The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca]0 and [K]0 in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

Potassium activity and changes in glial and neuronal membrane potentials during initiation and spread of afterdischarge in cerebral cortex of cat

Trains of electrical stimuli of increasing intensity were applied to the surface of the anterior suprasylvian gyrus to produce afterdischarges (AD) that remained localized or spread to a recording site 2 cm posteriorly in the same gyrus. The local afterdischarge was associated with a negative steady potential (SP) shift and increases in [K⁺]₀ that were maximal at or near the surface and gradually decreased in magnitude at deeper layers of the cortex.During spreading AD, recordings at the stimulus site showed a secondary increase in both the SP shift and [K⁺]₀ at about 400 to 1400 μm below the cortical surface. As the AD invaded the distant recording site it was associated with a comparable negative SP shift and increase in [K+]o. Neither the appearance of local AD nor its spread to the distant recording site were contingent upon critical elevations of [K⁺]₀.During secondary increases in [K+]o glial depolarizations were less than would be predicted if the membrane potential were determined solely by changes in the ratio of intra- to extracellular [K⁺]. Smaller deviations from the Nernst equation also occurred during the repolarizing phase of glial depolarization produced by weaker stimuli that did not produce a secondary increase in [K⁺]₀. Only immediately after the stimulus train did the relationship between glial depolarization and [K⁺]₀ approach the expected slope of a K⁺ electrode.Simultaneous intracellular recording of neurons and [K⁺]₀ did not show an increase in neuronal firing rate or membrane depolarization to account for the additional increase in [K⁺]₀ during spreading AD.The possible sources of the secondary increase in [K⁺]₀ and the significance of the failure of glia to depolarize to levels predicted by the Nernst equation are discussed.     

Effects of brain oxygenation on metabolic, hemodynamic, ionic and electrical responses to spreading depression in the rat

The effect of cortical spreading depression (CSD) on oxygen demand (extracellular K(+)), oxygen supply (cerebral blood flow - CBF) and oxygen balance (mitochondrial NADH) was studied by a special multiprobe assembly (MPA), during hypoxia and partial ischemia. The MPA was constructed and applied to monitor the CSD wave from its front line until complete recovery, continuously and simultaneously. CSD under hypoxia or partial ischemia led to an initial increase in NADH levels and a further decrease in CBF during the first phase of the CSD wave, indicating a decrease of tissue capability to compensate for an increase in oxygen demand. Furthermore, the special design of the MPA enabled identifying the close interrelation between oxygen demand, supply and balance during CSD propagation. In conclusion, brain oxygenation was shown to have a clear effect on tissue responses to CSD.     

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells

Two glutamate receptor agonists, NMDA (N-methyl-d-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na⁺/K⁺-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na⁺/K⁺-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na⁺/K⁺-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.     

Intracellular energy failure does not underlie hyperthermic spreading depressions in immature rat hippocampal slice

Hyperthermic spreading depression (HSD) in immature rat hippocampal slices is mediated by Na+/K(+)-ATPase failure. Here, we test whether depleting intracellular ATP serves as a possible mechanism for HSD genesis. Results indicate that (1) pre-incubation with 3 mM creatine for 3 h failed to prevent hyperthermic spreading depression occurrence; and (2) intracellular ATP concentration doubled during experimental hyperthermia. This study suggests that HSD is not be mediated by depletion of intracellular ATP during hyperthermia.     

Stimulation induced changes in extracellular free calcium in normal cortex and chronic alumina cream foci of cats

Changes in extracellular [Ca²⁺]₀ (δ Ca) were measured with ion selective microelectrodes in the sensorimotor cortex of cats, surrounding alumina cream lesions and in the contralateral homotopic cortex. The lesions were produced by topical application of alumina cream 6 months-6 years prior to experiments. In normal cortex, stimulus induced reductions of [Ca²⁺]₀ were found to be maximal (up to 0.45 mM) in depths of 200–300 μm below the cortical surface. At depths of 600 μm and more below the cortical surface, [Ca²⁺]₀ usually rose by up to 0.2 mM above baseline. In the vicinity of the chronic lesion as well as in contralateral cortex [Ca²⁺]₀ fell initially during stimulation in all depths. close to the lesion δ Ca was as high as 0.8 mM and sites of maximal δ Ca were found to be located deeper in the cortex. About 5 mm from the scar as well as in the contralateral homotopic cortex, maximum δ Ca levels were found in a depth of 200–300 μm. It is suggested that Ca²⁺ dependent mechanisms are involved in epileptogenesis in chronic epileptic foci.     

Regional changes in tissue pH and glucose content during cortical spreading depression in rat brain

After eliciting a single passage of spreading depression in rat brain cortex, regional alterations in glucose content and tissue pH were studied in relation to changes in cortical steady potential. Migration of spreading depression into non-invaded cortex was accompanied by a drastic decrease in glucose concentration and tissue pH which persisted longer than the transient depolarization of cortical cells. This delay in metabolic recovery is probably accounted for by severe tissue acidosis which results from the stimulation of anaerobic glycolysis.     

Erythrocyte membrane sodium — potassium adenosine triphosphatase activity in affective disorders

Summary Erythrocyte membrane Na⁺,K⁺-ATPase activity was studied in drug naive patients with bipolar (BP) mania (n=62) and unipolar (UP) depression (n=60) and normal controls (n=66). Compared to controls there was a significantly decreased Na⁺,K⁺-ATPase activity in UP depressives but no change in BP manics. However, lithium treatment caused a significant increase in Na⁺,K⁺-ATPase activity although there was no correlation between plasma lithium levels and enzyme activity. Plasma cortisol correlated inversely with Na⁺,K⁺-ATPase in UP depressives. Interestingly, the lithium responders [<50% Beck Rafaelson's Mania Rating Scale (BRMS) score] showed a significant increase in Na⁺,K⁺-ATPase activity compared to lithium nonresponders (>50% BRMS score). These observations indicate that monitoring of Na⁺,K⁺-ATPase activity during lithium therapy is useful to predict a therapeutic response.     

NaV1.5 sodium channels in a human microglial cell line

Microglial cells are the major immuno-competent cells in the mammalian brain where they play a crucial role in maintaining the CNS environment in the face of various potentially pathological insults. We have used electrophysiological and pharmacological methods to study a microglial cell line (C13-NJ) derived from the human CNS. In whole-cell patch clamp experiments we identified an inward current that exhibited biophysical hallmarks of a classical voltage-gated Na⁺ channel. This identification was confirmed by further experiments in which the current was eliminated by removal of Na⁺ from the bathing medium. Relatively weak inhibition by TTX (30 ± 3% at 500 nM) and sensitivity to 100 μM Zn²⁺ suggested that this current was predominantly mediated by the cardiac sodium channel isoform Na_V1.5. Sodium current density was not altered by treatment with either lipopolysaccharide or beta-amyloid 1–42. The presence of the Na_V1.5 subunit in microglial cells is discussed with respect to its reported roles in phagocytosis, proliferation and migration of other non-cardiac cells.     

Factors affecting the oxygen balance in the awake cerebral cortex exposed to spreading depression

Oxygen balance was evaluated in the cerebral cortex, using the surface fluorometry technique of the intramitochondrial NADH redox state, exposed to various physiological and pathological situations. Using flexible fiber optic light guide, connected to the brain surface via a cemented holder, the measurements were done continuously from the awake rat and gerbil. In few experiments the NADH redox state was correlated with the electrical and ionic activity (measured by surface K⁺ and DC electrodes). Three different animal models were used in the study: the adult rat, the very young rat (20 g) and the adult gerbil. Those 3 models were used in studying the effect of hypoxia, partial ischemia, and anesthesia on the metabolic and ionic activities measured from the awake brain. Spreading cortical depression (elicited by topical KCl solution) was used as a standard stimulation of the ionic and metabolic activities of the cerebral cortex. Two typical metabolic responses to spreading depression (SD) were recorded, namely ‘oxidation cycle’ and ‘reduction cycle’ depending upon the ability of the tissue to compensate for the extra amount of oxygen needed for the higher mitochondrial activity.It was found that the adult rat brain showed oxidation cycles in most conditions (besides partial ischemia), while the young rat and the gerbil brains were much more sensitive to the various perturbations of the brain and exhibited reduction cycle (as a response to SD) under all pathological situations tested.We conclude from our detailed studies that the type of response to SD, as measured by NADH surface fluorometry, represents the oxygen balance which exists in the tissue under various conditions.     

Electroencephalographic spreading depression and concomitant behavioral changes induced by intrahippocampal injections of ACTH1–24 andd-Ala-met-enkephalinamide in the rat

ACTH_1–24 (0.5or10 μg 0.17or3.45nmol) andd-Ala²-Met-enkephalinamide (DAME; 10 μg 17.05nmol) were injected unilaterally into the hippocampus of freely moving rats to examine their effects on EEG activity, DC potentials and behavior. In 85% of the rats DAME elicited spreading depression (SD) with epileptiform discharges preceding and following the wave of SD. The following behavioral changes were recorded. DAME- and KCl-induced SD were accompanied by an increase in locomotor activity and wet-dog shaking behavior, which occurred only during the period of SD. After a wave of SD induced by DAME a biphasic pattern of activity, consisting of an initial depression in locomotion followed by hyperactivity, appeared in 59% of the rats.ACTH_1–24 elicited SD in 13% of the rats tested. Neither the dosage of ACTH_1–24 nor the strain of rats influenced the occurrence of SD and the incidence of ACTH-induced grooming behavior. SD induced by KCl also resulted in excessive grooming comparable to that induced by ACTH_1–24. In the case of KCl-induced SD, grooming began directly after the injection of KCl and was frequently interrupted by short periods of locomotion. ACTH-induced grooming had a later onset and episodes of stretching and yawning were observed. It can be concluded that the behavioral effects of the injection of DAME are unspecific responses to SD and seizure activity. However, ACTH-induced grooming is not solely a byproduct of SD, since it occurred also in the absence of SD.     

A pharmacological model for studying the role of Na gradients in the modulation of synaptosomal free [Ca]i levels and energy metabolism

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Axonal transport of the voltage-dependent Na channel protein identified by its tetrodotoxin binding site in rat sciatic nerves

Na+ channels levels were measured in different segments of rat vagus and sciatic nerves by in vitro binding using a tritiated ethylene-diamine tetrodotoxin derivative ([3H]en-TTX). Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve indicating an anterograde and retrograde axoplasmic transport of Na+ channels. Accumulation of Na+ channels at the ligature was time-dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of a ligature was also visualized by autoradiographic studies in longitudinal sections of sciatic nerves using [3H]en-TTX.     

Quisqualate, kainate and NMDA can initiate spreading depression in the turtle cerebellum

This study evaluated the role of excitatory amino acid (EAA) receptor activation in spreading depression (SD), using the in vitro turtle cerebellum as a model system. SD was triggered by electrical stimulation or by elevated K+ after the cerebellum had been conditioned for at least 30 min with physiological saline in which most of the chloride had been replaced by propionate. SD was recognized as a transient (1-3 min) negative shift of extracellular potential accompanied by depression of evoked potentials (15-30 min) and an increase of extracellular K+ up to 60 mM, which spread across the cerebellum at rates of 1-7 mm/min. SD usually commenced in the granular layer, which apparently contains the 3 major EAA receptor subtypes, quisqualate, kainate and N-methyl-D-aspartate (NMDA), then subsequently spread to the molecular layer, which is largely free of NMDA receptors. Glutamate, aspartate, NMDA, kainate and quisqualate all triggered SD. Kynurenic acid and 2-aminophosphonovaleric acid (APV) inhibited SD under certain conditions further suggesting involvement of EAA receptors. The initiation of SD was blocked by high Mg2+ and facilitated in low extracellular Mg2+, which also eliminated the delay in molecular layer SD onset. Our data suggest that no one EAA receptor subtype is singly responsible for SD.     

CNS control of active sodium transport in muscle during progressive hypokalemia in the rat

The degree of plasma hypokalemia was graded with the duration of a potassium deficient diet. The intracellular Na⁺ and K⁺ contents ([Na]_i and [K]_i) of the innervated soleus muscle of hypokalemic rats were determined and plotted as a function of plasma K⁺ concentration following a potassium deficient diet during 1–8 weeks. The progression of plasma hypokalemia was highly correlated with both [Na]_i accumulation and [K]_i loss in the muscle. Denervation of muscles in the hypokalemic rat resulted in a rapid activation of the Na-pump in the denervated soleus muscle regardless of the degree of plasma hypokalemia. This pump activation in the denervated muscle was chronically maintained throughout the hypokalemia. Restoration of a normal, K⁺ containing, diet for 4 days in hypokalemic rats resulted in a complete recovery of normal Na⁺ and K⁺ contents in both the innervated muscle and plasma, while the [Na]_i and [K]_i in the denervated muscle was unaffected. The relationship between the CNS-induced inhibition of the muscle Na-pump and the plasma K⁺ levels in hypokalemic rats is discussed.     

Block of (Na,K)ATPase with ouabain induces spreading depression-like depolarization in hippocampal slices

We used ouabain (100 microM) to block Na+,K(+)ATPase of in vitro rat hippocampal slices. This treatment was sufficient to cause the sudden depolarization that is the hallmark of both spreading depression (SD) and of the SD-like anoxic depolarization (AD). This depolarization was accompanied by a large and sudden increase in [K](o), also reminiscent of that observed during both SD and AD. Ouabain-induced SD did not require a complete inactivation of Na+,K(+)ATPase, as it occurred when the enzyme was still capable of providing recovery of both V(o) and [K](o). The data indicate that functional inactivation of Na+,K(+)ATPase per se initiates events that lead to an SD-like AD. This ouabain-induced depolarization was not affected by block of synaptic transmission, instead it was abolished by hyperosmolarity of the extracellular space. The possible relevance of these findings to the pathophysiology of AD is discussed.     

Regional changes of blood flow, glucose, and ATP content determined on brain sections during a single passage of spreading depression in rat brain cortex

Hemodynamic and biochemical substrate changes are associated with cortical spreading depression (CSD). Regional methods were used to measure blood flow, and glucose and ATP concentrations in intact brain sections in rats undergoing a single passage of cortical spreading depression. Changes were expressed as the percentages of the contralateral homotopic area of the unaffected cortex. A depression in tissue ATP content preceded the negative DC potential shift and ATP was reduced by 12% (P less than 0.01) despite unaltered blood flow and glucose concentration. When the negative shift of DC potential reached its maximum, glucose content decreased to 72% of control (P less than 0.01) and was accompanied by a further ATP decrease to 54%. When the cortical steady potential declined, blood flow was elevated twofold (P less than 0.01). The ATP content gradually returned to normal; however, cortical glucose concentrations remained at 55% of control values. The relationship of blood flow and glucose and ATP concentration with other known changes during spreading depression are discussed. With the advantage of higher resolution the provided techniques may be a useful tool for studies on hemodynamic and biochemical changes of other pathophysiologic conditions.     

Rat optic nerve: Disruption of gliogenesis with 5-azacytidine during early postnatal development

The normal sequence of gliogenesis in the rat optic nerve was disrupted by neonatal treatment with the mitotic inhibitor 5-azacytidine (5-AZ). This protocol caused a marked reduction in the number of glial cells, especially oligodendrocytes, seen in 11- and 14-day-old animals. Myelin formation was also greatly reduced in animals of this age compared to controls, but optic nerve axons appeared to be well preserved. Electrophysiological studies demonstrated similar excitability properties and activity-evoked [K+] changes in normal and 5-AZ-treated nerves prior to 5 days of age. However, in older nerves there were striking changes in the compound action potential and activity-dependent K+ accumulation in 5-AZ-treated nerves compared to controls. This simple model of disrupted central nervous system gliogenesis should prove useful in analyzing a variety of questions regarding neuroglial interactions including the role of glial cells in ionic homeostasis of brain extracellular space.     

Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.