Peculiarities of synaptic transmission between photoreceptors and horizontal cells

A previously advanced hypothesis, according to which the transmitter which depolarizes the membrane of horizontal cells is continually liberated in the dark, and ceases to be liberated in the light, is tested experimentally. The data presented show that a current acting on presynatic receptor endings evokes a depolarizing response in horizontal cells to short current impulses passing through the retina (anode on receptor surface, cathode in vitreous body). These receptor endings are depolarized, which evidently leads to liberation of the transmitter from the receptors. Experiments with electrical stimulation of the retina have shown that treatment of the retina with potassium cyanide disrupts synaptic transmission between the receptor and horizontal cell. A potential equal to their membrane potential is established in horizontal cells in bright light; this potential is evidently the true rest potential of these cells. The relative stability of the membrane potential of horizontal cells in light with change in temperature is evidence in support of this assumption. In the dark, the membrane potential increases considerably with increase in temperature; this effect is possibly due to a rise in the rate of decomposition of the depolarizing transmitter. Evidence in support of this hypothesis is the rise in steepness of the falling phase of the response of the horizontal cells to electrical stimulation observed on elevation of the temperature.Institute for Problems of Information Transmission of the Academy of Sciences of the USSR, and Institute of Higher Nervous Activity and Neurophysiology of the Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 79–86, January–February, 1970.     

Modulation of synaptic transfer between retinal cones and horizontal cells by spatial contrast

We studied the influence of steady annular light on the kinetics and sensitivity of horizontal cell (HC) responses to modulation of the intensity of small concentric spots in the turtle retina. As shown by previous investigators, when the intensity of the annulus was equal to the mean spot intensity, spot response kinetics were the same as those for the modulation of spatially uniform light. Turning off the annulus attenuated dramatically high-frequency flicker sensitivity and enhanced somewhat low-frequency sensitivity. This phenomenon reflects a modulation of synaptic transfer between cones and second-order neurons that is mediated by cones, and it will be referred to as cone-mediated surround enhancement (CMSE). Our main results are as follows: (a) The change in test-spot response sensitivity and kinetics upon dimming a steady surrounding annulus is a consequence of the change in spatial contrast rather than change in overall light level. (b) Introduction of moderate contrast between the mean spot intensity and steady surrounding light intensity causes a marked change in spot response kinetics. (c) The dependence of spot response kinetics on surrounding light can be described by a phenomenological model in which the steady state gain and the time constant of one or two single-stage, low-pass filters increase with decreasing annular light intensity (d) The effect of surrounding light on spot responses of a given HC is not determined by change in the steady component of the membrane potential of that cell. (e) Light outside the receptive field of an HC can affect that cell's spot response kinetics. (f) In an expanding annulus experiment, the distance over which steady annular light affects spot response kinetics varies among HCs and can be quite different even between two cells with closely matched receptive field sizes. (g) The degree of CMSE is correlated with HC receptive field size. This correlation suggests that part of the enhancement mechanism is located in the HC. Taken together, our results suggest the involvement of the inner retina in CMSE.     

突触后钙通路有助于视锥与L型水平细胞间的突触可塑性

我们实验室以前发现,视网膜视锥与亮度型水平细胞（luminosity—type horizontal cell,LHC）之间的突触传递效率具有可塑性。重复性刺激红敏视锥增加了LHC对红光的超极化反应幅度,而且这种增强作用是可逆的。在本文中,我们运用细胞内记录技术和药理学分析的方法来考察重复性红光刺激引起的反应增强的可能机制。当通过胞内注射Ca^2＋的螯合剂EGTA来降低LHC内的Ca^2＋浓度后,重复性红光引起的反应增强被抑制,提示突触后钙信号是反应增强的一个重要因素。另外,反应增强现象还可以被钙离子通透的AMPA受体（Ca^2＋-permeable AMPA receptor,CP-AMPAR）的拈抗剂阻断,说明通过钙离子通透的谷氨酸受体内流的Ca^2＋与胞内Ca^2＋浓度的改变有关。进一步发现,胞外灌流ryanodine或caffeine也可以消除反应增强现象,说明由钙诱导的钙释放（calcium—induced calcium release,CICR）引起的钙信号可能也参与了反应增强现象的产生。结果提示,CICR和CP—AMPAR与重复性红光刺激引起的LHC对红光的反应增强有关。     

A positive feedback synapse from retinal horizontal cells to cone photoreceptors

Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca(2+) and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca(2+), whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement.     

Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells

It has recently been reported that relatively short‐term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF‐induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow‐up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition‐related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 µg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild‐type mice given periocular injections of 5 µg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases. J. Cell. Physiol. 224:262–272, 2010 © 2010 Wiley‐Liss, Inc.     

Model feedback mechanism between horizontal cells and photoreceptors of the vertebrate retina

A model with nonlinearity of the photoreceptor presynaptic membrane as its important distinguishing feature was created on the basis of the hypothesis that feedback between the horizontal cells and photoreceptors is effected by a current generated by the subsynaptic membrane of the horizontal cells and leaking partly into the photoreceptors. Measurements with the model also reproduced experimental observations such as depolarization of the cone during hyperpolarization of the horizontal cell in response to the showing of a ring of light or passage of an electric current, and also certain special features of the current-voltage curves of the cones.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 86–94, January–February, 1977.     

Cadherin is required for dendritic morphogenesis and synaptic terminal organization of retinal horizontal cells

Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina.     

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca²⁺) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.     

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20?%) as the number of retinal ganglion cells (19?%). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.     

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.     

Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.     

Action of serotonin and norepinephrine on spinal motoneurones following blockade of synaptic transmission

The actions of serotonin and norepinephrine were investigated on spinal motoneurones in isolated, hemisected rat and frog spinal cords. Serotonin and norepinephrine induced slowly developing depolarizations of spinal motoneurones which were frequently preceded by brief, low amplitude hyperpolarizations. Neither the depolarizations nor the hyperpolarizations were attenuated by 20 mM Mg2+ or tetrodotoxin, although synaptic transmission was blocked in both cases. It thus appears unlikely that the action of serotonin and norepinephrine on spinal motoneurone polarization and results from an indirect action via interneurones.     

Carboxypeptidase E is required for normal synaptic transmission from photoreceptors to the inner retina

Defects in the gene encoding carboxypeptidase E (CPE) in either mouse or human lead to multiple endocrine disorders, including obesity and diabetes. Recent studies on Cpe-/- mice indicated neurological deficits in these animals. As a model system to study the potential role of CPE in neurophysiology, we carried out electroretinography (ERG) and retinal morphological studies on Cpe-/- and Cpe fat/fat mutant mice. Normal retinal morphology was observed by light microscopy in both Cpe-/- and Cpe(fat/fat) mice. However, with increasing age, abnormal retinal function was revealed by ERG. Both Cpe-/- and Cpe fat/fat animals had progressively reduced ERG response sensitivity, decreased b-wave amplitude and delayed implicit time with age, while maintaining a normal a-wave amplitude. Immunohistochemical staining showed specific localization of CPE in photoreceptor synaptic terminals in wild-type (WT) mice, but in both Cpe-/- and Cpe fat/fat mice, CPE was absent in this layer. Bipolar cell morphology and distribution were normal in these mutant mice. Electron microscopy of retinas from Cpe fat/fat mice revealed significantly reduced spherule size, but normal synaptic ribbons and synaptic vesicle density, implicating a reduction in total number of vesicles per synapse in the photoreceptors of these animals. These results suggest that CPE is required for normal-sized photoreceptor synaptic terminal and normal signal transmission to the inner retina.     

pH changes in the invaginating synaptic cleft mediate feedback from horizontal cells to cone photoreceptors by modulating Ca2+ channels

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround-receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 microM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.     

Modulation of synaptic function in retinal amacrine cells

Amacrine cells are interneurons that have diverse functionsin retinal signal processing. In order to study signaling andmodulation in retinal amacrine cells, we employ a simplifiedculture system containing identifiable GABAergic amacrine cells.Immunocytochemistry experiments indicate that GABAergic amacrinecells express metabotropic glutamate receptor 5 (mGluR5), agroup I mGluR usually linked to the IP3 signaling pathway. Ca²⁺imaging experiments using an mGluR5-specific agonist indicatethat these receptors are functional and when activated, canstimulate temporally diverse Ca²⁺ elevations. To begin to establishthe role of these receptors in modulating amacrine cell function,we have used electrophysiological methods to ask whether ionchannels are the targets of mGluR5-dependent modulation. Herewe discuss our results indicating that activation of mGluR5leads to enhancement of currents through GABA_A receptors. Thisenhancement is dependent upon elevations in cytosolic Ca²⁺ andactivation of protein kinase C (PKC). To explore the consequencesof Ca²⁺ elevations in another context, we have used nitric oxide(NO) donors to mimic the effects of activating the Ca²⁺-dependentsynthetic enzyme for NO, neuronal nitric oxide synthase. Wefind that exposure to NO donors also enhances the amplitudeof currents through GABA_A receptors. Together, these resultsindicate that glutamate from presynaptic bipolar cells has thepotential to work through multiple mechanisms to regulate thefunction of amacrine-to-amacrine cell GABAergic synapses.