The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon’s contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.</jats:p

The Political De-Determination of Legal Rules and the Contested Meaning of the ‘No Bailout’ Clause

Traditional debates on legal theory have devoted a great deal of attention to the question of the determinacy of legal rules. With the aid of social sciences and linguistics, this article suggests a way out of the ‘determinate-indeterminate’ dichotomy that has dominated the academic debate on the topic so far. Instead, a dynamic approach is proposed, in which rules are deemed to undergo processes of political ‘de-determination’ and ‘re-determination’. To illustrate this, the article uses the example of Art. 125 of the Treaty on the Functioning of the European Union, the ‘no bailout’ provision, which played a major role in the management of the Euro-crisis. As will be shown, with the start of the crisis, this provision, whose meaning was once scarcely controversial, became the object of intense interpretative disagreement. As it became politically relevant, the rule also became the site of interpretative competitions, until the intervention of the European Court of Justice disambiguated and redefined its meaning

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin (Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/µg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates eight Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation

Unorthodoxy in legislation: The Hungarian experience

This paper deals with legal unorthodoxy. The main idea is to study the so-called unorthodox taxes Hungary has adopted in recent years. The study of unorthodox taxes will be preceded by a more general discussion of how law is made under unorthodoxy, and what are the special features of unorthodox legal policy. Unorthodoxy challenges equality before the law and is critical towards mass democracies. It also raises doubts on the operability of the rule of law, relying on personal skills, or loyalty, rather than on impersonal mechanisms arising from checks and balances as developed by the division of political power. Besides, for lack of legal suppositions, legislation suffers from casuistry and regulatory capture

Dynamic assembly of ribbon synapses and circuit maintenance in a vertebrate sensory system

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119- dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119- dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse

Alterations of Photoreceptor Synaptic Ribbons in the Retina of a Human Patient With Oculocutaneous Albinism Type 1 (OCA1)

Purpose: Albino (Tyrc-2J/Tyrc-2J) C57BL/6J mice carry a mutation in the tyrosinase gene and are known to display alterations of photoreceptor synaptic ribbons. In the present study, we wanted to test whether similar alterations exist in oculocutaneous albinism type 1 (OCA1), a human disease that also results from mutations in the tyrosinase gene.Methods: In the present study, we assessed the morphology of a human OCA1 retina in comparison to control human retinas. We analyzed the retina of a 35-year-old OCA1 patient by immunolabeling at light and electron microscopic levels, conventional transmission electron microscopy, and by genomic DNA sequencing of the RIBEYE/CtBP2 gene in comparison to normal human controls.Results: The morphological analyses revealed an overall surprisingly normal appearance of the retina, except for the presence of strikingly abnormal photoreceptor synaptic ribbons. Synaptic ribbons are presynaptic specializations of the continuously active retinal ribbon synapses and mainly consist of the RIBEYE protein. In the OCA1 patient, photoreceptor synaptic ribbons were very small and reduced to small fragments that were either still associated with the active zone transmitter release site or floating in the cytosol. The RIBEYE gene appeared to be unaltered in the OCA1 patient, except for some single nucleotide polymorphisms (SNPs) that were also present in controls.Conclusions: The OCA1 patients displayed similar defects of photoreceptor synaptic ribbons as previously observed in the albinotic mice with a defect in the tyrosinase gene. The observed alterations of synaptic ribbons are not due to mutations in the RIBEYE gene but are likely indirect consequences of the deficient melanin biosynthesis in the OCA1 patient