Ryota Adachi

Redistribution of Kv1 and Kv7 enhances neuronal excitability during structural axon initial segment plasticity

Structural plasticity of the axon initial segment (AIS), the trigger zone of neurons, is a powerful means for regulating neuronal activity. Here, we show that AIS plasticity is not limited to structural changes; it also occurs as changes in ion-channel expression, which substantially augments the efficacy of regulation. In the avian cochlear nucleus, depriving afferent inputs by removing cochlea elongated the AIS, and simultaneously switched the dominant Kv channels at the AIS from Kv1.1 to Kv7.2. Due to the slow activation kinetics of Kv7.2, the redistribution of the Kv channels reduced the shunting conductance at the elongated AIS during the initiation of action potentials and effectively enhanced the excitability of the deprived neurons. The results indicate that the functional plasticity of the AIS works cooperatively with the structural plasticity and compensates for the loss of afferent inputs to maintain the homeostasis of auditory circuits after hearing loss by cochlea removal.

Activity-dependent and activity-independent development of the axon initial segment.

The axon initial segment (AIS) is the site of spike initiation in neurons. Previous studies revealed that spatial distribution of the AIS varies greatly among neurons to meet their specific needs. However, when and how this differentiation arises is unknown. Neurons in the avian nucleus laminaris (NL) are binaural coincidence detectors for sound localization and show differentiation in the distribution of the AIS, with shorter length and a more distal position from the soma with an increase in tuning frequency. We studied these characteristics of the AIS in NL neurons of the chicken during development and found that the AIS differentiates in its distribution after initial formation, and this is driven by activity-dependent and activity-independent mechanisms that differentially regulate distal and proximal boundaries of the AIS. Before hearing onset, the ankyrinG-positive AIS existed at a wide stretch of proximal axon regardless of tuning frequency, but Na+ channels were only partially distributed within the AIS. Shortly after hearing onset, Na+ channels accumulated along the entire AIS, which started shortening and relocating distally to a larger extent in neurons with higher tuning frequencies. Ablation of inner ears abolished the shortening of the AIS without affecting the position of its proximal boundary, indicating that both distal and proximal AIS boundaries are disassembled during development, and the former is dependent on afferent activity. Thus, interaction of these activity-dependent and activity-independent mechanisms determines the cell-specific distribution of the AIS in NL neurons and plays a critical role in establishing the function of sound localization circuit.

Plasticity of the Axonal Trigger Zone

The axon initial segment (AIS) is a specialized axonal compartment that is involved in conversion of synaptic potentials into action potentials. Recent studies revealed that structural properties of the AIS, such as length and position relative to the soma, are differentiated in a cell-specific manner and shape signal processing of individual neurons. Moreover, these structural properties are not fixed but vary in response to prolonged changes of neuronal activity, which readjusts action potential threshold and compensates for the changes of activity, indicating that this structural plasticity of the AIS works as a homeostatic mechanism and contributes to maintain neuronal activity. Neuronal activity plays a crucial role in formation, maintenance, and refinement of neural circuits as well as in pathogenesis and/or pathophysiology of diseases. Thus, this plasticity should be a key to understand physiology and pathology of the brain.

In vivo calcium imaging of OFF-responding ASK chemosensory neurons in C. elegans

BACKGROUND How neurons and neuronal circuits transform sensory input into behavior is not well understood. Because of its well-described, simple nervous system, Caenorhabditis elegans is an ideal model organism to study this issue. Transformation of sensory signals into neural activity is a crucial first step in the sensory-motor transformation pathway in an animals nervous system. We examined the properties of chemosensory ASK neurons of C. elegans during sensory stimulation. METHOD A genetically encoded calcium sensor protein, G-CaMP, was expressed in ASK neurons of C. elegans, and the intracellular calcium dynamics of the neurons were observed. RESULTS After application of the attractants l-lysine or food-related stimuli, the level of calcium in ASK neurons decreased. In contrast, responses increased upon stimulus removal. Opposite responses were observed after application and removal of a repellent. CONCLUSION The observed changes in response to external stimuli suggest that the activity of ASK neurons may impact stimulus-evoked worm behavior. The stimulus-ON/activity-OFF properties of ASK neurons are similar to those of vertebrate retinal photoreceptors. GENERAL SIGNIFICANCE Analysis of sensory-motor transformation pathways based on the activity and structure of neuronal circuits is an important goal in neurobiology and is practical in C. elegans. Our study provides insights into the mechanism of such transformation in the animal.

Genetic analysis of ryanodine receptor function in Caenorhabditis elegans based on unc-68 revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca2+-induced Ca2+ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca2+ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.

Uptake of Albumin is Coupled with Stretch-Induced Hypertrophy of Skeletal Muscle Cells in Culture

Abstract Hypertrophy is induced in skeletal muscle when mechanical overload, for example repetitive stretching, is presented. This is a well-known phenomenon and the molecular mechanism involved has been investigated from various aspects. In this study, with a system that enables periodic stretching of cultured skeletal muscle cells, myotubes, along the long cellular axis uni-directionally at a constant frequency, we examined the effects of stretching on skeletal muscle using mouse C2 myotubes in culture as a model. Significant hypertrophy was observed in the myotubes after several days of periodic stretching and this was accompanied by the accumulation of a protein of about 67kDa. This protein was identified with albumin, which was present in the culture medium, based on its antigenicity, size and pI. When bovine serum albumin tagged with biotin was added to the culture medium, it became detectable in the cytoplasm of the stretched myotubes. mRNA encoding albumin was not detectable in the myotubes by northern blotting irrespective of their stretching or non-stretching, indicating that transcription of the albumin gene was not induced in the stretched muscle cells. From these results, we conclude that the accumulation of albumin in stretched myotubes was due to uptake of the protein from the culture medium not to de novo synthesis of the protein in myotubes. We suggest that albumin uptake may be involved in skeletal muscular hyper-trophy.

Biochemical and Molecular Biological Analyses of space-flown nematodes in Japan, the First International Caenorhabditis elegans Experiment (ICE-First).

The first International Caenorhabditis elegans Experiment (ICE-First) was carried out using a Russian Soyuz spacecraft from April 19–30, 2004. This experiment was apart of the program of the DELTA (Dutch Expedition for Life science Technology and Atmospheric research) mission, and the space agencies that participate in the International Space Station (ISS) program formed international research teams. A Japanese research team that conducted by Japan aerospace Exploration Agency (JAXA) investigated the following aspects of the organism: (1) whether meiotic chromosomal dynamics and apoptosis in the germ cells were normal under microgravity conditions, (2) the effect of the space flight on muscle cell development, and (3) the effect of the space flight on protein aggregation. In this article, we summarize the results of these biochemical and molecular biological analyses.

Modulation of Caenorhabditis elegans chemotaxis by cultivation and assay temperatures.

The chemotaxis behaviors of the nematode Caenorhabditis elegans cultivated at various temperatures (15 degrees C, 20 degrees C and 25 degrees C) were examined at various temperatures (10 degrees C, 15 degrees C, 20 degrees C and 25 degrees C) to determine the multi-sensory integration of physical (thermal) and chemical sensory information within its nervous system. Chemotaxis behavior toward sodium acetate and ammonium chloride were differently affected by both assay and cultivation temperatures, suggesting that the temperature effect on chemotaxis is not general, but rather distinctive for each chemosensory pathway. Since thermosensory cues are likely encountered constantly in C. elegans, we supposed that the chemotaxis behaviors of worms are achieved by the integration of chemo- and thermosensory information. To verify the possible contribution of thermosensory function in chemotaxis, we examined the chemotaxis behaviors of ttx-1(p767) mutant worms with defective AFD thermosensory neurons. The chemotaxis behaviors toward sodium acetate or ammonium chloride of mutant worms cultivated at 20 degrees C and 25 degrees C were reduced relative to those of wild-type worms. These results indicate the important role of multi-sensory integration of chemosensory and thermosensory information in chemotaxis behavior of the C. elegans.

Phase-dependent preference of thermosensation and chemosensation during simultaneous presentation assay in Caenorhabditis elegans

BackgroundMulti-sensory integration is necessary for organisms to discriminate different environmental stimuli and thus determine behavior. Caenorhabditis elegans has 12 pairs of amphid sensory neurons, which are involved in generating behaviors such as thermotaxis toward cultivation temperature, and chemotaxis toward chemical stimuli. This arrangement of known sensory neurons and measurable behavioral output makes C. elegans suitable for addressing questions of multi-sensory integration in the nervous system. Previous studies have suggested that C. elegans can process different chemoattractants simultaneously. However, little is known about how these organisms can integrate information from stimuli of different modality, such as thermal and chemical stimuli.ResultsWe studied the behavior of a population of C. elegans during simultaneous presentation of thermal and chemical stimuli. First, we examined thermotaxis within the radial temperature gradient produced by a feedback-controlled thermoregulator. Separately, we examined chemotaxis toward sodium chloride or isoamyl alcohol. Then, assays for simultaneous presentations of 15°C (colder temperature than 20°C room temperature) and chemoattractant were performed with 15°C-cultivated wild-type worms. Unlike the sum of behavioral indices for each separate behavior, simultaneous presentation resulted in a biased migration to cold regions in the first 10 min of the assay, and sodium chloride-regions in the last 40 min. However, when sodium chloride was replaced with isoamyl alcohol in the simultaneous presentation, the behavioral index was very similar to the sum of separate single presentation indices. We then recorded tracks of single worms and analyzed their behavior. For behavior toward sodium chloride, frequencies of forward and backward movements in simultaneous presentation were significantly different from those in single presentation. Also, migration toward 15°C in simultaneous presentation was faster than that in 15°C-single presentation.ConclusionWe conclude that worms preferred temperature to chemoattractant at first, but preferred the chemoattractant sodium chloride thereafter. This preference was not seen for isoamyl alcohol presentation. We attribute this phase-dependent preference to the result of integration of thermosensory and chemosensory signals received by distinct sensory neurons.

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

Abstract: Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.