Effects of serotonin on extracellular potassium concentration in the rat hippocampal slice

The effects of serotonin (5-HT) on extracellular potassium concentration ([K⁺]₀) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2–4 mM rise in [K⁺]₀ in the stratum pyramidale of area CA1. 5-HT caused a 0.6–1.1 mM rise in [K⁺]₀. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K⁺]₀ was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K⁺]₀ changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale.GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K⁺]₀ in stratum pyramidale. Extracellular calcium concentration measured with a Ca²⁺-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K⁺]₀.     

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Storage and release of endogenous and labelled GABA formed from [H]Glutamine and [C]Glucose in hippocampal slices: Effect of depolarization

To study the effect of depolarization on the synthesis, storage and release of GABA, hippocampal slices were incubated in 0.25 mM [³H]glutamine and 2.5 mM [¹⁴C]glucose in the presence of 3 or 50 mM K⁺. Total and labelled glutamine, glutamate and GABA contents were measured by high-performance liquid chromatography. Depolarization in the presence of Ca²⁺ led to a two-fold increase of labelled glutamate and a 3-fold increase of labelled GABA content originating from both labelled precursors. In the absence of Ca²⁺ and in the presence of 10 mM Mg²⁺, depolarization failed to increase labelled glutamate content and labelled GABA formation was increased by only 30%. Following superfusion with unlabelled 0.25 mM glutamine and 2.5 mM glucose a second depolarization with 50 mM K⁺ released twice as much labelled GABA from slices that had been incubated in the presence of 50 mM K⁺, than from those incubated in 3 mM K⁺. This difference remained unchanged in slices that were superfused with 1 mM aminooxyacetic acid, an inhibitor of GABA synthesis. The contribution of labelled GABA, especially of GABA derived from [³H]glutamine, to released GABA was significantly higher than to GABA stored in the slices. Results suggest that depolarization in the presence of Ca²⁺ results in increased glutamate and GABA synthesis from both glutamine and glucose and that part of GABA released by high K⁺ originates from preformed GABA stores.     

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.     

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.     

Effects of metabolic fragments of [Arg]-vasopressin on nerve growth in cultured hippocampal neurons

The effects of metabolic fragments of [Arg⁸]-vasopressin (AVP), [pGlu⁴, Cyt⁶]AVP (AVP_4–9), and desglycinamide-[pGlu⁴, Cyt⁶]AVP (AVP_4–8) on the growth of hippocampal neurons in culture were investigated in comparison with those of AVP. AVP_4–9 caused a significant increase in filopodial length following 96 h of exposure at concentrations higher than 300 nM. AVP_4–9 was more potent than AVP. AVP_4–8 also induced an increase in filopodial length, but this effect was less than that of AVP. The selective V₁ agonist [Phe², Ile³, Orn⁸]-vasopressin caused a significant increase in filopodial length, whereas the selective V₂ agonist [deamino-Cys¹, -Arg⁸]-vasopressin showed no such effect. OPC-21268, a vasopressin V₁ antagonist, blocked AVP and AVP fragment-induced increases in filopodial length. However, the V₂ antagonist OPC-31260 showed no such effect. A23187, a representative Ca ionophore, also increased filopodial length, and the A23187-induced increase in filopodial length was potentiated by AVP and AVP fragments. These results indicated that AVP_4–9 and AVP_4–8 increased filopodial length in cultured hippocampal neurons by activating V₁ receptors. Both phenomena induced by AVP_4–9 and AVP_4–8 were associated with intracellular calcium mobilization.     

Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia

Mutations in the presenilin genes PS1 and PS2, the major cause of familial Alzheimer's disease (FAD), are associated with alterations in Ca2+ signalling. In contrast to the majority of FAD-linked PS1 mutations, which cause an overload of intracellular Ca2+ pools, the FAD-linked PS2 mutation M239I reduces Ca2+ release from intracellular stores [Zatti, G., Ghidoni, R., Barbiero, L., Binetti, G., Pozzan, T., Fasolato, C., Pizzo, P., 2004. The presenilin 2 M239I mutation associated with Familial Alzheimer's Disease reduces Ca2+ release from intracellular stores. Neurobiol. Dis. 15/2, 269-278]. We here show that in human FAD fibroblasts another PS2 mutation (T122R) reduces both Ca2+ release and capacitative Ca2+ entry. The observation, done in two monozygotic twins, is of note since only one of the subjects showed overt signs of disease at the time of biopsy whereas the other one developed the disease 3 years later. This finding indicates that Ca2+ dysregulation anticipates the onset of dementia. A similar Ca2+ alteration occurred in HeLa and HEK293 cells transiently expressing PS2-T122R. Based on these data, the "Ca2+ overload" hypothesis in AD pathogenesis is here discussed and reformulated.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Effects of Hg and CH3Hg on Ca fluxes in rat brain microsomes

A permanent increase in cytosolic Ca²⁺ levels seems to be associated with various pathological situations which may result in cell death. Hg²⁺ and CH₃Hg⁺ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca²⁺-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg²⁺ and CH₃Hg⁺ inhibited Ca²⁺-ATPase and Ca²⁺ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg²⁺ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca²⁺ efflux (measured in the absences of Ca²⁺-ATPase ligands) was increased significantly by CH₃Hg⁺ and Hg²⁺. Again, the potency of Hg²⁺ was higher than that of CH₃Hg⁺. Blockers of Ca²⁺ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca t+ efflux induced by mercury compounds, possibly indicating that Ca²⁺ release occurs through Ca²⁺-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on Ca Z+-transport. However, if the enzyme was first inhibited with Hg²⁺ or CH₃Hg⁺ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca t+-transport bind to mercury with an extremely high affinity.     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.     

Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca stores in NG108-15 cells

Following mobilization with the inositol 1,4,5-trisphosphate (IP₃)-generating agonist bradykinin, Ca²⁺ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca²⁺ to refill. The process by which this store refills with Ca²⁺ was characterized by recording bradykinin-induced intracellular free Ca²⁺ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca²⁺ ATPase inhibitor, reversibly depleted intracellular Ca²⁺ stores in these cells, but did not recruit detectable Ca²⁺ influx, suggesting that these cells lack substantial capacitative Ca²⁺ entry. The paucity of voltage-sensitive Ca²⁺ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca²⁺ entry in nonexcitable cells might assist refilling IP₃-sensitive Ca²⁺ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca²⁺ entry in nonexcitable cells might inhibit the refilling of the IP₃-sensitive store in NG108-15 cells was explored. The IP₃-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca²⁺ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca²⁺ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca²⁺ stores by a plasmalemmal Ca²⁺ leak, this leak shares a pharmacology similar to the capacitative Ca²⁺ entry pathway described for nonexcitable cells.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Differential effect of a dihydropyridine derivative to Ca entry pathways in neuronal preparations

Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca²⁺ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca²⁺-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheochromocytoma cells, while the high K^+- stimulated Ca²⁺ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K⁺-stimulated Ca²⁺ influx and transmitter releases. It was then suggested that the voltage-dependent Ca²⁺ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.     

Dopamine changes the shape of action potentials in hippocampal pyramidal cells

Dopamine was found to have two electrophysiological effects on CA1 pyramidal cells in rat hippocampal slices. It increased the slow afterhyperpolarisation caused by a slow Ca2+-activated K+ conductance and it had an effect on action potentials that is postulated to be due to an increase in a fast Ca2+-activated K+ conductance. A given CA1 cell showed either one or both of the two responses to dopamine, or no response.     

Acid-sensing by carotid body is inhibited by blockers of voltage-sensitive Ca channels

The membrane potential hypothesis that the responses to hypercapnia of carotid chemosensory activity is mediated by voltage-gated Ca²⁺ channels was investigated by measuring directly the chemosensory output from rat and cat carotid bodies, perfused and superfused in vitro. We found that the inorganic and organic blockers of voltage-gated Ca²⁺ channels suppressed the hypercapnic responses, thereby supporting the membrane potential hypothesis.     

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.     

Differential development of Ca dynamics in presynaptic terminal and postsynaptic neuron of the rat auditory synapse

Postnatal development of Ca²⁺ influx and Ca²⁺ clearance capacity were investigated in the synapse of medial nucleus of the trapezoid body (MNTB) of rat with fura-2 fluorimetry. In contrast to the presynaptic terminal, Ca²⁺ dynamics does not basically change in the postsynaptic principal neuron developmentally. This differential development of Ca²⁺ dynamics between pre- and postsynaptic neurons might be crucial for the organized formation and functional maturation of this synapse.     

Differences between rats and rabbits in NMDA receptor-mediated calcium signalling in hippocampal neurones

In vivo microdialysis combined with the measurement of (45)Ca(2+) efflux from prelabelled hippocampus demonstrated a pronounced N-methyl-D-aspartate (NMDA)-evoked (45)Ca(2+) release to the dialysate in the rat dentate gyrus (DG) and CA1, whereas in rabbit a slight release of (45)Ca(2+) was observed only in the DG. In vitro, we noticed that the NMDA-evoked increase in Fura-2 detected intracellular Ca(2+) concentration in synaptoneurosomes from the rat, but not from the rabbit hippocampus, was strongly inhibited by the ryanodine receptor (RyR) antagonists dantrolene and ryanodine. To establish the mechanism of these differences, we characterised their possible dependence on the expression of RyR and their co-localisation with the calcium binding protein calbindin D(28k). A pronounced expression of [(3)H]ryanodine binding sites in the rat DG, which is only slight in the CA1, was demonstrated whereas in rabbit they were only found in the DG. The pattern of expression of calbindin D(28k) immunoreactivity and RyR in the rat and rabbit hippocampus was similar. These results suggest that the functional role of RyR in the generation of the NMDA receptor-mediated intracellular Ca(2+) signalling in the rabbit hippocampal neurones is marginal when compared to the rat. These differences reflect a diverse expression of RyR in both species. The corresponding differences in calbindin D(28k) immunoreactivity are most probably secondary in nature.