Retinal Amino Acid Neurochemistry of the Southern Hemisphere Lamprey,Geotria australis

Lampreys are one of the two surviving groups of the agnathan (jawless) stages in vertebrate evolution and are thus ideal candidates for elucidating the evolution of visual systems. This study investigated the retinal amino acid neurochemistry of the southern hemisphere lamprey Geotria australis during the downstream migration of the young, recently-metamorphosed juveniles to the sea and during the upstream migration of the fully-grown and sexually-maturing adults to their spawning areas. Glutamate and taurine were distributed throughout the retina, whilst GABA and glycine were confined to neurons of the inner retina matching patterns seen in most other vertebrates. Glutamine and aspartate immunoreactivity was closely matched to Müller cell morphology. Between the migratory phases, few differences were observed in the distribution of major neurotransmitters i.e. glutamate, GABA and glycine, but changes in amino acids associated with retinal metabolism i.e. glutamine and aspartate, were evident. Taurine immunoreactivity was mostly conserved between migrant stages, consistent with its role in primary cell functions such as osmoregulation. Further investigation of glutamate signalling using the probe agmatine (AGB) to map cation channel permeability revealed entry of AGB into photoreceptors and horizontal cells followed by accumulation in inner retinal neurons. Similarities in AGB profiles between upstream and downstream migrant of G. australis confirmed the conservation of glutamate neurotransmission. Finally, calcium binding proteins, calbindin and calretinin were localized to the inner retina whilst recoverin was localized to photoreceptors. Overall, conservation of major amino acid neurotransmitters and calcium-associated proteins in the lamprey retina confirms these elements as essential features of the vertebrate visual system. On the other hand, metabolic elements of the retina such as neurotransmitter precursor amino acids and Müller cells are more sensitive to environmental changes associated with migration.     

N-Methyl-d-aspartate receptors in the retina

The vertebrate retina is a “genuine neural center” (Ramón y Cajal), in which glutamate is a major excitatory neurotransmitter. Both N-methyl-d-aspartate (NMDA) and non-NMDA receptors are expressed in the retina. Although non-NMDA receptors and/or metabotropic glutamate receptors are generally thought to be responsible for mediating the transfer of visual signals in the outer retina, there is recent evidence suggesting that NMDA receptors are also expressed in photoreceptors, as well as horizontal and bipolar cells. In the inner retina, NMDA receptors, in addition to other glutamate receptor subtypes, are abundantly expressed to mediate visual signal transmission from bipolar cells to amacrine and ganglion cells, and could be involved in modulation of inhibitory feedback from amacrine cells to bipolar cells. NMDA receptors are extrasynaptically expressed in ganglion cells (and probably amacrine cells) and may play physiological roles in a special mode. Activity of NMDA receptors may be modulated by neuromodulators, such as d-serine and others. This article discusses retinal excitotoxicity mediated by NMDA receptors.     

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells

Vitamin C is transported in the brain by sodium vitamin C co‐transporter 2 (SVCT‐2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT‐2 and the uptake and release of [¹⁴C] ascorbate in chick retinal cells. SVCT‐2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT‐2 was expressed in cultured retinal neurons, but not in glial cells. [¹⁴C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium‐free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate‐stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l ‐β‐threo‐benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [³H] d ‐aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2‐Carboxy‐3‐carboxymethyl‐4‐isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7‐initroquinoxaline‐2,3‐dione (DNQX) or (5R,2S)‐(1)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801). However, DNQX, but not MK‐801 or 2‐amino‐5‐phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non‐NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2‐bis (2‐aminophenoxy) ethane‐N′,N′,N′,N′,‐tetraacetic acid tetrakis (acetoxy‐methyl ester) (BAPTA‐AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT‐2, and the release can be stimulated by NMDA or non‐NMDA glutamate receptors.     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells.     

Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express beta2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of alpha7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express alpha7 nAChRs. At least three different populations of cone bipolar cells exhibited alpha7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of alpha7-positive amacrine cells. Some GABAergic amacrine cells also displayed alpha7 immunoreactivity; alpha7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of alpha7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on alpha7 nAChRs expressed by GCs themselves.     

Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels.

Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.     

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague-Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus)

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.     

GABA release induced by aspartate-mediated activation of NMDA receptors is modulated by dopamine in a selective subpopulation of amacrine cells

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS, including the retina. In the chick retina, GABA is located in horizontal and amacrine cells and in some cells in the ganglion cell layer. It has been shown that glutamate and its agonists, NMDA, kainate, and aspartate, promote the release of GABA from isolated retina and from cultured retinal cells. Dopamine, the major catecholamine in the retina, inhibits the induction of GABA release by NMDA. Two to seven-day-old intact chicken retinas were stimulated with different glutamatergic agonists and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to 100 μ M NMDA for 30 minutes resulted in 50% reduction in the number of GABA-immunoreactive amacrine cells. Aspartate (100 μ M) treatment also resulted in 60% decrease in the number of GABA-immunoreactive amacrine cells. The number of GABA-immunoreactive horizontal cells was not affected by either NMDA or aspartate. In addition, dopamine reversed by 50% the reduction of the number of GABA-immunoreactive amacrine cells exposed to NMDA or aspartate. Kainate stimulation promoted a 50% reduction in the number of both GABA-immunoreactive amacrine and horizontal cells. Dopamine did not interfere with the kainate effect. While in control and in non-stimulated retinas a continuous and homogeneous immunolabeling was observed throughout the inner plexiform layer, retinas exposed to NMDA, kainate and aspartate displayed only a faint punctate labeling in the inner plexiform layer. It is concluded that, under our experimental conditions, both NMDA and aspartate induce the release of GABA exclusively from amacrine cells, and that the release is modulated by dopamine. On the other hand, kainate stimulates GABA release from both amacrine and horizontal cells with no interference of dopamine.     

Glutamate-Induced Excitotoxicity in Retina: Neuroprotection with Receptor Antagonist,Dextromethorphan, but Not with Calcium Channel Blockers

The purpose of our studies was to evaluate different strategies for possible neuroprotection in glutamate-induced neurotoxicity in the retina. In a first set of experiments we attempted to determine if dextrorphan antagonism of glutamate action on NMDA receptors would protect against excitotoxic injury associated with secondary damage seen after surgical laser treatment in retina. In a second set of experiments, the effects of different calcium channel blockers in an in-vitro model of N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell excitotoxicity that utilized rabbit retinal explants were evaluated. Dextrorphan infusion prior to laser treatment of rabbit retina produced a significant decrease in the area of neural retinal damage. We attribute the apparent dextrorphan protection to attenuation of glutamate mediated excitotoxicity secondary to laser induced cell death. Preincubation of rabbit retinal explants with verapamil, nimodipine or -conotoxin MVIIA did not cause a significant change in NMDA induced cell death in the ganglion cell layer.     

Timing of quantal release from the retinal bipolar terminal is regulated by a feedback circuit

In isolation, a presynaptic terminal generally releases quanta according to Poisson statistics, but in a circuit its release statistics might be shaped by synaptic interactions. We monitored quantal glutamate release from retinal bipolar cell terminals (which receive GABA-ergic feedback from amacrine cells) by recording spontaneous EPSCs (sEPSCs) in their postsynaptic amacrine and ganglion cells. In about one-third of these cells, sEPSCs were temporally correlated, arriving in brief bursts (10-55 ms) more often than expected from a Poisson process. Correlations were suppressed by antagonizing the GABA(C) receptor (expressed on bipolar terminals), and correlations were induced by raising extracellular calcium or osmolarity. Simulations of the feedback circuit produced "bursty" release when the bipolar cell escaped intermittently from inhibition. Correlations of similar duration were present in the light-evoked sEPSCs and spike trains of sluggish-type ganglion cells. These correlations were suppressed by antagonizing GABA(C) receptors, indicating that glutamate bursts from bipolar terminals induce spike bursts in ganglion cells.     

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.     

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of³H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.Special issue dedicated to Dr. Frederick E. Samson     

Changes in Retinal Morphology,Electroretinogram and Visual Behavior after Transient Global Ischemia in Adult Rats

The retina is a light-sensitive tissue of the central nervous system that is vulnerable to ischemia. The pathological mechanism underlying retinal ischemic injury is not fully understood. The purpose of this study was to investigate structural and functional changes of different types of rat retinal neurons and visual behavior following transient global ischemia. Retinal ischemia was induced using a 4-vessel occlusion model. Compared with the normal group, the number of βIII-tubulin positive retinal ganglion cells and calretinin positive amacrine cells were reduced from 6 h to 48 h following ischemia. The number of recoverin positive cone bipolar cells transiently decreased at 6 h and 12 h after ischemia. However, the fluorescence intensity of rhodopsin positive rod cells and fluorescent peanut agglutinin positive cone cells did not change after reperfusion. An electroretinogram recording showed that the a-wave, b-wave, oscillatory potentials and the photopic negative response were completely lost during ischemia. The amplitudes of the a- and b-waves were partially recovered at 1 h after ischemia, and returned to the control level at 48 h after reperfusion. However, the amplitudes of oscillatory potentials and the photopic negative response were still reduced at 48 h following reperfusion. Visual behavior detection showed there was no significant change in the time spent in the dark chamber between the control and 48 h group, but the distance moved, mean velocity in the black and white chambers and intercompartmental crosses were reduced at 48 h after ischemia. These results indicate that transient global ischemia induces dysfunction of retinal ganglion cells and amacrine cells at molecular and ERG levels. However, transient global ischemia in a 17 minute duration does not appear to affect photoreceptors.     

Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors

The vanilloid receptor VR1 (TRPV1) is a temperature- and capsaicin-sensitive cation channel expressed by a class of primary afferents involved in nociception. To confirm the hypothesis that VR1-positive primary afferents are glutamatergic and contact spinal neurons that express the main classes of ionotropic glutamate receptors, we performed multiple immunofluorescent staining for VR1 and the glutamate transporter VGLUT2 (a specific marker for glutamatergic transmission) or AMPA and NMDA receptor subunits. VR1-positive cells in the dorsal root ganglion and boutons of their central afferent fibers in the dorsal horn expressed VGLUT2, and the latter contacted AMPA- or NMDA receptor-positive perikarya. Based on our previous observations of preferential targeting of VR1-positive primary afferents to spinal neurons that express the neurokinin receptor NK1 (Hwang et al., 2003), we further quantified the frequency of termination of VR1-positive afferents onto NK1-positive neurons co-expressing glutamate receptors. A larger fraction of NK1/NMDA receptors-positive than NK1/AMPA receptors-positive sites were contacted by VR1-positive boutons. We conclude that VR1-positive primary afferents in the rat use glutamate as neurotransmitter and contact postsynaptic sites that co-express NK1 and ionotropic glutamate receptors.