVA and AVD interneurons was required for habituation. These interneurons are a potential target for cell-specific exploration of the molecular substrates of memory.Caenorhabditis elegans | mechanosensory habituation | memory | optical quantification | neural circuit I dentification of the physical substrates of memory in the brain, namely neurons and molecules encoding memory, is a major challenge in neuroscience. Recent studies have revealed that the transcription factor cAMP response element-binding protein (CREB) (1) is crucial for determining which neurons in the lateral amygdala participate in encoding an auditory fear memory, and that neurons with higher CREB activity are preferentially allocated into a unique memory trace (memory engram) (2-6). Thus, it has been generally thought through the accumulated weight of evidence that memory is represented by a specific population of neurons in the brain that forms memory engrams: only a portion of eligible neurons are recruited into a specific memory. Although this CREB-dependent memory allocation model is established (5), the identification of specific neurons supporting a given memory remains a long-standing challenge. Moreover, the extreme complexity of nervous systems in model animals still hampers cell-specific genetic analyses; therefore, even if neurons encoding memory could be identified, it would be difficult to address the subsequent question of how these neurons store memory at the molecular level in situ.The nematode Caenorhabditis elegans is a genetically tractable model organism with a neural circuit composed of only 302 neurons. Moreover, the wiring of this nervous system has been completely determined. These unique features of the C. elegans nervous system have proven to be advantageous for various behavioral experiments, such as genetic rescue experiments, ectopic expression analysis, overexpression analysis, and RNA interference-mediated gene disruption, because all of these transgenic studies can be conducted in situ in a cell-specific manner...

ComparingCaenorhabditis elegansgentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

“…The first class is based around the plate tap reflex and comprises assays that use an impactor or actuator to induce vibration of the agar substrate, triggering a response mediated by the gentle touch receptors 14 . As in our assay, stimulus strength can be measured, usually with a MEMS accelerometer 7,33,34 or laser Doppler vibrometer 35 , and responses of freely moving animals can be recorded. However, the stimulus is not localized to any one part of the animal, preventing the study of touches to a subset of touch receptors.…”

Nanoscale Mechanical Stimulation Method for Quantifying C. elegans Mechanosensory Behavior and Memory

“…12,13 In this system, amplitude of the nonlocalized vibration could be roughly changed, whereas its frequency and duration could not. Therefore, instead of the previous system, we used a piezoelectric speaker to evoke nonlocalized vibration on an agar surface of an NGM plate (Figs.…”

“…12 At first, an acquired AVI format movie was transformed into Tiff-format sequential images using ImageJ software (NIH). This sequential image file was used for subsequent motion analysis of each worm.…”

“…10 Because the neural circuit underlying this behavior was completely determined, we can easily investigate neural and molecular mechanisms using C. elegans cell-specific genetic methods. [10][11][12] In general, the choice of technique to impose mechanical stimulation and to readout behavioral output is critical for understanding the physiology of animal behavior. In C. elegans, nonlocalized mechanical stimulation has been provided by tapping only a single Petri plate using a solenoid tapper 13 or a ROBO cylinder, 12 by manually dropping a plastic box containing multiple plates from a constant height 10 or by playing sound to evoke vibration of an agar surface of a plate using dual cone speaker.…”

“…[10][11][12] In general, the choice of technique to impose mechanical stimulation and to readout behavioral output is critical for understanding the physiology of animal behavior. In C. elegans, nonlocalized mechanical stimulation has been provided by tapping only a single Petri plate using a solenoid tapper 13 or a ROBO cylinder, 12 by manually dropping a plastic box containing multiple plates from a constant height 10 or by playing sound to evoke vibration of an agar surface of a plate using dual cone speaker. 14 However, there have been very few methods that allow for precisely changing the vibration properties such as frequency, amplitude and duration while also validating these properties quantitatively.…”