Daytime modulation of cortical spreading depression according to blood glucose levels

The aim of this study was to investigate the effects of daytime and blood glucose levels on the propagation of cortical spreading depression (SD). Thirty-nine male Wistar rats were used. Animals were housed 5 per cage with a 12-h, light–dark cycle (lights on at 0600 h). Food and water were available ad libitum, and animals were fasted the night before the experiments. Cortical SD was recorded continuously for 3 h using Ag–AgCl agar-Ringer electrodes placed on the parietal cortex. Every 20 min, SD was elicited by 2% KCl stimulation of the frontal cortex for 1 min. After 1 h of SD-recording, blood glucose levels were measured, and animals were injected intravenously either with glucose (40% solution, 1 mL), insulin (0.3 U/100 g of body weight), or mannitol (20% solution, 1 mL). In the middle of the light period, which corresponds to zeitgeber time (ZT)5, 8 animals received glucose, 7 received insulin, and 6 received mannitol. In another experimental set, glucose or insulin was administered at ZT12 (at the end of the light period); 12 rats received glucose, and 6 received insulin. All the animals that received glucose were hyperglycaemic (P < 0.01), and the hyperglycaemia was less pronounced in the ZT12 group (P < 0.05; Student's t-test). Insulin induced acute hypoglycaemia in animals of both groups (ZT5, P < 0.02; ZT12, P < 0.05; Student's t-test). Glucose injection at ZT5 reduced SD, whereas the insulin ZT5 group showed increased SD propagation (ANOVA, P < 0.05 and 0.01, respectively). Neither glucose nor insulin injection changed SD velocity at ZT12. We concluded that blood glucose levels change the velocity of SD propagation and that these effects are influenced by the daytime. Dark periods seemed to produce a resistance to cortical SD propagation.     

Pupillary paralysis evoked by thalamic spreading depression in rats

Summary Spreading depression elicited in the thalamus of albino rats anesthetized with Dial by microinjection of 0.2–1.0 l 25% KCl is accompanied by a maximal pupillary dilatation of the paralytic type. The mydriasis starts immediately after KCl application, culminates about 90 sec later and then slowly subsides with a half time of about 5 min. Although with lateralized KCl application thalamic spreading depression reaches the ipsilateral lateral geniculate body approximately 1 min earlier than the contralateral one, the pupillary reactions are strictly synchronous in both eyes. Participation of the sympathetic fibres was ruled out by the observation that thalamic spreading depression evokes the same mydriasis in the encéphale isolé rats. Cortical spreading depression, on the contrary, elicits only slight and shortlasting mydriasis. Comparison of the spatial march of spreading depression through the thalamus with the time course of the pupillary reaction indicates that the maximum mydriasis is probably caused by direct invasion of the Edinger-Westphal nucleus by the spreading depression process.     

Effects of local anaesthetics on retinal spreading depression

Summary 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.Research supported by CNPq, FINEP and CEPG/UFRJ     

Excitation and depression of cortical neurones during spreading depression

Summary Variations in the excitability of individual cortical neurones during the invasion of spreading depression (SD) have been monitored by observing the alterations of spontaneous and L-glutamate-induced firing. Invasion of many neurones during SD is marked by a brief burst of firing which occurs concurrently with the onset of the negative slow extracellular potential. Other neurones do not fire, although the microelectrode records a negative slow wave. Depression of glutamate-induced and spontaneous firing follows and may last for several minutes. The initial loss of excitability of those neurones that discharge during SD invasion may be due to excessive depolarization. This phase is rapidly succeeded by a period of depressed excitability, during which the neurones can be invaded by an antidromic spike or excited by increased amounts of L-glutamate. These findings indicate that SD propagation initially involves the release of an excitant substance, possibly glutamic acid. The continuing effects of SD are due to the reduction in cell excitability. As many neurones are depressed without undergoing an initial excitation, it appears that a depressant substance is also involved. This may be gamma-aminobutyric acid.     

Effects of short-term and long-term treatment with medium- and long-chain triglycerides ketogenic diet on cortical spreading depression in young rats

The ketogenic diet (KD) is a high fat and low carbohydrate and protein diet. It is used in the clinical treatment of epilepsy, in order to decrease cerebral excitability. KD is usually composed by long-chain triglycerides (LCT) while medium-chain triglycerides (MCT) diet is beginning to be used in some clinical treatment of disorders of pyruvate carboxylase enzyme and long-chain fatty acid oxidation. Our study aimed to analyze the effects of medium- and long-chain KD on cerebral electrical activity, analyzing the propagation of the phenomenon of cortical spreading depression (CSD). Three groups of weaned rats (21 days old) received, for 7 weeks, either a control (AIN-93G diet), or a MCT-KD (rich in triheptanoin oil), or a LCT-KD (rich in soybean oil). They were compared to another three groups (21 days old) receiving the same diets for just 10 days. CSD propagation was evaluated just after ending the dietary treatments. Results showed that short-term KD treatment resulted in a significant reduction of the CSD velocity of propagation (control group: 4.02 ± 1.04 mm/min; MCT-KD: 0.81 ± 1.46 mm/min and LCT-KD: 2.26 ± 0.41 mm/min) compared to the control group. However, long-term treatment with both KDs had no effect on the CSD velocity (control group: 3.10 ± 0.41 mm/min, MCT-KD: 2.91 ± 1.62 mm/min, LCT-KD: 3.02 ± 2.26 mm/min) suggesting that both short-term KDs have a positive effect in decreasing brain cerebral excitability in young animals. These data show for the first time that triheptanoin has an effect on central nervous system.     

The minimum volume of depolarized neural tissue required for triggering cortical spreading depression in rat

Summary Cortical spreading depression (CSD) was evoked in anaesthetized rats by intracortical microinjection of 3.4·10^–8 mol KCl (the single injection threshold T₁). With two simultaneous injections at 1 mm tip separation 59% T₁ had to be applied to each point to elicit CSD. For interfocal distances 2, 3 and 4 mm the double injection thresholds T_1,2 were 65%, 74% and 97% of T₁ respectively. The spatial summation effect was still significant at 3 mm and undetectable at 4 mm tip separation. Recording electrodes placed 1–3 mm from the point of injection detected local slow potential changes which attained with subthreshold KCl injections 16% of the maximum CSD negativity at the 1.5 mm distance. The threshold amount of KCl required to trigger CSD at different intervals (30–480 sec) after initial injection of 0.8 T₁, exponentially increased with time from 28.2% t₁ at 30 sec to 88.9% at 480 sec with the slope 16.8% T₁ for a twofold increase of the interinjection interval. Quantitative analysis of results based on equations describing diffusion from an instantaneous point source indicates that the critical volume of depolarization is reached 61 sec after injection. According to the value of the diffusion coefficient of potassium the critical K+ concentration lies between 45 and 12 mequ/l and is reached at a distance of 600–950 from the injection.Visiting scientist from Department of Physiology, Gifu University, School of Medicine, Gifu City, Japan.     

Effects of glycine receptor antagonism on spreading depression in the rat

Spreading depression (SD) in the rat brain is inhibited by (NMDA) receptor antagonists. Because the NMDA receptor glycine recognition site must be occupied for activation of the NMDA ionophore, we hypothesized that antagonism of the glycine receptor would also affect SD. In halothane anesthetized rats, SD was initiated by electrocortical stimulation. Both the initiation threshold and propagation rate of SD were recorded. Rats were then administered the glycine receptor antagonist ACEA-1021 (or vehicle only) or ketamine and the stimulus was repeated. Rats were then killed and terminal depolarization was observed for. Ketamine completely inhibited initiation of SD. In contrast, all rats treated with ACEA-1021 exhibited SD. While ACEA-1021 caused no difference in the stimulation threshold for SD, propagation rate was decreased in a dose-dependent fashion. Terminal depolarization occurred in all rats. Antagonism of glycine at the NMDA receptor recognition site did not inhibit initiation of SD but played a modulatory role in the mechanism of its propagation.     

Audiogenic seizures associated with a cortical spreading depression wave suppress spike-wave discharges in rats

To study the role of the cortex and sub-cortical structures in the generation of epileptic spike-wave discharges in more detail, cortical and striatal activity was eliminated by the induction of spreading depression in a non-invasive way. EEG and DC potentials were recorded from the cortex and striatum of WAG/Rij rats. Several of these rats show two forms of generalised epilepsy: spontaneously occurring non-convulsive absence seizures, together with convulsive audiogenic seizures. The latter can be evoked by a brief sound stimulation, provoking a fit of wild running, which is regarded as the first phase of an audiogenic seizure. In a majority of fits the cortical DC potential does not show main changes, while the spontaneously occurring spike-wave discharges are briefly suppressed for some minutes. In a minority of fits, however, audiogenic seizures are associated with a spreading depression wave, clearly expressed in the cortical DC potential. This wave is bilaterally initiated in the cortex and propagates to the caudate nucleus of the striatum. In these cases spontaneously occurring spike-wave discharges are fully suppressed for about 1 h. It is suggested that cortical spreading depression, triggered by a short audiogenic seizure, induces a long-lasting suppression of spike-wave discharges. These results are in line with the concept that spike-wave discharges are originally initiated in the cortex, as proposed by the 'cortical focus' theory. The precise role of the striatum remains less clear, although this structure seems not to play a pivotal role in spike-wave generation.     

Direct evidence of inter-hemispheric modulation by callosal fibers: a cortical spreading depression study in well-nourished and early-malnourished adult rats

Inter-hemispheric modulation has been extensively studied, as it is critical for human behavior and could be involved in the development of neuropsychiatric disorders, such as major depression, epilepsy and stroke. Malnutrition early in life can alter brain processes such as inter-hemispheric modulation and these alterations can persist into adulthood. Here, we used cortical spreading depression (CSD) as a neurophysiological parameter to investigate inter-hemispheric modulation in 19 well-nourished and 18 early-malnourished male adult rats. CSD was evoked on the right frontal region and monitored at two parietal points on the same hemisphere. After a 2 h baseline recording, fibers projecting from the left to the right hemisphere via the corpus callosum were cut with a longitudinal lesion in the contralateral cortex and recording continued for two more hours. The results show that (1) baseline CSD propagation velocities were higher in the malnourished rats, as compared to the respective well-nourished controls; (2) post-lesion velocities increased in both nutritional groups, as compared with the baseline values; (3) this CSD increase persisted 3–7 days after lesion, suggesting a lasting effect and (4) in the malnourished group the post-lesion CSD enhancement was of smaller amplitude. Furthermore, a midline lesion (callosotomy) in eight well-nourished rats similarly facilitated CSD, suggesting the involvement of callosal fibers on this effect. No differences were found in sham-operated rats (21 well-nourished and 21 malnourished), as well as in a superficial (1 mm deep) contralateral lesion group (8 well-nourished). Results support the hypothesis of a lasting inhibitory contralateral influence on CSD propagation, which is attenuated, but not abolished, by early malnutrition.     

Lateralization of learned taste aversion by cortical spreading depression

Male Sprague-Dawley rats which were taught a strong aversion to ingestion of lithium chloride under unilateral cortical spreading depression failed to show this aversion when spreading depression was changed to the opposite hemisphere. Control rats which learned the aversion with both hemispheres functional show a very strong aversion to LiCl in subsequent tests with either both hemispheres functional or one hemisphere under spreading depression. These results indicate that cortical participation is important for learning of a taste aversion.     

Time-varying spreading depression waves in rat cortex revealed by optical intrinsic signal imaging

This study aimed to investigate the variation of propagation patterns of successive spreading depression (SD) waves induced by K+ in rat cortex. SD was elicited by 1 M KCl solution in the frontal cortex of 18 Sprague-Dawley rats under alpha-chloralose/urethane anesthesia. We applied optical intrinsic signal imaging (OISI) at an isosbestic point of hemoglobin (550 nm) to examine regional cerebral blood volume (CBV) changes in the parieto-occipital cortex. In 6 of the 18 rats, OISI was performed in conjunction with DC potential recording of the cortex. CBV changes appeared as repetitive propagation of wave-like hyperemia at a speed of 3.7+/-0.4 mm/min, which was characterized by a significant negative peak (-14.3+/-3.2%) in the reflectance signal. Among the observed 186 SDs, the first wave always propagated through the entire imaged cortex in every rat, whereas following waves were likely to bypass the medial area of the imaged cortex (partially propagated waves, n=65, 35%). Correspondingly, DC potential shifts showed non-uniform in the medial area, and they seemed closely related to the changes in reflectance. For partially propagated SD waves, the mean time interval to the previous SD wave (217.0+/-24.3 s) was significantly shorter than for fully propagated SD waves (251.2+/-29.0 s). The results suggest that the propagation patterns of a series of SD waves are time-varying in different regions of rat cortex, and the variation is related to the interval between SD waves.     

Learned taste aversion induced by cortical spreading depression.

Male albino rats were cannulated and placed on a 24 hr water deprivation schedule. The animals were allowed 10 min access to water in a large animal cage for 5 days. On the sixth day of deprivation the animals were randomly divided into 6 groups and given either 12 percent KCl, 25 percent KCl, or Ringers solution applied unilaterally or bilaterally to the cortex immediately after access to 8 percent sucrose. On the seventh day of deprivation, each rat was placed in a two-choice situation with the sucrose solution and water. Only the unilateral and bilateral 12 percent KCl groups developed an aversion to the sucrose. These results indicate that CSD has aversive as well as amnesic properties, there exists a gradient of amnesia, dependent on concentration, and that the cortex is not necessary for learning a taste aversion.     

Effects of cortical spreading depression on behaviors elicited by hypothalamic stimulation in rats

Eating or drinking was elicited via electrical stimulation of the lateral hypothalamus of rats. To determine the effects of single waves of cortical spreading depression on elicited behavior, 1,1 animals were tested with 10 sec trains of stimulation at 20 sec intervals and administered 1–5 μl of 6% KCl through cannulae onto the frontal or occipital cortices of the contralateral or ipsilateral hemispheres. Under single waves of bilateral spreading depression the elicited consummatory behavior was blocked, but other signs of arousal were intact. Single waves of unilateral depression increased the onset-latencies and decreased the duration of elicited behavior, depending on the magnitude of the electrical stimulation. Anterior cortical injection blocked elicited behavior earlier than occipital injection. No differences were apparent between ipsilateral and contralateral injection of KCl. In some animals single waves of unilateral depression were accompanied by a reversible shifting from one elicited consummatory response to another, usually from eating to drinking. The results were interpreted to reflect cortico-hypothalamic interactions in the control of motivated behavior.     

Memory confinement versus generalization decrement on retrograde amnesia produced by cortical spreading depression

An examination of memory confinement vs. generalization decrement under unilateral cortical spreading depression was undertaken using a licking suppression to a tone previously paired with a shock. In Experiment I, rats that were trained and tested under normal cortical conditions showed an impairment of memory when KCl-induced depression was given to an unilateral hemisphere within 15 min after the training. In Experiment II, where rats were trained under normal cortical conditions followed by unilateral depression and tested under unilateral cortical depression, the degree of impairment was more severe when the test was performed under depression shifted to the other hemisphere than under depression to the same hemisphere. The results taken together support the memory confinement theory.     

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 ∼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. Received: 1 August 1996 / Accepted: 5 November 1996     

The influence of repeated spreading depression-induced calcium transients on neuronal viability in moderately hypoglycemic rats

The calcium transients which are associated with spreading depression (SD) do not lead to neuronal necrosis, even if the SDs are repeated over hours. We have previously shown that a restriction of energy production by moderate hypoglycemia prolongs the calcium transients during SD. In the present experiments, we explored whether such prolonged transients lead to neuronal necrosis. To that end, SDs were elicited for 2 h by topical application of KC1 in anesthetized rats at plasma glucose concentrations of 6, 3, and 2 mM. The animals were then allowed to recover, and they were studied histopathologically after 7 days. In two other groups, hypoglycemic coma of 5 min duration (defined in terms of the d.c. potential shift) was induced either without or with a preceding train of SDs. These animals were also evaluated with respect to histopathological alterations. SDs elicited for 2 h did not give rise to neuronal damage when elicited at plasma glucose concentration of 6 mM, and, of the animals maintained at 3 and 2 mM, only a few animals showed (mild) damage. In general, therefore, repeated SDs with calcium transients of normal or increased duration fail to induce neuronal damage. The results suggest that, if calcium transients are responsible for a gradual extension of the infarct into the penumbra zone of a focal ischemie lesion some additional pathophysiological factors must be present, such as overt energy failure, acidosis, or microvascular damage. A hypoglycemia-induced calcium transient of 5 min duration gave no or only moderate neuronal damage. However, if a series of SDs were elicited in the precoma period, the damage was exaggerated. The results demonstrate that, normally, brain tissues can tolerate a hypoglycemic calcium transient of up to 5 min duration without incurring neuronal necrosis. They also demonstrate that calcium transients preceding a subsequent insult involving calcium influx into cells exaggerate the damage incurred. It is tentatively concluded that the priming transients alter membrane properties in such a way that cellular calcium homeostasis is perturbed.     

Involvement of GABA and ACh in retinal spreading depression: Effects of “low calcium — high magnesium” solutions

Summary 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 supervising 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.     

Pavlovian conditioning of eating induced by spreading depression in cortex, striatum and hippocampus of rats.

A single wave of unilateral spreading depression in the cortex, hippocampus or caudate nucleus of rats elicits eating after 2-6 min. The present experiments provide evidence that such spreading depression-induced eating can be classically conditioned to a complex conditioned stimulus. A wave of spreading depression was triggered by injection of 0.5-2.0 mul of 25 percent KCl solution. In a first experiment successful conditioning was demonstrated in 20 rats, involving 7 cortical, 5 caudate and 8 hippocampal spreading depression sites. Four animals failed to show any conditioned eating. A control group of 8 animals, in which cortical spreading depression did not induce eating, showed no increase in eating in the presence of the CS after conditioning trials. A second experiment, which included pseudoconditioning and NaCl control groups, confirmed the results obtained in Experiment 1. Classical conditioning was successful in 11 animals, involving 6 cortical and 5 caudate spreading depression sites. Neither the pseudoconditioning (14 animals, involving 8 cortical and 6 caudate spreading depression sites) not the NaCl control group (8 animals, all with cortical sites), showed eating in the presence of the CS. In both experiments the conditioned eating underwent gradual extinction.     

在体皮层扩散性抑制动物模型的建立

目的：用KCl和针刺分别在鼠皮层诱导皮层扩散性抑制（CSD），建立大鼠CSD动物模型，为进一步研究奠定基础。方法:采用Spangue-Dawley大鼠25只，随机分为针刺诱导组（n=10）、KCl诱导组（n=10）和NaCl对照组（n=5），在大鼠颅骨上分别钻磨出诱导窗口和观测窗口，分别在诱导窗口用针刺和滴加KCl诱导大鼠脑CSD，对照组在诱导窗口滴加NaCl，并用电生理记录和光学成像的方法来描记CSD的产生与传播，并分析其特点。结果:针刺诱导组和KCl诱导组在观测窗口均可以观察到向外周扩散的弧形波，观测窗口均可以观察到明暗相间的弧形波向远处扩散，针刺诱导的CSD波呈典型的同心圆样向四周均匀扩散，KCl诱导的CSD波则是呈不规则的圆弧形波向外周扩散，针刺组每次只诱发1次CSD波，而KCl组则可以诱导多次CSD波。在产生CSD波的同时，伴随着去极化电位的产生，而在NaCl对照组没有这一现象的发生。结论:用本方法制作CSD动物模型，简单易行，可以用OISI直观观测CSD的产生和发展，并与电生理观测到的去极化波一致，适于在体研究CSD，为进一步研究CSD的发生机制及其可能的作用提供了一种有效手段。