Simultaneous recording of behavioral and neural responses of free-moving nematodes C. elegans.

To reveal the neural mechanisms that control animal behavior, it is necessary to link the neural responses to behavioral changes and interpret them. We have developed a protocol to simultaneously record the behavior and neural activity of freely moving C. elegans by combining a microfluidic device and a tracking stage. Here we detail the protocol for the experiment, with an example of behavioral and neural responses of nematodes to salt concentration changes. For complete details on the use and execution of this protocol, please refer to Sato et al. (2021).

Glutamate signaling from a single sensory neuron mediates experience-dependent bidirectional behavior in Caenorhabditis elegans.

Orientation and navigation behaviors of animals are modulated by past experiences. However, little is known about the mechanisms by which sensory inputs are translated into multi-directional orientation behaviors in an experience-dependent manner. Here, we report a neural mechanism for bidirectional salt-concentration chemotaxis of Caenorhabditis elegans. The salt-sensing neuron ASE right (ASER) is always activated by a decrease of salt concentration, while the directionality of reorientation behaviors is inverted depending on previous salt experiences. AIB, the interneuron postsynaptic to ASER, and neurons farther downstream of AIB show experience-dependent bidirectional responses, which are correlated with reorientation behaviors. These bidirectional behavioral and neural responses are mediated by glutamate released from ASER. Glutamate acts through the excitatory glutamate receptor GLR-1 and inhibitory glutamate receptor AVR-14, both acting in AIB. These findings suggest that experience-dependent reorientation behaviors are generated by altering the magnitude of excitatory and inhibitory postsynaptic signals from a sensory neuron to interneurons.

Presynaptic MAST kinase controls opposing postsynaptic responses to convey stimulus valence in Caenorhabditis elegans.

Presynaptic plasticity is known to modulate the strength of synaptic transmission. However, it remains unknown whether regulation in presynaptic neurons can evoke excitatory and inhibitory postsynaptic responses. We report here that the Caenorhabditis elegans homologs of MAST kinase, Stomatin, and Diacylglycerol kinase act in a thermosensory neuron to elicit in its postsynaptic neuron an excitatory or inhibitory response that correlates with the valence of thermal stimuli. By monitoring neural activity of the valence-coding interneuron in freely behaving animals, we show that the alteration between excitatory and inhibitory responses of the interneuron is mediated by controlling the balance of two opposing signals released from the presynaptic neuron. These alternative transmissions further generate opposing behavioral outputs necessary for the navigation on thermal gradients. Our findings suggest that valence-encoding interneuronal activity is determined by a presynaptic mechanism whereby MAST kinase, Stomatin, and Diacylglycerol kinase influence presynaptic outputs.

A new platform for long-term tracking and recording of neural activity and simultaneous optogenetic control in freely behaving Caenorhabditis elegans.

BACKGROUND: Real-time recording and manipulation of neural activity in freely behaving animals can greatly advance our understanding of how neural circuits regulate behavior. Ca2+ imaging and optogenetic manipulation with optical probes are key technologies for this purpose. However, integrating the two optical approaches with behavioral analysis has been technically challenging. NEW METHOD: Here, we developed a new imaging system, ICaST (Integrated platform for Ca2+ imaging, Stimulation, and Tracking), which combines an automatic worm tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. We optimized different excitation wavelengths for the concurrent use of channelrhodopsin-2 and G-CaMP, a green fluorescent protein (GFP)-based, genetically encoded Ca2+ indicator. RESULTS: Using ICaST in conjunction with an improved G-CaMP7, we successfully achieved long-term tracking and Ca2+ imaging of the AVA backward command interneurons while tracking the head of a moving animal. We also performed all-optical manipulation and simultaneous recording of Ca2+ dynamics from GABAergic motor neurons in conjunction with behavior monitoring. COMPARISON WITH EXISTING METHOD(S): Our system differs from conventional systems in that it does not require fluorescent markers for tracking and can track any part of the worm's body via bright-field imaging at high magnification. Consequently, this approach enables the long-term imaging of activity from neurons or nerve processes of interest with high spatiotemporal resolution. CONCLUSION: Our imaging system is a powerful tool for studying the neural circuit mechanisms of C. elegans behavior and has potential for use in other small animals.

Calcium dynamics regulating the timing of decision-making in C. elegans.

Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+]i), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.

In actio optophysiological analyses reveal functional diversification of dopaminergic neurons in the nematode C. elegans.

Many neuronal groups such as dopamine-releasing (dopaminergic) neurons are functionally divergent, although the details of such divergence are not well understood. Dopamine in the nematode Caenorhabditis elegans modulates various neural functions and is released from four left-right pairs of neurons. The terminal identities of these dopaminergic neurons are regulated by the same genetic program, and previous studies have suggested that they are functionally redundant. In this study, however, we show functional divergence within the dopaminergic neurons of C. elegans. Because dopaminergic neurons of the animals were supposedly activated by mechanical stimulus upon entry into a lawn of their food bacteria, we developed a novel integrated microscope system that can auto-track a freely-moving (in actio) C. elegans to individually monitor and stimulate the neuronal activities of multiple neurons. We found that only head-dorsal pair of dopaminergic neurons (CEPD), but not head-ventral or posterior pairs, were preferentially activated upon food entry. In addition, the optogenetic activation of CEPD neurons alone exhibited effects similar to those observed upon food entry. Thus, our results demonstrated functional divergence in the genetically similar dopaminergic neurons, which may provide a new entry point toward understanding functional diversity of neurons beyond genetic terminal identification.

Regulation of experience-dependent bidirectional chemotaxis by a neural circuit switch in Caenorhabditis elegans.

The nematode Caenorhabditis elegans changes its chemotaxis to NaCl depending on previous experience. At the behavioral level, this chemotactic plasticity is generated by reversing the elementary behaviors for chemotaxis, klinotaxis, and klinokinesis. Here, we report that bidirectional klinotaxis is achieved by the proper use of at least two different neural subcircuits. We simulated an NaCl concentration change by activating an NaCl-sensitive chemosensory neuron in phase with head swing and successfully induced klinotaxis-like curving. The curving direction reversed depending on preconditioning, which was consistent with klinotaxis plasticity under a real concentration gradient. Cell-specific ablation and activation of downstream interneurons revealed that ASER-evoked curving toward lower concentration was mediated by AIY interneurons, whereas curving to the opposite direction was not. These results suggest that the experience-dependent bidirectionality of klinotaxis is generated by a switch between different neural subcircuits downstream of the chemosensory neuron.