Recording cerebellar neuron activities in swimming goldfish

ABSTRACT:   Neuronal activities were investigated in the cerebellum of immobilized and swimming goldfish Carassius auratus . Extracellularly recorded neural activities of the cerebellum in immobilized goldfish were characterized and classified into five types. Based on the waveforms and recording depths, these five neural activity types were estimated to originate from three identified classes of cerebellar neurons: Purkinje cells, eurydendroid cells, and granule cells. Chronic recording of cerebellar neuron activities in unrestrained goldfish was performed for more than 100 h. During the chronic recordings, a submersible amplifier attached to the goldfish head, and a multielectrode array developed for the present study were used. Neuronal activities in the cerebellum of free-swimming fish could also be classified into five types as in the immobilized condition. Firing patterns of two neurons identified as Purkinje cells and eurydendroid cells were analyzed during turning movements of the goldfish. The firing patterns of these neurons changed in relation to turning movements. Although some improvements are required, the chronic recording method developed in the present study can be applied to further investigations concerning the direct relationship between brain neural activities and certain behavior.     

Validation of the DNDC-Rice model by using CH4 and N2O flux data from rice cultivated in pots under alternate wetting and drying irrigation management

The DNDC (DeNitrification-DeComposition)-Rice model, one of the most advanced process-based models for the estimation of greenhouse gas emissions from paddy fields, has been discussed mostly in terms of the reproducibility of observed methane (CH₄) emissions from Japanese rice paddies, but the model has not yet been validated for tropical rice paddies under alternate wetting and drying (AWD) irrigation management, a water-saving technique. We validated the model by using CH₄ and nitrous oxide (N₂O) flux data from rice in pots cultivated under AWD irrigation management in a screen-house at the International Rice Research Institute (Los Baños, the Philippines). After minor modification and adjustment of the model to the experimental irrigation conditions, we calculated grain yield and straw production. The observed mean daily CH₄ fluxes from the continuous flooding (CF) and AWD pots were 4.49 and 1.22?kg?C?ha^?1?day^?1, respectively, and the observed mean daily N₂O fluxes from the pots were 0.105 and 34.1?g?N?ha^?1?day^?1, respectively. The root-mean-square errors, indicators of simulation error, of daily CH₄ fluxes from CF and AWD pots were calculated as 1.76 and 1.86?kg?C?ha^?1?day^?1, respectively, and those of daily N₂O fluxes were 2.23 and 124?g?N?ha^?1?day^?1, respectively. The simulated gross CH₄ emissions for CF and AWD from the puddling stage (2 days before transplanting) to harvest (97 days after transplanting) were 417 and 126?kg?C?ha^?1, respectively; these values were 9.8% lower and 0.76% higher, respectively, than the observed values. The simulated gross N₂O emissions during the same period were 0.0279 and 1.45?kg?N?ha^?1 for CF and AWD, respectively; these values were respectively 87% and 29% lower than the observed values. The observed total global warming potential (GWP) of AWD resulting from the CH₄ and N₂O emissions was approximately one-third of that in the CF treatment. The simulated GWPs of both CF and AWD were close to the observed values despite the discrepancy in N₂O emissions, because N₂O emissions contributed much less than CH₄ emissions to the total GWP. These results suggest that the DNDC-Rice model can be used to estimate CH₄ emission and total GWP from tropical paddy fields under both CF and AWD conditions.     

Growth of recombinant equine herpesvirus 1 (EHV-1) replaced with passage-induced mutant gene 1 and gene 71 derived from an attenuated EHV-1 in cell cultures and in the lungs of mice

The relationship of passage-induced mutant genes 1 and 71 of an attenuated equine herpesvirus 1 (EHV-1) with virulence was analysed by constructing nine recombinant EHV-1 viruses by homologous recombination. Gene 1 or/and gene 71 of a virulent EHV-1 strain, HH1, was replaced by a mutant gene 1 or/and 71 of an attenuated HH1 strain, BK343, respectively. The beta-galactosidase gene of Escherichia coli was inserted within the gene 1 or 71 coding sequence of HH1 to inactivate the genes. Virus replications of these recombinant viruses in cell cultures were similar, but release of the gene 71-inactivated virus from infected cells was delayed compared to that of the other viruses. Plaque sizes of the recombinant viruses were similar to those of HH1, but those of BK343 were significantly smaller, indicating an effect of some unknown factor(s) on viral cell-to-cell spread. The growth abilities of the recombinant viruses with a mutant gene 1 or/and 71 in lungs of mice were similar to those of HH1, but those of gene 71-inactivated viruses were reduced to the level of BK343 and the titers were about 100-times lower than those of the other recombinant viruses. These results indicate that the mutant genes 1 and 71 of BK343 might not confer an attenuated nature to EHV-1.     

Presence and ontogeny of intestinal and pancreatic phospholipase A2-like proteins in the Red Sea bream,Pagrus major. An immunocytochemical study

We have studied the location and the ontogeny of the digestive enzyme, phospholipase A₂ (PLA₂) immunohistochemically in the adult and larvae/juvenile of the red sea breamPagrus major by using an antiserum against theNaja naja venom PLA₂. The antiserum reacts with at least one enzyme among the PLA₂s purified from the fish hepatopancreas or intestine. Although the reactivities were comparatively low, it labelled zymogen granules of the pancreatic acinar cells and secretory materials of certain epithelial cells in the depths of epithelial crypts in the pyloric caeca of the adult. The immunoreactivities of PLA₂s were investigated in the viscera of larvae and juveniles of the 0 to 85^th day after hatch. In the larvae of the 13^th day, accumulation of PLA₂-positive zymogen granules in the pancreatic acinar cells were first recognized by the immunostaining. The intensity of the labelling subsequently became stronger and dramatically increased between the 20^th and 30^th day. This increase appeared to be one of the physiological changes associated with the transition to a new benthic life as juveniles. Lack of PLA₂ in the pancreas before the 13^th day may suggest the possibility that larvae utilized exogenous PLA₂, inherent in their prey, to digest the phospholipids. On the other hand, no reactivity was found in the intestine until the 85^th day.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Dynamic response of wall-floor joints of wooden light-frame constructions under forced harmonic vibrations

Shaking table tests of the wall-floor joints of wooden light-frame constructions under forced harmonic vibrations are conducted in this study so as to observe the dynamic responsive characteristics. The principal results are as follows: The responsive characteristics of timber constructions under strong earthquakes cannot be directly correlated with their resonant frequencies under free or forced vibrations with low input accelerations, because they behave as continuous bodies when the input accelerations are less than the apparent frictional limits of structural joints. The apparent frictional limits are reduced by periodic fluctuation of the effective vertical loads as a result of the vertical motion of the specimens. The characteristic dynamic responses of wall-floor joints depend clearly upon the frequency and input accelerations of forced vibrations. These dependencies arise from the nonlinear load-slip relationship of the wall-floor joints. The equivalent stiffness in their successive transient phases decreases as joint slip increases, which gradually changes the resonant frequencies of the wall-floor joints. This indicates that the frequency components dominant to ultimate or safety-limit resistance should be distinguished from those dominant to allowable or serviceability-limit resistance.     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

Generation of monoclonal antibody against canine neural-cell adhesion molecule

A monoclonal antibody, K9BYU, was generated using Escherichia coli recombinant extracellular domain of canine neural-cell adhesion molecule (N-CAM) as an antigen. Immunoreactivity of K9BYU to insect cell recombinant canine N-CAM was demonstrated by Western blotting using Sf9 insect cells transfected with the canine N-CAM gene. In Western blotting against canine brain tissue, K9BYU detected three isoforms of N-CAM that correspond to three major isoforms of human and mouse N-CAM (N-CAM-120, -140, and -180). From these results, K9BYU was considered to be a useful tool for research of canine N-CAM.     

Two new species,Phytophthora nagaii sp. nov. and P. fragariaefolia sp. nov., causing serious diseases on rose and strawberry plants,respectively, in Japan

A new disease of rose was noticed in Chiba Prefecture of Japan in 1968, and the pathogen was initially identified as Phytophthora megasperma based on morphological characteristics. Similar Phytophthora isolates have since been collected from rose plants in Chiba, Kanagawa, and Shizuoka Prefectures. In 2005, several Phytophthora isolates were recovered from crowns of strawberry plants in Hokkaido Prefecture. These were considered to be members of a new species. In this study, we re-examined all these isolates using morphological and physiological studies and a multilocus phylogenetic analysis. The rose and strawberry isolates were mostly similar morphologically and physiologically, with some exceptions. The rose isolates differed significantly from P. megasperma sensu stricto and other related Phytophthora species. The rose and strawberry isolates had external proliferation of sporangia, characteristic funnel-shaped oogonia, predominantly paragynous antheridia, and fast growth rates of 10.5 mm/24 h at an optimum temperature of 28 °C. In the multilocus phylogenetic tree constructed using sequences from the rDNA ITS regions, rDNA LSU, and the translation elongation factor 1-α, β-tubulin and coxI genes, they formed a distinct monophyletic group in clade 7 with strong bootstrap support. The rose and strawberry isolates separated into two distinct groups. The results indicate that the rose and strawberry isolates constitute two separate species, designated here as Phytophthora nagaii and P. fragariaefolia.