Calcium regulates vesicle replenishment at the cone ribbon synapse.

Cones release glutamate-filled vesicles continuously in darkness, and changing illumination modulates this release. Because sustained release in darkness is governed by vesicle replenishment rates, we analyzed how cone membrane potential regulates replenishment. Synaptic release from cones was measured by recording postsynaptic currents in Ambystoma tigrinum horizontal or OFF bipolar cells evoked by depolarization of simultaneously voltage-clamped cones. We measured replenishment after attaining a steady state between vesicle release and replenishment using trains of test pulses. Increasing Ca(2+) currents (I(Ca)) by changing the test step from -30 to -10 mV increased replenishment. Lengthening -30 mV test pulses to match the Ca(2+) influx during 25 ms test pulses to -10 mV produced similar replenishment rates. Reducing Ca(2+) driving force by using test steps to +30 mV slowed replenishment. Using UV flashes to reverse inhibition of I(Ca) by nifedipine accelerated replenishment. Increasing [Ca(2+)](i) by flash photolysis of caged Ca(2+) also accelerated replenishment. Replenishment, but not the initial burst of release, was enhanced by using an intracellular Ca(2+) buffer of 0.5 mm EGTA rather than 5 mm EGTA, and diminished by 1 mm BAPTA. This suggests that although release and replenishment exhibited similar Ca(2+) dependencies, release sites areCa(2+) channels but replenishment sites are \u3e200 nm away. Membrane potential thus regulates replenishment by controlling Ca(2+) influx, principally by effects on replenishment mechanisms but also by altering releasable pool size. This in turn provides a mechanism for converting changes in light intensity into changes in sustained release at the cone ribbon synapse

Feedback from horizontal cells to rod photoreceptors in vertebrate retina.

Retinal horizontal cells (HCs) provide negative feedback to cones, but, largely because annular illumination fails to evoke a depolarizing response in rods, it is widely believed that there is no feedback from HCs to rods. However, feedback from HCs to cones involves small changes in the calcium current (I(Ca)) that do not always generate detectable depolarizing responses. We therefore recorded I(Ca) directly from rods to test whether they were modulated by feedback from HCs. To circumvent problems presented by overlapping receptive fields of HCs and rods, we manipulated the membrane potential of voltage-clamped HCs while simultaneously recording from rods in a salamander retinal slice preparation. Like HC feedback in cones, hyperpolarizing HCs from -14 to -54, -84, and -104 mV increased the amplitude of I(Ca) recorded from synaptically connected rods and caused hyperpolarizing shifts in I(Ca) voltage dependence. These effects were blocked by supplementing the bicarbonate-buffered saline solution with HEPES. In rods lacking light-responsive outer segments, hyperpolarizing neighboring HCs with light caused a negative activation shift and increased the amplitude of I(Ca). These changes in I(Ca) were blocked by HEPES and by inhibiting HC light responses with a glutamate antagonist, indicating that they were caused by HC feedback. These results show that rods, like cones, receive negative feedback from HCs that regulates the amplitude and voltage dependence of I(Ca). HC-to-rod feedback counters light-evoked decreases in synaptic output and thus shapes the transmission of rod responses to downstream visual neurons

A Synaptotagmin Isoform Switch during the Development of an Identified CNS Synapse

Various Synaptotagmin (Syt) isoform genes are found in mammals, but it is unknown whether Syts can function redundantly in a given nerve terminal, or whether isoforms can be switched during the development of a nerve terminal. Here, we investigated the possibility of a developmental Syt isoform switch using the calyx of Held as a model synapse. At mature calyx synapses, fast Ca2+-driven transmitter release depended entirely on Syt2, but the release phenotype of Syt2 knockout (KO) mice was weaker at immature calyces, and absent at pre-calyceal synapses early postnatally. Instead, conditional genetic inactivation shows that Syt1 mediates fast release at pre-calyceal synapses, as well as a fast release component resistant to Syt2 deletion in immature calyces. This demonstrates a developmental Syt1-Syt2 isoform switch at an identified synapse, a mechanism that could fine-tune the speed, reliability, and plasticity of transmitter release at fast releasing CNS synapses

Robo3-Driven Axon Midline Crossing Conditions Functional Maturation of a Large Commissural Synapse

SummaryDuring the formation of neuronal circuits, axon pathfinding decisions specify the location of synapses on the correct brain side and in correct target areas. We investigated a possible link between axon midline crossing and the subsequent development of output synapses formed by these axons. Conditional knockout of Robo3 in the auditory system forced a large commissural synapse, the calyx of Held, to be exclusively formed on the wrong, ipsilateral side. Ipsilateral calyx of Held synapses showed strong transmission defects, with reduced and desynchronized transmitter release, fewer fast-releasable vesicles, and smaller and more variable presynaptic Ca2+ currents. Transmission defects were not observed in a downstream inhibitory synapse, and some defects persisted into adulthood. These results suggest that axon midline crossing conditions functional maturation of commissural synapses, thereby minimizing the impact of mislocalized synapses on information processing. This mechanism might be relevant to human disease caused by mutations in the ROBO3 gene

An Alien Divalent Ion Reveals a Major Role for Ca2+ Buffering in Controlling Slow Transmitter Release

Ca2+-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca2+ buffers and Ca2+ sensors in shaping release kinetics are still controversial. Replacing extracellular Ca2+ by Sr2+ causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr2+ uncaging and made quantitative Sr2+ - and Ca2+ -imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca2+ sensors and Ca2+ buffers in the control of fast and slow release. We show that Sr2+ activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr2+ also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr2+ was less efficient than Ca2+ in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr2+ than for Ca2+ (similar to 5-fold). This, together with an enhanced Sr2+ permeation through presynaptic Ca2+ channels (similar to 2-fold), admits a drastically higher spatially averaged Sr2+ transient compared with Ca2+. Together, despite the lower affinity of Sr2+ at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr2+ transients explain the enhanced late release. This also allows us to conclude that Ca2+ buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca2+

Antiaritmiás és proaritmiás mechanizmusok elemzése = Analysis of antiarrhythmic and proarrhythmic mechanism

Szívizomsejtben a K+-áramok antiaritmiás ''repolarizációs tartalékot'' tartanak fenn: Az IKr, IKs és IK1 akár egyikének gátlása súlyos proaritmiás repolarizáció megnyúlást okoz és letális kamrafibrillációhoz vezethet. - Alloxán-diabeteses kutyán az Ito és IKs csökkenése ugyanilyen hatású, amit a Kv4.3 és Mink csatorna fehérje expresszió egyidejű gátoltsága kisér. - Szívbillyentyű átültetés céljából használt, egészséges donor szivekből izolált humán kamrai miocitákon az IKs csökkenése szintén növeli a proaritmia és a hirtelen szívhalál rizikóját. - A K+-csatorna alegység expresszió mértékének sorrendje: KvLQT1 és ERG1-nél: ember>nyúl>tengerimalad, Mink-nél: tengerimalac>ember>nyúl. - Az INa+/Ca2+ csereáram gátlása kutyán antiaritmiás hatású, csökkenti az utódepolarizációt és a triggerelt aktivitást. - A leghatékonyabb új antiaritmikumok ''többszörös (tedisamil) ill. ''hibrid'' (azimilid, terikalant) ioncsatorna-gátlók. A pitvarszelektiv antiaritmiás szerek hatásmódja: az IKAch, IKur és IK1 izolált blokkolása. - A prekondicionálás antiaritmiás hatásában kutyán a NO trigger és mediátor, prosztaciklin- és szabadgyök-képződés viszont nem vesz részt ebben. | In the myocardial cell, there is a 'repolarization reserve' maintained by K+ currents: inhibition any of the IKr, IKs and IK1 results in severe proarrhythmic prolongation of repolarization and may cause lethal ventricular fibrillation. - The decrease of IKs and also Ito exhibits the same effect in alloxan-diabetic dogs which is accompanied by concomitant retardation of the expression of the K+ channel proteins Kv4.3 and Mink. - In undiseased human ventricular myocites obtained from donors for valve transplant surgery diminution of IKs also increases the risk of proarrhythmia and suddenc cardiac death. The extent of the order of expression of K+ channel subunits: human>rabbit>guinea pig with KvLQT1 and ERG1, and guinea pig>human>rabbit with Mink. - Inhibition of the INa+/Ca2+ exchange current is antiarrhythmic in dogs; it decreases the afterdepolarization and triggered activity. - The most efficacious new antiarrhythmics are ''multiple'' (tedisamil) or 'hybrid' (azimilid, terikalant) ion-channel blockers. - The mode of action of atrial-selective antiarrhythmics: isolated block of IKAch or IKur or IKs. - In the antiarrhythmic action of preconditioning in dogs nitrogen oxide acts as trigger and mediator, whereas formation of prostacyclin and free radicals are not involved in this effect

Nucleic acid and non-nucleic acid-based reprogramming of adult limbal progenitors to pluripotency

Reprogramming somatic cells to a pluripotent state by nucleic acid based (NAB) approaches, involving the ectopic expression of transcription factors, has emerged as a standard method. We recently demonstrated that limbal progenitors that regenerate cornea are reprogrammable to pluripotency by a non-NAB approach through simple manipulation of microenvironment thus extending the possible therapeutic use of these readily accessible cells beyond the proven treatment of corneal diseases and injury. Therefore, to determine the validity and robustness of non-cell autonomous reprogramming of limbal progenitors for a wider clinical use, here, we have compared their reprogramming by non-NAB and NAB approaches. We observed that both approaches led to (1) the emergence of colonies displaying pluripotency markers, accompanied by a temporal reciprocal changes in limbal-specific and pluripotency gene expression, and (2) epigenetic alterations of Oct4 and Nanog, associated with the de-novo activation of their expression. While the efficiency of reprogramming and passaging of re-programmed cells were significantly better with the NAB approach, the non-NAB approach, in contrast, led to a regulated reprogramming of gene expression, and a significant decrease in the expression of Hormad1, a gene associated with immunogenic responses. The reprogramming efficiency by non-NAB approach was influenced by exosomes present in conditioned medium. Cells reprogrammed by both approaches were capable of differentiating along the three germ lineages and generating chimeras. The analysis suggests that both approaches are effective in reprogramming limbal progenitors but the non-NAB approach may be more suitable for potential clinical applications by averting the risk of insertional mutagenesis and immune responses associated with the NAB approach

An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses

Novel Neuroprotective Strategies in Ischemic Retinal Lesions

Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i) urocortin 2; (ii) a mitochondrial ATP-sensitive K+ channel opener, diazoxide; (iii) a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv) a novel poly(ADP-ribose) polymerase inhibitor (HO3089). The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques