Extrasynaptic NMDA Receptors on Rod Pathway Amacrine Cells: Molecular Composition, Activation, and Signaling.

In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca2+ in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity.SIGNIFICANCE STATEMENT Glutamate is the most important excitatory neurotransmitter in the CNS, but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties determined by their specific subunit composition. These receptors can be found at both synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, with different and complementary GluN2 subunits. The localization of GluN2A-containing receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for differential modulation of excitability and signaling in this retinal microcircuit.

Localization of the zinc binding tubulin polymerization promoting protein in the mice and human eye.

Tubulin Polymerization Promoting Protein (TPPP/p25) modulates the dynamics and stability of the microtubule network by its bundling and acetylation enhancing activities that can be modulated by the binding of zinc to TPPP/p25. Its expression is essential for the differentiation of oligodendrocytes, the major constituents of the myelin sheath, and has been associated with neuronal inclusions. In this paper, evidence is provided for the expression and localization of TPPP/p25 in the zinc-rich retina and in the oligodendrocytes in the optic nerve. Localization of TPPP/p25 was established by confocal microscopy using calbindin and synaptophysin as markers of specific striations in the inner plexiform layer (IPL) and presynaptic terminals, respectively. Postsynaptic nerve terminals in striations S1, S3 and S5 in the IPL and a subset of amacrine cells show immunopositivity against TPPP/p25 both in mice and human eyes. The co-localization of TPPP/p25 with acetylated tubulin was detected in amacrine cells, oligodendrocyte cell bodies and in synapses in the IPL. Quantitative Western blot revealed that the TPPP/p25 level in the retina was 0.05-0.13 ng/µg protein, comparable to that in the brain. There was a central (from optic nerve head) to peripheral retinal gradient in TPPP/p25 protein levels. Our in vivo studies revealed that the oral zinc supplementation of mice significantly increased TPPP/p25 as well as acetylated tubulin levels in the IPL. These results suggest that TPPP/p25, a microtubule stabilizer can play a role in the organization and reorganization of synaptic connections and visual integration in the eye.

An unconventional glutamatergic circuit in the retina formed by vGluT3 amacrine cells.

In the vertebrate retina, glutamate is traditionally thought to be released only by photoreceptors and bipolar cells to transmit visual signals radially along parallel ON and OFF channels. Lateral interactions in the inner retina are mediated by amacrine cells, which are thought to be inhibitory neurons. Here, we report calcium-dependent glutamate release from vGluT3-expressing amacrine cells (GACs) in the mouse retina. GACs provide an excitatory glutamatergic input to ON-OFF and ON direction-selective ganglion cells (DSGCs) and a subpopulation of W3 ganglion cells, but not to starburst amacrine cells. GACs receive excitatory inputs from both ON and OFF channels, generate ON-OFF light responses with a medium-center, wide-surround receptive field structure, and directly regulate ganglion cell activity. The results reveal a functional glutamatergic circuit that mediates noncanonical excitatory interactions in the retina and probably plays a role in generating ON-OFF responses, crossover excitation, and lateral excitation.

Activation of the Nrf2/HO-1 antioxidant pathway contributes to the protective effects of Lycium barbarum polysaccharides in the rodent retina after ischemia-reperfusion-induced damage.

Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are protective to retina after ischemia-reperfusion (I/R). The antioxidant response element (ARE)-mediated antioxidant pathway plays an important role in maintaining the redox status of the retina. Heme oxygenase-1 (HO-1), combined with potent AREs in its promoter, is a highly effective therapeutic target for the protection against neurodegenerative diseases, including I/R-induced retinal damage. The aim of our present study was to investigate whether the protective effect of LBP after I/R damage was mediated via activation of the Nrf2/HO-1-antioxidant pathway in the retina. Retinal I/R was induced by an increase in intraocular pressure to 130 mm Hg for 60 minutes. Prior to the induction of ischemia, rats were orally treated with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. For specific experiments, zinc protoporphyrin (ZnPP, 20 mg/kg), an HO-1 inhibitor, was intraperitoneally administered at 24 h prior to ischemia. The protective effects of LBP were evaluated by quantifying ganglion cell and amacrine cell survival, and by measuring cell apoptosis in the retinal layers. In addition, HO-1 expression was examined using Western blotting and immunofluorescence analyses. Cytosolic and nuclear Nrf2 was measured using immunofluorescent staining. LBP treatment significantly increased Nrf2 nuclear accumulation and HO-1 expression in the retina after I/R injury. Increased apoptosis and a decrease in the number of viable cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in the I/R retina, which were reversed by LBP treatment. The HO-1 inhibitor, ZnPP, diminished the LBP treatment-induced protective effects in the retina after I/R. Taken together, these results suggested that LBP partially exerted its beneficial neuroprotective effects via the activation of Nrf2 and an increase in HO-1 protein expression.

Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory.

BACKGROUND: The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates. METHODS: The expression of genes expressed in the sensory vesicle of the ascidian Ciona intestinalis including those encoding components of the monoaminergic neurotransmission systems, was analyzed by in situ hybridization or in vivo transfection of the corresponding regulatory elements driving fluorescent reporters. Modulation of photic responses by monoamines was studied by electrophysiology combined with pharmacological treatments. RESULTS: We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins). CONCLUSION: We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina.

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography.

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.