Fragile X Mental Retardation Protein expression in the retina is regulated by light.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick.

Calcium-induced calcium release contributes to synaptic release from mouse rod photoreceptors.

We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 microM ryanodine evoked Ca(2+) increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca(2+)-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 microM) reduced the electroretinogram (ERG) b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 microM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodine's effects is further indicated by the finding that ryanodine (100 microM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 microM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons.

Localization of the alpha(1F) calcium channel subunit in the rat retina.

PURPOSE: The molecular identity of the calcium channels that mediate glutamate release from photoreceptors is unknown. Mutations in the recently identified, retina-specific alpha(1F) calcium channel subunit cause incomplete X-linked congenital stationary night blindness (CSNB2), the phenotype of which is consistent with a defect in neurotransmission within the retina. The purpose of this study was to determine the cellular distribution of the alpha(1F) subunit in the retina. METHODS: Antibodies were raised against a unique peptide from the human alpha(1F) sequence. Rat retina sections were labeled with affinity-purified alpha(1F) antibodies and the immunofluorescence analyzed by confocal microscopy. The alpha(1F) staining was compared with that obtained with a pan-alpha(1) antibody, used to reveal the distribution of known voltage-gated calcium channels in the retina. Some sections were double labeled for alpha(1F) and the photoreceptor synaptic ribbon marker, bassoon. RESULTS: Staining of retina sections with anti-alpha(1F) resulted in strong punctate labeling in the outer plexiform layer (OPL) and weak punctate labeling in the inner plexiform layer (IPL), consistent with a synaptic localization. Staining was also observed in the outer nuclear layer. Within the OPL, alpha(1F) immunoreactivity was clustered in discrete, horseshoe-shaped patches, the shape and dimensions of which are characteristic of rod active zones. Similar structures were labeled with the pan-alpha(1) antibody. Localization of alpha(1F) immunoreactivity to rod active zones was confirmed by double labeling for bassoon, a component of photoreceptor synaptic ribbons. CONCLUSIONS: The distribution of alpha(1F) immunoreactivity in the OPL suggests that calcium influx through alpha(1F) or alpha(1F)-like channels mediates glutamate release from rod photoreceptors.

Expression of the alpha1F calcium channel subunit by photoreceptors in the rat retina.

PURPOSE: The CACNA1F gene encodes a voltage-gated calcium channel alpha1 subunit, alpha1F, which is expressed in the human retina. Mutations in this gene cause incomplete X-linked congenital stationary night blindness (CSNB2). The aim of this study was to obtain the sequence of the rat alpha1F cDNA and localize the encoded polypeptide in the rat retina. METHODS: The full-length rat alpha1F sequence was compiled from sequencing of overlapping alpha1F PCR fragments amplified from rat retinal cDNA. Antiserum was raised against a human alpha1F peptide. It was found that the human alpha1F peptide used to generate the antiserum was conserved at only 11 out of 19 residues in the cloned rat sequence. Therefore, antibodies were affinity purified against either the human alpha1F peptide or the equivalent rat peptide and used for immunofluorescent staining of rat retina sections. RESULTS: The rat alpha1F amino acid sequence was found to be 91% and 95% identical to the human and mouse alpha1F sequences, respectively. Antibodies affinity purified against the human alpha1F peptide stained both the outer nuclear layer (ONL) and outer plexiform layer of rat retina sections. In contrast, staining with antibodies affinity purified against the corresponding rat alpha1F peptide was restricted to the ONL. CONCLUSIONS: The rat alpha1F amino acid sequence is highly homologous to the human and mouse sequences. The immunohistochemical results indicate the existence of distinct alpha1F isoforms or alpha1F-like channels, which are differentially distributed in the cell bodies and synaptic terminals of photoreceptors in the rat retina.

Localization of voltage-sensitive L-type calcium channels in the chicken retina.

L-type calcium channels have been associated with synaptic transmission in the retina, and are a potential site for modulation of the release of neurotransmitters. The present study documents the immunohistochemical localization of neuronal alpha1 subunits of L-type calcium channels in chicken retina, using antibodies to the alpha1c, alpha1d and alpha1f subunits of L-type calcium channels. The alpha1c-like subunits were localized to Müller cells, with predominantly radial processes, and a prominent band of horizontal processes in the outer plexiform layer. The antibody to alpha1d subunits labelled most, if not all, cell bodies. The antibody to a human alpha1f subunit strongly labelled photoreceptor terminals. Fainter immunoreactivity was detected in the inner segments of the photoreceptors, a subset of amacrine cells, two bands of labelling in the inner plexiform layer and many ganglion cells. The differential cellular distributons of these alpha1-subunits suggests subtle functional differences in their roles at different cellular locations.

Neurotransmitter release at ribbon synapses in the retina.

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.

Presynaptic proteins of ribbon synapses in the retina.

The synapses of photoreceptors and bipolar cells in the retina are characterized ultrastructurally by the presence of an electron-dense bar, the synaptic ribbon, lying perpendicular to the plasma membrane at the active zone and extending about 0.5 microm into the cytoplasm. Hence, these synapses are known as ribbon synapses. All neurons that make ribbon synapses release neurotransmitter tonically. That is, neurotransmitter is released continuously from these neurons and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven, phasic release from other neurons. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but several differences that may be important determinants of tonic transmitter release have been identified in the retina by immunohistochemistry. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.

Calcium channel heterogeneity among cone photoreceptors in the tree shrew retina.

Retinal photoreceptors are depolarized in darkness and release neurotransmitter tonically. They respond to light by hyperpolarization and a concomitant reduction in transmitter release. The calcium-dependent release of transmitter is coupled to graded changes in membrane potential by L-type calcium channels in the photoreceptor terminals. This paper reports the immuno-localization of an L-type channel alpha1D subunit to most, but not all, synaptic terminals of cones in the tree shrew retina. Double labelling for the alpha1D subunit and the plasma membrane Ca2+-ATPase, which has been shown to be present in all tree shrew cones, revealed a subpopulation of cone terminals that did not react with the alpha1D antibody. The nonimmunoreactive synaptic terminals represented approximately 5.8% of the total and formed a highly regular array across the retina reminiscent of the blue cones. Double-staining for the alpha1D subunit and blue cone opsin confirmed that these are the blue cones. The observed differences in calcium channel immunoreactivity between long and short wavelength cones points to previously unsuspected heterogeneity in the molecular machinery governing transmitter release from spectrally different cone types.

Calcium extrusion from mammalian photoreceptor terminals.

Ribbon synapses of vertebrate photoreceptors constantly release glutamate in darkness. Transmitter release is maintained by a steady influx of calcium through voltage-dependent calcium channels, implying the presence of a mechanism that is able to extrude calcium at an equal rate. The two predominant mechanisms of intracellular calcium extrusion are the plasma membrane calcium ATPase (PMCA) and the Na+/Ca2+-exchanger. Immunohistochemical staining of retina sections revealed strong immunoreactivity for the PMCA in rod and cone terminals, whereas staining for the Na+/Ca2+-exchanger was very weak. The PMCA was localized to the plasma membrane along the sides of the photoreceptor terminals and was excluded from the base of the terminals where the active zones are located. The amplitude of a calcium-activated chloride current was used to monitor the intracellular calcium concentration. An upper limit for the time required to remove intracellular free calcium is obtained from two time constants measured for the calcium-activated chloride current tail currents: one of 50 msec and a second of 190 msec. Calcium extrusion was inhibited in the absence of intracellular ATP or in the presence of the PMCA inhibitor orthovanadate, but was unaffected by replacement of external Na+ with Li+. The data indicate that the PMCA, rather than the Na+/Ca2+-exchanger, is the predominant mechanism for calcium extrusion from photoreceptor synaptic terminals.

A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina.

In contrast to conventional synapses, which release neurotransmitter transiently, ribbon synapses formed by photoreceptors and bipolar cells of the retina release neurotransmitter continuously and modulate the rate in response to light. Both modes of release are mediated by synaptic vesicles but probably differ in the regulation of docking and fusion of synaptic vesicles with the plasma membrane. We have found that syntaxin 1, an essential component of the core fusion complex in conventional synapses, is absent from ribbon synapses of the retina, raising the possibility that these synapses contain a different type of syntaxin or syntaxin-like protein. By immunoprecipitating syntaxin 1-depleted retina and brain extracts with a SNAP-25 antibody and microsequencing the precipitated proteins, syntaxin 3 was detected in retina complexed with SNAP-25, synaptobrevin, and complexin. Using an anti-syntaxin 3 antiserum, syntaxin 3 was demonstrated to be present at high levels in retina compared to brain. Immunofluorescent staining of rat retina sections confirmed that syntaxin 3 is expressed by photoreceptor and bipolar cells in the retina. Thus, in the retina, expression of syntaxin 3 is correlated with ribbon synapses and may play a role in the tonic release of neurotransmitter.

Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, in the mammalian retina.

Synaptophysin and synaptoporin are homologous proteins that are among the most abundant synaptic vesicle proteins. Despite their high degree of sequence similarity, they are differentially distributed in the brain. The distribution of synaptophysin and synaptoporin was examined in the adult rat and rabbit retina by using single- and double- labeling immunocytochemistry with conventional light microscopy and confocal laser scanning microscopy. In the rat retina, synaptophysin immunoreactivity was found in the outer plexiform layer in terminals of photoreceptors and was homogeneously distributed throughout the inner plexiform layer. Synaptoporin immunoreactivity, however, was restricted to the inner plexiform layer. Labeling was most prominent in three distinct bands of the inner plexiform layer separated by two bands of very low synaptoporin immunoreactivity. In the rabbit retina, synaptophysin and synaptoporin immunoreactivity were found in the inner and outer plexiform layers. In the inner plexiform layer, labeling for both vesicle proteins was homogeneous, with no detectable stratification. In the outer plexiform layer, synaptophysin was present in photoreceptor terminals, and synaptoporin was present in horizontal cells. Staining of isolated rabbit retinal cells confirmed that both the axonless A type and the axon-bearing B type horizontal cells are immunoreactive for synaptoporin. In addition, electron microscopy of synaptoporin-immunostained rabbit retinas revealed no labeling of photoreceptor terminals but of putative synaptic sites in horizontal cells in the outer plexiform layer. No functional correlation was found in the expression of either synaptic vesicle protein with the type of neuron or synapse (ribbon or conventional).

The plasma membrane protein SNAP-25, but not syntaxin, is present at photoreceptor and bipolar cell synapses in the rat retina.

The two neuronal plasma membrane proteins synaptosomal associated protein 25 kDa (SNAP-25) and syntaxin form, in current models of synaptic vesicle exocytosis, a docking complex on the presynaptic plasma membrane. This docking complex interacts with the integral synaptic vesicle protein, synaptobrevin. We have examined, in the rat retina, the distribution of SNAP-25 and syntaxin using light and electron microscopic immunocytochemistry. SNAP-25 immunoreactivity was present in the outer and inner nuclear layers at the membranes of photoreceptor and amacrine cell somata, in the outer plexiform layer in the terminals of photoreceptor cells and throughout the inner plexiform layer in the terminals of bipolar and amacrine cells. In contrast, syntaxin immunoreactivity was not found in the terminals of photoreceptor and bipolar cells, but only in the terminals of amacrine cells. Because of the absence of detectable syntaxin from the terminals of photoreceptor and bipolar cells, we propose that either a novel syntaxin isoform or a syntaxin-like protein exists at the ribbon synapses formed by these neurons.

AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia.

Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH-terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Müller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Müller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10-15, correlating strongly with the onset of retinal function.

Generation of truncated brain AE3 isoforms by alternate mRNA processing.

AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA processing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-terminal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic detergent, suggesting a possible association with the cytoskeleton.

Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin.

The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by co-immunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was co-immunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.

Modulation of the cytotoxic mechanism of 6-thioguanine by 4-amino-5-imidazolecarboxamide.

Previous evidence has indicated that either purine starvation or incorporation into DNA may be the dominant biochemical effect of the antileukemic agent 6-thioguanine (TG), depending on exposure conditions. Furthermore, it has been suggested that the paradoxical decrease in TG-induced cytotoxicity at high drug concentrations may be due to an antagonistic interaction between these two mechanisms, in which purine starvation inhibits DNA synthesis and, therefore, incorporation of TG into DNA. In this report we test the hypothesis that by concurrent treatment of L1210 cells with TG and the purine precursor 4-amino-5-imidazolecarboxamide (AIC) it is possible to alleviate DNA synthesis inhibition caused by high concentrations of TG, thus enhancing TG incorporation into DNA and TG-induced cell kill. Both the cytotoxic and cytokinetic results presented support this hypothesis. However, gross incorporation of TG into DNA was not increased by AIC under conditions in which a significant enhancement of cytotoxicity (i.e., 1 log) was observed. These findings suggest that the potentiating effect of AIC may be most prominent on the subpopulation of cells that are resistant to treatment with TG alone, and they demonstrate that the cytotoxic effects of TG treatments are more accurately reflected by observing specific cytokinetic changes (delayed late S/G2 arrest) than by measuring the average extent of TG incorporation into DNA within a given population. Finally, we propose that it may be possible to select conditions for administration of TG that favor one or the other cytotoxic mechanism, depending on whether the clinical objective is induction of remission (where rapid cell lysis due to purine starvation would be desired) or eradication of subclinical disease during remission (where proliferation-dependent cytotoxicity due to DNA incorporation should be more effective.

Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger.

We have isolated AE3, a novel gene expressed primarily in brain neurons and in heart. The predicted AE3 polypeptide shares a high degree of identity with the anion exchange and cytoskeletal binding domains of the erythrocyte band 3 protein. Expression of AE3 cDNA in COS cells leads to chronic cytoplasmic acidification and to chloride- and bicarbonate-dependent changes in intracellular pH, confirming that this gene product is an anion exchanger. Characterization of an AE3 mutant lacking the NH2-terminal 645 amino acids demonstrates that the COOH-terminal half of the polypeptide is both necessary and sufficient for correct insertion into the plasma membrane and for anion exchange activity. The NH2-terminal domain may play a role in regulating the activity of the exchanger and may be involved in the structural organization of the cytoskeleton in neurons.

Comparison of in vivo and in vitro effects of continuous exposure of L1210 cells to 6-thioguanine.

In this study the cytokinetic and antitumor effects of 12-h continuous treatment with 6-thioguanine (TG) were studied in L1210 cells in vivo and in vitro. Loss of clonogenicity in vitro was maximized at a drug concentration of 0.2 microM. Higher drug concentrations produced less cell kill, and the surviving fraction observed after exposure to 25 microM TG was 1 log higher than at 0.2 microM (2% versus 0.2% of control cloning efficiency, respectively). Delayed G2 arrest in vitro was also found to be most pronounced at 0.2 microM, with G1 arrest more predominant at higher concentrations. Studies in vivo were conducted using C57BL X DBA/2 F1 mice, with or without advanced L1210 ascites tumor. In initial experiments performed on animals without tumor, the 50% lethal dose for 12-h s.c. infusions of TG was approximately 0.8 mumol/kg/min. Correlation of steady-state TG plasma levels with infusion rate revealed a linear relationship up to 0.62 mumol/kg/min, above which the TG plasma concentration increased disproportionately to input rate. Total body clearance of TG, calculated from the linear portion of this curve, was 123 ml/kg/min. The antitumor effects of TG infusions were correlated with steady state plasma concentrations achieved in each individual animal, and it was found that dose rates yielding levels from 1 to 10 microM increased survival time by about 40%, with no apparent optimum plasma level in this range. Examination of the cytokinetic effects caused by TG infusions at the low and high ends of this maximally therapeutic range showed that, as was the case in vitro, lower concentrations of TG caused delayed G2 arrest, while higher concentrations induced more rapid G1 arrest. On the basis of these, as well as previous findings, we propose that the operative mechanism of cell kill by TG in vivo may be dose dependent and may be reflected by the relative degree of G2 versus G1 arrest. We also suggest that the appropriate strategy for the clinical use of TG is to determine the drug concentration which produces maximum G2 arrest of tumor cells, and to infuse continuously at a rate to achieve that level for the maximum time tolerated by the patient, rather than to select an arbitrary length of infusion followed by escalation to maximum tolerated drug concentration.