Chang-Hyun Park (Department of Biological Sciences, 3rd Year Doctoral Student / Currently Postdoctoral Fellow, Institute of Basic Sciences (IBS), Korea)

Shinji Kanda (Associate Professor, Department of Biological Sciences, Atmosphere and Ocean Research Institute)

Yuichi Iino (Professor, Department of Biological Sciences)

Hirofumi Kunitomo (Associate Professor, Department of Biological Sciences )

Key points of the presentation

• We found that the ClC-type anion channel ^{(Note 1)} regulates the taste-seeking behavior of C. elegans ^{(Note 2} ).
• ClC channels contribute to the sensing of salt concentration changes by taste neurons ^{(Note 3} ) via the transport of chloride ions.
• Genes for ClC channels are conserved across species and are responsible for genetic disorders in humans. In addition to clarifying the role of ClC channels in nerves, the results of this study are expected to lead to a better understanding of these diseases.

Summary of Presentation

Animals use their sense of smell and taste as cues to determine the type and location of food in the environment, and sometimes move to those locations through trial and error. It remains unclear what genes regulate the functions of the brain and nervous system during such exploratory behavior.

A research group led by Associate Professor Hirofumi Kunitomo of the Graduate School of Science at The University of Tokyo and Associate Professor Shinji Kanda of the Atmosphere and Ocean Research Institute at The University of Tokyo has discovered that ClC-type anion channels are involved in the regulation of behavior based on taste information, using the nematode worm, which is often used for neural research. The study of the behavior and neural activity of nematodes with mutated genes revealed that ClC channels contribute to the response of gustatory neurons to salt stimuli through the regulation of intracellular chloride ion concentrations. This is a new finding indicating that ClC channels modulate neuronal responsiveness and, in turn, are involved in the control of behavior. The results of this study are expected to contribute to our understanding of the relationship between ClC channel function and disease.

Announcement

For animals living in nature, obtaining food is a critical factor directly related to survival. In addition to olfactory and gustatory information obtained from the environment, animals must make use of their past experiences to efficiently move toward their feeding grounds. At this point, the brain/nervous system coordinates sensory information with memory and directs movement toward the destination. There are still many unresolved questions as to what genes are involved in this complex process of information processing.

Although C. elegans is a simple organism with a brain and nervous system consisting of only about 300 neurons, it is possible to observe learning that is dependent on experience to change behavior. One example is taste learning. Wild-type nematodes memorize the concentration of salt (sodium chloride, henceforth simply called salt) in their feeding environment and exhibit exploratory behavior (salt chemotaxis) toward the memorized salt concentration for a while afterwards. Associate Professor Hirofumi Kunitomo, Professor Yuichi Iino, Graduate School of Science, The University of Tokyo, and their colleagues are working to elucidate the mechanisms of stimulus perception by sensory neurons, information processing in neural circuits, and memory and learning using taste learning in C. elegans as a model. Previous studies have shown that taste learning requires that taste neurons called ASERs correctly detect changes in salt concentration. It is also known that the neural circuits that process information from the ASERs correct the direction of movement when the animal is moving toward the wrong salt concentration, and the animal then moves toward the correct salt concentration. However, it was not well understood how the responsiveness of the ASER to salt is regulated and how this response is reflected in the control of behavior.

In this study, Chang-Hyun Park, a third-year PhD student (at the time of the research) and his colleagues obtained mutants showing abnormal salt chemotaxis and identified the causative gene in order to find a new gene required for taste learning. They found that two different mutants both had mutations in the ClC-type anion channel gene ( clh-1 gene). The fact that multiple mutations of the same gene were obtained suggested that this gene may play an important role. Surprisingly, however, the loss-of-function mutant, which completely lost the function of the clh-1 gene, did not show abnormal salt chemotaxis, indicating that the clh-1 gene was altered to have a different function in the above two mutants. Detailed examination of the salt chemotaxis of the mutants also revealed that the mutants showed a weakened chemotaxis toward lower salt concentrations compared to the wild type, but no significant abnormalities in learning.

The clh-1 gene is expressed in most tissues of the body, not just nerves. Experiments to restore gene function in individual tissues and cells revealed that normal clh-1 gene function in ASER is important for salt chemotaxis; ASER senses changes in environmental salt concentration, activates, and transmits this information to downstream interneurons. Comparing wild-type and clh-1 mutants, we found that the mutant ASER failed to respond properly to changes in salt concentration, particularly in response to repeated stimulation (Fig. 1, top and middle panels). We also found that when ASER is excited, the concentration of intracellular chloride ions increases, and the increase is greater in clh-1 mutants. These results indicate that the clh-1 gene contributes to the responsiveness of ASER to changes in salt concentration through regulation of cellular ion concentrations. Furthermore, in clh-1 mutants, the activity of interneurons that receive information from ASER is weakened, making it more difficult for them to change direction toward lower salt concentrations (Figure 1, lower panel).

Figure 1: Function of the clh-1 gene in Caenorhabditis elegans salt chemotaxis.
C. elegans senses changes in salt concentration with ASER taste neurons in the head (upper panel). clh-1 mutants have dysregulation of cellular anion concentration, resulting in a reduced ASER response to salt stimuli (middle panel). As a result, the mutant has reduced chemotaxis toward lower salt concentrations (lower panel).

ClC channel genes are widely conserved throughout the living world, from bacteria to humans, and are widely involved in the regulation of intracellular ion concentrations. Therefore, mutations in the ClC gene are known to disrupt cellular homeostasis and cause several genetic disorders in humans. In addition, mammals have nine genes of the ClC family ^{(Note 4)}, each of which is known to play a different role in tissues and cells. On the other hand, C. elegans has six genes of the ClC family, which are thought to play similar roles to those of mammals, but have not been investigated in detail. In the current study,only thefirstclh-1mutation showed a significant abnormality in salt chemotaxis, suggesting that each gene does indeed have a different function. Interestingly, C. elegans with loss of function in all six genes also survived and produced offspring. This result indicates that ClCfamilygenes are not essential for individual survival and that the function of regulatingcellularion concentrations may be replaced by other genes.may provide insights into human disease.

Published Journals

Journal Title	eLife
Title of paper	Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of C. elegans
Author(s)	Chanhyun Park, Yuki Sakurai , Hirofumi Sato , Shinji Kanda , Yuichi Iino*, Hirofumi Kunitomo* (*co-senior author)
DOI Number	10.7554/eLife.55701
Abstract URL	https://elifesciences.org/articles/55701

Terminology

1 ClC-type anion channel

All cells exchange substances in and out of the cell via transport proteins on the plasma membrane, and ClC-type anion channels are one such transport protein that allows anions _{such as} chloride ion (Cl-) and bicarbonate ion ( _HCO3-) to pass through. Ion channel function is essential for maintaining a constant ionic environment in the cell, and in neurons (nerve cells), ion channels change the membrane potential and excite the cell. ↑up

Note 2: Caenorhabditis elegans

The scientific name is " Caenorhabditis elegans. It is a linear animal with an adult body length of approximately 1 mm and feeds on bacteria. It is often used as a model organism in developmental biology and neuroscience research because of its advantages such as its transparent body, which allows internal observation under a microscope, rapid alternation of generations, and compact brain and nervous system. ↑up

Note 3 Taste neurons

Each nematode neuron has a unique name. Neurons called ASER, located in the head of the individual, function as taste sensors that detect changes in the salt concentration of the environment. ↑up

Note 4 ClC family

Genes with similar functions other than anion channels are also included in the ClC family. ↑up