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In contrast to the knowledge of chemical synapses, little is known regarding the properties of gap junction-mediated electrical synapses in developing zebrafish, which provide a valuable model to study neural function at the systems level. Identifiable "mixed" (electrical and chemical) auditory synaptic contacts known as "club endings" on Mauthner cells (2 large reticulospinal neurons involved in tail-flip escape responses) allow exploration of electrical transmission in fish. Here, we show that paralleling the development of auditory responses, electrical synapses at these contacts become anatomically identifiable at day 3 postfertilization, reaching a number of ∼6 between days 4 and 9. Furthermore, each terminal contains ∼18 gap junctions, representing between 2,000 and 3,000 connexon channels formed by the teleost homologs of mammalian connexin 36. Electrophysiological recordings revealed that gap junctions at each of these contacts are functional and that synaptic transmission has properties that are comparable with those of adult fish. Thus a surprisingly small number of mixed synapses are responsible for the acquisition of auditory responses by the Mauthner cells, and these are likely sufficient to support escape behaviors at early developmental stages.
- connexin 36
- connexin 35
- Mauthner cell
- auditory development
- escape response
because of its experimental accessibility, developing zebrafish has become an increasingly popular organism for not only the study of genetic and developmental aspects of the vertebrate nervous system (Roussigne et al. 2012; Stuckenholz et al. 2005; Ulrich et al. 2011), but also the understanding of diverse neural processes at the systems level (Ahrens et al. 2012; Beck et al. 2004; Bianco et al. 2012; Burgess and Granato 2007; McLean et al. 2007). Consequently, a good understanding of the properties of various forms of synaptic communication operating between larval zebrafish neurons is required to appreciate the network mechanisms underlying these processes. Whereas chemical excitatory (Patten and Ali 2007, 2009; Patten et al. 2010; Wen and Brehm 2005; Wen et al. 2013) and inhibitory (Legendre and Korn 1994, 1995; Legendre 1999) transmissions have been the focus of several studies, significantly less is known regarding the properties and molecular composition of electrical synapses in developing zebrafish. Moreover, a better understanding of the mechanisms underlying electrical transmission in developing zebrafish will contribute to a better characterization of neural processes that heavily rely on this modality of transmission, including retinal mechanisms (Li et al. 2012; Morris et al. 2008; Wei et al. 2012; Wong et al. 2004) and motor behaviors (Ahrens et al. 2012; Koyama et al. 2011; McLean and Fetcho 2009; Saint-Amant and Drapeau 2001).
Developing zebrafish offer a convenient model to study the development of the auditory system (Kohashi and Oda 2008; Nicolson 2005; Tanimoto et al. 2009). Auditory "mixed" (electrical and chemical) synapses terminating on the teleost Mauthner cell (M-cell), known as "large myelinated club endings" or simply club endings (CEs), constitute a valuable model for the study of vertebrate electrical transmission, as they are amenable to correlation of their ultrastructural and biochemical features with their in vivo physiological properties (Pereda et al. 2004). The M-cell network, centered on the properties and spinal connectivity of these colossal reticulospinal neurons, mediates auditory-evoked tail-flip escape responses in teleost fish (Faber and Pereda 2011). A wealth of data support the primary role of CEs in these behaviors (Curti and Pereda 2010; Mu et al. 2012). Recent evidence (Tanimoto et al. 2009) indicates that the functional maturation of the inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by second-order neurons located in the central nervous system (CNS) such as the M-cell (Tanimoto et al. 2009). However, the developmental properties of auditory synapses themselves on the M-cell remain unknown.
To gain insight into the maturation and properties of electrical synapses in developing zebrafish, we took advantage of our knowledge of the properties (Lin and Faber 1988a; Pereda et al. 2004) and molecular determinants (Flores et al. 2008; Pereda et al. 2003; Rash et al. 2013) of electrical transmission at CEs in adult goldfish. Here, we show that, parallel to the development of auditory responses in the M-cell of larval zebrafish (Tanimoto et al. 2009), CEs become identifiable by immunohistochemistry after day 3 postfertilization (dpf) to reach a number of ∼6/M-cell between 4 and 9 dpf. As in goldfish CEs, gap junctions at these terminals are formed by homologs of the mammalian connexin 36 (Cx36; Condorelli et al. 2000), Cx35, and Cx34.7 (O'Brien et al. 1998) and contain the connexin-associated protein zonula occludens-1 (ZO-1; Flores et al. 2008), suggesting that gap-junction channels are supported by a similar molecular scaffold. We also demonstrate using electrophysiological approaches that these mixed synapses exhibit functional properties that are comparable with those of adult goldfish, indicating that a relatively small number of auditory terminals (∼6 vs. ∼85 in adult goldfish; Furukawa and Ishii 1967) is sufficient to support escape responses (Tanimoto et al. 2009) at these early developmental stages.
Zebrafish husbandry and mutants.
AB zebrafish were maintained under a 13:11-h light-dark cycle at 28°C using standard laboratory practices (Westerfield 2000). Zebrafish embryos were obtained by natural mating and were staged according to number of dpf and confirmed according to standard morphological criteria (Kimmel et al. 1995). All procedures were performed with approval from the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine.
Immunohistochemistry and confocal imaging.
Zebrafish at 2–9 dpf were fixed in either 4% formaldehyde made from freshly depolymerized paraformaldehyde (PFA) or 2% TCA at 4°C overnight or at room temperature for 3 h, dehydrated into methanol using a graded series (20, 40, 60, 80, and 100% methanol), and stored overnight or longer at −20°C. Before staining, fixed larvae were rehydrated into PBSTx (PBS with 0.5% Triton X-100) through a graded methanol series (100, 80, 60, 40, 20, and 0% methanol) and washed in PBSTx three times for 5, 10, and 15 min, respectively. Larvae were then individually transferred onto a glass slide for dissection. The brains were carefully removed using tweezers and sharp tungsten needles under a bright-field microscope. In some larvae, the forebrain, the midbrain, and the cerebellum were also removed to reduce the thickness of the sample. The isolated brains were collected and washed in PBSTx, incubated at room temperature in blocking solution (10% newborn goat serum in PBSTx) for at least 1 h, and then transferred into blocking solution containing the primary antibodies at the following dilutions: Cx35 monoclonal antibody (MAB3043; EMD Millipore), 1:300; ZO-1 monoclonal antibody (cat. no. 33-9100; Invitrogen), 1:200; and ZO-1 rabbit polyclonal antibody (cat. no. 61-7300; Invitrogen), 1:200. The following day, brains were washed in PBSTx five times (5, 10, 15, and 30 min). After the washes, the brains were transferred into the blocking solution containing secondary antibodies at 1:200 dilutions. Secondary antibodies included: Cy5-tagged goat-anti-mouse IgG (A-10524; Invitrogen), Cy2-tagged goat-anti-rabbit IgG (cat. no. 111-225-003; Jackson ImmunoResearch Laboratories), Alexa Fluor 488-tagged goat-anti-mouse IgG (A11001; Invitrogen), and Alexa Fluor 594-tagged goat-anti rabbit IgG (A11012; Invitrogen). The brains were incubated in the secondary antibody solution in the dark at room temperature for 4 h or at 4°C overnight and handled in the dark. Then, the brains were washed three times for 5, 10, and 15 min, transferred to methanol using a graded series (20, 40, 60, 80, and 100%), and stored in methanol until imaging (usually the same day of experiment). To mount the brains on a slide, each brain was transferred onto a slide in the dark. Brains were mounted on a slide with a drop of BBA (33% benzyl benzoate and 67% benzyl alcohol) and covered with a coverslip, which was glued onto the slide with cyanoacrylate and sealed with nail polish. Samples were imaged using LSM 510 and LSM 710 Zeiss microscopes. Control sections were routinely incubated with secondary antibodies in the absence of primary antibodies. Images were analyzed using ZEN 2010 (Zeiss) and Photoshop CS3 (Adobe) software.
Image analysis.
For estimates of the number of Cx35-labeled puncta at individual CEs, images were analyzed using ZEN 2009, Volocity 6.3 (PerkinElmer), and AutoQuant X1.4.1 (Media Cybernetics) software. Regions of interest corresponding to individual CEs were initially identified in ZEN 2009. These areas were cropped to include only individual CEs (4–8 sections each of 0.37- to 0.45-μm thickness). Cropped z-stack images underwent 3-dimensional deconvolution using AutoQuant (manual counting) and Volocity (automatic counting), respectively. For image analysis with AutoQuant, a process called adaptive point spread function deconvolution was used, and deconvolved images were manually thresholded to remove background (threshold level was kept constant for all analyzed images). Puncta approximately two- to threefold brighter than background were then manually quantified. For image analysis with Volocity, iterative restoration was used with a calculated point spread function determined by the refractive index of the medium, numerical aperture of the objective used, and the emission wavelength of rhodamine and secondary antibodies. In Volocity, images were automatically thresholded, and puncta were quantified using the "Find Objects" function with a minimum object size of 0.027 μm3 (cubic estimate of the practical resolution limit of the confocal microscope).
Backfilling of reticulospinal neurons.
Zebrafish at 2–9 dpf were anesthetized with 0.03% MS-222 and embedded in 2% low-melting-point agarose (Bio-Rad). A tungsten needle, gently inserted between the spinal cord and the notochord at approximately the 10th somite, was quickly moved through the spinal cord to create an incision. Then, the tip of a second tungsten needle, coated with crystals of tetramethylrhodamine-dextran (70,000 mol wt; D1818; Invitrogen), was quickly applied to the incision for ∼10 s. Backfilled larvae were removed from the agarose 5 min after this procedure and allowed to recover in egg water at 28°C for 3–4 h. Larvae were then fixed in 4% formaldehyde with 1% sucrose in PBSTx at room temperature for 3 h and processed for immunolabeling as described above.
Freeze-fracture replica immunogold labeling.
Backfilling with tetramethylrhodamine was used to identify the position of the M-cells in the replica. Brains from zebrafish larvae 5–6 dpf were dissected, frozen, freeze-fractured, and replicated according to our detailed procedures (Pereda et al. 1994). A gold index grid was bonded to the frozen sample by using 3% Lexan plastic dissolved in dichloroethane; the samples were thawed and "grid-mapped" by confocal microscopy, and cellular material was removed by gentle washing with SDS detergent. For freeze-fracture replica immunogold labeling (FRIL), residual connexin proteins adhering to the replica after SDS washing (Fujimoto 1995, 1997) were labeled with rabbit anti-Cx34.7-I (JOB 2930-2), chicken anti-Cx34.7-CT (JOB), and mouse monoclonal antibody directed against Cx35 (MAB3043 and MAB3045; Chemicon) and counterlabeled with goat gold-conjugated anti-mouse (5- and 20-nm gold beads for Cx35; BBI), anti-rabbit secondary antibodies (10- and 30-nm gold beads for Cx34.7; BBI), and donkey anti-chicken antibodies (12-nm gold beads for Cx34.7; Jackson ImmunoResearch Laboratories). Replicas were examined using a JEOL 2000 EX-II transmission electron microscope operated at 100 kV.
Zebrafish 4–7 dpf were used for electrophysiology recordings. To expose the brain, the larvae were first paralyzed in a solution containing 10 μM d-tubocurarine (Sigma) in a solution consisting of (in mM): 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES glucose, pH 7.8, 274 mosM (Koyama et al. 2011). Larvae were placed on their backs and held with pins in a Sylgard-coated petri dish, and the brain stem was accessed ventrally following the procedure described in Koyama et al. (2011). The dissected larvae were transferred to the recording setup and maintained with the same external solution. M-cells were identified using far-red differential interference contrast (DIC) under ×40 water-immersion objective and recorded using a whole cell recording configuration under current-clamp. The electrode (5 MΩ) internal solution consisted of (in mM): 105 CH3O3SK, 10 HEPES, 0.1 EGTA, 2 MgCl2·6H2O, 4 Na2ATP, 0.4 Tris-GTP, 10 K2-phosphocreatine, 23 mannitol, adjusted to pH 7.2, 274 mosM (Koyama et al. 2011). The junction potential was estimated to be ∼12 mV. Resting potentials ranged from 60 to 72 mV; M-cell action potential ranged from 73 to 80 mV; the input resistance of the M-cell ranged from 5 to 7 MΩ. Bipolar stimulation of the ear was obtained using a "theta" (septated) glass pipette filled with external solution. This pipette was positioned under visual guidance in the posterior macula in the ear, near the hair cells. In vivo intradendritic recordings (250–350 μm from the M-cell soma) of mixed synaptic responses in adult goldfish were done using the experimental approaches that were described previously (Smith and Pereda 2003). Population synaptic responses were obtained by stimulating the posterior branch of the VIIIth nerve. Single terminal synaptic responses were obtained by recording from individual VIIIth nerve afferents in the posterior VIIIth nerve outside of the brain while simultaneously recording the lateral dendrite of the M-cell.
Labeling of auditory afferents.
4-dpf Tol-056 enhancer trap transgenic zebrafish [where M-cells are tagged with green fluorescent protein (GFP); kindly provided by Dr. Michael Granato] were fixed overnight at 4°C in 4% PFA with 4% sucrose. Following washing with PBSTx, the fish were mounted on 3% agar and covered with 1.5% low-melting-point agarose. A glass micropipette containing 1.0% 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; D282; Invitrogen) in chloroform was inserted into the ear directly dorsal to the posterior otolith, and DiI was applied by pressure (40 psi during 12 ms) using a Picospritzer (Picospritzer III; Parker Automation). Following this procedure, larvae were removed from the agar plate and placed at room temperature in 1× PBS overnight. Brains were then removed, stained with mouse anti-Cx35 and rabbit recombinant monoclonal anti-GFP antibodies (G10362; Invitrogen) at 1:300 dilutions, and finally imaged using the procedures described above.
Labeling of dopaminergic fibers.
The relative position of dopaminergic fibers to the M-cells was revealed using the Tg(dat:EGFP) transgenic zebrafish expressing green fluorescent protein in dopaminergic neurons of the ventral diencephalon (kindly provided by Dr. Mark Ekker; Xi et al. 2011). dat:EGFP zebrafish 4–5 dpf were backfilled with Texas red dextran (D-3328; Invitrogen). After fixation, the larvae were stained with mouse anti-Cx35 and rabbit recombinant monoclonal anti-GFP antibodies (G10362; Invitrogen) at 1:300 dilution.
Identification of CEs in developing zebrafish.
CEs are terminations of auditory afferents originating in the teleost sacculus (Fig. 1A). They comprise a relatively homogeneous morphological group of terminals that are tightly clustered on the distal portion of the lateral dendrite of the M-cell (Bartelmez and Hoerr 1933). These endings can be unambiguously identified in the lateral dendrite of the goldfish M-cell by immunolabeling with anti-Cx35 (Fig. 1B; Flores et al. 2008; Pereda et al. 2003), as the approximately 60–100 gap-junction plaques contained at each of these unusually large contacts are homogenously distributed throughout the whole appositional surface (Fig. 1, B and C). Thus we first determined that CEs in developing zebrafish could also be identified by Cx35 labeling. For this purpose, reticulospinal neurons including the M-cell and MiD2cm and MiD3cm cells of 4- to 8-dpf zebrafish were backfilled by spinal application of tetramethylrhodamine-dextran, and the intact brains were labeled with monoclonal Cx35 antibody (Fig. 2A). As illustrated in Fig. 2, immunofluorescence was observed along the distal portion of the M-cell lateral dendrite at large ovoid areas that, because of their relative size, ovoid shape, and location, were presumed to correspond to CEs. As in adult goldfish, Cx35-labeled ovoid areas were defined by multiple fluorescent patches (Fig. 2, B–D). To confirm that they correspond to auditory afferents, we performed DiI applications in the vicinity of the posterior macula of the ear (thought to give rise to the saccular macula; Nicolson 2005), which resulted in the labeling of axons that terminated on these large ovoid areas (Fig. 3, A–C). Other, much smaller Cx35 immunoreactive puncta were found in more proximal areas of the lateral dendrite and soma (Fig. 2B), as observed in goldfish (Flores et al. 2008; Pereda et al. 2003), which probably correspond to other octavolateralis inputs to the M-cell (Szabo et al. 2007). This labeling is also consistent with recent reports indicating the presence of Cx35 in the M-cell dendrite of developing zebrafish (Jabeen and Thirumalai 2013). Finally, numerous immunoreactive puncta also were observed along the hindbrain, in particular at areas corresponding to the developing ear (Fig. 2B).
Developmental profile of CEs.
The ability to identify CEs with Cx35 labeling provided us with the opportunity to examine the developmental appearance of these contacts. Zebrafish at 3–9 dpf were examined with anti-Cx35, and their diameter, number, and relative size (estimated as the diameter of the CE divided by the diameter of the lateral dendrite of the M-cell) were quantified (Fig. 3D). Although fewer and smaller in diameter than those of adult goldfish, these large Cx35-labeled areas became identifiable at day 3, reaching a stable number of ∼6/M-cell between days 4 and 9: the average number of CEs was 6.2 ± 0.68 SE (n = 10 dendrites) in 5 dpf and 6.02 ± 0.41 (n = 4 dendrites) in 9-dpf zebrafish (P = 0.40, 1-tailed t-test comparing 5 vs. 9 dpf; Fig. 3E). Their diameter and relative size were also found to be relatively stable between days 4 and 9 (Fig. 3, F and G). The average CE diameter was 1.90 μm ± 0.15 in 4-dpf larvae (n = 11), 1.81 ± 0.04 in 5-dpf larvae (n = 74), 1.77 ± 0.08 in 6-dpf larvae (n = 13), and 1.90 ± 0.06 in 9-dpf larvae (n = 24; Fig. 3F); these measurements were not significantly different from each other (P > 0.05). The CE relative size at different ages was also not significantly different (P > 0.05), averaging 1.01 ± 0.06 in 4 dpf (n = 11), 0.94 ± 0.04 in 5 dpf (n = 74), 1.06 ± 0.04 in 6 dpf (n = 12), and 1.01 ± 0.06 in 9-dpf zebrafish (n = 24).
We next compared these parameters with those of adult goldfish (7–12 cm long) and adult zebrafish (3–4 cm long; Fig. 4, A–C). The average diameter of these Cx35-labeled areas was 6.46 μm ± 0.22 in adult goldfish (n = 45), 4.5 μm ± 0.23 in adult zebrafish (n = 21), and 1.83 μm ± 0.04 in 4- to 9-dpf zebrafish (n = 98; Fig. 4E). The relative size was 0.27 in adult goldfish, 0.27 in adult zebrafish, and 0.97 in 4- to 9-dpf zebrafish (Fig. 4F). The small number of CEs identified in larval zebrafish markedly contrasted with those observed in adult goldfish (Furukawa and Ishii 1967) and other teleosts (Bartelmez and Hoerr 1933; Bodian 1937), which are estimated to be ∼85/M-cell (Fig. 4D), a number that is also likely to apply to adult zebrafish (see discussion). Thus, from the comparative point of view, CEs in 4- to 9-dpf larval zebrafish are fewer in number, but their relative size is larger (Fig. 4G), suggesting that a number of ∼6 terminals/M-cell is sufficient to support their function during these developmental stages.
Number and molecular composition of gap junctions in CEs of larval zebrafish.
We then determined the number of gap junctions and gap-junction channels that were present at a single larval zebrafish CE. It has been established that the number of Cx35 puncta at single goldfish CEs (Fig. 1B) correlates with the number of gap junctions present at these terminals (Flores et al. 2008). To improve visualization of these puncta at single larval zebrafish CEs, we performed deconvolution analysis of confocal images (Fig. 5, A and B; AutoQuant software). The number of manually counted puncta averaged 17.72 ± 0.99 (SE; n = 22). A second, automatic deconvolution analysis (Volocity software) of the same set of images yielded a similar number, averaging 17.82 ± 2.12 puncta (see methods for details), suggesting that 4- to 6-dpf zebrafish CEs contain on average ∼18 gap-junction plaques (Fig. 5C). This suggestion was further supported by ultrastructural analysis with FRIL of two probable, partially reconstructed CEs. For their identification in the replica, the M-cells were retrogradely labeled by application of tetramethylrhodamine-dextran in the spinal cord (see methods). Because of their unusually large size (1.3- to 1.9-μm diameter and 1.5- to 3-μm2 area of apposition) and presence of multiple (>10) gap junctions/axon terminal and based on the presence of multiple large synapses on the same neuron as well as the anatomic location of these cells in hindbrain, these mixed (electrical and chemical) synapses are identified as CEs. CEs are the only axon terminals of that size that we found labeled with Cx35 under confocal microscopy in this area of the larval zebrafish hindbrain (Fig. 2), and their distinctively larger size is consistent with previous ultrastructural findings (Kimmel et al. 1981). The size of the gap-junction plaques was highly variable (0.001–0.239 μm2), and 2 CEs examined by FRIL (Fig. 6D) contained approximately 1,866 and 1,498 connexons, respectively. Because in both cases the fracture plane was diverted from the CE plasma membrane into the M-cell cytoplasm at an area of perpendicular membrane apposition (Fig. 6, D and E, lower right), additional gap junctions may have existed outside of the area that was exposed. Moreover, FRIL revealed gap junctions that were too small to be detected by immunofluorescence microscopy [see Kamasawa et al. (2006) for quantitative documentation]. If so, the confocal and FRIL data could represent an underestimate of both the number of gap junctions and number of connexon channels. Nevertheless, assuming that single terminals contain 18 gap junction plaques on average, we estimate that each CE contains between approximately 2,000 and 3,000 gap junction channels.
Recent data indicate that in addition to Cx35 a 2nd homolog of Cx36, Cx34.7, is present at these terminals (Rash et al. 2013). To examine for the presence of a homolog of Cx34.7 in CEs of larval zebrafish, we performed double-labeling experiments with anti-Cx35 and anti-Cx34.7 antibodies. As illustrated in Fig. 6, A–C, both antibodies were colocalized at CEs in the lateral dendrite of the M-cell. Confirming this observation, labeling for Cx34.7 was observed by FRIL of the M-cell side of gap junctions at probable CE/M-cell synapses (Fig. 6C). For their identification in the replica, the M-cells were retrogradely labeled by application of tetramethylrhodamine-dextran in the spinal cord (see methods). As in adult goldfish (Rash et al. 2013), 22 of the 25 gap junctions found in postsynaptic hemiplaques in these 2 adjacent CEs were labeled almost exclusively for Cx34.7 [113 of 116 gold beads with 3 beads/2.5% of intermediate diameter (25 nm), reflecting either 2.5% of gold beads labeling for Cx35 or, more likely, the unavoidable but minor variability in sizes of gold beads (4.8% coefficient of variation; product data sheet; BBInternational)]. The other 7 unlabeled gap junctions were small (4–85 connexons) and therefore may have gone unlabeled due to the moderate labeling efficiency (1 gold bead/30 connexons) and the stochastic nature of immunogold labeling or represent evidence for a few neuronal gap junctions composed of another as yet unidentified connexin other than Cx34.7 and Cx35. (Four additional potential gap junctions were not positively identified because they were not shadowed with platinum but coated only with carbon. Combined with steep viewing angle of the carbon coat, the connexons were not clearly resolvable. Thus, because the size of those probable gap junctions and their number of connexons could not be determined accurately, the data from these 4 possible gap junctions were not included in the calculations above.) Combined with our confocal immunocytochemistry showing strong colocalization of both Cx35 and Cx34.7 at CE/M-cell synapses, our FRIL demonstration of essentially pure Cx34.7 in M-cell hemiplaques suggests that gap-junction channels in larval zebrafish, as in adult goldfish (Rash et al. 2013), are heterotypical, with Cx35 primarily or exclusively presynaptic and Cx34.7 primarily or exclusively postsynaptic. In support, gold beads for Cx35 (5- and 20-nm gold beads without 10-, 12-, or 30-nm gold beads for Cx34.7) were found in three presynaptic hemiplaques of unidentified neuronal gap junctions located >50 μm from the M-cell, demonstrating the efficacy of the Cx35 antibodies in this replica. Gold beads for Cx34.7 but not for Cx35 were detected in hemiplaques of an additional seven unidentified neuronal gap junctions that were >50 μm from the M-cell (Fig. 6) and, because of the location and sizes, presumably were on neighboring vestibulospinal or reticulospinal neurons. Finally, one gap junction contained gold beads for both Cx35 and Cx34.7, suggesting that, in contrast to CEs, the observed asymmetry in the distribution of connexins might not apply to all neuronal gap junctions at this developmental stage. Overall, at 5 dpf, both Cx34.7 and Cx35 occur abundantly at CE/M-cell and other neuronal gap junctions, with Cx34.7 found by FRIL only in the postsynaptic hemiplaques and Cx35 found almost exclusively in presynaptic hemiplaques.
ZO-1 is a member of the MAGUK family of proteins that was reported to interact with several connexins (Hervé et al. 2004), including Cx36 (Li et al. 2004). Recent work indicates that ZO-1 also colocalizes and interacts with Cx35 in goldfish CEs (Flores et al. 2008). Because of its extensive colocalization with Cx35 and direct interaction with the COOH terminus of Cx36-like connexins, ZO-1 is expected to play an important structural and functional role in CEs (Flores et al. 2008, 2010). Therefore, we asked whether ZO-1 is also present in CEs of larval zebrafish. In double-labeling experiments with anti-Cx35 and anti-ZO-1 antibodies, we found that these proteins colocalize in larval zebrafish CEs (Fig. 7A). The pattern and extent of colocalization between these two proteins were similar to those observed in adult goldfish CEs (Fig. 7B). Thus the results suggest that gap-junction channels in larval zebrafish CEs are formed by the fish homologs of Cx36 found in adult goldfish and supported by a similar molecular scaffold.
Proximity to regulatory elements.
Electrical (and chemical) synaptic transmission in CEs has been shown to be under the regulation of dopamine (Cachope et al. 2007; Pereda et al. 1992, 1994), which is released from varicosities located at dopaminergic fibers in the vicinity of CEs (Cachope et al. 2007; Pereda et al. 1992). Recent data indicate that auditory responses to the M-cell in larval zebrafish are also under the regulatory control of dopamine (Mu et al. 2012). Therefore, we investigated the presence of dopaminergic fibers near the M-cell lateral dendrite and their proximity to CEs. In Tg(dat:EGFP) transgenic zebrafish expressing green fluorescent protein in dopaminergic neurons of the ventral diencephalon (Xi et al. 2011), fixed and labeled with anti-GFP antibodies, we observed the presence of dopaminergic fibers in the vicinity of the lateral dendrite (Fig. 8A). Furthermore, in double-immunolabeling with anti-Cx35, varicose fibers were intimately associated with CEs (Fig. 8B), indicating that dopaminergic fibers of larval zebrafish and adult goldfish (Cachope et al. 2007; Pereda et al. 1992) are similarly arranged, suggesting they serve similar functional roles.
Functional properties of synapses at larval zebrafish CEs.
CEs in 4- to 9-dpf zebrafish are identifiable by labeling for gap-junction and scaffold proteins. On the other hand, the presence of these molecules does not necessarily imply that electrical synapses at these endings are functional. To explore the physiological properties of larval CEs, we performed whole cell recordings of the M-cell in 5- to 7-dpf zebrafish (Fig. 9A) while monitoring the responses evoked by a bipolar stimulating electrode placed in the vicinity of the hair cells in the posterior macula (Fig. 9B). Stimulation of the dendritic end of auditory afferents to the M-cell (for which the central endings could be identified by DIC optics on the lateral dendrite; Fig. 9C) evoked a low-threshold synaptic response composed of a brief and large early component followed by a delayed and longer-lasting component (Fig. 9D). These responses were indistinguishable from the mixed (electrical and chemical) synaptic response observed in adult goldfish in response to stimulation of CEs in the posterior branch of the VIIIth nerve (Fig. 9E), where the first component was shown to represent the electrical coupling produced by presynaptic action potentials in CEs and the second the release of neurotransmitter from these terminals (Furshpan 1964; Lin and Faber 1988b). Stimulation with higher strength evoked additional delayed responses, likely representing the activation of higher threshold and lower conducting afferents to the M-cell (data not shown). Remarkably, the time of arrival of the electrical component to the M-cell was similar in both adult goldfish and larval zebrafish (∼200 μs; compare Fig. 8, E and F) and was usually "riding" on the stimulus artifact of the ear stimulation. The short delay cannot be ascribable to the relatively shorter distance between the ear and the M-cell in larval zebrafish, as stimulation of other afferent inputs by simply reversing the polarity of the stimulating electrode evoked synaptic responses with considerable longer synaptic delays (Fig. 8F), indicating that axons in larval zebrafish are capable of conducting action potentials at different speeds and that CEs are relatively fast conducting.
The two components of the mixed synaptic response also could be observed in isolation (Fig. 9G); whereas the first component was always present, the second component, representing the probabilistic release of neurotransmitter, could be absent from trial to trial and was facilitated by paired pulses, a stimulation protocol known to facilitate transmitter release in adult goldfish CEs (Fig. 9, G and H; Curti et al. 2008; Lin and Faber 1988a,b). Spontaneous synaptic responses, usually lacking chemical transmission, were observed during whole cell recordings (Fig. 10A). These responses were reminiscent of unitary CE responses obtained in adult goldfish during paired pre- and postsynaptic recordings (compare Fig. 10, B and C) and had similar amplitude (0.50 mV ± 0.10 SD, n = 65) to that observed by "minimal stimulation" (all-or-none response obtained with minimal stimulation strength) obtained by extracellular stimulation of the ear (0.56 mV ± 0.01 SD, n = 9; Fig. 10, D and E), indicating that they represent the natural activation of single terminals.
CEs become identifiable by Cx35 labeling at day 3 to reach a stable number of ∼6 CEs/M-cell between days 4 and 9; the gradual increase in number suggests that not all CEs are at the same developmental and functional stage. Stimulation of the ear evoked graded responses that usually had six clear discernible steps (Fig. 10D). Increasing the stimulus strength did not further enhance the early response but rather resulted in the activation of components with longer synaptic delays (data not shown). This evoked "maximal" response was of 2.99 mV ± 0.65 SD, n = 9. Because the unitary responses are ∼0.5 mV (spontaneous and evoked minimal; see above), it indicates, confirming the graded responses (Fig. 10D), that the maximal response represents the activation of six terminals, each contributing ∼0.5 mV (Fig. 10E). In summary, both components of the mixed synaptic response are present in CEs of larval zebrafish, indicating that gap junctions at these terminals are functional at these developmental stages. Furthermore, all CEs are functional and seem to provide timing information (arrival time to the M-cell) comparable with those in adult goldfish.
The teleost CEs: a unique model to study vertebrate electrical transmission.
Since their description by Bartelmez (Bartelmez 1915; Bartelmez and Hoerr 1933), CEs have provided an invaluable window for the study of vertebrate synaptic transmission. In his classic paper describing these terminals, Bodian (1937) provided conclusive evidence for the absence of direct protoplasmic continuity between the pre- and postsynaptic elements. By applying electron microscopy to goldfish CEs, Robertson et al. (1963) provided early evidence of the structures we know today as gap junctions. Parallel electrophysiological studies by Furshpan (1964) in CEs provided unequivocal evidence for the presence of electrical transmission in vertebrates. More recently, it was found that gap junctions at CEs contain teleost homologs of Cx36 (Pereda et al. 2003; Rash et al. 2013) and, like their chemical counterparts, electrical synapses at CEs undergo activity-dependent potentiation (Cachope et al. 2007; Pereda and Faber 1996; Pereda et al. 2004; Yang et al. 1990). Investigations on CEs unmasked the intimate functional relationship between glutamatergic and electrical transmission that are responsible for triggering activity-dependent changes (Cachope et al. 2007; Pereda et al. 2004; Smith and Pereda 2003), which recently also were shown to occur at thalamic inhibitory interneurons (Landisman and Connors 2005) and in the inferior olive (Mathy et al. 2014; Turecek et al. 2014). We show here that these CE terminals also can be identified in larval zebrafish and, moreover, their molecular composition and physiological properties are similar to those of adult goldfish. The present characterization will allow this unique model synapse to be used for new levels of analysis that combine electrophysiology, in vivo imaging, and genetic manipulations.
Properties of electrical transmission in larval zebrafish.
In contrast to chemical transmission (Legendre and Korn 1994, 1995; Legendre 1999; Patten and Ali 2007, 2009; Patten et al. 2010; Wen and Brehm 2005), less is known regarding the properties of electrical transmission in developing zebrafish. We report here the molecular composition and functional properties of electrical synapses in developing CEs. We found that contacts between CEs and the M-cells in larval zebrafish each contain ∼18 gap junctions, representing in total between 2,000 and 3,000 channels/CE. Interestingly, assuming that values of presynaptic action potentials and input resistance are similar to those measured in goldfish, the average amplitude of the recorded unitary electrical synaptic responses, together with measurements of M-cell input resistance, allowed tentative calculations of gap-junctional conductance, which suggested that only 2.5% of channels present at each larval zebrafish CE are functional [see Rash et al. (2013) for rationale and methods]. Although this highly speculative estimate needs to be confirmed, such a small fraction of functional channels would be consistent with that estimated for adult goldfish CEs (1–4%; Flores et al. 2012; Lin and Faber 1988b). Gap junctions at larval zebrafish contain two homologs of the mammalian Cx36, Cx35 and Cx34.7, which were also identified by immunochemical means at gap junctions between CEs and the M-cell in adult goldfish (Rash et al. 2013). Because of its widespread brain distribution and preferred neuronal expression (Belluardo et al. 1999), Cx36 could be considered the main synaptic connexin in mammals and was reported to mediate electrical transmission in most CNS structures (Connors and Long 2004). Our labeling for Cx35 in CEs is consistent with recent reports indicating the presence of this connexin in zebrafish brain (Carlisle and Ribera 2014; Jabeen and Thirumalai 2013). As in adult goldfish, we found that Cx34.7 labeling was restricted to postsynaptic hemiplaques, at least by 5 dpf, suggesting that gap junctions between CEs and the M-cells in larval zebrafish are also made by the apposition of hemichannels, most of which are formed by two homologs of the mammalian Cx36 (Rash et al. 2013). Interestingly, several isoforms of these connexins have been recently identified in zebrafish. Given the presence of several Cx35 (Cx35.1 and Cx35.5) and Cx34 (Cx34.1 and Cx34.7) genes/proteins in zebrafish (Adam Miller and John O'Brien, personal communication), it is unclear whether the antibodies used in this study recognize two or more orthologs. However, our previous work (Rash et al. 2013) has found that the antibody used against Cx35 does not recognize Cx34.7, whereas the antibody against Cx34.7 does not recognize Cx35. Additionally, based on the epitopes for which these antibodies were raised, we expect that the Cx35 antibody will not recognize either of the Cx34 proteins and the Cx34.7 antibody will not recognize either of the Cx35 proteins. These channels seem to be supported by a similar molecular scaffold, as we detected the presence of the scaffolding protein ZO-1 at larval zebrafish CEs. ZO-1 is a member of the MAGUK family of proteins that, because of its extensive colocalization and interaction with Cx36-like connexins, was suggested to play an important structural and functional role in goldfish CEs (Flores et al. 2008). Thus our results indicate that gap junctions at larval zebrafish and adult goldfish CEs share a similar molecular composition and have similar functional properties.
Both components of the mixed synaptic response were observed at CEs of larval zebrafish. Mixed synaptic responses in the M-cell evoked by electrical stimulation of CEs were indistinguishable from the mixed synaptic responses observed in adult goldfish, where the first component was established to correspond to the electrical coupling potential of a presynaptically occurring action potential (Lin and Faber 1988a), and the delayed and longer-lasting component results from the release of glutamate from these terminals (Pereda et al. 2004; Wolszon et al. 1997). Although glutamatergic transmission was not the emphasis of this study, it appeared to exhibit kinetics and release properties (paired-pulse facilitation) that were consistent with those observed in adult goldfish (Wolszon et al. 1997). The electrical and chemical components of the mixed unitary synaptic responses were also indistinguishable from goldfish responses, indicating that gap junctions (and glutamatergic synapses) at these terminals are fully functional at the developmental stages under study.
The presence of large and abundant gap junctions at CEs guarantees fast synaptic transmission needed for an escape behavior. Nevertheless, whereas CEs are thought to provide critical timing information, the sensory input required to trigger an escape response is likely multimodal and requires not only auditory, but also vestibular and lateral line information (Casagrand et al. 1999; Mirjany and Faber 2011). In addition, gap junctions support a mechanism of lateral excitation in which the depolarization produced by some active CE afferents spreads back to neighboring, inactive CE terminals to increase their excitability (Curti and Pereda 2004; Pereda et al. 1995). This mechanism is thought to promote the coordinated activity of this population of afferents, increasing the probability of initiating an escape response. Lateral excitation is supported by the rectification properties of gap junctions. Because rectification relies on the existence of heterotypical Cx35/Cx34.7 channels (Rash et al. 2013), lateral excitation is also likely to operate in larval zebrafish, whose gap-junction channels are similarly constructed.
Thus our results indicate that these developing synapses have properties that are comparable with those of adult teleosts. Consistent with this conclusion, a recent report (Mu et al. 2012) indicates that CEs in larval zebrafish, as those in adult goldfish (Cachope et al. 2007; Pereda et al. 1992, 1994), exhibit dopamine-dependent potentiation of the mixed synaptic response. This finding is consistent with the proximity of dopaminergic fibers to CEs that we describe here, which exhibit a pattern similar to that observed in adult goldfish (Cachope et al. 2007; Pereda et al. 1992). Altogether, the data suggest that electrical synapses in developing zebrafish have properties similar to those observed in adult zebrafish.
CEs and the development of auditory responses in the M-cell.
Developing zebrafish have recently become a convenient organism for the study of the development of the auditory system (Nicolson 2005). We describe here the properties of a central auditory synapse in larval zebrafish, whose participation is thought to be critical for an essential escape behavior. Because of its experimental accessibility, the development of auditory responses in the M-cell has provided a model to correlate the development of the external ear and primary auditory afferents with an easily measureable animal behavior (Kohashi and Oda 2008; Kohashi et al. 2012; Tanimoto et al. 2009). The developmental profile of CEs (as detected by Cx35 labeling) is consistent with the acquisition of auditory responses by the M-cell (Tanimoto et al. 2009), and the switch to auditory evoked escape response by the M-cell system occurs at ∼75 h postfertilization (Kohashi et al. 2012). Our results are also consistent with previous ultrastructural data (Kimmel et al. 1981) demonstrating at these developmental stages the presence of large gap-junction-containing single synaptic contacts in the distal portion of the M-cell lateral dendrite, on which myelinated axons originating in the ear form synapses.
The mixed synaptic response evoked by CEs in larval zebrafish is indistinguishable from that of adult goldfish, where both seem to provide similar timing information (arrival time) to the M-cell, suggesting that their anatomic and physiological properties are scaled to produce the same function despite the striking size differences between larval zebrafish and adult goldfish. Interestingly, the degree of myelination reported by Kimmel et al. (1981) seems to be much less than that observed in adult goldfish CEs (Robertson et al. 1963). Remarkably, our results indicate that only a small number of terminals are required to support escape responses during these early developmental stages: only ∼6/M-cell in 4- to 9-dpf larval zebrafish vs. ∼85/M-cell in adult goldfish (Furukawa and Ishii 1967) with similar large numbers likely in adult zebrafish. That is, although we were not able to estimate rigorously the number of CEs in adult zebrafish, their number and density seem consistent with those observed in other teleosts such as bullhead (Bodian 1937) and goldfish (Furukawa and Ishii 1967). The presence of a higher number of CEs in older larvae was reported for other teleosts (Bartelmez and Hoerr 1933), suggesting that the number of these terminals gradually increases during development to reach the number observed in the adult. In summary, we show here that electrical synapses at CEs in 4- to 9-dpf zebrafish exhibit properties that are comparable with those of adult goldfish, suggesting they are at least partially responsible for the acquisition of auditory responses in the M-cell.
This research was supported by National Institutes of Health Grants DC-03186, DC-011099, R21-NS-055726, and NS-0552827 to A. E. Pereda and S10-RR-05831, S10-RR-08329, NS-044395, and NS-044010 to J. E. Rash.
No conflicts of interest, financial or otherwise, are declared by the author(s).
C.Y., J.E.R., and A.E.P. conception and design of research; C.Y., K.G.V., M.D., V.E., A.G.L., C.S.-R., T.Y., and A.E.P. performed experiments; C.Y., K.G.V., V.E., A.G.L., C.S.-R., J.E.R., and A.E.P. analyzed data; C.Y., K.G.V., A.G.L., J.E.R., and A.E.P. interpreted results of experiments; C.Y., K.G.V., M.D., V.E., A.G.L., T.Y., and A.E.P. prepared figures; C.Y., K.G.V., J.E.R., and A.E.P. drafted manuscript; C.Y., K.G.V., M.D., J.E.R., and A.E.P. edited and revised manuscript; C.Y., K.G.V., J.E.R., and A.E.P. approved final version of manuscript.
We are deeply indebted to Florence Marlow for help and guidance. We also thank Yoichi Oda, Adam Miller, and John O'Brien for comments on the manuscript and Monica Zell for the goldfish images in Fig. 1.
- Copyright © 2014 the American Physiological Society


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Echelon Custom Interior
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Rio Olympics might be a mess, but Tokyo 2020 has some cool high-tech plans
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CANCER June 22nd - July 23rd
The prospect of confronting a difficult relative is frightening. You want to be respectful towards older people. Some individuals make this impossible. There's no reason you should be gracious to someone who is unkind, stubborn and demanding. Instead of trying to make them happy, satisfy your own needs instead. Do the bare minimum for your family member. If they complain, don't make excuses. Simply explain you have more pressing matters to attend. Don't get drawn into a row.
Ten reasons we want to be on TOWIE!
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J P Produce Inc
9396 Pinion Dr., Lake Worth, FL 33467
(561) 740-9938
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According to our records, this business is located at 9396 Pinion Dr. in Lake Worth (in Palm Beach County), Florida 33467, the location GPS coordinates are: 26.630377 (latitude), -80.196266 (longitude). J P Produce Inc is categorized under Fruit And Vegetable Markets (SIC code 5431). Known organization owner is Jerome Nielb. You can contact the company by phone at (561) 740-9938.
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US urges aerial spraying amid jump in Puerto Rico Zika cases
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Immaculate One Level Home in Tellico Village
222 Tommotley Drive, Loudon, TN 37774
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A rare find! Immaculate 2,500 sq. ft. home on 1 level, in desirable north end of Tellico Village. Awesome golf course and lake neighborhood. Some of the fantastic features of this brick & vinyl home include: hardwood floors; tall trey ceilings; double-sided gas fireplace (between great room & hearth room); large formal dining room opens to the great room; split bedroom design; kitchen boasts 42'' oak cabinets with lots of counter & cabinet space; kitchen opens to hearth room; a main level bonus room with it's own private ''gallery'' hallway makes a great artist studio, sewing/craft room or study; covered deck on north side of house; crawl space has dry concrete storage pad; front yard & driveway level, gentle slope in back; common property in back for added privacy; lovely landscaping; all kitchen appliances + washer/dryer convey with the home; too many great features to list here!
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Tommotley Greens-Tellico Village
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Highway 111 & Avenue 68, Mecca, CA 92254Request info Add to list
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V.20 No.23 | 6/9/2011
Music to Your Ears
By Summer Olsson
It's Sexy Squeezebox Time
The accordion is one of my favorite instruments. Its sound can be peppy, polka-y, haunting, mournful and, yes, even sexy. As such, I am predisposed to like this show, and I think it might turn you on, too. At Winning Coffee Co. on Wednesday, June 15, let the spirit of the sexy accordion take you. Two sultry accordion-toting performers from California will join Burque's own Zoltan Orkestar in squeezing out vaudeville, cabaret, traditional French stuff and more.
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Deutsch Collegiate Professor of Comparative Politics and German Studies at the University of Michigan, Ann Arbor Andrei Markovits is the Arthur F. Help us improve Please don't include any personal or financial information, for example your National Insurance or credit card numbers. There are guys who will have me for breakfast when it comes to this stuff, as they were in this business way before I knew what a span was.
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text: http://www.adaction.org/Campaign2004/2004PR_1/2004pr_1.HTM http://www.ada-sepa.org/ Look it up for yourself. Kerry will use spin doctors to make himself look moderate but judging from records like this McGovern could be considered a right wing extremist! The more people learn about Kerry I think the less chance he is going to be elected but in the even he does get elected we can perhaps blame the more right wing purists who think that unless Bush is 100% voting their way then they'd rather have an ultra liberal in office appointing federal and supreme court justices. With this sort of attitude the right will become as marginalized as the Greens.


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Found 21 Microsoft Project tutors in Macon, GA 31201 – page 1 of 3.
Macon 31206 · 3 miles from downtown · $35/hour · teaches Microsoft Access -
I am passionate about helping others succeed. I believe we all have the capacity to be as great as our ambition and am honored to help others reach their potential.certified
Macon 31201 · 0 miles from downtown · $50/hour · teaches Microsoft Excel -
Hello Prospective Students and Parents! My name is Ellen and I just finished my Master of Science in Physics from Georgia State University. I earned my undergraduate degrees...certified
Forsyth 31029 · 25 miles from 31201 · $35/hour · teaches Microsoft Excel -
Greetings! It's the start of beginnings, new experiences, and, most of all, and new knowledge. My name is Brittany E. and I will be your and/or your child's tutor.certified
Warner Robins 31088 · 16 miles from 31201 · $50/hour · teaches Microsoft Access -
Hey there! I'm Jonathan! I absolutely love teaching. Before I joined the military, I was studying to be a Spanish teacher. Teaching Spanish is definitely my passion but moreover...certified
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Warner Robins 31088 · 16 miles from 31201 · $35/hour · teaches Microsoft Access -
I previously worked for Central Georgia Technical College as an adjunct instructor teaching business and computer classes for over 15 years. For ten of those years...certified
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Six of the 12 persons named in the FIR registered in the murder of Northbrook Jute Mill CEO H K Maheshwari at Bhadrehwar in Hooghly have been arrested since last night.
Sources in the police said Md Azharuddin, Mukunda Gupta, Md Aslam, Bikash Tanti, Rabindra Sarkar and Swapan Gupta were produced in court today and other than Tanti, who was sent in jail custody of 14 days, the rest have been awarded a police custody of five days. The sections under which they have been charged are 120 (B), 302, 307, 326 and 147 of the IPC.
Maheswari was lynched by agitated mill workers Sunday when he rejected their demand for increase in weekly working hours so that they were paid more.
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Among relatives of H K Maheshwari who have arrived for the last rites are his daughters Payal Pasaria and Anjali Lakhotia and their husbands. His two brothers, P K Maheshwari and J K Maheshwari, have also reached.
"His (Maheshwari's) son who is in Chicago would reach today at 9.30 pm. The last rites would be performed at Babughat tomorrow morning," said Pradeep Pasaria, one of Maheshwari's sons-in-law.
He also said the family would appeal to the government and administration for speedy justice.
District Trinamool Congress president Tapan Dasgupta, TMC MLA of Champdani Muzaffar Khan and Champdani municipality chairman Suresh Mishra visited Maheshwari's family during the day.
"The CM gave a statement in the assembly that those involved in this incident, irrespective of their political affiliations, would be booked," said Khan and added that the BJP-backed Bharatiya Mazdoor Sangh (BMS) and Centre of Indian Trade Unions (CITU) were behind the killing in order to malign the reputation of the state government that has maintained a harmonious relationship across all industries.
State BJP general secretary Samik Bhattacharya along with a team of BJP workers spoke to mill workers and also met the family members of the deceased.
Responding to the allegations of the state government, he said, "There is no BJP labour wing in the first place. Besides, BMS leader Rajendra Yadav was not in town for the last 10 days or so."
He added: "What the ruling party is doing can only be termed as cheap politics. We want an unbiased and speedy probe into the matter. "
Tapan Dasgupta termed the BJP team's visit a drama. "They have killed Maheshwari and now they come to put up a drama here," he said.
Meanwhile, the company has put up a "supension of work with immediate effect" notice at the mill gate citing labour unrest. A few mill workers present outside were reluctant to talk. Many of them who stay in the vicinity have left their home since last night fearing arrest. "My husband works at the mill. He has left this place," said the wife of a mill worker who lives in a slum behind the mill.


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As an illustration, low male development hormone can tank your sexual desire, while lower levels of progesterone are related to insomnia along with different sleep disruptions. the first time i saw pw was a style council concert at royal albert hall, so yes, i'm that old as well. The former Leicester City defender, 23, only joined the Bees on a non contract basis at the beginning of February.
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Ahmad Rahami in custody after shooting police officer in Linden, NJ, source say - NBC
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Which sentence is a paradox? a. The comedian was seriously funny. b. This is a genuine imitation of that designer watch. c. A small crowd gathered to watch
the concert. d. Nobody goes to that restaurant because it is usually crowded. e. Do you have the original copies we asked for?1


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5 911 jobs
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text:
Martin to tour South Africa with Sussex Academy
JASON MARTIN will spend three weeks in South Africa this winter as part of the Sussex Academy squad that is touring the country.
JASON MARTIN will spend three weeks in South Africa this winter as part of the Sussex Academy squad that is touring the country.
FOUR uncapped players have been named in Guernsey's 13-man squad for Saturday's inter-insular at Grainville.
DECLAN MARTEL hit a crucial 60 that helped Guernsey return to winning ways yesterday and boost their hopes of progressing from the ICC Under-19 World Cup Qualifier in the Netherlands.
GRIFFINS have won the Rozel Shield and become Evening League champions for the first time in the club's history.
WINNING the group and earning a spot in the next stage of World Cup qualifying is the aim for Guernsey's under-19s team in Amsterdam this weekend.
ELIZABETH COLLEGE bowlers at the College Field on Saturday afternoon felt the full force of two of the young men who in the coming years could become the backbone of the island team on the international stage.
TWO rounds of matches into the Odey Premiership and Griffins are already the team to catch.
ON TOP of the world at the start of the day, in danger of leaving it by the end. That was the situation facing Guernsey after their seven-wicket beating by Jersey at Farmers Field yesterday.
GUERNSEY returned to the top of ICC World Cricket League Division Five as they made it three wins from three in Jersey on Tuesday.
MATT STOKES enjoyed a well-earned day off from World Cricket League action along with his island colleagues yesterday. From next week, though, the young all-rounder will be heading to the Midlands for a summer's cricket with County Championship club Leicestershire.
GUERNSEY eased to an eight-wicket win over Tanzania on Sunday to make it a perfect weekend in ICC World Cricket League Division Five in Jersey.
GUERNSEY began their ICC World Cricket League Division Five campaign with a four-wicket victory over Vanuatu in a rain-hit game at FB Fields on Saturday.
FINISHING in the bottom three and being relegated from the World Cricket League would be a 'failure' in the eyes of the Guernsey captain.
GRIFFINS sent out a huge statement of intent in the opening game of the Odey Wealth Evening League Premier season by blowing away Cobo last night.
MAX ELLIS was the star as his superb five-wicket haul spun Guernsey to an emphatic victory on their Sussex League debut in Bognor Regis.
A TRIP to Los Angeles awaits the top two teams from World Cricket League Division Five next month.
GUERNSEY will have an invaluable opportunity to learn from as many as three former international cricketers this weekend as their World Cricket League preparations gather pace in Sussex.
GH SMIT returns to the Guernsey squad that will look for back-to-back World Cricket League promotions in their Division Five campaign in Jersey next month.
GUERNSEY now know what lies ahead of them in May after the ICC yesterday confirmed the fixtures for the World Cricket League Five tournament to be held in Jersey.
SURINAME'S fate after an ICC enquiry into the legitimacy of their success in last September's ICC World Cricket League Division Six should be known soon.
FUNDING for Guernsey cricket has been slashed by the International Cricket Committee and a 'disappointed' GCB chief executive, Mark Latter, has criticised the world body by saying 'it is no way to run a business'.


text:
Sage buys Spanish biz for £55m
Muy bien
Sage is buying Logic Control, a Spanish accounting software vendor, for £55m in cash.
Logic Control has 20,000 customers and takes Sage's Spanish operations into bigger businesses. Logic Control also develops software for accountants.
The company had revenues of £28.2m and an operating profit of £20.6m for the year to 31 December 2004. Net assets at the end of the year were £20.6m. ®
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text:
gimme that ice cream
good op
better to read. no screen adaptation can do justice to nabokov's prose
Pictures that would make good album covers.
maybe but you'd have to fuck with the exposure or whatever to make it more artsy
i always get irrationally annoyed at these threads
then ignore them
/bleep/ general
tuesday blues edition
>tfw no qt3.14 grime gf
>you both will never shitpost together on /mu/ while blasting grime at tinitus level on your cheap ghetto department surrounded by crumbling ikea furniture
new rabit is on m8
You're living on borrowed time
do you think krystal would like krautrock
Have you ever meet a /mu/tant IRL? pic unrelated
No, thankfully.
Yes, person just bluntly asked if I browse /mu/ because I had mentioned Death Grips and Swans before.
Was an experience I hope to never have again.
I have IRL friends who browse /mu/ and I hate it.
Im just wondering, why is Death Grips stuff usually listed under the genre rap? I figured with all of the yelling and guitars and hard drums, they would listed under metal or something of the sort.
>MC Ride
You haven't listened to much of either genre, have you?
Also, a good chunk of Death Grip's material lacks guitars anon.
>listed under rap
well, it is rap
rap music is any music with RAPPING
its not hip hop mostly, i agree, but i depends on the album
Let's talk about slowcore, /mu/.
Favorites/h8s/recs/shares or whatever
Slowcore general.
i'm not sure why this is so well-regarded.
i mean, it's okay but even low's or codeine's inferior albums are more interesting than this. am i missing something? just seems to middle-of-the-road
whats the best codeine record. I love duster but maybe i should codeine a listen
Duster reunion when?
What upcoming 2015 releases are you looking forward to?
So far, this year has been really bland. Last year started off a bit stronger.
>Jenny Death
>I Don't Like Shit
>So Help Me God
>Mr. Wonderful
>Jenny Death
>Earl Sweatshirt
>Snoop Dogg
>A$AP Rocky
>Chance The Rapper
>Casey Veggies
Comment too long. Click here to view the full text.
I dunno man, I was really liking Juggernaut. I'm looking forward to Coma Ecliptic.
Great choices.
>qt looks at iPod
>pray she doesn't find the metal or vidya soundtracks
>implying grills have good enough taste to call you out on it
>Listening to either
ayyy lmao
>tfw a bitch checks your iphone
ITT: Albums that were ahead of their time
Apart from all the nu disco albums that came before it, right?
Or Jamiroquai, who had been doing it for a decade.
Anon, I love this album. but you have to give that honor to Discovery/Homework
ok, somebody else will start then
peace and understanding edition
March 27th m8s
I want peace for the kpg.
I really like Hani despite what I may have put off
She's a really cool idol, , burping in cars and falling asleep and wanting to leave idol life when her contract expires.
Name a better female singer.
Protip: you can't.
Justin bieber
>Name a better female singer.
Neko Case
poor man's kate bush
pardners and chavs unite
fuck the krauts
pardners go die
pardners go die
>i actually call bleeps "uk music" and delude myself into believing the uk invented anything
kek tbh LADS
Post best festival line-ups in your great nation, I wanna see the best thing I'm gonna miss out on this year
I don't think there even is a decent festival in the UK, we're generally quite good for getting musicians over here anyway year round
pic in the OP is a more than decent festival in the UK, wouldn't you say?
That's in the UK? Fair enough but it's not exactly a mindblowing lineup
post the band you're sure you've listened to more than anyone else
i listen to hella discographty three times a day fight me faggot
m-me too


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TAXILA: Ahle-Sunnat Wal Jamaat central leader Aurangzeb Farooqui has been arrested in Taxila by the police, ARY News reported.
The law enforcers did not disclose any reason for the arrest of ASWJ leader.
ASWJ chief Maulana Muhammad Ahmed Ludhanvi said his party strongly condemned the arrest of their leader.
He demanded immediate release of Farooqui.


text:
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Small Business
Store owners in Christchurch's central city Re:Start shopping hub are in limbo with one shop closing and others unsure whether they can stay on next year.
Construction of a $70 million retail and office complex to replace the pop-up mall on Cashel St was supposed to start in January this year.
However, with no construction in sight, Re:Start tenants say they do not know if they can stay in their premises beyond March.
Re:Start chairman John Suckling said the mall had permission to stay until then, but the trustees were "pushing very hard" to get an extension until January 2015.
"We believe that a decision is very close, but negotiations are tricky at present," he said.
The landowners were not sure when they would start building on the site and did not want to commit too far ahead, Suckling said.
City Mall landlord Nick Hunt said he would not comment as no agreement had been reached about the extension.
Hunt owns half a Cashel St block with plans for an office and shopping precinct.
He told The Press earlier this year that he was struggling to secure office tenants for his project because the rents needed to fund construction costs were too high.
Minnie Cooper shoe shop owner Sandy Cooper said she would not renew her lease at the beginning of January.
Cooper did not want to keep shipping stock to Christchurch not knowing how long she would be able to stay. She said the boutique might return later to another location, but no decision had been made yet.
Rosie Suyker and grandmother Valerie Glass were visiting from Methven yesterday. Glass said it was sad to see the shop go.
Hapa design store co-owner Maureen Taane said Re:Start tenants had been left in the dark about the extent of their leases.
"It's incredibly frustrating to not have this certainty."
She said she would love to stay one more year as her shop had been doing really well. It would also give her time to find a permanent location.
Suckling said it was very difficult for retailers to plan ahead as there was no long-term lease on the land. However, most were keen to stay one more year, he said.
Amanda Teear, of Maher shoe shop, said she would stay in the temporary mall until the end. She said the uncertainty was part of the deal and that tenants had to "go with the flow".
Jane McNabb was shopping for shoes at the Re:Start yesterday. She said she would like the shops to stay at least one more year.
"I wouldn't like to go to the mall instead. They need to keep it without stopping construction projects," she said.
Suckling said Re:Start had been built as a temporary facility and would not stay long term.
"The whole purpose of the Re:Start was to provide a transitional bridge between the earthquakes and new premises starting."
The cluster of shipping containers would close down as soon as tenants would be able to move into new buildings.
"Re:Start doesn't want to compete with new retail facilities."
Central City Development Unit director Warwick Isaacs said there "may be provision" to extend Re:Start's lease beyond March.
Developers Lichfield Holdings and Colliers International revealed in 2012 the low-rise retail and office complex would cover 7500 square metres off City Mall.
The complex would have an 80-metre frontage opposite Ballantynes.
- The Press
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The 1957 Class of Gallatin High School met for lunch at Cycles Café in Gallatin and attended the alumni banquet.
Pictured, left ro right, front row, Shirley (Yates) Woody, Belton; Janice (Warden) Baslee, Excelsior Springs; Mary (Eads) Holbrook, Gallatin; Norma (Hughs) Fletcher, Gallatin; Mildred (Bradley) McNeely, Jameson; Carol (Wilkerson) Foster, Chillicothe; Rita (Gatenby) Beard, Boulder City; back row, Connie Walton, Gallatin; R.L. Woody, Belton; Donnie Wathen, Cameron; Darlene (Dunnington) Fuller,
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Missed the live Ask the Expert event?
Not to worry. Check out the archived thread of the Q&A with Ken Hutcheson, President of U.S. Lawns, and the LawnSite community in the Franchising forum .
Got my Walker back from shop.
Discussion in 'Homeowner Assistance Forum' started by tonym, May 1, 2005.