Yong-Mei Zhong

Activation of the sigma receptor 1 suppresses NMDA responses in rat retinal ganglion cells

The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions.

Expression of glycine receptor and transporter on bullfrog retinal Müller cells

The expression of the glycine receptor (GlyR) alpha1, alpha2 and beta subunits and glycine transporter (GlyT) on Müller cells was studied in bullfrog retina using double immunofluorescence labeling and confocal scanning microscopy. Double labeling of glial fibrillary acidic protein (GFAP), a specific marker for Müller cells, and the GlyR subunits showed that almost all Müller cells moderately expressed GlyR alpha1 and weakly GlyR beta, whereas no immunoreactivity for GlyR alpha2 was observed. The labeling for GlyR alpha1 and GlyR beta appeared in somata, major processes, endfeet and branchlets of the Müller cells. Müller cells were also GlyT1-labeled. Consistent with previous electrophysiological results, these findings suggest that Müller cells may be involved in modulation of glycinergic transmission by reciprocal interactions with retinal neurons through GlyR and GlyT.

Expression of somatostatin receptor subtype 5 in rat retinal amacrine cells

Somatostatin (SRIF), as a neuroactive peptide in the CNS, exerts its actions via five subtypes of specific receptors (ssts). In this work, the localization of sst(5) was studied immunocytochemically in rat retinal amacrine cells (ACs). Labeling for sst(5) was diffusely distributed throughout the full thickness of the inner plexiform layer (IPL) and formed two distinct fluorescence bands in the distal part of the IPL. Double labeling experiments showed that sst(5) was expressed in GABAergic ACs. It was further shown that labeling for sst(5) was observed in both dopaminergic and cholinergic ACs, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively. The immunostaining appeared mainly on the cell membranes and somatodendritic compartments of these ACs. For the cholinergic ACs, weak sst(5)-immunoreactivity was also observed in the processes terminating in the IPL. In contrast, no sst(5)-immunoreactivity was found in glycinergic AII ACs, stained by parvalbumin (PV). Furthermore, labeling for SRIF was co-localized with sst(5) in both dopaminergic and cholinergic ACs. These results suggest that sst(5) may serve as an autoreceptor or conventional receptor in retinal ACs.

Gene expression and protein distribution of orexins and orexin receptors in rat retina.

Orexins, composed of orexin A and orexin B, are identified as endogenous ligands of two orphan G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). Orexins are implicated in regulating wake/sleep states, feeding behaviors, etc. Using reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling, we investigated the distributions of orexin A, orexin B, OX1R and OX2R in rat retina. RT-PCR analysis revealed the presence of mRNAs of prepro-orexin, OX1R and OX2R in rat retina. Immunostaining for orexin A and orexin B was observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina, horizontal cells, labeled by calbindin, and bipolar cells, labeled by homeobox protein Chx10, were orexin A- and orexin B-positive. In the inner retina, two orexins were both found in GABAergic amacrine cells (ACs), including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase and choline acetyltransferase respectively. Glycinergic ACs, including AII ACs, also expressed orexins. Weak to moderate labeling for orexin A and orexin B was diffusely distributed in the inner plexiform layer. Additionally, orexins were expressed in almost all ganglion cells (GCs) retrogradely labeled by cholera toxin B subunit. Specifically, double-labeling experiments demonstrated that melanopsin-positive GCs (intrinsically photosensitive retinal GCs, ipRGCs) were labeled by two orexins. Morever, OX1R immunoreactivity was observed in most of GCs and all dopaminergic ACs, as well as in both outer and inner plexiform layers. In contrast, no obvious OX2R immunostaining was detectable in the rat retina. These results suggest that orexins may modulate the function of neurons, especially in the inner retina. We further hypothesize that the orexin signaling via ipRGCs may be involved in setting the suprachiasmatic nucleus (SCN) circadian clock.

Cellular localization of P2Y6 receptor in rat retina

Extracellular nucleotides exert their actions via two subfamilies of purinoceptors: P2X and P2Y. Eight mammalian P2Y receptor subtypes (P2Y(1,2,4,6,11,12,13,14)) have been identified. In this work, the localization of P2Y(6) was studied in rat retina using double immunofluorescence labeling and confocal scanning microscopy. Immunostaining for P2Y(6) was strong in the outer plexiform layer and was diffusely distributed throughout the full thickness of the inner plexiform layer. In addition, P2Y(6) immunoreactivity was clearly observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina photoreceptor terminals, labeled by VGluT1, and horizontal cells, labeled by calbindin, were P2Y(6)-positive. However, no P2Y(6) immunostaining was detected in bipolar cells, labeled by homeobox protein Chx10. In the inner retina P2Y(6) was localized to most of GABAergic amacrine cells, including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively. Some of glycinergic amacrine cells, but not glycinergic AII amacrine cells, were also labeled by P2Y(6). Moreover, P2Y(6) immunoreactivity was seen in almost all ganglion cells, labeled by Brn3a. In Müller glial cells, stained by cellular retinaldehyde binding protein (CRALBP), however, no P2Y(6) expression was found in both somata and processes. We speculate that P2Y(6) may be involved in retinal information processing in different ways, probably by regulating the release of transmitters and/or modulating the radial flow of visual signals and lateral interaction mediated by horizontal and amacrine cells.

Distinctive morphology of hippocampal CA1 terminations in orbital and medial frontal cortex in macaque monkeys

The fine morphology of hippocampal connections to the orbital and medial frontal cortex (OMFC) was investigated by placing injections of anterograde tracers in the CA1 in two monkeys. The axons terminated mainly in layers 2 and 3, of areas 11, 13, 14c, 25, and 32, and were widely divergent in these layers, traversing 2–4xa0mm. Boutons were scattered along the main axon, but also occurred as distinctive small, spherical clusters of terminations (“mini-clusters”; diameter <50xa0μm). Occasional larger terminal arbors were observed in layer 3 and these were often unusually tortuous or convoluted. These features may imply a specialized microcircuitry of hippocampal-OMFC connections, including an origin from a particular subpopulation of CA1 projection neurons.

Natriuretic peptides are localized to rat retinal amacrine cells

Natriuretic peptides (NPs) may act as neuromodulators through activation of three specific receptor subtypes (NPRs). In the present study we examined the expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on different subtypes of retinal amacrine cells (ACs) in rat by immunofluorescence double labeling. All three NPs were moderately expressed in dopaminergic and cholinergic ACs, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively. The immunostaining appeared on the membrane, cytoplasm and somatodendritic compartments of these ACs. In AII glycinergic ACs, labeled by parvalbumin (PV), however, only faint punctate staining, if any, was seen. These results suggest that NPs could be produced in ACs and play a neuromodulatory role in the inner retina. Together with a previous immunocytochemical study, showing that NPR-B is present in cultured rat GABAergic ACs, our results further suggest that NPs produced in ACs may also modulate their own activity.

Localization of zinc in the outer retina of carp: A light- and electron-microscopic study

Many lines of evidence suggest that zinc may play an important neuromodulatory role in the central nervous system, including the retina. In this work, localization of zinc in the outer retina of carp was studied, using the silver amplification method, by light and electron microscopy. Reaction products (silver grains) were widely distributed throughout the retina, including photoreceptors, the outer and inner nuclear layers (ONL and INL), the ganglion cell layer (GCL), as well as in both outer and inner plexiform layers (OPL and IPL). Generally, staining for zinc was stronger in the outer retina than in the inner retina, and grains were aggregated with the greatest density in the OPL and the outer limiting membrane (OLM). Silver precipitates were also detected in the inner segments, axons, but not outer segments of photoreceptors. At the ultrastructural level, zinc was localized to myoid regions, mitochondria in the inner segments, internuclear space and nuclei of photoreceptors. In addition, silver grains were found in the terminals of photoreceptors, cone pedicles, and rod spherules, as well as in some processes in the OPL, which might be dendrites of horizontal cells. The presence of zinc in the terminals of photoreceptors suggests that zinc might be released from photoreceptor terminals and play modulatory roles in the outer retina. Synapse 62:352–357, 2008.

Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells

Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

Orexin-B modulates synaptic transmission of rod bipolar cells in rat retina

ABSTRACT Orexin‐A, ‐B play a crucial role in arousal and feeding by activating two G‐protein‐coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Orexins, along with orexin receptors, are expressed in retinal neurons, and they have been shown to differentially modulate excitatory AMPA receptors of amacrine and ganglion cells in the inner retina. In this work we report that orexin‐B modulates the activity of rod bipolar cells (RBCs) located in the outer retina of rat. Intravitreal injection of orexin‐B increased the amplitude of the scotopic electroretinographic b‐wave, a reflection of RBC activity, recorded in vivo. Patch clamp recordings in rat retinal slices showed that orexin‐B did not change glutamatergic excitatory component of the RBC response driven by photoreceptors. Effects of orexin‐B on GABA receptor‐mediated synaptic transmission of RBCs were then examined. In retinal slice preparations orexin‐B suppressed GABA receptor‐mediated inhibitory postsynaptic currents of RBCs in the inner plexiform layer. Furthermore, using whole‐cell recordings in isolated RBCs it was shown that orexin‐B suppressed GABAC receptor‐, but not GABAA receptor‐, mediated currents of the RBCs, an effect that was blocked by OX1R and OX2R antagonists. The orexin‐B‐induced inhibition of GABAC currents was likely mediated by a Gi/o/PC‐PLC/Ca2+‐independent PKC signaling pathway, as such inhibition was absent when each step of the above‐pathway was blocked with GDP‐&bgr;‐S/pertussis toxin (for Gi/o), D609 (for PLC), bisindolylmaleimide IV (for PKC)/rottlerin (for PKC&dgr;), respectively. The orexin‐B‐induced potentiation of RBC activity may improve visual acuity and contrast sensitivity of the animal during the dark period (wake phase). HighlightsOrexin‐B potentiates the rod‐driven responses of rat retinal bipolar cells (BCs).Such modulation is induced by reducing GABAergic feedback from amacrine cells to BCs.Orexin‐B suppresses GABAergic inhibitory postsynaptic currents (IPSCs) of RBCs.Orexin‐B suppresses GABA currents of RBCs via OX1R and OX2R activation.Gi/o/PC‐PLC/Ca2+‐independent PKC signaling pathway mediates the orexin effect on BCs.