Kazumi Sakata

Functional expression of GABA ϱ 3 receptors in Xenopus oocytes

Abstract Homomeric rat GABA ϱ 3 receptors were expressed in Xenopus oocytes, and their pharmacological profile was investigated electrophysiologically. GABA activated the ϱ 3 receptors with an EC 50 value of 7.5 μM and a Hill coefficient of 1.6. The GABA-induced current was not antagonized by bicuculline (100 μM), but was blocked by picrotoxin (IC 50 : 0.68 μ M for 100 μM GABA). The current was almost insensitive to pentobarbital, diazepam and a neurosteroid, 3α-OH-DHP. Many of the pharmacological properties of the p 3 subunit were similar to those of the previously reported rat ϱ 1 and ϱ 2 subunits and GABA c receptors.

Neural network model to generate head swing in locomotion of Caenorhabditis elegans

Computer simulation of the neural network composed of the head neurons of Caenorhabditis elegans was performed to reconstruct the realistic changes in the membrane potential of motoneurons in swinging the head for coordinated forward locomotion. The model neuron had ion channels for calcium and potassium, whose parameters were obtained by fitting the experimental data. Transmission properties of the chemical synapses were set as graded. The neural network involved in forward movement was extracted by tracing the neuronal activity flow upstream from the motoneurons connected to the head muscles. Simulations were performed with datasets, which included all combinations of the excitatory and inhibitory properties of the neurons. In this model, a pulse input entered only from motoneuron VB1, and activation of the stretch receptors on SAA neurons was necessary for the periodic bending. The synaptic output property of each neuron was estimated for the alternate contraction of the dorsal and ventral muscles. The AIB neuron was excitatory, RIV and SMD neurons seemed to be excitatory and RMD and SAA neurons seemed to be inhibitory. With datasets violating Dales principle for the SMB neuron, AIB neuron was excitatory and RMD neuron was inhibitory. RIA, RIV and SMD neurons seemed to be excitatory.

A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans.

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.

Calcium inhibits willardiine-induced responses in kainate receptor GluR6(Q)/KA-2.

A number of studies have demonstrated that willardiine [(S)-1-(2-amino-2-carboxyethyl) pyrimidine-2,4-dione] is a useful agonist for the activation of AMPA/kainate receptors. Here we examine the effect of extracellular calcium on currents evoked by willardiine in HEK 293 cells expressing the GluR6(Q)/KA-2 kainate receptor subunits. At a concentration of 1.8 mM, Ca2+ inhibited the currents induced by 100 μM willardiine by ∼50%. When extracellular Na+ ions were replaced with Ca2+ ions there were no measurable inward currents. We conclude that Ca2+ inhibition of the willardiine-induced response is concentration dependent.

Development of new optical imaging systems of oxygen metabolism and simultaneous measurement in hemodynamic changes using awake mice

BACKGROUND PET allows the measurement of CBF, CBV and CMRO2 in human and plays an important role in the diagnosis of pathologic conditions and clinical research. On the other hand, in animal studies, there is no optical imaging system for evaluating changes in CBF and CBV, and oxygen metabolism, from the same brain area under awake condition. NEW METHOD In the present study, we developed a simultaneous measurement system of LSI and IOSI, which was verified by LDF. Moreover, to evaluate oxygen metabolism, FAI was performed from the same brain area as LSI and IOSI measurements. RESULTS The change in CBF according to LSI was correlated with that by LDF. Similarly, the change in CBV obtained by IOSI was also correlated with RBC concentration change measured by LDF. The change in oxygen metabolism by FAI was associated with that in CBF obtained by LSI, although the change in CBF was greater than that in oxygen metabolism. COMPARISON WITH EXISTING METHOD(S) We revealed that the relationship between oxygen metabolism and CBF as measured by our system was in good agreement with the relationship between CMRO2 and CBF in human PET studies. CONCLUSIONS Our measurement system of CBF, CBV and oxygen metabolism is not only useful for studying neurovascular coupling, but also easily corroborates human PET studies.

Navigational choice between reversal and curve during acidic pH avoidance behavior in Caenorhabditis elegans

BackgroundUnder experimental conditions, virtually all behaviors of Caenorhabditis elegans are achieved by combinations of simple locomotion, including forward, reversal movement, turning by deep body bending, and gradual shallow turning. To study how worms regulate these locomotion in response to sensory information, acidic pH avoidance behavior was analyzed by using worm tracking system.ResultsIn the acidic pH avoidance, we characterized two types of behavioral maneuvers that have similar behavioral sequences in chemotaxis and thermotaxis. A stereotypic reversal-turn-forward sequence of reversal avoidance caused an abrupt random reorientation, and a shallow gradual turn in curve avoidance caused non-random reorientation in a less acidic direction to avoid the acidic pH. Our results suggest that these two maneuvers were each triggered by a distinct threshold pH. A simulation study using the two-distinct-threshold model reproduced the avoidance behavior of the real worm, supporting the presence of the threshold. Threshold pH for both reversal and curve avoidance was altered in mutants with reduced or enhanced glutamatergic signaling from acid-sensing neurons.ConclusionsC. elegans employ two behavioral maneuvers, reversal (klinokinesis) and curve (klinotaxis) to avoid acidic pH. Unlike the chemotaxis in C. elegans, reversal and curve avoidances were triggered by absolute pH rather than temporal derivative of stimulus concentration in this behavior. The pH threshold is different between reversal and curve avoidance. Mutant studies suggested that the difference results from a differential amount of glutamate released from ASH and ASK chemosensory neurons.

The Caenorhabditis elegans R13A5.9 gene plays a role in synaptic vesicle exocytosis

The Caenorhabditis elegans R13A5.9 gene encodes a putative membrane protein with homologs in mammals. When the R13A5.9 protein was fused to different fluorescent proteins, signal was observed in or near synaptic vesicles; thus, we sought to determine whether this gene plays a role in synaptic vesicle formation, function, or exocytosis. R13A5.9 mutant worms exhibited low sensitivity to aldicarb (an acetylcholinesterase inhibitor), which suggested that vesicular loading or release, or acetylcholine synthesis, was disrupted in these organisms. This was supported by the observation that an R13A5.9 mutant strain exhibited an excessive accumulation of synaptic vesicles. Collectively, these results suggest a functional role for R13A5.9 in synaptic vesicle exocytosis.

Imaging of Neuronal Activity in Awake Mice by Measurements of Flavoprotein Autofluorescence Corrected for Cerebral Blood Flow

Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

Inhibitory effects of caffeine on gustatory plasticity in the nematode Caenorhabditis elegans

The effects of caffeine on salt chemotaxis learning were investigated using the nematode Caenorhabditis elegans. To estimate the degree of salt chemotaxis learning, nematodes were placed in a mixed solution of NaCl and caffeine, and then the chemotaxis index of NaCl was obtained from the nematodes placed on agar medium after pre-exposure to caffeine concentrations of 0.01, 0.1, 0.3, and 1.0%. Locomotor activity and preference behavior for caffeine were also estimated under these caffeine conditions. Nematodes pre-exposed to 0.3% caffeine showed inhibition of salt chemotaxis learning. Additional experiments indicated that nematodes showed a preference response to the middle concentration of caffeine (0.1%), with preference behavior declining in the 0.3% caffeine condition. Stable locomotor activity was observed under 0.01–0.3% caffeine conditions. These results suggest that salt chemotaxis learning with 0.3% caffeine is useful for investigating the effects of caffeine on learning in nematodes. Graphical abstract We developed the nematode as a model animal for studying the effects of caffeine on learning. Nematodes pre-exposed to 0.3% caffeine showed inhibition of learning.

Expression of GABA receptor ϱ2 and ϱ3 subunits in rat brain

TAROU OGURUSU, RYOZO SHINGAI, KAZUYA YANAGI, KAZUMI SAKATA Department of Computer and Information Science, Faculty of Engineering, lwate University, Morioka, lwate Japan GABAc receptors have been reported to gate bicuculline-insensitive Clcurrents in various parts of the vertebrate brain and retina. It has been suggested GABAc receptors in rat retina are composed partially or entirely of GABA receptor p subunits. However, little is known about subunit compositions of GABAc receptors in brain. Analysis of clones derived from PCR amplification of adult, early postnatal (P8), and embryonic (E16) rat brain cDNA (96 clones each) using mixed oligonucleotides primers derived from amino acid sequeces in TM2 and TM4 of p subunits produced p2 sequence. Next, the RT-PCR was performed with oligonucleotides discriminating among all three p subunit encoding cDNAs. No PCR products corresponding to pl subunit were observed. In contrast, p2 (adult> P8 > El 6) and p3 (El 6> P82 adult) mRNAs were detected in rat brain. Expression of p2 and p3 mRNAs were detected in many brain regions by RT-PCR. Coexpression of p2 and p3 subunits in Xenopus oocytes resulted in heteromeric p2ip3 receptors which were less sensitive to picrotoxinin than homomeric p3 receptors. These results suggest that part of p2 subunits, whrch do not form functional homomeric receptors, form heterooligomeric receptors with p3 subunits in adult rat brain.