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1.
Summary Glycine is one of the essential neurotransmitters modulating visual signals in retina. Glycine activates Cl- permeable receptors that conduct either inhibitory or excitatory actions, depending on the Cl electrical–chemical gradient (E Cl) positive or negative to the resting potential in the cells. Interestingly, both glycine-induced inhibitory and excitatory responses are present in adult retinas, and the effects are confined in the inner and outer retinal neurons. Glycine inhibits glutamate synapses in the inner plexiform layer (IPL), resulting in shaping light responses in ganglion cells. In contrast, glycine excites horizontal cells and On-bipolar dendrites in the outer plexiform layer (OPL). The function of glycinergic synapse in the outer retina represents the effect of network feedback from a group of centrifugal neurons, glycinergic interplexiform cells. Moreover, immunocytochemical studies identify glycine receptor subunits (α1, α2, α3 and β) in retinas, forming picrotoxin-sensitive α-homomeric and picrotoxin-insensitive α/β-heteromeric receptors. Glycine receptors are modulated by intracellular Ca2+ and protein kinas C and A pathways. Extracellular Zn2+ regulates glycine receptors in a concentration-dependent manner, nanomolar Zn2+ enhancing glycine responses, and micromolar Zn2+ suppressing glycine responses in retinal neurons. These studies describe the function and mechanism of glycinergic synapses in retinas.  相似文献   

2.
Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca2+ permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca2+-activated Cl channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl]i above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na+-K+-2Cl co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl. To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl]i was decreased by bathing ORNs in low Cl solution. Both manipulations reduced only the Cl component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.  相似文献   

3.
We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn2+ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl = SCN> Br>> I, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.  相似文献   

4.
The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+‐dependent Cl‐ and K+‐ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo‐electron microscopy structure of human NKCC1 in the substrate‐loaded (Na+, K+, and 2 Cl) and occluded, inward‐facing state that has also been observed for the SLC6‐type transporters MhsT and LeuT. Cl binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT‐fold scaffold and bundle domains. Cl‐ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl‐sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.  相似文献   

5.
The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca2+-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 μmol l−1), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 μmol l−1) and were independent of bath Cl, indicating that the stimulated ileal Isc does not involve electrogenic Cl secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl secretion. The cAMP- and Ca2+-dependent secretory responses were inhibited by the removal of HCO3 suggesting that these responses were due to electrogenic HCO3 secretion. We conclude that the ileum of the possum does not secrete Cl due to low levels of NKCC1 expression. It does however appear to secrete HCO3 . These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.  相似文献   

6.
The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.  相似文献   

7.
The cation‐chloride co‐transporters are important regulators of the cellular Cl homeostasis. Among them the Na+‐K+?2Cl? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K+‐Cl? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na+ action potentials but was mimicked by a Ca2+‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016  相似文献   

8.
Apoptosis is an essential process in organ development, tissue homeostasis, somatic cell turnover, and the pathogenesis of degenerative diseases. Apoptotic cell death occurs in response to a variety of stimuli in physiological and pathological circumstances. Efflux of K+ and Cl leads to apoptotic volume decrease (AVD) of the cell. Both mitochondrion-mediated intrinsic, and death receptor-mediated extrinsic, apoptotic stimuli have been reported to rapidly activate Cl conductances in a large variety of cell types. In epithelial cells and cardiomyocytes, the AVD-inducing anion channel was recently determined to be the volume-sensitive outwardly rectifying (VSOR) Cl channel which is usually activated by swelling under non-apoptotic conditions. Blocking the VSOR Cl channel prevented cell death in not only epithelial and cardiac cells, but also other cell types, by inhibiting the induction of AVD and subsequent apoptotic events. Ischemia-reperfusion-induced apoptotic death in cardiomyocytes and brain neurons was also prevented by Cl channel blockers. Furthermore, cancer cell apoptosis induced by the anti-cancer drug cisplatin was recently found to be associated with augmented activity of the VSOR Cl channel and to be inhibited by a Cl channel blocker. The apoptosis-inducing VSOR Cl channel is distinct from ClC-3 and its molecular identity remains to be determined.  相似文献   

9.
A key feature of signal processing in the mammalian retina is parallel processing, where the segregation of visual information, e.g., brightness, darkness, and color, starts at the first synapse in the retina, the photoreceptor synapse. These various aspects are transmitted in parallel from the input neurons of the retina, the photoreceptor cells, through the interconnecting bipolar cells, to the output neurons, the ganglion cells. The photoreceptors and bipolar cells release a single excitatory neurotransmitter, glutamate, at their synapses. This parsimony is contrasted by the expression of a plethora of glutamate receptors, receptor subunits, and isoforms. The detailed knowledge of the synaptic distribution of glutamate receptors thus is of major importance in understanding the mechanisms of retinal signal processing. This review intends to highlight recent studies on the distribution of glutamate receptors at the photoreceptor synapses of the mammalian retina.  相似文献   

10.
Effect of endothelin-1 and chemically induced hypoxia on Na+−K+−Cl cotransport activity in cultured rat brain capillary endothelial cells was examined by using86Rb+ as a tracer for K+; bumetanide-sensitive K+ uptake was defined as Na+−K+−Cl cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na+−K+−Cl cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na+−K+−Cl cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca2+. Oligomycin, sodium azide, or antimycin A increased Na+−K+−Cl cotransport activity by 80–200%. Oligomycin-induced Na+−K+−Cl cotransport activity was reduced by an intracellular Ca2+ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca2+, and not protein kinase C, in hypoxia-induced Na+−K+−Cl cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.  相似文献   

11.
The balance of K+, Na+, and Cl fluxes across the cell membrane with the Na+/K+ pump, ion channels, and Na+K+2Cl (NKCC) and Na+-Cl (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K+, Na+, and Cl fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K+, Na+, and Cl concentrations in cell water; total Cl flux; total K+ influx; and the ouabain-inhibited pump component of the Rb+(K+) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na+ channels, whereas K+ and Cl channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K+, Na+, and Cl account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.  相似文献   

12.
We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl -channel function is required for activation. We used basilaterally α-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 μM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl current ( ) through CFTR was blocked by: 1) Cl removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 μM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous ΔF508 CF ducts). However, Cl gradients in the direction of absorption supported, while Cl gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated through CFTR in the direction of absorption (Cl gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing through CFTR in the direction of secretion (Cl gradient from cell to lumen) prevents ENaC activation even in the presence of through CFTR. An erratum to this article is available at .  相似文献   

13.
Intracellular Cl concentrations ([Cl]i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl is accumulated by the Na+-K+-2Cl cotransporter 1 (NKCC1), resulting in a [Cl]i above electrochemical equilibrium and a depolarizing Cl efflux upon Cl channel opening. Here, we investigate the [Cl]i and function of Cl in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl]i of WT TG neurons indicated active NKCC1-dependent Cl accumulation. Gamma-aminobutyric acid (GABA)A receptor activation induced a reduction of [Cl]i as well as Ca2+ transients in a corresponding fraction of TG neurons. Ca2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca2+ channels (VGCCs). Ca2+ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1−/− TG neurons, but elevated under conditions of a lowered [Cl]o suggesting a Cl-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca2+-activated Cl channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca2+ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca2+-activated Cl-dependent signal amplification mechanism in TG neurons that requires intracellular Cl accumulation by NKCC1 and the activation of CaCCs.  相似文献   

14.
The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl-HCO3 exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO3 secretion; Na+ electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl-HCO3 exchanger, Cl channel, and Na+-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues. Min-Hwang Chang, Consuelo Plata, and Kambiz Zandi-Nejad have contributed equally to this work.  相似文献   

15.
The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl/HCO3 exchangers and Cl channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO3, we investigated the relation between cisplatin-induced apoptosis and Cl channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.  相似文献   

16.
In the vertebrate CNS, glycine acts as an inhibitory neurotransmitter and as the obligatory coagonist of glutamate at N-methyl-d-aspartate receptors. These roles depend on extracellular glycine levels, regulated by Na+/Cl-dependent transporters GLYT1, present mainly in glial cells, and GLYT2, predominantly neuronal. In Bergmann glia, GLYT1 mediates both, glycine uptake and efflux, which, in turn, influences excitatory neurotransmission at Purkinje cell synapses. The biochemical properties of GLYTs and their regulation by signaling pathways in these cells are largely unknown. We characterized Gly uptake in confluent primary cultures of Bergmann glia from chick cerebellum. Transport was found to be energy- and Na+-dependent, and was resolved into a high (Km=25 μM) and a low affinity (Km=1.1 mM) components identified as GLYT1 and transport System A, respectively. Results show that high affinity transport by GLYT1 is regulated by calcium from intracellular stores, calmodulin, and myosin light chain kinase through an actin cytoskeleton-mediated action. Special issue dedicated to Dr. Simo S. Oja  相似文献   

17.
The presence of basolateral Cl channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl channels via a cGMP-dependent pathway. Anion replacement and 36Cl flux studies showed that NO affected both Cl and HCO 3 secretion. Two different types of Cl channels are known to be present in the basolateral membrane of epithelial cells: Zn2+-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl conductance in the presence of Zn2+ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.  相似文献   

18.
Inhibitory drives in the nervous system are provided by synapses operating mostly through Cl ion channels. We describe a novel CFP-YFP-based fluorescence Cl indicator, Cl-sensor, characterized by a high sensitivity, which can be successfully used as a tool for monitoring intracellular Cl in various biological preparations. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 380–381, July–October, 2007.  相似文献   

19.
In this review, we discuss the function and modulation of chloride-selective glycine receptor (GlyR) channels, some genetic diseases originated from dysfunction of GlyRs, and modulation of glycinergic synapses by intracellular calcium (Ca2+) with particular attention on the motoneurons of the hypoglossal nucleus. This motor nucleus is a brainstem structure implicated in the command of coordinated movements during oral behavioral phenomena, including feeding, drinking, grooming, and respiration. In this nucleus, more than 90% of its cells are motoneurons. These hypoglossal motoneurons (HMs) are involved in a variety of motor functions and exhibit two remarkable features: (i) a low endogenous Ca2+ buffering capacity, which determines the rapid dynamics of cytosolic intracellular Ca2+, and (ii) powerful glycinergic inputs, which determine the main inhibitory drive on the above cells in adult animals. Glycine receptors belong to the superfamily of Cys-loop ligand-gated ion channels. They are capable of forming functional homo-or heteromeric chloride-selective channels. Dysfunction of GlyRs results in a genetic neurological motor disorders, including hyperekplexia. These diseases originate from mutations in the GlyR gene, leading to a decrease in single channel conductance, a lower affinity to the neurotransmitter, or a low level of GlyR expression. The function of glycinergic synapses is modulated during developmental changes and strictly controlled by several feedback mechanisms at pre-and post-synaptic levels. The developmental modulation consists in switch in the GlyR subunit composition and change in the chloride homeostasis during the synaptic maturation and formation of inhibitory networks. Retrograde signalling plays an important role in the synaptic function of HMs; it provides post-synaptic neurons with efficient tools for controlling pre-synaptic afferents. Glycine receptors and glycinergic synapses are also regulated by intracellular Ca2+. The mechanisms of these modulations are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 338–349, July–October, 2007.  相似文献   

20.
Schultz  K.  Goldman  D. J.  Ohtsuka  T.  Hirano  J.  Barton  L.  Stell  W. K. 《Brain Cell Biology》1997,26(10):651-666
L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1–4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength (“red”) and intermediate-wavelength (“green”) cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength (“blue”) cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at Mr=110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.  相似文献   

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