A simple biological way to screen dopaminergic agonists.

The velocity of propagation of the in vitro retinal model of spreading depression is very sensitive to changes in the ionic composition of the extracellular medium and also to the the addition of different drugs. 10 microM of SKF 38393, a D1 agonist, increases the velocity of propagation of the wave while 10 microM of Quinpirole, a D2 agonist, decreases it. Both changes are blocked by their specific antagonists, SCH23390 and 1-sulpiride respectively. This assay can biologically screen potential dopaminergic drugs indicating its physiological D1 and/or D2 preferred effect in the tissue for future analysis by other different methodologies.

Cortical slow potential changes during convulsions induced by maximal electroshock or penicillin focus.

We have described the occurrence in freely moving gerbils of slow potential changes (SPC) in two different models of experimental epilepsy: 1) maximal electroshock and 2) bilateral epileptic foci induced by penicillin. SPC is considered a by-product of epileptiform activity in both models and correlates to the SPC which occurs during spreading depression. In the first model there develops a cortical SPC simultaneous with a depression of EEG activity, although there is no propagation of the wave. We suggest that a non-propagated multifocal depression (MD) occurs in the MES model. In the model of focal epilepsy, all requirements are fulfilled, and the SPC is characterized as the one which occurs during spreading depression propagating with an average velocity of 8 mm/min.

Transient changes in energy metabolites and intracellular pH during spreading depression in the chick retina.

The in vitro preparation of the chick retina can be used to show the occurrence of transient changes in the intracellular pH and of energy metabolites which occurs during spreading depression (SD). There is an initial increase in intracellular pH associated with elevated values for ADP, P-Creatine, lactate and pyruvate, an intermediary acid shift with increases in ATP values and decreases in ADP, and a late alkaline rebound where P-Creatine levels are reduced and the content of ADP and lactate are elevated. These transient changes in intracellular pH observed during SD, when correlated to the levels of energy metabolites, supports the hypothesis that the intracellular pH can be used by the tissue as a mechanism to rapidly modify the metabolic activities of neurons and glial cells. We suggest that the first alkaline shift is caused by glial cells and the intermediary acid shift by neurons. However, a specific cell could not be pointed out as responsible for the late alkaline shift but it could explain the refractoriness of the neurons during the 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.

Opioid mechanisms involved in the slow potential change and neuronal refractoriness during cortical spreading depression.

The slow potential change (spc) accompanying spreading depression (SD) was studied in rats and in a seizure-sensitive strain of Mongolian gerbil under three different experimental paradigms, each involving the use of naloxone. Gerbils undergoing electroconvulsive shock treatment displayed SD during the post-ictal phase, which was blocked by the intraperitoneal (i.p.) administration of naloxone (20-50 mg kg-1). Topical application of naloxone to the exposed cortex of the anaesthetized gerbil and rat blocked the spc of SD evoked by KCl. Microiontophoretic ejection of naloxone during extracellular recordings reversed cell refractoriness following the spc, demonstrated by the observation of a maintained sensitivity to iontophoretic pulses of glutamate. The results suggest a possible involvement of naloxone-sensitive processes in the mechanism responsible for cortical SD.

Light-induced changes in energy metabolites, guanine nucleotides, and guanylate cyclase within frog retinal layers.

Freeze-dried sections were prepared from retinas of frogs which were dark-adapted or exposed to varying periods of light. Samples of the discrete layers were dissected, weighed, and analyzed for energy metabolites, guanylate compounds, and the enzyme guanylate cyclase. ATP and P-creatine were measured in both dark- and light-adapted retinas. There was a gradient in ATP and P-creatine levels in dark-adapted retinas, with the lower concentrations in the photoreceptors, and increasing concentrations in the inner retina. After light adaptation, concentrations increased, an observation which supports the concept that transmitter release occurs in the dark and ceases in the light. The sum of GTP plus GDP, GDP, and cyclic GMP were analyzed in dark-adapted retinas and after exposure to 2 min or 2 h of room light. GDP was rather uniformly distributed in the retinal layers, was increased by 2 min of light in all layers but the outer nuclear, and remained elevated at 2 h in the inner retina. GTP values showed a marked localization in the outer nuclear layer, which increased after 2 min or 2 h of illumination; in all other layers GTP was decreased by light. Cyclic GMP in the dark was highest in the photoreceptor cells, decreasing to one-third after 2 min of light; there were significant increases in the outer plexiform and inner nuclear layers at this time. Cyclic GMP remained low in the photoreceptor cells even after 2 h of light, while the inner layers returned to dark values. Guanylate cyclase, like cyclic GMP, was largely confined to the photoreceptor cells and showed a maximal increase after 2 min of light exposure.

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.