During fast movements in vertebrates, slow motor units are thought to be deactivated due to the mechanical demands of muscle contraction, but the associated neuronal mechanisms for this are unknown. ...Here, we perform functional analyses of spinal V1 neurons by selectively killing them in larval zebrafish, revealing two functions of V1 neurons. The first is the long-proposed role of V1 neurons: they play an important role in shortening the cycle period during swimming by providing in-phase inhibition. The second is that V1 neurons play an important role in the selection of active sets of neurons. We show that strong inhibitory inputs coming from V1 neurons play a crucial role in suppressing the activities of slow-type V2a and motor neurons, and, consequently, of slow muscles during fast swimming. Our results thus highlight the critical role of spinal inhibitory neurons for silencing slow-component neurons during fast movements.
The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment ...via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.
Aims
We compared the protective effects of sodium glucose co‐transporter (SGLT) 2 inhibitor luseogliflozin on pancreatic β‐cells between early and advanced stages of diabetes and between short‐ and ...long‐term use.
Materials and methods
Diabetic db/db mice were treated with luseogliflozin for 2 weeks in an early stage of diabetes (7‐9 weeks of age) and an advanced stage of diabetes (16‐18 weeks) for a longer period of time (7‐18 weeks). We performed various morphological analyses of pancreatic islets and examined gene expression profiles in islets after such treatment.
Results
In diabetic db/db mice, insulin biosynthesis and secretion were markedly increased by luseogliflozin in an early stage of diabetes but not in an advanced stage. In addition, β‐cell mass was preserved by luseogliflozin only in an early stage. Furthermore, when db/db mice were treated with luseogliflozin for a longer period of time, starting from an early stage, β‐cell function and mass were markedly preserved even after a longer period of time compared to untreated db/db mice.
Conclusion
Luseogliflozin exerts more protective effects in an early stage of diabetes compared to an advanced stage, and longer‐term use of luseogliflozin exerts more beneficial effects on pancreatic β‐cells compared to short‐term use.
The developing spinal cord is subdivided into distinct progenitor domains, each of which gives rise to different types of neurons. However, the developmental mechanisms responsible for generating ...neuronal diversity within a domain are not well understood. Here, we have studied zebrafish V0 neurons, those that derive from the p0 progenitor domain, to address this question. We find that all V0 neurons have commissural axons, but they can be divided into excitatory and inhibitory classes. V0 excitatory neurons (V0-e) can be further categorized into three groups based on their axonal trajectories; V0-eA (ascending), V0-eB (bifurcating), and V0-eD (descending) neurons. By using time-lapse imaging of p0 progenitors and their progeny, we show that inhibitory and excitatory neurons are produced from different progenitors. We also demonstrate that V0-eA neurons are produced from distinct progenitors, while V0-eB and V0-eD neurons are produced from common progenitors. We then use birth-date analysis to reveal that V0-eA, V0-eB, and V0-eD neurons arise in this order. By perturbing Notch signaling and accelerating neuronal differentiation, we predictably alter the generation of early born V0-e neurons at the expense of later born ones. These results suggest that multiple types of V0 neurons are produced by two distinct mechanisms; from heterogeneous p0 progenitors and from the same p0 progenitor, but in a time-dependent manner.
The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal ...network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.
During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that ...many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive.
We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically.
These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.
► Optogenetical activation of hindbrain V2a neurons elicits swimming in zebrafish ► Optogenetical silencing of hindbrain V2a neurons stops swimming ► Hindbrain V2a neurons provide both tonic and phasic excitation to locomotor circuits
Together with the worldwide
Washoku
(traditional Japanese foods and drinks) boom, interest in sake, a traditional Japanese alcoholic drink, is increasing around the world. There are few scientific ...analyses and studies on the production of sake or the final product itself. We show the diversity of bacterial contaminants during sake production and investigated the effects of different ingredients on sake (for example, amino acids). The
koji
mold
Aspergillus oryzae
converts rice starch into sugars, and then, the sake yeast
Saccharomyces cerevisiae
converts the sugars to ethanol. Comparative studies of the bacterial flora of different sakes have shown that various bacterial species are detected, but that there are few frequently detected bacteria. In addition, the bacterial flora does not vary much during the process of sake brewing, after the
koji
(steamed rice covered with
koji
mold) and
moto
(fermentation starter) are mixed, suggesting that most bacteria contaminate the sake during the process of
koji
and
moto
production. Thus, there is the possibility that the contaminating bacteria may grow due to a relationship with the
koji
mold and/or the sake yeast. The flavor, taste, and quality of sakes differ, even between the same brands of sakes, which may be attributed to variations in the contaminating bacteria during sake production.
Developmental maturation occurs in slow swimming behavior in larval zebrafish; older larvae acquire the ability to perform slow swimming while keeping their head stable in the yaw dimension. A class ...of long-distance descending commissural excitatory V0v neurons, called MCoD neurons, are known to develop in a later phase of neurogenesis, and participate in slow swimming in older larvae. We hypothesized that these MCoD neurons play a role in coordinating the activities of trunk muscles in the diagonal dimension (e.g., the rostral left and the caudal right) to produce the S-shaped swimming form that contributes to the stability of the head. Here, we show that MCoD neurons do indeed play this role. In larvae in which MCoD neurons were laser-ablated, the swimming body form often adopted a one-sided (C-shaped) bend with reduced appearance of the normal S-shaped bend. With this change in swimming form, the MCoD-ablated larvae exhibited a greater degree of head yaw displacement during slow swimming. In mice, the long-distance descending commissural V0v neurons have been implicated in diagonal interlimb coordination during walking. Together with this, our study suggests that the long-distance descending commissural V0v neurons form an evolutionarily conserved pathway in the spinal locomotor circuits that coordinates the movements of the diagonal body/limb muscles.
Guidelines and recommendations developed and/or endorsed by the American College of Rheumatology (ACR) are intended to provide guidance for particular patterns of practice and not to dictate the care ...of a particular patient. The ACR considers adherence to these guidelines and recommendations to be voluntary, with the ultimate determination regarding their application to be made by the physician in light of each patient’s individual circumstances. Guidelines and recommendations are intended to promote beneficial or desirable outcomes but cannot guarantee any specific outcome. Guidelines and recommendations developed or endorsed by the ACR are subject to periodic revision as warranted by the evolution of medical knowledge, technology, and practice.
The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron subtypes: V2a and V2b. Delta-Notch-mediated cell-cell interactions between postmitotic immature neurons have been ...implicated in the segregation of neuron subtypes. However, lineage relationships between V2a and V2b neurons have not been reported. We address this issue using Tgvsx1:GFP zebrafish, a model system in which high GFP expression is initiated near the final stage of p2 progenitors. Cell fates were followed in progeny using time-lapse microscopy. Results indicate that the vast majority, if not all, of GFP-labeled p2 progenitors divide once to produce V2a/V2b neuron pairs, indicating that V2a and V2b neurons are generated by the asymmetric division of pair-producing progenitor cells. Together with evidence that Notch signaling is involved in the cell fate specification process, our results strongly suggest that Delta-Notch interactions between sister cells play a crucial role in the final outcome of these asymmetric divisions. This mechanism for determining cell fate is similar to asymmetric divisions that occur during Drosophila neurogenesis, where ganglion mother cells divide once to produce distinct neurons. However, unlike in Drosophila, the divisional axes of p2 progenitors in zebrafish were not fixed. We report that the terminal division of pair-producing progenitor cells in vertebrate neurogenesis can reproducibly produce two distinct neurons through a mechanism that may not depend on the orientation of the division axis.
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