The instructive role of binocular vision in the Xenopus tectum

This review presents the fascinating neurobiology underlying the development of the frog optic tectum, the brain structure where the two separate inputs from the two eye are combined into a single, integrated map. In the species Xenopus laevis, binocular visual information has a dramatic impact on axon growth and connectivity, and the formation of binocular connections in this system provides a rich basis for both theoretical and experimental investigations.     

Xenopus exhibits seasonal variation in retinotectal latency but not tecto-isthmo-tectal latency

1. The tectum of Xenopus receives visuotopic input from both eyes. The contralateral eye's projection reaches the tectum directly, via the optic nerve. The ipsilateral eye's projection reaches the tectum indirectly, via the nucleus isthmi and isthmo-tectal projection. 2. Because of the multi-synaptic nature of the ipsilateral pathway, there is an inherent delay between the time that information from the contralateral eye reaches the tectum and the time that information from the ipsilateral eye arrives at the tectum. The length of the intertectal delay is a function of the latencies of the contralateral and ipsilateral pathways. 3. The length of this intertectal delay has functional, as well as developmental, implications with regard to the role of N-methyl-D-aspartate receptors in tectal cell activity and development of orderly synaptic connections. 4. We have found that the latencies of the contralateral and ipsilateral pathways exhibit a seasonal variation, increasing during the winter months. The increases of both latencies during the winter were of similar magnitude, indicating that there were no significant changes in intertectal delay. The seasonal alteration in contralateral latency was not affected by dark-rearing and was affected to only a minor extent by a week-long alteration of ambient temperature.     

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K_v1.5, Na_v1.5, Hcn4, Ca_v3.2, Ca_v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca²⁺ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca²⁺ transients were recapitulated by a simulation model. Although a Ca²⁺ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca²⁺ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.     

Binocular maps in Xenopus tectum: Visual experience and the development of isthmotectal topography

Xenopus frogs have a prominent binocular field that develops as a consequence of the migration of the eyes during the remodeling of the head during and after metamorphosis. In the optic tectum, a topographic representation of the ipsilateral eye develops during this same period. It is relayed indirectly, via the nucleus isthmi. In the early stages of binocular development, the topographic matching of the ipsilateral input to the retinotectal input from the contralateral eye is largely governed by chemical cues, but the ultimate determinant of the ipsilateral map is binocular visual input. Visual input is such a dominant factor that abnormal visual input resulting from unilateral eye rotation can induce isthmotectal axons to alter their trajectories dramatically, even shifting their terminal zones from one pole of the tectum to the other. This plasticity normally is high only during a 3-4-month critical period of late tadpole-early juvenile life, but the critical period can be extended indefinitely by dark-rearing. N-methyl-D-aspartate (NMDA) receptors are involved in this process; plasticity can be blocked or promoted by chronic treatment with NMDA antagonists or agonists, respectively. Cholinergic nicotinic receptors on retinotectal axons are likely to play an essential role as well. Modifications in the polysialylation of neural cell adhesion molecule are correlated with the state of plasticity. The circuitry underlying binocular plasticity is not yet fully understood but has proved not to be a simple convergence of ipsilateral and contralateral inputs onto the same targets.     

Acceleration by NMDA treatment of visually induced map reorganization in juvenile Xenopus after larval eye rotation

Each tectal lobe of Xenopus forgs receives two topographic maps, one via the ipsilateral eye and one via the contralateral eye. The alignment of the ipsilateral map with the contralateral map depends upon bincoular visual input during a critical period that extends from late tadpole to early juvenile stages. Rotation of one eye during the critical period leads to reorganization of the ipsilateral map, which eventually comes back into alignment with the contralateral map despite the abnormal eye position. The ipsilateral eye's map initially develops as if there had been no alteration in eye position; there is a delay of 4–6 weeks before reorganization can be detected by electrophysiological mapping. In this paper, the possible role of the NMDA receptor in the delay in reorganization is addressed. The degree of NMDA receptor activation may need to be above some threshold level to trigger reorganization. If NMDA receptor activation normally is below that level until after the first month postmetamorphosis, then exogenous NMDA might boost the process sufficiently to start the reorganization process sooner than usual. In order to test this possibility, the left eye of tadpoles was rotated and NMDA was applied to the right tectal lobe for 3–5 weeks, starting at 1 week postmetamorphosis. Electrophysiological mapping demonstrated that reorganization takes place more rapidly than in untreated forgs or frogs treated with vehicle only. This result is consistent with the interpretation that the activation of the NMDA receptor is a rate-limiting step in the activity-dependent matching of binocular maps in Xenopus tectum. 1994 John Wiley & Sons, Inc.     

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations—E344K, ΔK814, Δ2864-2865GC, Q998E, and T1046M—were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 μM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.     

Connections between the nucleus isthmi and the tectum in larval and post-metamorphic axolotls

The nucleus isthmi (NI) is the primary relay for the frog's ipsilateral visuotectal projection. Using electrophysiological methods, ipsilateral visuotectal activity has been recorded in thyroxine-treated, postmetamorphic axolotls but not in larval axolotls. In order to determine whether changes in isthmotectal projections are responsible for this change in electrophysiological responsiveness, we have investigated the connections between the tectum and the NI using horseradish peroxidase. Our results indicate that the axolotl's isthmotectal pathways are strikingly similar to those of the frog NI, and that the NI sends bilateral projections to the tecta in both larval and thyroxine-treated, postmetamorphic axolotls. Thus, the anatomical connections underlying the ipsilateral visuotectal projection are present during larval stages, despite the lack of electrophysiological evidence for the larval ipsilateral visuotectal projection. We hypothesize that thyroxine-induced metamorphosis produces changes in the terminal arborizations of the crossed isthmotectal projection which allow them to be detected by presynaptic electrophysiological techniques.     

Application of modified supercritical carbon dioxide extraction to microbial quinone analysis

Supercritical carbon dioxide (scCO₂) was applied to extract microbial quinones from activated sludge. Identification and analysis was then performed using high-performance liquid chromatography (HPLC) equipped with ultraviolet–visible (UV–Vis) detector and photodiode array detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scCO₂ were similar to that of the conventional method (organic solvent extraction).     

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.