Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K⁺ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [³H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or ₁-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [³H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1M atropine but not by 1M prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 M atropine. The peptide calcium channel blocker -conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.     

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.     

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.     

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.     

Activation of muscarinic receptors inhibits beta-amyloid peptide-induced signaling in cortical slices

Deposition of fibrillar aggregates of the beta-amyloid peptide (Abeta) is a key pathologic feature during the early stage of Alzheimer's disease. The initial neuronal responses to Abeta in cortical circuits and the regulation of Abeta-induced signaling remain unclear. In this study, we found that exposure of cortical slices to Abeta(1-42) or Abeta(25-35) induced a marked increase in the activation of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII), two enzymes critically involved in a variety of cellular functions. Activation of M1 muscarinic receptors, but not nicotinic receptors, significantly inhibited the Abeta activation of PKC and CaMKII. Increasing inhibitory transmission mimicked the M1 effect on Abeta, whereas blocking GABA(A) receptors eliminated the M1 action. Moreover, electrophysiological evidence shows that application of Abeta to cortical slices induced action potential firing and enhanced excitatory postsynaptic currents, whereas muscarinic agonists potently increased inhibitory postsynaptic currents. These results suggest that Abeta activates PKC and CaMKII through enhancing excitatory activity in glutamatergic synaptic networks. Activation of M1 receptors inhibits Abeta signaling by enhancing the counteracting GABA(ergic) inhibitory transmission. Thus the muscarinic reversal of the Abeta-induced biochemical and physiological changes provides a potential mechanism for the treatment of Alzheimer's disease with cholinergic enhancers.     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Trimethyltin-induced loss of NMDA and kainate receptors in the rat brain

Summary Adult rats exposed acutely to trimethyltin (TMT) manifest a number of behavioral alterations, in conjunction with neuronal degeneration in the limbic system. In the present study, changes in³H-TCP binding to N-methyl-D-aspartate (NMDA) receptors and³H-kainic acid (KA) binding to kainate receptors were studied by autoradiographic methods following TMT exposure (8 mg/kg, i.p.) in adult Sprague Dawley rats. No significant alterations were found at 4 hours after exposure. An extensive loss of³H-TCP and³H-KA binding was seen in the hilar region of the CA3 field at 2 and 12 weeks after TMT exposure. Also, the³H-TCP binding was decreased in piriform cortex and in striatum. Thus, TMT exposure leads to a major and regional selective loss of NMDA and kainate receptors in the limbic system, alterations that may be involved in the neuropathology and behavioral sequelae of TMT toxicity.Abbreviations TMT trimethyltin - NMDA N-methyl-D-aspartate - KA Kainic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine     

Excitotoxic damage of the hippocampus in the rat: involvement of N-methyl-D-aspartate receptors

The intrahippocampal injection of two agonists of excitatory aminoacid (EAA) receptors elicited neuronal damages localized in CA1 and dentate gyrus for N-methyl-D-aspartate (NMDA) (20 nmol) and extended to the various hippocampal areas, except dentate gyrus for kainic acid (KA) (2.5 nmol). The pretreatment of the animals with N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) (20 mg/kg), a noncompetitive NMDA-receptor antagonist, prevented the neuronal injury induced by NMDA and KA in CA1. The distribution of neuronal damages and of TCP-protected areas closely correlated to that of EEA-receptors and of TCP binding sites in the hippocampus.     

Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity

Over‐activation of the N‐methyl‐d ‐aspartate (NMDA) receptor results in a Ca²⁺‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca²⁺ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca²⁺ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced ⁴⁵Ca²⁺ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and ⁴⁵Ca²⁺ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to ⁴⁵Ca²⁺ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca²⁺ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures. Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.     

Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes

Local, global and propagating calcium (Ca(2+)) signals provide the substrate for glial excitability. Here we analyse Ca(2+) permeability of NMDA and P2X(1/5) receptors expressed in cortical astrocytes and provide evidence that activation of these receptors trigger astroglial Ca(2+) signals when stimulated by either endogenous agonists or by synaptic release of neurotransmitters. The Ca(2+) permeability of the ionotropic receptors was determined by reversal potential shift analysis; the permeability ratio P(Ca)/P(K) was 3.1 for NMDA receptors and 2.2 for P2X(1/5) receptors. Selective stimulation of ionotropic receptors (with NMDA and α,β-methyleneATP) in freshly isolated cortical astrocytes induced ion currents associated with transient increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)). Stimulation of neuronal afferents in cortical slices triggered glial synaptic currents and [Ca(2+)](i) responses, which were partially blocked by selective antagonists of NMDA (D-AP5 and UBP141) and P2X(1/5) (NF449) receptors. We conclude that ionotropic receptors contribute to astroglial Ca(2+) signalling and may provide a specific mechanism for fast neuronal-glial signalling at the synaptic level.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

Epileptiform postsynaptic currents in primary culture of rat cortical neurons: Calcium mechanisms

In this study we demonstrate that the primary culture of rat cortical neurons is a convenient model for investigations of epileptogenesis mechanisms and specifically, of the postsynaptic epileptiform currents (EC) reflecting periodical asynchronous glutamate release. In particular, we have revealed that in primary culture of cortical neurons EC can appear spontaneously or can be triggered by the withdrawal of magnesium block of NMDA receptor channels or by shutting down GABAergic inhibition. EC were found to depend on intracellular calcium oscillations. The secondary calcium release from intracellular stores was needed for EC synchronization. EC were suppressed by the influences causing either neuronal calcium overload or decrease of intracellular calcium concentration. Calcium entry into neurons in the case of NMDA receptor hyperactivation or in the case of calcium ionophore ionomycin treatment eliminated EC. The suppression of EC also occurred after a decrease of intracellular calcium concentration induced by BAPTA loaded into the neurons or by stimulation of calcium removal from cells via Na⁺/Ca²⁺ exchanger by 1 nM ouabain. Partial dependence of EC on action potential generation was found. Thus, EC in neurons are activated by intracellular periodic calcium waves within a limited concentration window.     

Effect of pyrithiamine treatment on potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enhanced Na+/K+ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K+ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K+ was monitored directly with a K+-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K+ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K+ upon tetrodotoxin addition was inversely related to the extracellular Ca2+ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.     

Changes in autophagy proteins in a rat model of controlled cortical impact induced brain injury

Autophagy has been implicated in several neurodegenerative diseases and recently its role in acute brain injury has received increased interest. In our study, we investigated the profiles of autophagy-linked proteins (MAP-LC3 (Atg8), beclin-1 (Atg6) and the beclin-1-binding protein, bcl-2, following controlled cortical impact injury in rats—a model for moderate-to-severe traumatic brain injury. We observed significant increases in the levels of the processed form of LC3 (LC3-II) in the ipsilateral cortex 2 h to 2 days after injury when compared to sham. Furthermore, the beclin-1/bcl-2 ratio in the ipsilateral cortex was found to have increased from 1 and 2 days after injury. Since both of these changes are established autophagy-enabling events, and, based on these data, we propose that autophagy, plays a role in the manifestation of cell injury following brain trauma.     

Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons

We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 μM to 1 mM with an apoptosis-like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4-dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca²⁺ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca²⁺ levels, in the rat brain.     

Effects of antidepressant drugs on inositol phospholipid hydrolysis in rat cerebral cortical slices

The effects of several different types of antidepressant drugs on phosphoinositide hydrolysis by slices of rat cerebral cortex was investigated by prelabeling inositol phospholipids with [³H]inositol and then measuring the formation of [³H]inositol phosphates (a total fraction consisting of the mono-and poly-phosphates was collected) in the presence of 10 mM LiCl. All of the drugs tested (amitriptyline, trimipramine, mianserin, desipramine, tranylcypromine, and citalopram) inhibited NE-stimulated [³H]inositol phosphate formation. This inhibition appeared to be due to antagonism of ₁-receptors. In addition to inhibiting the effects of NE, the tricyclic antidepressants themselves were able to stimulate [³H]inositol phosphate formation. This stimulation occurred at drug concentrations higher than that needed to inhibit stimulation by NE. Stimulatory effects of the antidepressants themselves were not blocked by the ₁-antagonist, prazosin. An examination of the types of inositol phosphates formed revealed that formation of inositol monophosphate was stimulated, but that inostiol biphosphate production was decreased by tricyclic antidepressants compared to control.