Ketamine: The best partner for isoflurane in neonatal anesthesia?

Isoflurane is one of the most commonly used inhalation anesthetic in neonatal anaesthesia. It has been suggested that isoflurane can induce caspase activation and apoptosis when applied in a clinically relevant concentration in the developing brain. Recent researches have indicated that a clinically relevant isoflurane treatment may induce neurodegeneration and apoptosis by activating the endoplasmic reticulum (ER) membrane inositol 1,4,5-trisphosphate (IP(3)) receptor, producing excessive calcium release from ER to the cytoplasm and triggering apoptosis. Although the exact mechanism by which isoflurane induces apoptosis still needs further study, it is generally accepted that the increase of cytosolic free calcium levels is the major risk factor. Previous studies have found that during early postnatal life, activation of gamma-aminobutyric acid (GABA(A)) receptor reduces the voltage-dependent Mg(2+) block of N-methyl-d-aspartate (NMDA) channels in neurons and increases cytosolic calcium levels by potentiated the Ca(2+) influx through NMDA channels; while in the adult, it may enhance the voltage-dependent Mg(2+) block of NMDA channels and decrease the Ca(2+) influx through NMDA channels. Since isoflurane acts at the GABA(A) receptor in an agonistic manner, here we presume that isoflurane increases intracellular calcium in neonatal neurons not only by activating IP(3) receptors in the endoplasmic reticulum (ER) membrane, but also by activating the GABA(A) receptor and depolarizing the postsynaptic membrane enough to facilitate NMDA receptor-mediated Ca(2+) influx. Meanwhile, we hypothesized that ketamine, a widely used pediatric anesthetic, acts as a noncompetitive antagonist of the NMDA type of glutamate receptors, which may be the best partner for isoflurane in neonatal anesthesia for it may attenuate isoflurane-induced caspase activation and apoptosis in the neonatal neurons by inhibiting the isoflurane-induced elevation in cytosolic calcium not only by blocking the NMDA receptors, but also by suppressing inositol triphosphate formation in the cytoplasm.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

Kappa opioid receptor activation in the nucleus accumbens inhibits glutamate and GABA release through different mechanisms

Through their actions in the nucleus accumbens (NAc), kappa opioid (KOP) receptors and their endogenous ligand, dynorphin, modify behaviors associated with the administration of drugs of abuse and are regulated by exposure to such drugs. Despite their demonstrated behavioral significance, the synaptic actions of KOP receptor ligands in the NAc are not clearly understood. Using whole-cell voltage-clamp recordings of NAc medium spiny neurons, we have found that, in addition to suppressing glutamate release, the KOP receptor agonist also inhibits GABA release. Interestingly, the mechanism of inhibition of the release of glutamate differs from that controlling GABA. reduces the frequency of Ca(2+)-independent miniature excitatory postsynaptic currents, but not miniature inhibitory postsynaptic currents. Furthermore, while the inhibition of GABAergic transmission is blocked by the N-type Ca(2+) channel blocker omega-CgTx, the inhibition of excitatory glutamatergic transmission by is unaffected by N-type Ca(2+) channel blockade. These results indicate that KOP receptor activation inhibits GABA release by reducing Ca(2+) influx, but inhibits glutamate release at a step downstream of Ca(2+) entry.     

The effects of isoproterenol on abnormal electrical and contractile activity and diastolic calcium are attenuated in myocytes from aged Fischer 344 rats

We investigated whether the age-related decrease in sensitivity of the heart to catecholamines was accompanied by changes in Ca(2+) homeostasis and abnormal electrical and contractile activity caused by beta-adrenergic receptor (beta-AR) stimulation. Ventricular myocytes were isolated from young adult (3 months) and aged (24 months) male Fischer 344 rats. Unloaded cell shortening was measured in field-stimulated myocytes (2Hz, 37 degrees C); membrane currents and action potentials were measured with microelectrodes. Contractile responses to the non-selective beta-AR agonist, isoproterenol were significantly decreased in aged myocytes compared to younger myocytes and aged myocytes were less sensitive to isoproterenol. In contrast, Ca(2+) transients measured simultaneously with contractions were similar between groups. Isoproterenol increased sarcoplasmic reticulum Ca(2+) stores in both groups, but the increase was larger in aged cells. However, signs of Ca(2+) overload induced by isoproterenol were reduced with age. Diastolic Ca(2+) accumulation, contracture and the incidences of transient inward current, oscillatory afterpotentials (OAPs), aftertransients and aftercontractions induced by isoproterenol also were reduced with age. These results demonstrate that aged myocytes exhibit fewer signs of Ca(2+) overload in response to isoproterenol than young adult myocytes. These age-related changes in intracellular Ca(2+) may protect the aging heart against induction of arrhythmias initiated by OAPs.(1).     

GABAergic actions on cholinergic laterodorsal tegmental neurons: implications for control of behavioral state

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl(-) -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABA(A) receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca(2+) solutions. THIP, a selective GABA(A) receptor agonist with a preference for δ-subunit containing GABA(A) receptors, induced inward currents, suggesting the existence of extrasynaptic GABA(A) receptors. LDT cells also possess GABA(B) receptors as baclofen-activated a TTX- and low Ca(2+)-resistant outward current that was attenuated by the GABA(B) antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a G(IRK) mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABA(B) receptors linked to the same G(IRK) activated in these cells by muscarinic receptor stimulation. Activation of GABA(B) receptors inhibited Ca(2+) increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca(2+) were unaltered by prior emptying of intracellular Ca(2+) stores, but were abolished by low extracellular Ca(2+) and pre-application of nifedipine, indicating that activation of GABA(B) receptors inhibits influx of Ca(2+) involving L-type Ca(2+) channels. Presence of GABA(C) receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABA(C) receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABA(A, B and C) receptors, including extrasynaptically located GABA(A) receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT.     

Age-related changes in the subtypes of voltage-dependent calcium channels in rat brain cortical synapses

Age-related changes in the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to depolarization-induced Ca(2+) influx and in the density of VDCC subtypes in cortical synapses were investigated using synaptosomes and their membrane preparations from brain cortices of Wistar rats. The relative contribution of VDCC subtypes to Ca(2+) influx was determined by measuring the inhibition of depolarization-induced Ca(2+) influx with four VDCC subtype-specific peptide blockers. In adult rat synaptosomes, L-, N-, P- and Q-type channels accounted for 24, 32, 27 and 12% of the total Ca(2+) influx, respectively. Brain aging significantly reduced the relative contributions of N- and P-type channels and increased the contribution of the channels resistant to the four blockers used. The densities of VDCC subtypes, determined by binding experiments using radiolabeled PN200 -110, omega-conotoxin GVIA and omega-conotoxin MVIIC, were found to be significantly decreased in aged synaptic plasma membranes. On the contrary, the dissociation constants of the blockers were not changed except for PN200-110-sensitive L-type channels. These results suggest that aging alters the relative contributions of each VDCC subtype to depolarization-induced Ca(2+) influx and decreases the number of VDCCs in rat brain cortical synapses. These changes in VDCCs may lead to age-related hypofunction of synaptic neurotransmission in brain cortices.     

Direction-selective adaptation in fly visual motion-sensitive neurons is generated by an intrinsic conductance-based mechanism

Motion-sensitive neurons in the blowfly brain present an ideal model system to study the cellular mechanisms and functional significance of adaptation to visual motion. Various adaptation processes have been described, but it is still largely unknown which of these processes are generated in the motion-sensitive neurons themselves and which originate at more peripheral processing stages. By input resistance measurements I demonstrate that direction-selective adaptation is generated by an activity-dependent conductance increase in the motion-sensitive neurons. Based on correlations between dendritic Ca(2+) accumulation and slow hyperpolarizing after-potentials following excitatory stimulation, a regulation of direction-selective adaptation by Ca(2+) has previously been suggested. In the present study, however, adaptation phenomena are not evoked when the cytosolic Ca(2+) concentration is elevated by ultraviolet photolysis of caged Ca(2+) in single neurons rather than by motion stimulation. This result renders it unlikely, that adaptation in fly motion-sensitive neurons is regulated by bulk cytosolic Ca(2+).     

17beta-estradiol benzoate decreases the AHP amplitude in CA1 pyramidal neurons

Disruption of Ca(2+) homeostasis is hypothesized to mediate several electrophysiological markers of brain aging. Recent evidence indicates that estradiol can rapidly alter Ca(2+)-dependent processes in neurons through nongenomic mechanisms. In the current study, electrophysiological effects of 17beta-estradiol benzoate (EB) on the Ca(2+)-activated afterhyperpolarization (AHP) were investigated using intracellular sharp electrode recording in hippocampal slices from ovariectomized Fischer 344 female rats. The AHP amplitude was enhanced in aged (22-24 mo) compared with young (5-8 mo) rats and direct application of EB (100 pM) reduced the AHP in aged rats. The age-related difference was due, in part, to the increased AHP amplitude of aged animals, since an EB-mediated decrease in the AHP could be observed in young rats when the extracellular Ca(2+) was elevated to increase the AHP amplitude. In aged rats, bath application of EB occluded the ability of the L-channel blocker, nifedipine (10 microM), to attenuate the AHP. The results support a role for EB in modifying hippocampal Ca(2+)-dependent processes in a manner diametrically opposite that observed during aging, possibly through L-channel inhibition.     

Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex

Regulation of the efficacy of synaptic transmission by activity-dependent processes has been implicated in learning and memory as well as in developmental processes. We previously described transient potentiation of excitatory synapses onto layer 2/3 pyramidal neurons in the visual cortex that is induced by coincident presynaptic stimulation and postsynaptic depolarization. In the adult visual cortex, activation of N-methyl-d-aspartate (NMDA) glutamate receptors is necessary to induce this plasticity. These receptors act as coincidence detectors, sensing presynaptic glutamate release and postsynaptic depolarization, and cause an influx of Ca(2+) that is necessary for the potentiation. In the neurons of the neonatal visual cortex, on the other hand, coincident presynaptic stimulation and postsynaptic depolarization induce stable long-term potentiation (LTP). In addition, reduced but significant LTP can be induced in many neurons in the presence of the NMDA receptor (NMDAR) antagonist, 2-amino-5-phosphonovaleric acid despite the Ca(2+) requirement. Therefore there must be an alternative postsynaptic Ca(2+) source and coincidence detection mechanism linked to the LTP induction mechanism in the neonatal cortex operating in addition to NMDARs. In this study, we find that in layer 2/3 pyramidal neurons, release of Ca(2+) from inositol trisphosphate (InsP(3)) receptor-mediated intracellular stores and influx through voltage-gated Ca(2+) channels (VGCCs) provide alternative postsynaptic Ca(2+) sources. We hypothesize that InsP(3)Rs are coincidence detectors, sensing presynaptic glutamate release through linkage with group I metabotropic glutamate receptors (mGluRs), and depolarization, through VGCCs. We also find that the downstream protein kinases, PKA and PKC, have a role in potentiation in layer 2/3 pyramidal neurons of the neonatal visual cortex.     

Na(+)/Ca(2+) exchanger in GABAergic presynaptic boutons of rat central neurons

Rat Meynert neurons were acutely isolated using a dissociation technique that maintains functional GABAergic presynaptic boutons. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded under voltage-clamp conditions using whole cell patch-clamp recordings. Using the frequency of mIPSCs as a measure of presynaptic terminal excitability, the existence of a Na(+)/Ca(2+) exchanger (NCX) in these GABAergic nerve terminals was clearly demonstrated. Both the frequency and the amplitude of mIPSCs were unaffected by replacement of extracellular Na(2+). However, in this Na(+)-free external solution, ouabain could now induce a transient increase of mIPSCs frequency, which was not inhibited by adding Cd(2+) or cyclopiazonic acid but was inhibited by removing external Ca(2+). This indicates that this transient potentiation was dependent on external Ca(2+), but that this Ca(2+) influx was not via voltage-dependent Ca(2+) channels. KB-R7943, an inhibitor of NCX, at a concentration of 3 x 10(-6) M, reduced this transient increase of mIPSCs frequency without affecting mIPSCs amplitude and the response to exogenous GABA. These results demonstrate the existence of NCX in these GABAergic nerve terminals. In zero external Na(+), ouabain causes an accumulation of intraterminal Na(+) and a resultant influx of Ca(2+) through the reversed mode operation of NCX. However, under more physiological conditions, NCX may also operate in a forward mode and serve to maintain low intracellular [Ca(2+)] in nerve terminals.     

Mechanisms of unmodified CdSe quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons

Quantum dots (QDs) have shown great promise for applications in biology and medicine, which is being challenged by their potential nanotoxicity. Reactive oxygen species (ROS) produced by QDs are believed to be partially responsible for QD cytotoxicity. Cytoplasmic Ca(2+) plays an important role in the development of ROS injury. Here we found unmodified cadmium selenium (CdSe) QDs could elevate cytoplasmic calcium levels ([Ca(2+)](i)) in primary cultures of hippocampal neurons, involved both extracellular Ca(2+) influx and internal Ca(2+) release. More specifically, verapamil and mibefradil (L-type and T-type calcium channels antagonists, respectively) failed to prevent extracellular Ca(2+) influx under QD insult, while omega-conotoxin (N-type antagonist) could partially block this Ca(2+) influx. Surprisingly, this Ca(2+) influx could be well blocked by voltage-gated sodium channels (VGSCs) antagonist, tetrodotoxin (TTX). QD-induced internal Ca(2+) release could be avoided by clonazepam, a specific inhibitor of mitochondrial sodium-calcium exchangers (MNCX), and also by TTX. Furthermore, dantrolene, an antagonist of ryanodine (Ry) receptors in endoplasmic reticulum (ER), almost abolished internal Ca(2+) release, while 2-APB [inositol triphosphate (IP(3)) receptors antagonist] failed to block this Ca(2+) release, indicating that released Ca(2+) from mitochondria, which was induced by extracellular Na(+) influx, further triggered much more Ca(2+) release from ER. Our results imply that more research on the biocompatibility and biosafety of QD is both warranted and necessary.     

Excitatory and inhibitory actions of isoflurane in bovine chromaffin cells

Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing CNS function. Many anesthetics, including isoflurane, are thought to modulate and/or directly activate GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors. We previously showed that activation of the GABA(A) receptors, with specific agonists, leads to cellular excitation resulting from the depolarized anion equilibrium potential. In this study, our goal was to determine whether isoflurane mimicked this response and to explore the functional consequences of this activation. Furthermore, we sought to study the actions of isoflurane on nicotinic acetylcholine receptors (nAChRs) as they mediate the "sympathetic drive" in these cells. For these studies the Ca(2+)-indicator dye fura-2 was used to assay [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release. We show that bovine adrenal chromaffin cells were excited by isoflurane at clinically relevant concentrations. Isoflurane directly activated GABA(A) receptors found in chromaffin cells, which depolarized the cells and elevated [Ca(2+)](i). Application of isoflurane directly to the chromaffin cells elicited catecholamine secretion from these cells. At the same time, isoflurane suppressed activation of nAChRs, which presumably blocks "sympathetic drive" to the chromaffin cells. These latter results may help explain why isoflurane produces the hypotension observed clinically.     

The activity of the Na+/Ca2+ exchanger largely modulates the Ca2+i signal induced by hypo-osmotic stress in rat cerebellar astrocytes. The effect of osmolarity on exchange activity

We recently demonstrated that rat cerebellar Type-1 astrocytes express a very active Na(+)/Ca(2+) exchanger highly colocalized with ryanodine receptors (RyRs), which in turn play a key role in glutamate-induced Ca(2+) signaling through a calcium-induced calcium release (CICR) mechanism. In this work we have explored whether the Na(+)/Ca(2+) exchanger has any role in the Ca(2+)(i) signal induced by hypo-osmotic stress in these cells, using microspectrofluorometric measurements with Fura-2, pharmacological tools, and confocal microscopy image analysis. We present evidence for the first time that the increase in [Ca(2+)](i) in rat cerebellar Type-1 astrocytes, resulting from moderate hypotonic shock, is mediated by Ca(2+) release from ryanodine-operated Ca(2+)(i) stores, and that the magnitude of the intracellular Ca(2+) signal induced by hypotonicity in the short term (up to 240 s) is small and controlled by the activity of the Na(+)/Ca(2+) exchanger operating in its extrusion mode. With longer times in the hypotonic medium, intracellular Ca(2+) store depletion leads to Ca(2+) entry through store-operated Ca(2+) channels. We found it interesting that the activity of the Na(+)/Ca(2+) exchanger measured during this reverse mode operation (Ca(2+) entry in exchange for internal Na(+)) was found to be greatly increased in hypotonic solutions and decreased in hypertonic ones. The buffering of the [Ca(2+)](i) rise induced by hypo-osmotic stress may prevent excessive increases in [Ca(2+)](i), which otherwise might impair the normal function of this glial cell.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

The volatile anesthetic isoflurane suppresses spontaneous calcium oscillations in vitro in rat hippocampal neurons by activation of adenosine A1 receptors

Primary cultures of rat hippocampal neurons were loaded with the Ca(2+)-indicator fluo-3 and studied with a confocal laser microscope. In Mg(2+)-free medium the cultures showed spontaneous synchronized calcium oscillations. These oscillations derived from excitatory signal transmission by N-methyl-D-aspartate and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and were modulated by gamma-aminobutyric acid(A) receptors. The oscillations were dose-dependently depressed by adenosine (IC50=2 microM) or by 2-chloro-N6-cyclopentyladenosine a specific adenosine A1 receptor agonist (IC50=40 nM). These effects were reverted by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific adenosine A1 receptor antagonist. The volatile anesthetic isoflurane also depressed these spontaneous calcium oscillations in a dose dependent manner (IC50=0.25 MAC, Minimum Alveolar Concentration). The isoflurane-induced inhibition was partly reversed in 29-38% of the neurons by DPCPX, indicating that the anesthetic activates this receptor possibly by increasing the extracellular concentration of adenosine.     

Enhancement of excitatory synaptic transmission in spiny neurons after transient forebrain ischemia

Spiny neurons in the neostriatum are highly vulnerable to ischemia. Enhancement of excitatory synaptic transmissions has been implicated in ischemia-induced excitotoxic neuronal death. Here we report that evoked excitatory postsynaptic currents in spiny neurons were potentiated after transient forebrain ischemia. The ischemia-induced potentiation in synaptic efficacy was associated with an enhancement of presynaptic release as demonstrated by an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a decrease in the paired-pulse ratio. The amplitude of inward currents evoked by exogenous application of glutamate did not show significant changes after ischemia, suggesting that postsynaptic mechanism is not involved. The ischemia-induced increase in mEPSCs frequency was not affected by blockade of voltage-gated calcium channels, but it was eliminated in the absence of extracellular calcium. Bath application of ATP P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly reduced mEPSC frequency in ischemic neurons but had no effects on the control ones. Furthermore, the inhibitory effect of PPADS on ischemic neurons was abolished in Ca2+-free external solution. These results indicate that excitatory synaptic transmissions in spiny neurons are potentiated after ischemia via presynaptic mechanisms. Activation of P2X receptors and the consequent Ca2+ influx might contribute to the ischemia-induced facilitation of glutamate release.     

Changes in the calcium dependence of glutamate transmission in the hippocampal CA1 region after brief hypoxia-hypoglycemia.

Using the model of hypoxia-hypoglycemia (HH) in rat brain slices, we asked whether glutamate transmission is altered following a brief HH episode. The HH challenge was conducted by exposing slices to a glucose-free medium aerated with 95% N2-5% CO2, for approximately 4 min, and glutamate transmission in the hippocampal CA1 region was monitored at different post HH times. In slices examined 相似文献   

Age-dependence of effects of A1 adenosine receptor antagonism in rat hippocampal slices.

1. Population excitatory postsynaptic potentials (EPSPs) and population spikes evoked in area CA1 of hippocampal slices from aged Fischer 344 rats were significantly smaller in amplitude than responses obtained in slices from young Fischer 344 rats. 2. The A1 adenosine receptor antagonist 8-cyclopentyltheophylline (8-CPT) produced a concentration-dependent increase in synaptic potentials in slices from both young and aged rats. Low concentrations (1 nM) of 8-CPT were effective in producing increases in both population spike amplitudes and population EPSP slopes in young and aged rat slices. Response increases were maximized by 100 nM 8-CPT in slices from rats of both age groups. 3. Adenosine antagonism produced greater average increases in synaptic responses in hippocampal slices from aged rats at all concentrations tested (1.0 nM-1.0 microM). A qualitative age-related difference in the response to 8-CPT was also observed; 8-CPT produced a late component, consisting of multiple population spikes, in evoked responses in slices obtained from aged but not young rats. 4. Adenosine antagonism significantly increased the maximum evocable response (both spike amplitude and EPSP slope) in slices from aged rats, relative to increases observed in slices from young rats. This suggested that smaller synaptic potentials seen in slices from aged rats were in part due to greater levels of "tonic" adenosinergic inhibition. 5. Slices from young and aged rats were incubated in the adenosine reuptake inhibitor soluflazine (R64719; 1.0, 10, and 100 microM) and the inhibition of population EPSPs was observed for 60 min. No difference was observed in the rate of inhibition or the maximal level of inhibition produced by soluflazine, in slices from rats of either age group. 6. Application of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclo-hepten- 5,10-imine hydrogen maleate (MK-801) and 2-amino-5-phosphonopentanoic acid (2-AP5), antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors, reduced the late multiple population spike component in slices from aged rats incubated in 8-CPT. A smaller direct effect of the NMDA antagonists was observed in slices from aged rats in the absence of 8-CPT treatment at maximal response levels. No effect of NMDA receptor antagonism was observed in slices from young rats under either condition. 7. Hippocampal tissue, from young and old rats utilized in the electrophysiological experiments, was assayed for A1 adenosine binding site density with a saturating concentration of radiolabeled agonist and antagonist. Guanine nucleotide modulation of agonist binding was also measured.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subtypes of voltage-sensitive calcium channels in cultured rat brain neurons

Subtypes of voltage-sensitive calcium channels have been investigated in cultured rat brain neurons using two classes of specific probes, dihydropyridine compounds and omega-conotoxin. Membranes prepared from cultured neurons contain specific binding sites for [3H]PN200-110, a dihydropyridine antagonist, and for 125I-omega-conotoxin with a stoichiometry of about 1:1. A depolarization induced 45Ca2+ influx into intact brain neurons was partially inhibited by a dihydropyridine antagonist, nifedipine and stimulated by a dihydropyridine agonist, Bay K8644. This dihydropyridine sensitive 45Ca2+ flux was insensitive to omega-conotoxin at concentrations which saturate the specific toxin binding sites indicating that in cultured brain neurons, dihydropyridine-sensitive calcium channels are not sensitive to omega-conotoxin.     

N-Methyl-D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.