Ganglion cells in larval zebrafish retina integrate inputs from multiple cone types.

We recently showed the presence of seven physiological cone opsins-R1 (575 nm), R2 (556 nm), G1 (460 nm), G3 (480 nm), B1 (415 nm), B2 (440 nm), and UV (358 nm)-in electroretinogram (ERG) recordings of larval zebrafish (Danio rerio) retina. Larval ganglion cells (GCs) are generally thought to integrate only four cone opsin signals (red, green, blue, and UV). We address the question as to whether they may integrate seven cone spectral signals. Here we examined the 127 possible combinations of seven cone signals to find the optimal representation, as based on impulse discharge data sets from GC axons in the larval optic nerve. We recorded four varieties of light-response waveform, sustained-ON, transient-ON, ON-OFF, and OFF, based on the time course of mean discharge rates to all stimulus wavelengths combined. Modeling of GC responses revealed that each received 1-6 cone opsin signals, with a mean of 3.8 ± 1.3 cone signals/GC. Most onset or offset responses were opponent (ON, 80%; OFF, 100%). The most common cone signals were UV (93%), R2 (50%), G3 (55%), and G1 (60%). Seventy-three percent of cone opsin signals were excitatory, and 27% were inhibitory. UV signals favored excitation, whereas G3 and B2 signals favored inhibition. R1/R2, G1/G3, and B1/B2 opsin signals were selectively associated along a nonsynergistic/opponent axis. Overall, these results suggest that larval zebrafish GC spectral responses are complex and use inputs from the seven expressed opsins.NEW & NOTEWORTHY Ganglion cells in larval zebrafish retina have complex spectral responses driven by seven different cone opsin types. UV cone inputs are significant and excitatory to ganglion cells, whereas green and blue cone inputs favor inhibition. Most dramatic are the pentachromatic cells. These responses were identified at 5-6 days after fertilization, reflecting an impressive level of color processing not seen in older fish or mammals.

Using zebrafish to assess the effect of chronic, early developmental exposure to environmentally relevant concentrations of 5-fluorouracil and leucovorin.

Environmental contaminants can deleteriously affect aquatic animals. One such contaminant is 5-fluorouracil (5-FU), a long-prescribed chemotherapeutic drug. Leucovorin (LV) is co-administered with 5-FU, potentiating its effects. Zebrafish (Danio rerio) larvae were reared in ng/L treatments of either 5-FU, LV, or a combined 5-FU/LV mixture for 8 dy. Survival was measured daily and swimming behavior assessed every other day. After 8 dy, larval length was measured, and densitometry of p53-labeled cryostat sections determined the extent of apoptosis. No significant differences in survival or apoptosis were found; larvae in the highest concentrations were largest. Changes in behavior of 5-FU-treated larvae were based on exposure duration; changes in LV-treated larvae were affected by drug concentration and duration. Larvae co-exposed to 5-FU/LV had responses like 5-FU-treated larvae. Overall, early developmental exposure of zebrafish larvae to environmentally-relevant concentrations of 5-FU and LV did not adversely affect survival, growth, and behavior suggesting realistic concentrations are sublethal and non-toxic.

Cone signals in monostratified and bistratified amacrine cells of adult zebrafish retina.

Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light-response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic-sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid-stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. "C-type" physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532-1557, 2017. © 2016 Wiley Periodicals, Inc.

Variability in mitochondria of zebrafish photoreceptor ellipsoids.

Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter.

The first pulse of the extremely bright GRB 130427A: a test lab for synchrotron shocks.

Gamma-ray burst (GRB) 130427A is one of the most energetic GRBs ever observed. The initial pulse up to 2.5 seconds is possibly the brightest well-isolated pulse observed to date. A fine time resolution spectral analysis shows power-law decays of the peak energy from the onset of the pulse, consistent with models of internal synchrotron shock pulses. However, a strongly correlated power-law behavior is observed between the luminosity and the spectral peak energy that is inconsistent with curvature effects arising in the relativistic outflow. It is difficult for any of the existing models to account for all of the observed spectral and temporal behaviors simultaneously.

Fermi-LAT observations of the gamma-ray burst GRB 130427A.

The observations of the exceptionally bright gamma-ray burst (GRB) 130427A by the Large Area Telescope aboard the Fermi Gamma-ray Space Telescope provide constraints on the nature of these unique astrophysical sources. GRB 130427A had the largest fluence, highest-energy photon (95 GeV), longest γ-ray duration (20 hours), and one of the largest isotropic energy releases ever observed from a GRB. Temporal and spectral analyses of GRB 130427A challenge the widely accepted model that the nonthermal high-energy emission in the afterglow phase of GRBs is synchrotron emission radiated by electrons accelerated at an external shock.

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio.

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.

Bipolar cells in the zebrafish retina.

Zebrafish are an existing model for genetic and developmental studies due to their rapid external development and transparent embryos, which allow easy manipulation and observation of early developmental stages. The application of the zebrafish model to vision research has allowed for examination of retinal development and the characteristics of different retinal cell types, including bipolar cells. In particular, bipolar cell development, including differentiation, maturation, and gene expression, has been documented, as has physiological properties, such as voltage- and ligand-gated currents, and neurotransmitter receptor and ion channel expression. Mutant strains and transgenic lines have been used to document how bipolar cell connections and/or development may be altered, and toxicological studies examining how environmental factors may impact bipolar cell activity have been performed. The purpose of this paper was to review the existing literature on zebrafish bipolar cells, to provide a comprehensive overview of current information pertaining to this retinal cell type.

A relativistic type Ibc supernova without a detected gamma-ray burst.

Long duration gamma-ray bursts (GRBs) mark the explosive death of some massive stars and are a rare sub-class of type Ibc supernovae. They are distinguished by the production of an energetic and collimated relativistic outflow powered by a central engine (an accreting black hole or neutron star). Observationally, this outflow is manifested in the pulse of gamma-rays and a long-lived radio afterglow. Until now, central-engine-driven supernovae have been discovered exclusively through their gamma-ray emission, yet it is expected that a larger population goes undetected because of limited satellite sensitivity or beaming of the collimated emission away from our line of sight. In this framework, the recovery of undetected GRBs may be possible through radio searches for type Ibc supernovae with relativistic outflows. Here we report the discovery of luminous radio emission from the seemingly ordinary type Ibc SN 2009bb, which requires a substantial relativistic outflow powered by a central engine. A comparison with our radio survey of type Ibc supernovae reveals that the fraction harbouring central engines is low, about one per cent, measured independently from, but consistent with, the inferred rate of nearby GRBs. Independently, a second mildly relativistic supernova has been reported.

Non-adherence to statin therapy: a major challenge for preventive cardiology.

BACKGROUND: Hypercholesterolemia is a major risk factor for atherosclerosis and cardiovascular disease, the leading cause of death worldwide. In the last twenty years, effective lipid-lowering therapies, particularly statins, have become widely available to prevent and reverse the progression of disease. However, there is a significant gap between expected and actual benefits; this may be attributed to poor adherence to statin therapy. OBJECTIVE: To define the extent, causes (including psychological aspects), consequences and management of non-adherence to statins. METHODS: Literature using PubMed and Medline up to and including 30 July 2009. RESULTS: Adherence to statin therapy is suboptimal in both primary and secondary prevention of cardiovascular disease. Causes vary, and include patient factors (e.g., comorbidities, financial constraints, psychological issues), practitioner factors (e.g., poor knowledge of adherence, time constraints, poor communication skills and patient-doctor working alliance) and system factors (e.g., medication costs, lack of clinical monitoring, drug side effects). Non-adherence is associated with adverse health outcomes and increased costs of healthcare. A framework, based on a multidisciplinary approach, for addressing non-adherence, including managing the statin-intolerant patient, is presented. CONCLUSIONS: Non-adherence to statins is a significant issue for the prevention and treatment of cardiovascular disease. Increased awareness of the causes and solutions for overcoming non-adherence, including safer prescribing, improvement in physician-patient alliance and reduction in drug costs, will enhance the cost-effectiveness of the use of statins and significantly improve patient care and outcomes.

A limit on the variation of the speed of light arising from quantum gravity effects.

A cornerstone of Einstein's special relativity is Lorentz invariance-the postulate that all observers measure exactly the same speed of light in vacuum, independent of photon-energy. While special relativity assumes that there is no fundamental length-scale associated with such invariance, there is a fundamental scale (the Planck scale, l(Planck) approximately 1.62 x 10(-33) cm or E(Planck) = M(Planck)c(2) approximately 1.22 x 10(19) GeV), at which quantum effects are expected to strongly affect the nature of space-time. There is great interest in the (not yet validated) idea that Lorentz invariance might break near the Planck scale. A key test of such violation of Lorentz invariance is a possible variation of photon speed with energy. Even a tiny variation in photon speed, when accumulated over cosmological light-travel times, may be revealed by observing sharp features in gamma-ray burst (GRB) light-curves. Here we report the detection of emission up to approximately 31 GeV from the distant and short GRB 090510. We find no evidence for the violation of Lorentz invariance, and place a lower limit of 1.2E(Planck) on the scale of a linear energy dependence (or an inverse wavelength dependence), subject to reasonable assumptions about the emission (equivalently we have an upper limit of l(Planck)/1.2 on the length scale of the effect). Our results disfavour quantum-gravity theories in which the quantum nature of space-time on a very small scale linearly alters the speed of light.

A light and transmission electron microscope study of the distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the zebrafish eye.

The distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the eyes of the zebrafish, Danio rerio (Hamilton), were investigated using light and transmission electron microscopy. A comparative study of the quality of preservation provided by three different fixation procedures revealed no consistently striking general differences. However, somewhat subjectively, the fixative containing Millonig's buffer did consistently provide better fixation of myelin. Overall, nerve processes, depending on the site studied, were distributed as either (1) bundles (in the choroid near the optic nerve head and in the choroid adjacent to the limbus), (2) linear arrays (in the junction between the sclera and cartilage and in the choroid adjacent to the retina) or (3) individual units (in the choroid under the cartilage or in the sclera). Both myelinated and unmyelinated processes were identified in these locations. Myelinated processes usually contained both neurofilaments and neurotubules, but a few apparently contained only neurofilaments. Unmyelinated processes usually contained mainly neurotubules, but a few apparently contained only neurofilaments. Taken together, these findings indicate innervation of extra-retinal structures, as seen in zebrafish, is highly conserved among vertebrates, further supporting the use of zebrafish as a model for the vertebrate visual system.

Induction of hyperglycaemia in zebrafish (Danio rerio) leads to morphological changes in the retina.

Diabetes affects over 16 million Americans yearly, resulting in hyperglycaemia and microvascular complications, including retinopathy, neuropathy and nephropathy. Animal models have been developed to examine the immunological aspects of type 1 diabetes and the pathogenic mechanisms associated with diabetic retinopathy, but the methods of diabetes induction raise concerns regarding these models. Zebrafish (Danio rerio) have been used extensively to study developmental processes and mutant zebrafish strains have been used to examine vision disease present in humans. In this paper, we have induced hyperglycaemia in zebrafish by alternately immersing the fish in glucose solution or water. Eyes from untreated fish or fish exposed to alternating glucose/water solutions for 28 days were dissected, sectioned and stained to visualise cell bodies in the retina. In untreated fish retinas, the inner plexiform layer (IPL) and inner nuclear layer (INL) were approximately the same thickness, whereas in fish repeatedly exposed to glucose solutions the IPL was approximately 55% the thickness of the INL. Both the IPL and INL were significantly reduced in retinas of treated fish, compared to untreated fish, similar to that seen in other animal models of diabetes and in diabetic patients. These results suggest that zebrafish may be used as an animal model in which to study diabetic retinopathy.

Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina.

Horizontal, bipolar, and amacrine cells in the zebrafish retina were morphologically characterized using DiOlistic techniques. In this method, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-coated microcarriers are shot at high speed onto the surfaces of living retinal slices where the DiI then delineates axons, somata, and dendrites of isolated neurons. Zebrafish retinal somata were 5-10 microm in diameter. Three horizontal cell types (HA-1, HA-2, and HB) were identified; dendritic tree diameters averaged 25-40 microm. HA somata were round. Cells classified as HA-2 were larger than HA-1 cells and possessed an axon. HB somata were flattened, without an axon, although short fusiform structure(s) projected from the soma. Bipolar cells were separated into 17 morphological types. Dendritic trees ranged from 10 to 70 microM. There were six B(on) types with axon boutons only in the ON sublamina of the inner plexiform layer (IPL), and seven B(off) types with axon boutons or branches only in the OFF sublamina. Four types of bistratified bipolar cells displayed boutons in both ON and OFF layers. Amacrine cells occurred in seven types. A(off) cells (three types) were monostratified and ramified in the IPL OFF sublamina. Dendritic fields were 60-150 microM. A(on) pyriform cells (three types) branched in the ON sublamina. Dendritic fields were 50-170 microM. A(diffuse) cells articulated processes in all IPL strata. Dendritic fields were 15-90 microM. These findings are important for studies examining signal processing in zebrafish retina and for understanding changes in function resulting from mutations and perturbations of retinal organization.

Zebrafish retinal slice preparation.

This paper describes the protocol for generating thin (approximately 100 microm) slices of the zebrafish retina. Retinal slices retain the cytoarchitecture and synaptic contacts found in vivo, allowing neurons to be identified prior to physiological recordings. These characteristics distinguish retinal slices from both isolated cell and eyecup preparations. Studies using the zebrafish retinal slice have classified different retinal cell types, documented voltage- and ligand-gated current responses in distal bipolar neurons, and correlated physiological responses with neuronal morphology. Data collected using this protocol have provided baseline information about retinal circuitry that can be directly applied to behavioral studies examining visual function and/or mutants with visual system defects.

The expression of GAD67 isoforms in zebrafish retinal tissue changes over the light/dark cycle.

We show the levels of glutamic acid decarboxylase (GAD), the enzyme catalyzing the conversion of glutamic acid to GABA, changes in zebrafish retinal tissue during the light/dark cycle. Further, we identify two transcripts of the GAD67 gene, full-length GAD67 and the truncated 25 kDa alternative splice variant (ES), as the major GAD isoforms in this tissue. GAD-positive neurons were identified immunocytochemically by probing retinal sections with K2, an antibody to the GAD67 isoform, and with an antibody specific for the 25 kDa splice variant. For both antibodies, GAD-immunoreactivity was observed in horizontal cells in the distal retina and amacrine cells in the proximal retina, with both cell bodies and processes labeled. No apparent difference in K2 labeling pattern was observed in tissue harvested 8 hrs after light offset or onset, whereas ES label was identified in more structures in dark tissue. Quantification of GAD levels was determined by densitometry of Western Blots. The protein content of GAD67 and ES varied between tissue harvested during the light and the dark. ES expression was up-regulated in dark tissue; whereas, full-length GAD67 expression increased in light tissue. In vivo GABA content, measured with high performance liquid chromatography (HPLC), was found to increase in light tissue, paralleling the expression of full-length GAD67 transcripts. Expression of ES did not correlate with measured GABA levels, suggesting this isoform, which lacks the catalytic domain necessary for enzymatic activity, may have a different physiological role in retinal tissue. The inverse expression patterns of full-length GAD67 and ES suggest that alternative splicing of GAD67 may be triggered by the light and/or dark cycle, resulting in a change in inhibitory neurotransmitter content in retinal tissue.

Axonal stratification patterns and glutamate-gated conductance mechanisms in zebrafish retinal bipolar cells.

1. Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. 2. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). 3. OFF-BCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. 4. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev approximately 0 mV, mimicked by L-(+)-2-amino-4-phosphonobutyric acid (APB) or trans-1-amino-1, 3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev >= ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. 5. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. 6. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL.