Mutations in ap1b1 Cause Mistargeting of the Na(+)/K(+)-ATPase Pump in Sensory Hair Cells.

The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+)/K(+)-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+) levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells

Forward Genetic Analysis of Visual Behavior in Zebrafish

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders

Modeling Brain Resonance Phenomena Using a Neural Mass Model

Stimulation with rhythmic light flicker (photic driving) plays an important role in the diagnosis of schizophrenia, mood disorder, migraine, and epilepsy. In particular, the adjustment of spontaneous brain rhythms to the stimulus frequency (entrainment) is used to assess the functional flexibility of the brain. We aim to gain deeper understanding of the mechanisms underlying this technique and to predict the effects of stimulus frequency and intensity. For this purpose, a modified Jansen and Rit neural mass model (NMM) of a cortical circuit is used. This mean field model has been designed to strike a balance between mathematical simplicity and biological plausibility. We reproduced the entrainment phenomenon observed in EEG during a photic driving experiment. More generally, we demonstrate that such a single area model can already yield very complex dynamics, including chaos, for biologically plausible parameter ranges. We chart the entire parameter space by means of characteristic Lyapunov spectra and Kaplan-Yorke dimension as well as time series and power spectra. Rhythmic and chaotic brain states were found virtually next to each other, such that small parameter changes can give rise to switching from one to another. Strikingly, this characteristic pattern of unpredictability generated by the model was matched to the experimental data with reasonable accuracy. These findings confirm that the NMM is a useful model of brain dynamics during photic driving. In this context, it can be used to study the mechanisms of, for example, perception and epileptic seizure generation. In particular, it enabled us to make predictions regarding the stimulus amplitude in further experiments for improving the entrainment effect

Loss of eyes in zebrafish caused by mutation of chokh/rx3

The vertebrate eye forms by specification of the retina anlage and subsequent morphogenesis of the optic vesicles, from which the neural retina differentiates. chokh (chk) mutant zebrafish lack eyes from the earliest stages in development. Marker gene analysis indicates that retinal fate is specified normally, but optic vesicle evagination and neuronal differentiation are blocked. We show that the chk gene encodes the homeodomain-containing transcription factor, Rx3. Loss of Rx3 function in another teleost, medaka, has also been shown to result in an eyeless phenotype. The medaka rx3 locus can fully rescue the zebrafish mutant phenotype. We provide evidence that the regulation of rx3 is evolutionarily conserved, whereas the downstream cascade contains significant differences in gene regulation. Thus, these mutations in orthologous genes allow us to study the evolution of vertebrate eye development at the molecular level

Pain management in zebrafish : Report from a FELASA Working Group.

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe

<i>ap1b1</i> mutants have deficits in HC mechanotransduction.

<p><i><b>A–C,</b></i> FM 1-43 label of neuromast HCs in WT, <i>tm246a</i> and <i>t20325</i> mutants at 5 dpf. Scale bars, 5 µm. <b><i>D,</i></b> Average intensity (A.U.) of FM 1-43 label in <i>tm246a</i> and <i>t20325</i> mutants quantified at 3 dpf (<i>tm246a</i>: WT n = 18, mutant n = 5; <i>t20325</i>: WT n = 16, mutant n = 8 neuromasts) and 5 dpf (<i>tm246a</i>: WT n = 9, mutant n = 10; <i>t20325</i>: WT n = 12, mutant n = 11 neuromasts) from at least 3 larvae along with WT, age-matched siblings. <b><i>E,</i></b> The proportion of HCs displaying calcium transients in response to a water-jet stimulus (solid) compared to those that do not respond (nr = non-responders, hatched lines). The percent of non-responding HCs in the <i>t20325</i> mutants is greater than the percent non-responders in WT at all stages of development assayed; Chi Squared test, p<0.0001. <b><i>F,</i></b> Trace representing the average calcium responses to a 2 sec water-jet stimulus from 5 dpf WT and <i>t20325</i> mutant larvae (n = 20 HCs). The grey box indicates the timing of the water-jet stimulus. <b><i>G,</i></b> Dot plot showing calcium transients in WT and <i>t20325</i> larvae at 2, 3 and 5 dpf (non-responders were excluded). Each point represents an individual HC. Error bars represent SEM and statistical analysis was performed using a Mann-Whitney U-test.</p

Increase of intracellular Na⁺ levels in mutant HCs.

<p><i><b>A–C,</b></i> Sodium Green label in WT, <i>tm246a</i> and <i>t20325</i> mutant neuromasts. Dotted magenta circles outline HCs that were used for quantification in that plane of view. Scale bar, 5 µm. <b><i>D,</i></b> Quantification of Sodium Green label in <i>ap1b1</i> mutant, <i>pcdh15^th263b,</i> and corresponding WT HCs. (<i>tm246a</i>: WT n = 124, mutant n = 90; <i>t20325</i>: WT n = 125, mutant n = 74; <i>th263b</i>: WT n = 100, mutant n = 84 HCs). Error bars represent SEM. Statistical analysis performed with a Mann-Whitney U-test.</p

Decreased cell integrity and an increased number of intracellular membrane compartments in <i>ap1b1</i> mutant HCs.

<p><i><b>A, B,</b></i> Comparable sections of the anterior macula of WT and <i>tm246a</i> mutant at 5 dpf. An asterisk indicates a breakdown of cytoplasm in a <i>tm246a</i> mutant HC. Scale bars, 3 µm. <b><i>C,</i></b> Example of an apical bleb extruding from a mutant HC containing several vesicular compartments and a multivesicular body. Scale bar, 1 µm. <b><i>D–E,</i></b> Comparable close-ups of WT and <i>tm246a</i> mutant HCs just below the cuticular plate. Scale bar, 1 µm. In <i>tm246a</i> mutant HCs, more multivesicular bodies (arrowheads) were present compared to WT HCs. <b><i>F,</i></b> Quantification of the observed number of multivesicular bodies in WT sibling and <i>tm246a</i> mutant HCs. <b><i>G–H,</i></b> Sections of HCs near the tight junctions showing an increased number of large vesicles in the mutants compared to WT. Vesicles in WT are indicated with arrows. <b><i>I,</i></b> Observed sizes of vesicles in WT sibling and <i>tm246a</i> mutant HCs. For quantification in <b><i>F</i></b> and <b><i>I</i></b>, WT: n = 15, <i>tm246a</i>: n = 20 HCs.</p

Stereociliary bundles of <i>ap1b1</i> mutant.

<p><i><b>A–C,</b></i> Representative confocal images of neuromast hair bundles in WT, <i>tm246a</i> and <i>t20325</i> mutants at 5 dpf. Bundles were viewed from a top-down angle and actin was labeled with phalloidin-Alexa 488. This view shows the planar cell polarity of hair-bundles. Scale bar, 1 µm. <b><i>D, E,</i></b> Side view of stereocilia from the lateral cristae of 5 dpf WT and <i>t20325</i> mutants in the <i>Tg(myo6b:βactin-GFP)</i> background (z-projections, 2 µm thick). Scale bar, 5 µm.</p

Positional cloning of <i>skylab</i> mutations and expression of <i>ap1b1</i>.

<p><i><b>A,</b></i> A diagram of the 330 kb <i>skylab</i> critical interval (striped region) obtained through mapping of the <i>tm246a</i> allele. The critical interval encompasses the coding regions of five annotated genes as well as part of two other genes. <b><i>B</i></b><b>,</b> An exon diagram of the <i>ap1b1</i> gene. The coding region is depicted in grey and the 5′ and 3′ UTRs are depicted in white. The locations of the <i>t20325</i> C-T transition the <i>tm246a</i> 23 bp deletion between exons 8 and 9 are indicated. <b><i>C</i></b><b>,</b> The nucleotides deleted from the splice acceptor site between exons 8 and 9 in the <i>tm246a</i> mutant are highlighted in red in the WT transcript. The resulting translations are shown above the WT and <i>tm246a</i> transcripts. <b><i>D</i></b><b>,</b> Diagram showing the location of <i>tm246a</i> and <i>t20325</i> mutations in the Ap1b1 protein. <b><i>E,</i></b><i> ap1b1</i> is expressed ubiquitously at 24 hpf. <b><i>F,</i></b> Sense control for <i>ap1b1 in situ</i> experiments. <b><i>G,</i></b> Expression of <i>ap1b1</i> persists in the head at 48 hpf. Scale bars in E-G, 5 µm. <b><i>H, I,</i></b> Magnified images of the developing ear at 24 and 48 hpf, respectively. Scale bars in H and I, 10 µm. HB, hindbrain; OV, otic vesicle; AM, anterior macula; AC, anterior crista; LC lateral crista; PC, posterior crista.</p