Embryonic Exposure to Domoic Acid Increases the Susceptibility of Zebrafish Larvae to the Chemical Convulsant Pentylenetetrazole

BACKGROUND: Domoic acid (DA) is a neurotoxin produced by diatoms of the genus Pseudonitzschia that targets the limbic system to induce tonic–clonic seizures and memory impairment. In utero DA exposure of mice leads to a reduction in seizure threshold to subsequent DA exposures in mid-postnatal life, and similar studies have shown neurotoxic effects in rats that were delayed until adolescence. OBJECTIVE: We used in ovo microinjection of zebrafish (Danio rerio) to characterize the effect of embryonic exposure of DA on seizure-inducing agents later in life as an alternative species model to screen environmental contaminants that might induce a fetal-originating adult disease. METHODS: Embryos were microinjected within hours of fertilization to DA concentrations ranging from 0.12 to 1.26 ng/mg egg weight. Seven days later, the larval animals were characterized for sensitivity to the chemical convulsant pentylenetetrazole (PTZ), an agent that is well-defined in laboratory rodents and, more recently, in zebrafish. RESULTS: In ovo DA exposure, most significantly at 0.4 ng/mg, reduces the latency time until first PTZ seizure in larval fish and increases the severity of seizures as determined by seizure stage and movement parameters. The interaction between in ovo DA exposure and PTZ caused seizure behaviors to individually asymptomatic doses of PTZ (1.0 and 1.25 mM) and DA (0.13 and 0.22 ng/mg). CONCLUSION: These studies demonstrate that in ovo exposure to DA reduces the threshold to chemically induced seizures in larval fish and increases the severity of seizure behavior in a manner that is consistent with in utero studies of laboratory rodents

Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene

The aim of this study was to isolate cis-acting regulatory elements for the generation of transgenic zebrafish models of neurodegeneration. Zebrafish enolase-2 (eno2) showed neuronal expression increasing from 24 to 72 h post-fertilization (hpf) and persisting through adulthood. A 12 kb eno2 genomic fragment, extending from 8 kb upstream of exon 1 to exon 2, encompassing intron 1, was sufficient to drive neuronal reporter gene expression in vivo over a similar time course. Five independent lines of stable Tg(eno2 : GFP) zebrafish expressed GFP widely in neurons, including populations with relevance to neurodegeneration, such as cholinergic neurons, dopaminergic neurons and cerebellar Purkinje cells. We replaced the exon 2-GFP fusion gene with a cDNA encoding the 4-repeat isoform of the human microtubule-associated protein Tau. The first intron of eno2 was spliced with high fidelity and efficiency from the chimeric eno2-Tau transcript. Tau was expressed at ∼8-fold higher levels in Tg(eno2 : Tau) zebrafish brain than normal human brain, and localized to axons, neuropil and ectopic neuronal somatic accumulations resembling neurofibrillary tangles. The 12 kb eno2 promoter drives high-level transgene expression in differentiated neurons throughout the CNS of stable transgenic zebrafish. This regulatory element will be useful for the construction of transgenic zebrafish models of neurodegeneration

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish

Efficacy of Curl up Task As Abdominal Exercise Program for Diastasis Recti Abdominis

Purpose/Hypothesis: The separation between the rectus abdominal muscles caused by a widening and thinning linea alba, called diastasis recti abdominis (DRA), contributes to impairments in strength of the abdominal and trunk muscles. DRA is most frequently observed during pregnancy and may recede after childbirth but can be present at 12 months or later after giving birth. The purpose of this study was to determine if a focused 12 or 24 week exercise program improves inter-rectus distance and abdominal strength in women who have given birth. Number of Subjects: Forty-eight women 27-49 years (mean 37 years) were randomized into a 12 (n=16) or 24 week intervention group (n=17) or a control group (n=15). Materials and Methods: Outcome testing occurred at baseline, 12 and 24 weeks. The inter-rectus distance (IRD) was measured at rest at the level of umbilicus with digital calipers. Abdominal strength was assessed with manual muscle testing (MMT) as described by Kendall. The intervention groups were instructed in exercises to be performed 3x/week, 5 repetitions each: strengthening (Preactivation transversus abdominis (TrA) with curl-up exercise with verbal and palpation cues to monitor the tension in the linea alba), breathing education including lower rib mechanics and engaging the TrA, and postural education. The control group was not provided with exercise. An intention to treat analysis using the last measure carried forward was conducted. Within group change in IRD was analyzed with paired sample t-tests. Change in IRD and MMT was compared using ANCOVA, with time since delivery as a covariate. Significance was 0.05, a priori. Results: Subjects were similar at baseline on age, BMI, and number of pregnancies, however time since delivery was significantly longer (69 months, compared to 43 months in 24 week group, 32 months in 12 week group; p=0.016) in the control group. All groups saw a decrease of IRD from baseline to 12 and 24 weeks (p Conclusions: While there were decreases in the IRD among all groups, these were significantly better at 12 and 24 weeks in both exercise groups in comparison to control with the exercise groups showing clinically relevant change in comparison to control. Since both the 12 and 24 week exercise groups saw statistically and clinically significant change at 12 weeks, this may be long enough to encourage ongoing resolution of DRA. Meaningful strength changes, however, require a longer intervention of 24 weeks rather than 12. Clinical Relevance: This program challenges the conventional treatment of DRA by incorporating the curl-up exercise and emphasizing the importance of utilizing linea alba tension and transverse abdominis activation. The comprehensive approach aims to optimize outcomes for individuals with DRA.https://ecommons.udayton.edu/dpt_symposium/1043/thumbnail.jp

Information is power: The experiences, attitudes and needs of individuals who chose to have prenatal genomic sequencing for fetal anomalies

OBJECTIVE: This study sought to evaluate the experiences of individuals who chose to participate in a study and receive prenatal genomic sequencing (pGS) for fetuses with congenital structural anomalies. METHOD: Individuals who received research results of prenatal sequencing were invited to participate in semi-structured interviews about their experiences. A constructivist grounded theory approach was used to code and analyze interviews. RESULTS: Thirty-three participants from 27 pregnancies were interviewed. Participants were motivated to enroll in the study to find out more about their fetus\u27 condition and prepare for the future. The waiting period was a time of significant anxiety for participants. Most participants felt relief and closure upon receiving results, regardless of the category of result, and had a clear understanding of the implications of the results. CONCLUSION: Participants\u27 experiences with pGS were often intertwined with the experience of having a fetus with an abnormality. Participants were satisfied with the decision to participate in research and the support they received from the healthcare team, although waiting for results was associated with anxiety. The healthcare team plays an integral role in setting expectations and validating feelings of anxiety, fear and uncertainty

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. Here, we outline our protocol for embedding, serial sectioning, staining, and visualizing zebrafish embryos embedded in JB-4™ plastic resin – a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition we describe our procedures for staining plastic sections with Toluidine Blue or Hematoxylin and Eosin (H&E), and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and GFP signals in JB-4™ resin. The protocol we outline – from embryo preparation, embedding, sectioning and staining to visualization - can be accomplished in three days. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish