Neuronal splicing regulator RBFOX3 mediates seizures via regulating Vamp1 expression preferentially in NPY-expressing GABAergic neurons.

Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (Caenorhabditis elegans) 3 (RBFOX3)/NeuN, a neuronal splicing regulator. However, the mechanism of seizure mediation by RBFOX3 remains unknown. Here, we show that mice with deletion of Rbfox3 in gamma-aminobutyric acid (GABA) ergic neurons exhibit spontaneous seizures and high premature mortality due to increased presynaptic release, postsynaptic potential, neuronal excitability, and synaptic transmission in hippocampal dentate gyrus granule cells (DGGCs). Attenuating early excitatory gamma-aminobutyric acid (GABA) action by administering bumetanide, an inhibitor of early GABA depolarization, rescued premature mortality. Rbfox3 deletion reduced hippocampal expression of vesicle-associated membrane protein 1 (VAMP1), a GABAergic neuron-specific presynaptic protein. Postnatal restoration of VAMP1 rescued premature mortality and neuronal excitability in DGGCs. Furthermore, Rbfox3 deletion in GABAergic neurons showed fewer neuropeptide Y (NPY)-expressing GABAergic neurons. In addition, deletion of Rbfox3 in NPY-expressing GABAergic neurons lowered intrinsic excitability and increased seizure susceptibility. Our results establish RBFOX3 as a critical regulator and possible treatment path for epilepsy.

RTL1/PEG11 imprinted in human and mouse brain mediates anxiety-like and social behaviors and regulates neuronal excitability in the locus coeruleus.

RTL1/PEG11, which has been associated with anxiety disorders, is a retrotransposon-derived imprinted gene in the placenta. However, imprinting patterns and functions of RTL1 in the brain have not been well-investigated. We found Rtl1 was paternally, but not maternally, expressed in brain stem, thalamus, and hypothalamus of mice, and imprinting status of RTL1 was maintained in human brain. Paternal Rtl1 knockout (Rtl1m+/p-) mice had higher neonatal death rates due to impaired suckling, and low body weights beginning on embryonic day 16.5. High paternal expression of Rtl1 was detected in the locus coeruleus (LC) and Rtl1m+/p- mice showed an increased delay in time of onset for action potentials and inward currents with decreased neuronal excitability of LC neurons. Importantly, Rtl1m+/p- mice exhibited behaviors associated with anxiety, depression, fear-related learning and memory, social dominance, and low locomotor activity. Taken together, our findings demonstrate RTL1 is imprinted in brain, mediates emotional and social behaviors, and regulates excitability in LC neurons.

First Report of Phialocephala bamuru Causing Root Rot on Hard Fescue (Festuca brevipila) in the United States.

Turfgrasses are susceptible to a wide variety of ectotrophic root-infecting (ERI) fungi that cause root rot (Tredway et al., 2023). Among the root rot diseases, fairway patch, caused by Phialocephala bamuru P.T.W. Wong & C. Dong sp. nov., was recently identified and characterized in Australia infecting bermudagrass (Cynodon dactylon) and kikuyu (Pennisetum clandestinum) grass (Wong et al., 2015). Symptoms begin as small, 5-10 cm diameter patches of yellowed turf that may coalesce into larger areas of diseased grass. A characteristic sign of fairway patch is roots colonized by dark brown to black, ectotrophic mycelium. In June 2020, many tan colored, irregular-shaped patches ranging from 10-30 cm in diameter developed on a hard fescue (Festuca brevipila) cultivar 'Beacon' turfgrass field in North Brunswick, New Jersey, USA. The centers of these patches later died and became sunken or filled in partially by recovering hard fescue. The patches grew into tan irregular-shaped rings with diameters up to 3 m by Aug 2023. Symptoms were indicative of a root disease. Five 'Beacon' hard fescue soil cores at the interface of the symptomatic and non-symptomatic area were sampled in Aug 2023. Root and crown samples were observed under a dissecting microscope and dark ectotrophic hyphae were observed on both. Roots with visible ectotrophic mycelium were removed, rinsed in sterile water three times, cut into 5 mm pieces, and plated onto 10% potato dextrose agar amended with streptomycin and gentamicin at 100 mg/L (PDA+). The plates were incubated at 25°C in the dark for 5 days. The most abundant colonies being characteristic long, septate hyphae that were hyaline at the edge and dark brown to black in the center and resembled the fungus described in Wong et al., 2015. These colonies were subcultured onto PDA+ medium and selected for molecular identification. Other less abundant colonies could be identified using morphology after subcultured and had no record being pathogenic to turfgrass. To confirm the isolate's identity, its internal transcribed spacer (ITS) region was amplified in PCR using the ITS1F/ITS4 primers (Bellemain et al., 2010). The amplicon was then sequenced with both ITS1 and ITS4 primers by Sanger sequencing. Sequences were assembled (GenBank #PP000819). The consensus sequence was then BLASTn analyzed with default settings, and the result showed 99.64% sequence identity with P. bamuru (GenBank #MG195534.1). Koch's postulate was conducted in an environmentally controlled growth chamber. Six healthy 'Beacon' hard fescue plugs were sampled from the field. Three of the six plugs (treatment) were each inoculated with P. bamuru by placing 20 g of P. bamuru colonized millets beneath and around the plug before filling the pots with sand. The other three plugs (control) received the same treatment except the P. bamuru colonized millets were autoclaved. The pots were incubated in the growth chamber with a 16 h light period and 25/20°C day/night temperatures. Symptoms resembling those observed in the field appeared on the treatment pots after 21 days of incubation while the control pots remained healthy. The roots from the treatment pots were examined under the dissecting microscope to confirm the colonization of P. bamuru on the roots, and P. bamuru was reisolated and confirmed using the aforementioned morphological traits and molecular assays (GenBank #PP000820). This is the first report of a turfgrass root rot disease caused by P. bamuru in the United States. Further epidemiological, disease ecological, and pathogen biological studies are required to clarify the importance of this disease in the United States and establish proper disease containment or control measures.

Alternation of social behaviors for zebrafish (Danio rerio) in response to acute cold stress.

Low temperature is one of the most common abiotic stresses for aquatic ectotherms. Ambient low temperatures reduce the metabolic rate of teleosts, therefore, teleosts have developed strategies to modulate their physiological status for energy saving in response to cold stress, including behaviors, circulatory system, respiratory function, and metabolic adjustments. Many teleosts are social animals and they can live in large schools to serve a variety of functions, including predator avoidance, foraging efficiency, and reproduction. However, the impacts of acute cold stress on social behaviors of fish remain unclear. In the present study, we test the hypothesis that zebrafish alter their social behaviors for energy saving as a strategy in response to acute cold stress. We found that acute cold stress increased shoaling behavior that reflected a save-energy strategy for fish to forage and escape from the predators under cold stress. The aggressive levels measured by fighting behavior tests and mirror fighting tests were reduced by cold treatment. In addition, we also found that acute cold stress impaired the learning ability but did not affect memory. Our findings provided evidence that acute cold stress alters the social behaviors of aquatic ectotherms for energy saving; knowledge of their responses to cold is essential for their conservation and management.

Mouse hybrid genome mediates diverse brain phenotypes with the specificity of reciprocal crosses.

Hybrid species have more genetic diversity than their parents. However, the impact of the hybrid genome of reciprocal crosses on brain function remains largely unknown. We performed behavioral, molecular, and neuronal analyses on C57BL/6J mice (B6), CAST/EiJ mice (CAST), and hybrid mice resulting from reciprocal crosses of the two strains, B6/CAST F1i and B6/CAST F1r, respectively. Hybrid mice displayed greater motor strength and coordination, food grinding, social dominance, and less sociability compared to their parental strains. Parental origin influenced body weight, locomotor speed, and heat nociception of hybrid mice. Parental origin, cell type, and the interaction of both affected expression patterns of hybrid genomes including imprinted genes. There was a correlation between affected genes and corresponding behavioral phenotypes. Hybrid genomes mediated neuronal activity in the locus coeruleus, a brain region implicated in arousal, adaptive behaviors, and sleep-wake cycle due to its norepinephrine projections throughout the central nervous system. The comprehensive brain phenotypes in these hybrid mice reveal important functional readouts associated with interactions of hybrid genomes and impacts of parental genomes.

Molecular Physiological Evidence for the Role of Na+-Cl- Co-Transporter in Branchial Na+ Uptake in Freshwater Teleosts.

The gills are the major organ for Na+ uptake in teleosts. It was proposed that freshwater (FW) teleosts adopt Na+/H+ exchanger 3 (Nhe3) as the primary transporter for Na+ uptake and Na+-Cl- co-transporter (Ncc) as the backup transporter. However, convincing molecular physiological evidence to support the role of Ncc in branchial Na+ uptake is still lacking due to the limitations of functional assays in the gills. Thus, this study aimed to reveal the role of branchial Ncc in Na+ uptake with an in vivo detection platform (scanning ion-selective electrode technique, SIET) that has been recently established in fish gills. First, we identified that Ncc2-expressing cells in zebrafish gills are a specific subtype of ionocyte (NCC ionocytes) by using single-cell transcriptome analysis and immunofluorescence. After a long-term low-Na+ FW exposure, zebrafish increased branchial Ncc2 expression and the number of NCC ionocytes and enhanced gill Na+ uptake capacity. Pharmacological treatments further suggested that Na+ is indeed taken up by Ncc, in addition to Nhe, in the gills. These findings reveal the uptake roles of both branchial Ncc and Nhe under FW and shed light on osmoregulatory physiology in adult fish.

Sublethal ammonia induces alterations of emotions, cognition, and social behaviors in zebrafish (Danio rerio).

Ammonia pollutants were usually found in aquatic environments is due to urban sewage, industrial wastewater discharge, and agricultural runoff and concentrations as high as 180 mg/L (NH4+) have been reported in rivers. High ammonia levels are known to impair multiple tissue and cell functions and cause fish death. Although ammonia is a potent neurotoxin, how sublethal concentrations of ammonia influence the central nervous system (CNS) and the complex behaviors of fish is still unclear. In the present study, we demonstrated that acute sublethal ammonia exposure can change social behavior of adult zebrafish. The exposure to 90 mg /L of (NH4+) for 4 h induced a strong fear response and lower shoaling cohesion; exposure to 180 mg /L of (NH4+) for 4 h reduced the aggressiveness, and social recognition, while the anxiety, social preference, learning, and short-term memory were not affected. Messenger RNA expressions of glutaminase and glutamate dehydrogenase in the brain were induced, suggesting that ammonia exposure altered glutamate neurotransmitters in the CNS. Our findings in zebrafish provided delicate information of ammonia neurotoxicity in complex higher-order social behaviors, which has not been revealed previously. In conclusion, sublethal and acute ammonia exposure can change specific behaviors of fish, which might lead to reductions in individual and population fitness levels.

Rapid Detection of the Recently Identified Turfgrass Pathogen Magnaporthiopsis meyeri-festucae Using Recombinase Polymerase Amplification.

Magnaporthiopsis meyeri-festucae is a recently identified root-infecting pathogen of fine fescue (Festuca spp.) turfgrasses. Although it is phylogenetically similar to other root-infecting turfgrass pathogens such as M. poae, management of M. meyeri-festucae is distinct and highlights the need for fast and accurate identification. The objective of this study was to develop a rapid detection method for M. meyeri-festucae using recombinase polymerase amplification (RPA) to assist turfgrass managers in identifying the disease in the field and facilitate further epidemiological research on the pathogen. Three isolates of M. meyeri-festucae and eight isolates from four related Magnaporthiopsis species were used to test the specificity of the RPA assay targeting M. meyeri-festucae. Rapid visualization of the RPA assay results using a mixture of purified amplicon and SYBR-Safe fluorescence emitting asymmetrical cyanine dye showed that the assay was effective at detecting M. meyeri-festucae on turfgrass roots with no observed incidence of false positives or false negatives. The assay also differentiated between M. meyeri-festucae and other Magnaporthiopsis species, although overall sensitivity was lower compared with a PCR-based method. The RPA assay successfully detected M. meyeri-festucae following inoculation onto and grinding of turfgrass roots, indicating possible use as a rapid field diagnostic tool for turfgrass managers. The fast and accurate RPA M. meyeri-festucae detection method presented here will be used for additional field and laboratory applications that will help improve the management of this emerging pathogen.

In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in adult gills to investigate branchial H+-excreting functions in vivo. We removed the opercula from zebrafish and then performed long-term acid acclimation experiments. The results of Western blot and immunofluorescence showed that the protein expression of H+-ATPase (HA) and the number of H+-ATPase-rich ionocytes were increased under acidic situations. The SIET results proved that the H+ excretion capacity is indeed enhanced in the gills acclimated to acidic water. In addition, both HA and Na+/H+ exchanger (Nhe) inhibitors suppressed the branchial H+ excretion capacity, suggesting that H+ is excreted in association with HA and Nhe in zebrafish gills. These results demonstrate that SIET is effective for in vivo detection in fish gills, representing a breakthrough approach for studying the molecular physiology of fish ion regulation.

Hyperlocal Variation in Soil Iron and the Rhizosphere Bacterial Community Determines Dollar Spot Development in Amenity Turfgrass.

Dollar spot, caused by the fungal pathogen Clarireedia spp., is an economically important foliar disease of amenity turfgrass in temperate climates worldwide. This disease often occurs in a highly variable manner, even on a local scale with relatively uniform environmental conditions. The objective of this study was to investigate mechanisms behind this local variation, focusing on contributions of the soil and rhizosphere microbiome. Turfgrass, rhizosphere, and bulk soil samples were collected from within a 256-m2 area of healthy turfgrass, transported to a controlled environment chamber, and inoculated with Clarireedia jacksonii Bacterial communities were profiled by targeting the 16S rRNA gene, and 16 different soil chemical properties were assessed. Despite their initial uniform appearance, the samples differentiated into highly susceptible and moderately susceptible groups following inoculation in the controlled environment chamber. The highly susceptible samples harbored a unique rhizosphere microbiome with suggestively lower relative abundance of putative antibiotic-producing bacterial taxa and higher predicted abundance of genes associated with xenobiotic biodegradation pathways. In addition, stepwise regression revealed that bulk soil iron content was the only significant soil characteristic that positively regressed with decreased dollar spot susceptibility during the peak disease development stage. These findings suggest that localized variation in soil iron induces the plant to select for a particular rhizosphere microbiome that alters the disease outcome. More broadly, further research in this area may indicate how plot-scale variability in soil properties can drive variable plant disease development through alterations in the rhizosphere microbiome.IMPORTANCE Dollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3 to 5 cm), circular patches that develop in a highly variable manner within plot scale even under seemingly uniform conditions. The mechanism behind this variable development is unknown. This study observed that differences in dollar spot development over a 256-m2 area were associated with differences in bulk soil iron concentration and correlated with a particular rhizosphere microbiome. These findings provide interesting avenues for future research to further characterize the mechanisms behind the highly variable development of dollar spot, which may inform innovative control strategies. Additionally, these results suggest that small changes in soil properties can alter plant activity and hence the plant-associated microbial community, which has important implications for a broad array of agricultural and horticultural plant pathosystems.

Oxytocin Signaling Acts as a Marker for Environmental Stressors in Zebrafish.

The oxytocin system plays a role in stress responses and behavior modulation. However, the effects of oxytocin signaling on stress adaptation remain unclear. Here, we demonstrated the roles of oxytocin signaling as a biomarker under stress conditions in the peripheral tissues (the gills) and central nervous system (the brain). All the environmental stressors downregulated the expression of oxytocin receptors in the gills, and the alteration of the expression of oxytocin receptors was also found in the brain after the acidic (AC) and high-ammonia (HA) treatments. The number of oxytocin neurons was increased after double-deionized (DI) treatment. By transgenic line, Tg(oxtl:EGFP), we also investigated the projections of oxytocin neurons and found oxytocin axon innervations in various nuclei that might regulate the anxiety levels and aggressiveness of adult zebrafish under different environmental stresses. The oxytocin system integrates physiological responses and behavioral outcomes to ensure environmental adaptation in adult zebrafish. Our study provides insight into oxytocin signaling as a stress indicator upon environmental stressors.

Transient receptor potential vanilloid 4 modulates ion balance through the isotocin pathway in zebrafish (Danio rerio).

Isotocin controls ion regulation through modulating the functions of ionocytes (also called mitochondria-rich cells or chloride cells). However, little is known about the upstream molecule of the isotocin system. Herein, we identify transient receptor potential vanilloid 4 (TRPV4), which regulates the mRNA and protein expressions of isotocin and affects ion regulation through the isotocin pathway. Double immunohistochemical results showed that TRPV4 is expressed in isotocinergic neurons in the hypothalamus of the adult zebrafish brain. To further elucidate the roles of TRPV4, we manipulated TRPV4 protein expression and evaluated its ionoregulatory functions in zebrafish embryos. TRPV4 gene knockdown with morpholino oligonucleotides decreased ionic contents (Na+, Cl-, and Ca2+) of whole larvae and the H+-secreting function of larval skin of zebrafish. mRNA expressions of ionocyte-related transporters, including H+-ATPase, the epithelial Ca2+ channel, and the Na+-Cl- cotransporter, were also suppressed in trpv4 morphants. Numbers of ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells) and epidermal stem cells in zebrafish larval skin also decreased after trpv4 knockdown. Our results showed that TRPV4 modulates ion balance through the isotocin pathway.

Arginine Vasopressin Modulates Ion and Acid/Base Balance by Regulating Cell Numbers of Sodium Chloride Cotransporter and H+-ATPase Rich Ionocytes.

Arginine vasopressin (Avp) is a conserved pleiotropic hormone that is known to regulate both water reabsorption and ion balance; however, many of the mechanisms underlying its effects remain unclear. Here, we used zebrafish embryos to investigate how Avp modulates ion and acid-base homeostasis. After incubating embryos in double-deionized water for 24 h, avp mRNA expression levels were significantly upregulated. Knockdown of Avp protein expression by an antisense morpholino oligonucleotide (MO) reduced the expression of ionocyte-related genes and downregulated whole-body Cl- content and H+ secretion, while Na+ and Ca2+ levels were not affected. Incubation of Avp antagonist SR49059 also downregulated the mRNA expression of sodium chloride cotransporter 2b (ncc2b), which is a transporter responsible for Cl- uptake. Correspondingly, avp morphants showed lower NCC and H+-ATPase rich (HR) cell numbers, but Na+/K+-ATPase rich (NaR) cell numbers remained unchanged. avp MO also downregulated the numbers of foxi3a- and p63-expressing cells. Finally, the mRNA expression levels of calcitonin gene-related peptide (cgrp) and its receptor, calcitonin receptor-like 1 (crlr1), were downregulated in avp morphants, suggesting that Avp might affect Cgrp and Crlr1 for modulating Cl- balance. Together, our results reveal a molecular/cellular pathway through which Avp regulates ion and acid-base balance, providing new insights into its function.

The effectiveness of music therapy for individuals with Rett syndrome and their families.

BACKGROUND: Patients with Rett syndrome (RTT) present characteristic regression in communication and hand skills, which eventually leads to intellectual and physical disability. Moreover, caregivers of patients with RTT face stressors related to patients' medical and developmental concerns. Given the indications from case reports, this pilot study investigated the effectiveness of music therapy on RTT patients, as well as on parental stress for families of children with RTT. METHODS: Families in the study group were enrolled in a twice-weekly 120-minute music therapy program for 24 weeks (n = 11), whereas families in the control group did not receive music therapy (n = 12). Participants were administered the Vineland Adaptive Behavior Scales, Rett Syndrome Clinical Severity Scale, Rett Syndrome Motor Behavioral Assessment, and Parenting Stress Index for caregivers of RTT children before and after the music therapy program. RESULTS: Music therapy improved receptive language, verbal and non-verbal communication skills, and social interaction for RTT patients. In addition, purposeful hand function, breathing patterns, and eye contact were significantly improved. Of note, music therapy also decreased the frequency of epileptic seizures. Lastly, caregivers in the study group exhibited significantly lower stress following the program. CONCLUSION: The 24-week music therapy program was effective in improving social interaction, communication skills, eye contact, hand function, and reducing seizure frequency among RTT patients. Additionally, music therapy was effective in relieving parenting stress, which may help healthcare providers initiate early intervention strategies that can prevent parenting stress and reduce the risk of depression.

The Extracellular Zn2+ Concentration Surrounding Excited Neurons Is High Enough to Bind Amyloid-ß Revealed by a Nanowire Transistor.

The Zn2+ stored in the secretory vesicles of glutamatergic neurons is coreleased with glutamate upon stimulation, resulting in the elevation of extracellular Zn2+ concentration (CZn2+ex). This elevation of CZn2+ex regulates the neurotransmission and facilitates the fibrilization of amyloid-ß (Aß). However, the exact CZn2+ex surrounding neurons under (patho)physiological conditions is not clear and the connection between CZn2+ex and the Aß fibrilization remains obscure. Here, a silicon nanowire field-effect transistor (SiNW-FET) with the Zn2+ -sensitive fluorophore, FluoZin-3 (FZ-3), to quantify the CZn2+ex in real time is modified. This FZ-3/SiNW-FET device has a dissociation constant of ≈12 × 10-9 m against Zn2+ . By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the CZn2+ex elevated to ≈110 × 10-9 m upon stimulation with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Blockers against the AMPA receptor or exocytosis greatly suppress this elevation, indicating that the Zn2+ stored in the synaptic vesicles is the major source responsible for this elevation of CZn2+ex. In addition, a SiNW-FET modified with Aß could bind Zn2+ with a dissociation constant of ≈633 × 10-9 m and respond to the Zn2+ released from AMPA-stimulated neurons. Therefore, the CZn2+ex can reach a level high enough to bind Aß and the Zn2+ homeostasis can be a therapeutic strategy to prevent neurodegeneration.

Age-Dependent Effects of Prenatal Dexamethasone Exposure on Immune Responses in Male Rats.

Prenatal glucocorticoid therapy is indicated in preterm delivery to prevent respiratory distress. This study was designed to evaluate the age-dependent effects of prenatal dexamethasone (DEX) therapy on the immune system using a rat model. Pregnant Sprague-Dawley rats received an intraperitoneal injection of DEX (0.1 mg/kg/day) or saline (VEH) over gestational days 14-20. Male offspring were sacrificed at postnatal day 7 (D7; infant stage), D120 (young adult stage), and D180 (adult stage) for evaluation of leukocyte subsets and isolation of splenocytes. The production of innate and adaptive immune cytokines was assessed from the culture supernatants of splenocytes, stimulated with lipopolysaccharide and concanavalin A, respectively. For innate cytokines, the levels of interferon gamma inducible protein 10 were significantly higher, but those of tumor necrosis factor-α were significantly lower, in the culture medium of splenocytes prepared from the DEX group at D120 than those in the VEH group. For adaptive cytokines, the levels of interleukin-4 (IL-4) were significantly higher at D7 and those of IL-10 were significantly higher at D120 after prenatal exposure to DEX. We also showed that the expression level of IL-4 mRNA was significantly higher in splenocytes prepared from the DEX group at D7, compared with the VEH group. Importantly, the mRNA expression level of T-bet, a key transcription factor for immune cells, was greatly decreased in the spleen of the DEX group at D7, compared with the VEH group. In conclusion, prenatal dexamethasone exposure shows the greater impact on immune responses of their male offspring in early life.

Conserved expression of the zebrafish syt4 gene in GABAergic neurons in the cerebellum of adult fishes revealed by mammalian SYT4 immunoreactive-like signals.

Synaptotagmin 4 (syt4) belongs to the synaptotagmin protein family, which has 17 and 28 family members in human and zebrafish, respectively. In zebrafish and rodents, syt4 is known to express abundantly in the entire central nervous system in the early developmental stages. In adult rodents, the gene expression shifts to be predominant in the cerebellum, mostly in Purkinje cells, a type of GABAergic neurons. However, there is no report of the expression pattern of syt4 in the adult zebrafish brain. Therefore, we hypothesize that the expression of syt4 is conserved in adult zebrafish and is specific to the GABAergic neurons, likely Purkinje cells, in the cerebellum. To examine the hypothesis, we first show that only one copy of syt4 gene remains in the zebrafish genome, and it is orthologous to the gene in other vertebrates. We further observe mammalian SYT4 antibody immunoreactive-like (mSYT4-ir) signals in several structures in the hindbrain including the medial divisions of the valvula cerebelli and the corpus cerebelli. In addition, our observations indicate the presence of mSYT4-ir signals in GABAergic neurons, most notably in the Purkinje cell layer of the molecular layer in the aforementioned structures. Conversely, mSYT4-ir signals are not observed in glutamatergic or cholinergic neurons. Therefore, we deduce that the syt4 gene in zebrafish exhibits a homologous expression pattern to those of previously studied vertebrate species, which is revealed by the positive immunoreactive-like signals of mammalian SYT4 antibodies.

Zebrafish as an animal model to study ion homeostasis.

Zebrafish (Danio rerio) possesses several advantages as an experimental organism, including the applicability of molecular tools, ease of in vivo cellular observation and functional analysis, and rapid embryonic development, making it an emerging model for the study of integrative and regulatory physiology and, in particular, the epithelial transport associated with body fluid ionic homeostasis. Zebrafish inhabits a hypotonic freshwater environment, and as such, the gills (or the skin, during embryonic stages) assume the role of the kidney in body fluid ionic homeostasis. Four types of ionocyte expressing distinct sets of transporters have been identified in these organs: H(+)-ATPase-rich, Na(+)-K(+)-ATPase-rich, Na(+)-Cl(-) cotransporter-expressing and K(+)-secreting cells; these ionocytes perform transepithelial H(+) secretion/Na(+) uptake/NH4 (+) excretion, Ca(2+) uptake, Na(+)/Cl(-) uptake, and K(+) secretion, respectively. Zebrafish ionocytes are analogous to various renal tubular cells, in terms of ion transporter expression and function. During embryonic development, ionocyte progenitors develop from epidermal stem cells and then differentiate into different types of ionocyte through a positive regulatory loop of Foxi3a/-3b and other transcription factors. Several hormones, including cortisol, vitamin D, stanniocalcin-1, calcitonin, and isotocin, were found to participate in the control pathways of ionic homeostasis by precisely studying the target ion transport pathways, ion transporters, or ionocytes of the hormonal actions. In conclusion, the zebrafish model not only enhances our understanding of body fluid ion homeostasis and hormonal control in fish but also informs studies on mammals and other animal species, thereby providing new insights into related fields.

Effects of maternal cadmium exposure on female reproductive functions, gamete quality, and offspring development in zebrafish (Danio rerio).

Impacts of maternal Cd(2+) exposure on female zebrafish (Danio rerio) were observed in females as well as their offspring. In females, Cd disturbed fecundity and other reproductive functions. In their offspring, it retarded gamete development and growth and influenced gene expression. There was a positive relationship between Cd(2+) contents in ovaries of females and treatment doses of 0-8.9 µM of Cd(2+). The mating rate decreased by 60 % when females were exposed to 8.9-35.6 µM of Cd(2+) for 72 h compared with the control group. It was observed that growth is delayed by one somite stage in maternal-Cd(2+) embryos compared with control embryos, which grew at the sixth-somite stage. The ceratohyal angles of larvae of Cd-exposed adults (maternal Cd(2+)) at 72 h postfertilization (hpf) appeared to have a positive response after doses of maternal Cd. In addition, approximately 30 % of 96-hpf larvae that were treated with a dose of 35.6 µM of maternal Cd(2+) appeared to have pericardial edema. At the 5-hpf stage of maternal Cd(2+) exposure, embryos showed 33 and 37 target genes, respectively, that were significantly downregulated and upregulated as shown by cDNA microarray analysis. A major effect of maternal Cd(2+) exposure on zebrafish embryo genes is that 18.9% of transcription functions were upregulated. In addition, 33.3% of transcripts relative to the function of protein biosynthesis were downregulated. These results showed that maternal Cd(2+) exposure influenced the reproduction ability of females and also caused their embryos to develop with abnormal gene expression.

Retention of ion channel genes expression increases Japanese medaka survival during seawater reacclimation.

Euryhaline teleosts exhibit varying acclimability to survive in environments that alternate between being hypotonic and hypertonic. Such ability is conferred by ion channels expressed by ionocytes, the ion-regulating cells in the gills or skin. However, switching between environments is physiologically challenging, because most channels can only perform unidirectional ion transportation. Coordination between acute responses, such as gene expression, and long-term responses, such as cell differentiation, is believed to strongly facilitate adaptability. Moreover, the pre-acclimation to half seawater salinity can improve the survivability of Japanese medaka (Oryzias latipes) during direct transfer to seawater; here, the ionocytes preserve hypertonic acclimability while performing hypotonic functions. Whether acclimability can be similarly induced in a closed species and their corresponding responses in terms of ion channel expression remain unclear. In the present study, Japanese medaka pre-acclimated in brackish water were noted to have higher survival rates while retaining higher expression of the three ion channel genes ATP1a1a.1, ATP1b1b, and SLC12a2a. This retention was maintained up to 2 weeks after the fish were transferred back into freshwater. Notably, this induced acclimability was not found in its close kin, Indian medaka (Oryzias dancena), the natural habitat of which is brackish water. In conclusion, Japanese medaka surpassed Indian medaka in seawater acclimability after experiencing exposure to brackish water, and this ability coincided with seawater-retention gene expression.