Electrical impedance of isolated retina and its changes during spreading depression.

The electrical impedance of isolated chick and toad retinas were measured in the absence of, and during, spreading depression, using sinusoidal measuring currents ranging from 10 to 50,000 Hz. Data obtained in the absence of the reaction indicate an electric heterogeneity of the tissue and suggest that at least two groups of cells are responsible for the observed frequency distribution. Spreading depression is accompanied by impedance changes that depend on the frequency of the measuring current and composition of the Ringer's solution. In chick retinas immersed in standard solution, impedance magnitude (Z) as well as phase angle (0) change in phase, displaying an initial increase followed by a longer decrease; in the toad, these changes depend on the measuring frequency: at 10 Hz they are similar to the ones observed in chick retinas, whereas at intermediate and high frequencies they are out of phase. In low Cl- + sucrose Ringer's solution a decrease of impedance is observed in both chick and toad retinas. In low Cl- + Na2SO4 + sucrose Ringer's solution a decrease of impedance also predominates but the Z and 0 curves are biphasic and complex, having time courses which are frequency-dependent. In addition, these changes are different in chick and toad preparations. The experiments show that the impedance changes during the retinal reaction might be related both to extracellular as well as cellular currents. The extracellular current changes could be due to decrease of ionic content and volume of extracellular space, the latter variations being drastically reduced when retinas are immersed in low Cl- solutions. The results also suggest changes of current flow through cellular components which are frequency-dependent: at low frequencies the preferential path being probably through neuronal, and at high frequencies, through glial cells.

Retinal spreading depression induced by photoactivation: involvement of free radicals and potassium.

The threshold for spreading depression (SD) in chicken retina elicited by rose bengal photoactivation was raised by Trolox (water soluble vitamin E) suggesting the participation of reactive oxygen species in SD. The typical increases in K+ concentration associated with SD were preceded by small K+ oscillations that were more sensitive to photoactivation than was SD. It is hypothesised that all these phenomena could be accounted for by a free-radical mediated transient increase in membrane permeability and that this may be relevant to ischemic brain damage.

Elicitation of spreading depression by rose bengal photodynamic action.

Spreading depression refers to a slowly propagating depression of the ordinary electrical activity of the nervous tissue. It can be elicited by different types of physical or chemical non-specific stimuli. Various evidences suggest that transient alterations of cell membranes are involved. For this reason, and considering the action of free radicals on cell membranes, the elicitation of the reaction by dye photoactivation has been investigated. Isolated chick retina superfused in the dark with Ringer solution was able to regularly exhibit spreading depression when submitted to 1 microM rose bengal pulse of 5 min in duration, followed by 2.1 x 10(4) to 4.2 x 10(4) Jm-2 light pulse. The phenomenon was monitored either by visual inspection of the light-scattering milky wave that accompanies the reaction or by recording its characteristic slow voltage variation. The reaction was not triggered if the retina, superfused with the dye, was (a) maintained in the dark; (b) illuminated with red light (3.75 x 10(2) to 2.25 x 10(4) Jm-2), or (c) stimulated by white light but superfused with nitrogen-saturated solutions. It is concluded that, under the present conditions, the elicitation of spreading depression is contingent on the photoactivation of rose bengal in the presence of oxygen.

Spreading depression: a neurohumoral reaction.

1. The main characteristics of spreading depression are a decrease of spontaneous electrical activity, slow negative potential changes, transmembrane ion translocations, and an increase in tissue lactate. This is a general phenomenon in the central nervous system. 2. Retinal spreading depression, a very useful model for this phenomenon, has been extensively studied in terms of its optical, electrical and mechanical components. Ionic changes in the extracellular microenvironment have also been assessed and chemical substances liberated from tissue have been detected. 3. The velocity of propagation has been measured and some physical and chemical factors underlying the spread of the electrophysiological depression have been identified. Hypotheses about the nature of the reaction are discussed.

Biphasic effects of gap junctional uncoupling agents on the propagation of retinal spreading depression.

The spreading depression wave of Leão (SD) propagates in the nervous tissue at 3-10 mm/min and is accompanied by an increase of intracellular Ca2+ concentration and a decrease in the extracellular milieu. Recently the spread of Ca2+ waves with propagating velocities of the same order of magnitude has been detected in cultures of nerve cells. It has been suggested that these waves, which can be blocked by gap junctional agents, are related to SD. The present experiments describe some effects of heptanol and octanol (10 microM to 10 mM), well-known uncoupling agents of gap junctions, on the slow voltage changes and the velocity of propagation of Leão's phenomenon in isolated chick retina: 1) a 50 to 700 microM concentration of these alcohols in the superfusate solutions increased the velocity, whereas high concentrations (1 to 10 mM) decreased and subsequently halted the spread; 2) the recovery period of the slow voltage changes of SD was shorter in the presence of alcohol and its amplitude was larger during faster SD waves. These effects were observed in the retina during successive individual waves as well as in the course of circling SD. All of these effects were reversible, without any sign of damage to the retina. In the concentration range of 50 microM to 10 mM, methanol, ethanol, butanol and cyclo-hexanol had no effect on retinal SD. These data suggest the involvement of gap junctions in Leão's wave. This involvement is complex and its functional effects together with other factors that influence the velocity of propagation of SD are being investigated.

Elicitation of retinal spreading depression by barium ions.

The association of potassium ions with the occurrence and propagation of spreading depression is well known. The effects of barium ions, a potassium-channel blocker, on spreading depression in isolated retina are described. Pulses of 1-4 mM BaCl2, when applied to the retina, first induce the reaction, then hinder the propagation of the wave which finally stops. The threshold level which triggers the reaction is 0.4-0.6 mM BaCl2 but it varies with the composition of the superfusing Ringer solution. For example, in retinas superfused with low NaCl, or NaCl partially substituted by sodium isethionate, spreading depression may be evoked by barium ions at micromolar concentrations, without changing the velocity of spreading. The delayed blocking actions observed with higher doses may predominantly affect the recovery processes of the wave front, and are related to the refractory period of a preceding spreading depression.

Effect of Na+ and Cl- on the velocity of propagation of the spreading depression in chick retina.

The chick retina in vitro preparation was used to quantify the effect of Na+ and Cl- on the velocity of propagation of spreading depression (SD). The progressive reduction of chloride concentration in the superfusing Ringer solution, either by partial removal of NaCl or by its partial substitution with isethionate, caused a logarithmic increase in the velocity of propagation of SD. Substituting Tris for Na+ had no appreciable effect on propagation velocity, suggesting that the propagation of the reaction is not necessarily dependent on Na+. However, choline did not substitute for Na+ because it decreased propagation velocity and, at higher concentrations, even blocked the spread of the reaction.

Extracellular Ca2+ and retinal spreading depression.

The present study focuses on the influence of changes in extracellular Ca2+ on retinal spreading depression in vitro. It shows that changes in Ca2+ concentration alter the speed of the reaction. This effect is more pronounced for variations occurring within limits close to the physiological concentration level. The experiments also indicate that if the tissue is initially exposed to a lowered extracellular Ca2+ concentration, then the effects of transient variation in Ca2+ are significantly enhanced. Finally, a synergistic action of Ca2+ and K+ is observed with respect to the spreading of the reaction.

Spreading depression and the blood-brain barrier.

The propagation of spreading cortical depression was studied in rabbits during the reversible opening of the blood-brain barrier induced by hypertonic aqueous solutions of sodium chloride and sodium diatrizoate. The rupture of the blood-brain barrier was monitored by the leakage of Evans blue-albumin from pial vessels. Spreading depression was monitored by recording its characteristic slow voltage variations. Test substances were injected into the carotid artery or topically applied to the pia-arachnoid surface. Intracarotid injection of hypertonic solutions that open the blood-brain barrier do not block the propagation of spreading depression. Topical application of NaCl solutions can block the spread of the cortical depression reaction without opening the blood-brain barrier. Recurrent spreading depression waves at a mean frequency of one every 5 min, for 4 h do not cause the release of Evans blue-albumin complex from pial vessels.

The effect of diphenylhydantoin on spreading depression.

The present study examines the effect of diphenylhydantoin on the elicitation by K+ and propagation of spreading depression in chick retina preparations in vitro. A dose-related decrease in the velocity of propagation was observed, this effect being lessened by increasing the pH of the Ringer bathing the preparation. Changes in Ringer Cl- concentration also altered the efficacy of the drug, higher concentrations enhancing and lower concentrations reducing efficacy. The threshold concentration of K+ necessary to elicit the reaction was elevated by diphenylhydantoin.

Threshold determination of spreading depression evoking substances in the retina in vitro.

A technique for the determination of the threshold concentrations of chemical substances for elicitation of spreading depression is described. The technique minimizes the interference of mechanical stimulating effects and alterations in the susceptibility of the retina to spreading depression due to changes in unstirred layers at the liquid-tissue interphase. The following threshold concentrations were shown to elicit the wave: 8000-10000 microM KCl, 100-200 microM sodium glutamate, 5-10 microM sodium kainate, 10-20 microM sodium N-methyl-aspartate, 600-1200 microM (NH4)2SO4 and 400-600 microM BaCl2. Variations of K+ or Mg2+ concentration in the standard Ringer solution that may cause an increase or decrease of spreading depression velocity have an inverse effect on the threshold for elicitation of the reaction.

Cholinergic neurotransmission in retinal spreading depression.

Experiments performed to demonstrate the participation of cholinergic synapses in retinal spreading depression (SD) have shown that: (1) acetylcholine (ACh) is liberated during SD, its concentration increasing up to 10(-4) mM in the superfusate of one eye cup; (2) the susceptibility of the retina to SD and the velocity of propagation of circling SD are influenced by cholinergic and anticholinergic drugs. ACh 1 mM is effective in eliciting the reaction, whereas d-tubocurarine 0.01 mM and atropine 0.25 mM block this effect. Prostigmine 0.075 mM is able to trigger the reaction and also potentiates the effect of ACh. The velocity of propagation is decreased by ACh 0.25-1.0 mM and prostigmine 0.030 mM, which also potentiate this effect of ACh. Pulses of d-tubocurarine 0.005-0.030 mM and atropine 0.025-0.30 mM decrease the velocity of propagation. However, they block the effect of ACh if they are continuously present in the superfusing solution; (3) the effects of ACh are not blocked by tetrodotoxin 3.19 x 10(-3) mM.

Retinal spreading depression and the extracellular milieu.

We used isolated chick retina in vitro to study the participation of the extracellular milieu in the occurrence and propagation of spreading depression. The propagation was followed by visual observation or microphotometry and the ionic changes in the extracellular compartment were recorded with double-barreled ion-selective microelectrodes. The front of the spreading wave is accompanied by increased light scattering in the tissue and by decrease of Cl-, Na+, and Ca2+, increase of K+, and an alkaline-acid shift in the extracellular space, concomitant with the slow voltage changes characteristic of the wave. As the spread is related to the chemical steady-state of the extracellular milieu, the velocity of propagation is influenced by a balanced interplay of the chemical constituents of the superfusing solution, e.g., K+, HCO-3, and glucose facilitate, while Cl- and Mg2+ hinder the wave. Steady-state alterations induced by physical factors (temperature) or related to experimental conditions (speed and direction of superfusate flow) change markedly the velocity of propagation. Generally the procedures that cause increase of velocity augment the susceptibility of the preparation to the reaction and eventually may trigger it. Propagated spreading depression is considered as a chemical diffusion reaction pervading more intensively the inner plexiform layer of the retina.

Changes in fluid compartments and ionic composition in the isolated chick retina during SD.

Total content of water, extracellular spaces (ES), na+, K+, and C1- in the isolated chick retina were measured in the presence (test) or absence (control) of spreading depression (SD). During SD in medium with 0.5 mM or 2 mM MgSO4, there is an increase in the intracellular concentration of Na+ and C1- and a decrease in the intracellular concentration of K+. A decrease in the ES was only found in the medium with 2 mM MgSO4 together with a diminished outmovement of K+. We suggest that a decrease in the ES is due to an increased absorption of K+ by the Muller cells, causing its swelling and consequently a decrease of the ES. The addition of sucrose (17 mM) to the incubation medium as the extracellular marker markedly decreased the intracellular concentration of C1- in control retinas, blocked the inward movement of this ion to the tissue during SD and also changed the K+ movement during the phenomenon in medium with 2 mM MgSO4. We suggest that C1- is an important ion in the ionic balance of the Muller cells and that sucrose must have its site of action at these cells.

Perspectives on spreading depression.

Spreading depression (SD) consists of a transient suppression of all neuronal activity that spreads slowly across regions of gray matter. The paper is divided into three parts. Martins-Ferreira describes 30 years of research on SD in the isolated retina. Much of this work has relied on the prominent intrinsic optical signals that accompany SD in the retina. By inducing SD to propagate in circles with a velocity of 3.7 mm min(-1), it is possible to investigate the finely balanced electrochemical equilibrium that maintains the traveling wave. SD is accompanied by a slow negative extracellular voltage and ion movements that are greatest in the inner plexiform layer of the retina. Nedergaard discusses the role of astrocytes in SD propagation. Astrocytes mediate slowly moving waves of intracellular Ca(2+) increase, for which gap junctions are essential. SD is accompanied by entry of Ca(2+) into cells and fails when gap junctions are blocked. SD, however, is blocked by glutamate receptor antagonists but glial Ca(2+) waves are not. Astrocytic Ca(2+) waves are probably involved in the initiation of SD but other factors, including K(+), glutamate and purinergic receptors, are necessary for sustained propagation. Nicholson describes studies on the different preparations that helped clarify the role of extracellular space in SD. It has long been known that extracellular K(+) reaches levels of 50 mM or more during SD. Studies with ion-selective microelectrodes showed that extracellular Na(+) and Cl(-) fall by as much as 100 mM during SD, and water leaves the extracellular space. Further work showed that extracellular Ca(2+) falls 10-fold during SD and significant changes in extracellular pH and ascorbate occur. These studies imply that large perturbations of the extracellular milieu occur during SD and are an essential part of the interlocking cascade of events that produce this still mysterious phenomenon.

Involvement of GABA and ACh in retinal spreading depression: effects of "low calcium-high magnesium" solutions.

Experiments have been performed on isolated chick retinas to demonstrate the participation of gabaergic and cholinergic systems in spreading depression (SD). Gamma-aminobutyric acid (GABA) and acetylcholine (ACh) were measured in the effluent solution of superfused retinas. The influence of changes in the concentration of calcium/magnesium on the release of these neurotransmitters was studied. GABA and ACh are released in the superfusate of retinas during SD. Such release was observed during experimental periods longer than 2 h during which SD was elicited regularly at 15-20 min intervals. Decreasing calcium concentration from 1.0 to 0.5 mM and simultaneously increasing magnesium from 1.0 to 2.0-4.0 mM led to a decrease in GABA and ACh release during SD. Variations in light-scattering and increases in potassium concentration, usually occurring during SD, also decreased when superfusing with low calcium/high magnesium solutions. Lowering calcium concentration to 0.5 mM and increasing magnesium to 2.0 mM eventually turned the tissue refractory to SD. Sometimes a magnesium concentration of 2.0 mM was not effective in blocking SD. However, this blockage could be attained by increasing the concentration of magnesium to 4.0 mM. The effects of low calcium - high magnesium solutions on GABA and ACh release during SD suggests that the release of the substances is at least partially due to synaptic activity. It is not yet possible to establish whether GABA and ACh release is essential for the occurrence of SD. Nevertheless such release suggest that these neurotransmitters could influence the characteristics of SD manifestations in the retina.

Dual nature of the peaks of light scattered during spreading depression in chick retina.

In this work some physical properties of the optical concomitants of spreading depression (SD) are investigated in isolated chick retinas. The two peaks of light scattering during the phenomenon were studied at various wavelengths of the illuminating light. It was shown that the first fast increase in light scattering is highly dependent on the wavelength and seems to be a "blue" scattering. The second slow peak, not so dependent, behaves as a "white" scattering. This implies in different underlying physical mechanisms for the two peaks. Based in this dissimilar behavior, using appropriate optical device and adding some substances to the maintaining Ringer solution it could be observed that the death of tissue is accompanied by a light scattering change comparable to the first peak of SD. The opalescence of the second peak is more similar to the opalescence caused by addition of NaCl to the Ringer in which the fragment of retina is maintained.

Spreading depression in isolated spinal cord.

1. Isolated spinal cord of amphibians is able to express Leão's spreading depression (SD). 2. SD can be evoked when the spinal cord is in an appropriated medium. There are two conditions that favor its elicitation: hypotonicity and low [Cl-] levels in the extracellular microenvironment. 3. Spinal cord SD is accompanied by a stereotyped negative extracellular voltage transient of 17.3 +/- 4.9 (standard deviation) mV amplitude and 1.2 +/- 0.5 min duration concomitantly with a reversible [K+]o increase up to 21.1 +/- 4.6 mM in the extracellular fluid. 4. Potassium salt solution can trigger SD waves that spread at 12 +/- 4.7 mm/min. 5. SD waves occur "spontaneously" in preparations superfused by Ringer solutions with very low [Cl-] (< 20 mM). The frequency of these waves decreases, and they may stop if [Mg2+] is increased.

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.