Isolated Chick Retina Research Articles

The Tight Coupling and Non-Linear Relationship between the Macroscopic Electrical and Optical Concomitants of Electrochemical CNS Waves Reflect the Non-Linear Dynamics of Neural Glial Propagation

In isolated chick retina, the visualization of electrochemical self-organized patterns is possible due to the presence of macroscopic intrinsic optical signals (IOSs). Isolated circular waves, standing patterns, and self-sustained sequences of spirals are all easily obtained using an IOS approach. In this paper we present the tight coupling and non-linear relationship between optical and electrical wave concomitants, and potassium-induced whole tissue excitability changes. Elementary statistical methods and time series analyses were applied to two sets of data: 1) solitary circular retinal spreading depression waves, and 2) tissue response to exogenous potassium fast pulses. The results were interpreted from the point of view of non-linear thermodynamical concepts and volume phase transitions in polyanionic gels according to the Tasaki action potential model. From these and previous results, it is clear that the glial network and extracellular matrix contribute to the propagation and emergence of these patterns.

Temporal alteration of spreading depression by the glycine transporter type-1 inhibitors NFPS and Org-24461 in chicken retina

We used isolated chicken retina to induce spreading depression by the glutamate receptor agonist N-methyl-d-aspartate. The N-methyl-d-aspartate-induced latency time of spreading depression was extended by the glycineB binding site competitive antagonist 7-chlorokynurenic acid. Addition of the glycine transporter type-1 inhibitors NFPS and Org-24461 reversed the inhibitory effect of 7-chlorokynurenic acid on N-methyl-d-aspartate-evoked spreading depression. The glycine uptake inhibitory activity of Org-24461, NFPS, and some newly synthesized analogs of NFPS was determined in CHO cells stably expressing human glycine transporter type-1b isoform. Compounds, which failed to inhibit glycine transporter type-1, also did not have effect on retinal spreading depression. These experiments indicate that the spreading depression model in chicken retina is a useful in vitro test to determine activity of glycine transporter type-1 inhibitors. In addition, our data serve further evidence for the role of glycine transporter type-1 in retinal neurotransmission and light processing.

Inhibition of hypoxia-induced [ 3H]glycine release from chicken retina by the glycine transporter type-1 (GlyT-1) inhibitors NFPS and Org-24461

Chicken posterior eyecup lined by the retina were prepared, loaded with [ 3H]glycine and superfused in order to determine its release in various experimental conditions. Electrical field stimulation of the retina evoked [ 3H]glycine release with a voltage- and frequency-dependent manner and this release may be originated from glycinergic amacrine cell processes of the inner plexiform layer of the retina. Glycine released from an abundance of different amacrine cells may modulate retinal circuitry by activation of inhibitory glycine receptors and by acting as a coagonist on N-methyl- d-aspartate receptors on AII amacrine cells and retinal ganglion cells. The latter effect of glycine may be modulated by glycine transporter type-1. Cells with glycine transporter type-1 immunopositive staining were visualized in the inner nuclear layer and dens immunolabeling was also detected throughout the inner plexiform layer of chicken retina. Glycine and the substrate-type glycine transporter type-1 inhibitor sarcosine increased [ 3H]glycine release from glycinergic amacrine cells and/or glial cells by extrusion of glycine from cytoplasmic pools by homo- and heteroexchange mechanisms. Deprivation of oxygen and glucose from the buffer used for superfusion evoked a marked increase in [ 3H]glycine efflux, an effect probably due to reverse mode operation of glycine transporter type-1. The non-transportable glycine transporter type-1 inhibitors NFPS and Org-24461, which did not alter [ 3H]glycine efflux from isolated chicken retina by themselves in normoxic condition, inhibited oxygen and glucose deprivation-induced [ 3H]glycine release. It is concluded that reduction of the N-methyl- d-aspartate receptor coagonist glycine concentrations in hypoxic conditions by glycine transporter type-1 inhibitors may decrease N-methyl- d-aspartate receptor-mediated neuronal toxicity and cell death in retinal tissue.

Modulation of CNS excitability by water movement. the D<sub>2</sub>O effects on the non-linear neuron-glial dynamics

Macroscopic spatiotemporal patterns arising in grey matter may explain the clinical manifestations of several functional neurological syndromes (migraine aura, epilepsies). Detailed descriptions of these patterns in central grey matter and their physicochemical or pharmacological manipulations can be useful in many scientific fields ranging from drug design to functional brain imaging. These evanescent dynamic structures are electrochemical in nature and show macroscopic tissue polarization due to coupled and macroscopic flow of ions and water across, along and between neuronal and glial membranes. So far the importance of the water flow in the CNS functional syndromes has been examined by manipulations of water channels aquaporines (AQP). In this paper we show the result of substituting H2O for D2O in retinal spreading depression experiments. This inverts the present logic by changing the flow in the water channels in intact tissue and observing the evolution of electrochemical patterns and recording the optical profiles of excitation waves in isolated chick retinas. D2O flow through AQPs is ~20% slower than that of H2O. The slower flux disturbs the tight coupling between ion and water flows across membranes and slowdown the Na-KATPase rate of change with metabolic consequences for the tissue. The whole tissue excitability shifts in a non-stationary manner toward a non-excitable state.

The synergetic modulation of the excitability of central gray matter by a neuropeptide: two protocols using excitation waves in chick retina

The isolated chick retina provides an in vitro tissue model, in which two protocols were developed to verify the efficacy of a peptide in the excitability control of the central gray matter. In the first, extra-cellular potassium homeostasis is challenged at long intervals and in the second, a wave is trapped in a ring of tissue causing the system to be under self-sustained challenge. Within the neuropil, the extra-cellular potassium transient observed in the first protocol was affected from the initial rising phase to the final concentration at the end of the five-minute pulse. There was no change in the concomitants of excitation waves elicited by the extra-cellular rise of potassium. However, there was an increase on the elicited waves latency and/or a rise in the threshold potassium concentration for these waves to appear. In the second protocol, the wave concomitants and the propagation velocity were affected by the peptide. The results suggest a synergetic action of the peptide on glial and synaptic membranes: by accelerating the glial Na/KATPase and changing the kinetics of the glial potassium channels, with glia tending to accumulate KCl. At the same time, there is an increase in potassium currents through nerve terminals.

Retinal charge sensitivity and spatial discrimination obtainable by subretinal implants: key lessons learned from isolated chicken retina

In order to obtain functional parameters relevant to the designing of a subretinal implant, we carried out electrical stimulation experiments with isolated chicken retina. The median threshold for network activation with planar disc electrodes (diameter 10 µm) was 0.5 nC (625 µC cm−2) for anodal voltage impulses and 1.6 nC (2 mC cm−2) for cathodal impulses. Above threshold, the number of spikes evoked by a single voltage impulse increased up to saturation within a range of injected charge from 0.1 nC to 1 nC for anodal impulses and from 1 nC to 10 nC for cathodal impulses. Using needle electrodes with a tip diameter of 1 µm, we determined the electrical point spread function (EPSF) for subretinal stimulation. It had a half width in the range of 100 µm, which corresponds to a visual angle of 21′ and to a visual acuity of 20/417 in the human eye. It is reasonable to conclude that with subretinal implants the minimum separable will be of the same dimension.

Effect of ebselen and organochalcogenides on excitotoxicity induced by glutamate in isolated chick retina

In this study, we evaluated the effects of three simple organochalcogenides (diphenyl diselenide, diphenyl ditelluride and diphenyl telluride) and ebselen on the glutamate-driven 45Ca 2+ influx into chick embryonic retinal cells, as well as their effects on the excitotoxic injury in retina cells. None of the compounds tested interfered with basal 45Ca 2+ uptake. Diphenyl diselenide and diphenyl ditelluride had no effects on glutamate-driven 45Ca 2+ influx. Diphenyl telluride (100–400 μM) decreased and ebselen (100–400 μM) completely blocked the glutamate-driven 45Ca 2+ influx ( P < 0.01) into chick retinal explants. The assessment of neural injury was made spectrophotometrically by quantification of cellularly reduced MTT (3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) 24 h after the beginning of glutamate exposure (8 h). Ebselen had no effects on retinal MTT reduction when co-incubated with glutamate for 8 h. However, when ebselen (100 and 400 μM) was co-incubated for 8 h with glutamate and remained in the incubation media until MTT evaluation (24 h after the beginning of incubation), it protected retinal cells against the decrease in MTT reduction induced by glutamate. These data indicate that besides its capacity of interacting with Ca 2+ channels, other mechanisms are involved in the neuroprotection afforded by ebselen in this work, possibly its antioxidant properties.

Interactions of allosteric modulators of AMPA/kainate receptors on spreading depression in the chicken retina

The functional role of AMPA and kainate receptors in spreading depression (SD) was investigated in the isolated chicken retina. Competitive (NBQX) and non-competitive (GYKI 52466, GYKI 53405 and GYKI 53655) antagonists of the AMPA receptor inhibited AMPA-induced SD in a concentration-dependent manner. Concentrations of drugs caused 50% inhibition (IC 50 values) are 0.2, 16.6, 7.0 and 1.4 μM, respectively. AMPA receptor positive modulator cyclothiazide was more effective in the potentiation of SD evoked by AMPA than by kainate. Slight potentiation of either AMPA- or kainate-induced SD was observed only at high concentration (1 mg/ml) by the kainate receptor modulator concanavalin A. Compounds that positively modulate AMPA receptor function (cyclothiazide, IDRA-21, S 18986, 1-BCP and aniracetam) caused a concentration-dependent potentiation in SD. Concentrations of drugs that caused 50% potentiation (estimated EC 50 values) are 9, 135, 142, 450 and 1383 μM, respectively. Interaction between cyclothiazide, aniracetam or S 18986 administered with each other, or with GYKI 52466, respectively, was also investigated. When cyclothiazide and S 18986 were co-applied, their effects seemed to be additive. However, lack of additivity was obtained when S 18986 was added together with aniracetam. Positive modulators applied at equiactive concentrations reduced the inhibitory action of GYKI 52466 and differently shifted its concentration–response curve. In this respect, S 18986 was the most effective (IC 50 of GYKI 52466 changed from 16.6 to 51.9 μM). Our findings indicate the contribution of AMPA rather than kainate receptors in the mediation of retinal spreading depression. Our data further support the idea that multiple positive modulatory sites are present on the AMPA receptor complex in addition to a negative modulatory site.

Neuroprotective Effects of Lamotrigine and Remacemide on Excitotoxicity Induced by Glutamate Agonists in Isolated Chick Retina

The possible neuroprotective effects of two recently developed antiepileptic compounds, lamotrigine (LTG) and remacemide (REMA), against glutamate agonist-induced excitotoxicity have been investigated in the isolated chick embryo retina model. Retina segments from 15- or 16-day-old embryos were incubated in 1 ml of balanced salt solution, at 25°C for 30 min, in the presence or absence of N-methyl-d-aspartate (NMDA), kainic acid (KA), or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (10 to 200 μM). LTG, REMA, and the active desglycinyl metabolite of REMA (d-REMA) (10–200 μM) were added separately 5 min before glutamate agonists. Retina damage was assessed after 24 h (i) by measuring LDH activity present in the medium, expressed as percentage of total retina LDH activity, and (ii) by histological analysis of retina specimens through scoring for the presence or absence of edema, necrosis, nuclear pyknosis, and cell layer damage. LTG, REMA, and d-REMA reduced LDH release produced by NMDA 58–70% in a dose-dependent manner, with d-REMA being the most potent (EC50: d-REMA, 25.75 ± 3.27 μM; REMA, 64.75 ± 7.75 μM; LTG, 60.50 ± 6.80 μM; P < 0.001). The drugs had less effect on the LDH release produced by AMPA and KA. Histological analysis confirmed these biochemical results, with all three compounds reducing edema and the number of necrotic and pyknotic nuclei in the ganglion layer. d-REMA provided almost complete protection of the ganglion cell layer against damage produced by NMDA. Combinations of d-REMA and LTG showed additive rather than potentiative effects against NMDA-induced cell injury. The present data provide pharmacological evidence that LTG, REMA, and d-REMA decrease glutamate agonist-induced excitotoxicity in isolated chick retina, findings that might have therapeutic implications for various neurological disorders.

Electrical multisite stimulation of the isolated chicken retina

Visual prostheses such as subretinal implants are intended for electrical multisite excitation of the retinal network. To investigate relevant issues like spatial resolution and operational range, we have developed an in vitro method using microelectrode arrays to stimulate isolated retinae. Ganglion cell activity in the chicken retina evoked by distally applied spatial voltage patterns consisted of fast bursts, transient inhibition and delayed discharges, and depended on the amount, location and spatial pattern of the injected charge. The response was altered or disappeared when synaptic transmission was blocked. Our results indicate that shape perception and object location can be partially achieved with subretinal electrical multisite stimulation.

Calcium-independent release of [ [formula omitted]]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [ 3 H ]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [ 3 H ]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca 2+-independent manner. Inhibition of Na +/K +-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na + electrochemical gradients, since nigericin and veratrine did not induce release in Na + containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.

Light-induced changes of extracellular ions and volume in the isolated chick retina-pigment epithelium preparation.

To better understand the mechanisms of extracellular space volume regulation and their possible effects on retinal function, light-induced changes in the concentrations of the principal extracellular ions (Na+, K+, Ca2+, and Cl-) were measured with ion-sensitive microelectrodes in the chick retina-pigment epithelium-choroid preparation. Changes of extracellular space volume were assessed by measuring the concentration of an impermeant marker, tetramethylammonium. In the inner retina, transient ON/OFF Na+ decrease was about twice as large as K+ increase, and the charge difference was compensated by a decrease in Cl- concentration. The ion changes were accompanied by extracellular space-volume decreases here. In the subretinal space, [Na+]o increase was about twice as large as K+ decrease, yet [Cl-]o, also decreased; this was accompanied by a sustained extracellular space-volume increase. The ionic changes in the inner retina are consistent with a model of extracellular space-volume regulation which assumes that neuronal depolarization causes net uptake of NaCl, cell swelling, and extracellular space shrinkage. However, to prevent the apparent violation of electroneutrality in the subretinal space, our simple model should be expanded to include the involvement of unidentified anion(s). Substantial changes in the subretinal space volume may influence interaction between the neural retina and pigment epithelium. Among ionic changes, only the light-induced [K+]o decrease around the photoreceptors and the [Ca2+]o increase near the photoreceptor bodies and synaptic terminals are large enough (-25% and 7.5%, respectively) to be likely candidates for integrated intercellular signaling.

Lubeluzole shows neuroprotective effects in an “in-vitro”-model for neuronal lesions in the chicken retina

In this study, the isolated chicken retina was used as an “in-vitro”-model for investigation of neuronal lesions to show the neuroprotective effects of lubeluzole. Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in photochemical stroke models in rats. In the retina, the typical cell swelling of a developing lesion is accompanied by a very strong intrinsic optical signal (IOS), occurring simultaneous with the electrical signal which is based on changes in light scattering. In the presented model, lesions were elicited electrically with a tungsten microelectrode (0.1 MΩ). The degree of damage was evaluated with optical methods by measuring area and brightness of the affected tissue. Lubeluzole was much more effective in reducing the growth of the lesions than its R-isomer. However, both compounds enhanced the possibility of the neuronal tissue to recover after excitotoxic stimuli.

Sumatriptan Can Block Spreading Depression in Isolated Chick Retina

Sumatriptan Can Block Spreading Depression in Isolated Chick Retina

Sumatriptan blocks spreading depression in isolated chick retina.

Spreading depression is a neurohumoral phenomenon that has been related to the pathophysiology of migraine. The recently introduced 5HT1D agonist anti-migraine compound sumatriptan blocks neurogenic extravasation and induces cerebral vasoconstriction, but the actual mechanism of action against migraine remains obscure. Retinal spreading depression (RSD) velocity has been measured in isolated chick retinas in the presence of 0.05-2.00 mM sumatriptan. This drug reversibly blocks RSD in a concentration-dependent manner. Since the preparation is blood-vessel free, this effect must be related to the nervous tissue.

Sumatriptan in the retinal spreading depression

The mechanisms of Leao Spreading Depression (SD), a neurophysiological phenomenon described more than 50 years ago, remain obscure. The objective of the present study was to analyze the effects of sumatriptan and related compounds on S D in isolated chick retina […] Sumatriptan in the retinal spreading depression (abstract)

Self-induced splitting of spiral-shaped spreading depression waves in chicken retina

Spreading depression (SD) of electroencephalographic activity is a dynamic wave phenomenon in the central nervous system (CNS). The retina, especially the isolated chicken retina, is an excellent constituent of the CNS in which to observe the dynamic behavior of the SD wave fronts, because it changes its optical properties during a SD attack. The waves become visible as milky fronts on a black background. It is still controversial what the basic mechanistic steps of SD are, but certainly SD belongs to the self-organization phenomena occurring in neuronal tissue. In this work, spiral-shaped wave fronts are analyzed using digital video imaging techniques. We report how the inner end of the wave front, the spiral tip, breaks away repeatedly. This separation process is associated with a Z-shaped trajectory (extension approximately 1.2 mm) that is described by the tip over one spiral revolution (period 2.45+/-0.1 min). The Z-shaped trajectory does not remain fixed, but performs a complex motion across the retina with each period. This is the first time, to our knowledge, that established imaging methods have been applied to the study of the two-dimensional features of SD wave propagation and to obtaining quantitative data of their dynamics. Since these methods do not interfere with the tissue, it is possible to observe the intrinsic properties of the phenomenon without any external influence.

An improved model for examining delayed excitotoxic neurodegeneration in isolated chick retina

Extensive use of the embryonic chick retina model has been made for examining mechanisms of acute excitotoxicity. The standard retina model does not, however, address the concept of delayed excitotoxicity. In previous retina based studies extending the time course to 24 h has proven problematic owing to damage arising in control tissue. Recently a model for examining delayed excitotoxicity in isolated retina was proposed. This method relied on a significant reduction in incubating temperature to avoid the variable damage occurring in control tissue. The data in the present study suggest that a reduction in temperature results in N-methyl- d-aspartate (NMDA) receptor dysfunction, preventing accurate assessment of NMDA receptor mediated excitotoxicity. An improved model is proposed which permits incubation at 37°C without damage occurring in control tissue.

Gap junctions are required for the propagation of spreading depression

Spreading depression (SD) is a slowly propagating depression of cerebral neuronal activity and transmembrane ionic gradients, that arises in response to a variety of noxious stimuli. SD bears a strong resemblance to gap junction-mediated calcium waves among cultured astrocytes. Here, we show that gap junction-mediated intercellular diffusion is necessary for the generation of SD. Waves of SD in the isolated chicken retina were blocked by five different inhibitors of gap junctional coupling, which was assessed by the intercellular transit of Lucifer Yellow (LY). Each of these gap junction blockers inhibited both the migration of SD and the diffusion of LY in a dose-dependent manner. In contrast, glutamate-evoked calcium influx into retinal cells was not affected by these compounds. The results indicate that intercellular coupling through gap junctions is required for SD. Gap junction-mediated communication might therefore constitute an important mechanism in both normative and pathological brain function.

Effects of local anaesthetics on retinal spreading depression.

This study examines the effect of two local anaesthetics, benzocaine and lidocaine, on propagation, amplitude and duration of the slow potential change of spreading depression. The experiments were performed in isolated chick retina superfused with Ringer's solution. We observed, for both drugs, a dose-related decrease in all parameters analysed. The maximal effect on propagation was blockade of the reaction.