Paired-pulse depression at photoreceptor synapses.

Synaptic depression produced by repetitive stimulation is likely to be particularly important in shaping responses of second-order retinal neurons at the tonically active photoreceptor synapse. We analyzed the time course and mechanisms of synaptic depression at rod and cone synapses using paired-pulse protocols involving two complementary measurements of exocytosis: (1) paired whole-cell recordings of the postsynaptic current (PSC) in second-order retinal neurons and (2) capacitance measurements of vesicular membrane fusion in rods and cones. PSCs in ON bipolar, OFF bipolar, and horizontal cells evoked by stimulation of either rods or cones recovered from paired-pulse depression (PPD) at rates similar to the recovery of exocytotic capacitance changes in rods and cones. Correlation between presynaptic and postsynaptic measures of recovery from PPD suggests that 80-90% of the depression at these synapses is presynaptic in origin. Consistent with a predominantly presynaptic mechanism, inhibiting desensitization of postsynaptic glutamate receptors had little effect on PPD. The depression of exocytotic capacitance changes exceeded depression of the presynaptic calcium current, suggesting that it is primarily caused by a depletion of synaptic vesicles. In support of this idea, limiting Ca2+ influx by using weaker depolarizing stimuli promoted faster recovery from PPD. Although cones exhibit much faster exocytotic kinetics than rods, exocytotic capacitance changes recovered from PPD at similar rates in both cell types. Thus, depression of release is not likely to contribute to differences in the kinetics of transmission from rods and cones

Kinetics of exocytosis is faster in cones than in rods.

Cone-driven responses of second-order retinal neurons are considerably faster than rod-driven responses. We examined whether differences in the kinetics of synaptic transmitter release from rods and cones may contribute to differences in postsynaptic response kinetics. Exocytosis from rods and cones was triggered by membrane depolarization and monitored in two ways: (1) by measuring EPSCs evoked in second-order neurons by depolarizing steps applied to presynaptic rods or cones during simultaneous paired whole-cell recordings or (2) by direct measurements of exocytotic increases in membrane capacitance. The kinetics of release was assessed by varying the length of the depolarizing test step. Both measures of release revealed two kinetic components to the increase in exocytosis as a function of the duration of a step depolarization. In addition to slow sustained components in both cell types, the initial fast component of exocytosis had a time constant ofcones, \u3e10-fold faster than that of rods. Rod/cone differences in the kinetics of release were substantiated by a linear correlation between depolarization-evoked capacitance increases and EPSC charge transfer. Experiments on isolated rods indicate that the slower kinetics of exocytosis from rods was not a result of rod-rod coupling. The initial rapid release of vesicles from cones can shape the postsynaptic response and may contribute to the faster responses of cone-driven cells observed at light offset

Hyperpolarisation-activated current in glomerular cells of the rat olfactory bulb

Whole-cell patch-clamp recordings were carried out in visually identified periglomerular and external tufted cells of rat olfactory bulb. Most of the neurones showed a slowly developing hyperpolarisation-activated current with a threshold generally positive to resting potential and with a strongly voltage-dependent activation time constant. The current, identified as Ih, was sodium- and potassium-sensitive, suppressed by external caesium, and insensitive to barium. Under current-clamp conditions, perfusion with caesium induced a 10 mV hyperpolarisation and a marked reduction of the rate of low-frequency oscillations induced experimentally. It is concluded that most of the cells in the rat glomerular layer present a distinct h-current, which is tonically active at rest and which may contribute to the oscillatory behaviour of the bulbar network

Shaping of signal transmission at the photoreceptor synapse by EAAT2 glutamate transporters

Photoreceptor ribbon synapses tonically release glutamate. To ensure efficient signal transmission and prevent glutamate toxicity, a highly efficient glutamate removal system provided by members of the SLC1 gene family is required. By using a combination of biophysical and in vivo studies, we elucidate the role of excitatory amino acid transporter 2 (EAAT2) proteins in synaptic glutamate homeostasis at the zebrafish photoreceptor synapse. The main glutamate sink is provided by the glial EAAT2a, reflected by reduced electroretinographic responses in EAAT2a-depleted larvae. EAAT2b is located on the tips of cone pedicles and contributes little to glutamate reuptake. However, this transporter displays both a large chloride conductance and leak current, being important in stabilizing the cone resting potential. This work demonstrates not only how proteins originating from the same gene family can complement each other's expression profiles and biophysical properties, but also how presynaptic and glial transporters are coordinated to ensure efficient synaptic transmission at glutamatergic synapses of the central nervous system

Taurine provides neuroprotection against retinal ganglion cell degeneration.

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases

Taurine prevents the RGC death in NMDA-treated retinal explants.

<p>A) Digitalized reconstruction of a whole flat-mounted retinal explant immunolabeled with the POU4F1 (Brn3a) antibody. B–E) Representative enlarged fields from flat-mounted retinal explants acquired with the automated platform, showing POU4F1-immunopositive RGCs in a control untreated condition (Control; B) after 100 µM NMDA application (NMDA; C), after co-application of NMDA with taurine (1 mM; NMDA+Taurine; D) or after co-application of NMDA with MK801 (100 µM; NMDA+MK801; E) for 4 days. F) Quantification of RGC densities from whole flat-mounted retinal explants using the automated counting platform in Control group (n = 33, white bar); NMDA group: (n = 31, hatched bar); NMDA+Taurine group (n = 23, black bar) and Taurine group (n = 6, grey bar). G) Quantification of POU4F1-immunopositive RGC densities from whole flat-mounted explants under the control group (white bar), or the NMDA group (hatched bar), the NMDA plus MK 801 group (black bar), or finally the MK-801 group (grey bar) (n = 6 for each group). Data are expressed as means ± s.e.m. from n independent experiments. ***p<0.001, *p<0.05; One-way ANOVA followed by a Bonferroni post-hoc test. The scale bar represents 100 µm in panels (B–E).</p

Taurine stimulates the survival of pure adult retinal ganglion cells (RGCs) in culture.

<p>A–F) Purity of RGC cultures. Confocal representative images of pure RGC cultures immunolabeled with specific RGC markers, NF-200 (red in A) or βIII-Tubulin (red in B), as well as with a specific marker for microglia/macrophages, Cd11b/c (red in C), while all isolated cells were stained by the nuclear dye DAPI (blue in D–F). Note that most cells were immunolabeled with the RGC markers (NF-200 or βIII-Tubulin) (A,B,D,E) whereas a very few cells were positive for the macrophage marker (C,F). G–H) Effect of taurine on pure RGCs. Representative images showing viable cultured RGC labeled with calceinAM, after 6 days <i>in vitro</i> (6 DIV), in the negative control condition (Cont; G) and following 1 mM taurine application (Taur; H). I) Quantification of RGC densities after 6 DIV either in the control condition (Cont; white bar), with 1 mM taurine application (Taur; black bar), or with the B27 supplement, providing a positive control condition (Pos; grey bar). In each experiment, the respective RGC densities were expressed as a percentage of the negative control condition at 6 DIV. Illustrated data are means ± s.e.m. from 21 independent experiments. ***p<0.001, one-way ANOVA followed by a Dunns post-hoc test. Scale bars represent 100 µm in panels (A–H).</p

Taurine supplementation prevents RGC degeneration in glaucomatous Long-Evans rats following episcleral vein occlusion.

<p>A) Taurine plasmatic levels in Long-Evans rats drinking taurine-free water (Wat; green bar; mean ± s.e.m., n = 21) or taurine-supplemented water for 3 months (Taur; red bar; mean ± s.e.m., n = 24; **p<0.01, student’s t-test). B) Intraocular pressure (IOP) levels measured at regular time intervals after episcleral vein occlusions by cauterization on the operated right eyes (Caut) and unoperated left eyes (Cont) in rats drinking taurine-free water (Wat, green bars) or in taurine-treated rats (Taur; red bars). The difference between the IOP remained statically significant between the operated and unoperated eyes in both animal groups throughout the study (mean ± s.e.m., n>14 for all groups; ***p<0.001 as compared to water/control eyes, one-way ANOVA followed by a Bonferroni post-hoc test). C) Representative photopic electroretinograms (ERG) recorded from an operated right eye (Caut eye) and an unoperated left control eye (Cont eye) after 3 months of episcleral vein occlusion in taurine-treated rats (Taur, red traces) and in control rats drinking taurine-free water (Wat; green traces). D) Quantification of photopic ERG amplitudes measured from the left unoperated eyes (Cont eyes) and right operated eyes (Caut eyes) in taurine-supplemented rats (Taur; red bars; mean ± s.e.m., n = 24) or control rats drinking taurine-free water (Wat; green bars; mean ± s.e.m., n = 32 for control eyes and 40 for cauterized eyes) after 3 months of vein occlusion. E) Complete rat retinal section viewed with a digital fluorescence scanner (Nanozoomer) showing POU4F1 (Brn3a) immunolabeling (red) and nuclear staining with DAPI (blue). F-I) Representative confocal images of retinal cryo-sections showing the POU4F1 (Brn3a) positive RGC immunolabeling (red) and cell nuclei staining (DAPI; blue) performed in unoperated left eyes (Cont; F,H) and cauterized right eyes (Caut; G,I) in rats without (Wat; F, G) or with taurine supplementation (Taur; H, I) added to their drinking water. J) Quantification of POU4F1 (Brn3a) positive RCG densities in both unoperated left (Cont eye) and operated right (Caut eye) eyes from rats without (Wat; green bars; mean ± s.e.m., n = 11 and 10 for control and cauterized eyes, respectively) or with taurine supplementation (Taur; red bars; mean ± s.e.m., n = 11 and 9 for control and cauterized eyes, respectively). *p<0.05 and ***p<0.001 as compared to indicated group in (B, D, J), one-way ANOVA followed by a Bonferroni post-hoc test. Scale bars represent 4 mm in panel (E) and 50 µm in panels (F-I).</p

Taurine prevents RGC degeneration in P23H rats with blood vessel atrophy.

<p>A, B) Representative pictures showing the lectin staining of blood vessels at the periphery of flat-mounted retinae from a one-year old Sprague-Dawley rat (A) and a one-year old heterozygous P23H rat (B). C,D) Quantification of number of blood vessel branching points (C) and percentage of blood vessel coverage (D) obtained on the whole retina by an automated platform in Sprague-Dawley wild-type animals (WT; white bar), in untreated heterozygous P23H rats (P23H Wat; grey bar) and in taurine-supplemented P23H rats (P23H Taur; black bar). These quantifications demonstrated the absence of taurine effect on blood vessel atrophy (means ± s.e.m., n = 5 for each group). E) Quantification of POU4F1 (Brn3a) immunopositive RGCs in retinal cryosections from Sprague-Dawley wild-type animals (WT, white bar), from untreated heterozygous P23H rats (P23H Wat; grey bar) and from taurine supplemented P23H rats (P23H Taur; black bar). Data expressed as RGC per mm of retinal section, are means ± s.e.m. from n = 7 animals for each group. ***p<0.001 and *p<0.05 as compared to indicated groups, one-way ANOVA followed by a Bonferroni post-hoc test. Scale bar represents 100 µm in panels (A–B).</p