measurement-total-calcium-neurons-electron-probe-x-ray

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

National Institutes of Health

Mitochondria and endoplasmic reticulum (ER) are important modulators of intracellular calcium signaling pathways, but the role of these organelles in shaping synaptic calcium transients in dendrites of pyramidal neurons remains speculative. We have measured directly the concentrations of total Ca (bound plus free) within intracellular compartments of proximal dendrites of CA3 hippocampal neurons at times after synaptic stimulation corresponding to the peak of the cytoplasmic free Ca2+ transient (1 sec), to just after its decay (30 sec), and to well after its return to prestimulus levels (180 sec). Electron probe microanalysis of cryosections from rapidly frozen slice cultures has revealed that afferent mossy fiber stimulation evokes large, rapid elevations in the concentration of total mitochondrial Ca ([Ca](mito)) in depolarized dendrites. A single tetanus (50 Hz/1 sec) elevated [Ca](mito) more than fivefold above characteristically low basal levels within 1 sec of stimulation and >10-fold by 30 sec after stimulation. This strong Ca accumulation was reversible, because [Ca](mito) had recovered by 180 sec after the tetanus. Ca sequestered within mitochondria was localized to small inclusions that were distributed heterogeneously within, and probably among, individual mitochondria. By 30 sec after stimulation an active subpopulation of ER cisterns had accumulated more Ca than had mitochondria despite a approximately 1 sec delay before the onset of accumulation. Active ER cisterns retained their Ca load much longer (>3 min) than mitochondria. The complementary time courses of mitochondrial versus ER Ca2+ uptake and release suggest that these organelles participate in a choreographed interplay, each shaping dendritic Ca2+ signals within characteristic regimes of cytosolic Ca2+ concentration and time.

Correlated calcium uptake and release by mitochondria and endoplasmic reticulum of CA3 hippocampal dendrites after afferent synaptic stimulation.

&#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x5174;&#x594B;&#x6BD2;&#x6027;&#x523A;&#x6FC0;&#x8BF1;&#x5BFC;&#x7EBF;&#x7C92;&#x4F53;&#x9499;&#x8D85;&#x8F7D;&#x548C;&#x91CA;&#x653E;&#x7684;&#x4FC3;&#x51CB;&#x4EA1;&#x86CB;&#x767D;&#xFF0C;&#x54EA;&#x4E9B;&#x89E6;&#x53D1;&#x5668;&#x5EF6;&#x8FDF;&#x7EC6;&#x80DE;&#x6B7B;&#x4EA1;&#x3002;&#x7136;&#x800C;&#xFF0C;apoptogen &#x53D1;&#x5E03;&#x7684;&#x786E;&#x5207;&#x673A;&#x5236;&#x4ECD;&#x6709;&#x4E89;&#x8BAE;&#x3002;&#x7279;&#x70B9;&#x7EBF;&#x7C92;&#x4F53;&#x9499;&#x8D1F;&#x8F7D;&#x548C;&#x5355;&#x5143;&#x683C;&#x7684;&#x8106;&#x5F31;&#x6027;&#xFF0C;&#x4E4B;&#x95F4;&#x7684;&#x8054;&#x7CFB;&#x5E76;&#x6D4B;&#x8BD5;&#x53EA;&#x4E9A;&#x7FA4;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x53D7;&#x635F;&#x7684;&#x9499;&#x91CD;&#x8F7D;&#x7248;&#x672C; apoptogens &#x7684;&#x5047;&#x8BF4;&#xFF0C;&#x6211;&#x4EEC;&#x76F4;&#x63A5;&#x8861;&#x91CF;&#x4E2A;&#x522B;&#x7EBF;&#x7C92;&#x4F53;&#x4E2D;&#x603B; Ca &#xFF08;&#x514D;&#x8D39;&#x52A0;&#x4E0A;&#x7ED1;&#x5B9A;&#xFF09; &#x7684;&#x6D53;&#x5EA6;&#x5E76;&#x5728;&#x5E76;&#x884C;&#x7684;&#x7ED3;&#x6784;&#x53D8;&#x5316;&#x548C; pro &#x51CB;&#x4EA1;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c &#x5728;&#x4F53;&#x5916;&#x57F9;&#x517B;&#x6D77;&#x9A6C;&#x795E;&#x7ECF;&#x5143; NMDA &#x8FC7;&#x5EA6;&#x540E;&#x7684;&#x4E9A;&#x7EC6;&#x80DE;&#x5B9A;&#x4F4D;&#x76D1;&#x6D4B;&#x3002;&#x8D85;&#x51FA;&#x7684;&#x6E38;&#x79BB;&#x9499;&#x548C;&#x94A0;&#x7684;&#x6444;&#x52A8;&#x77AC;&#x6001;&#x63D0;&#x5347; + / K + &#x7A33;&#x6001;&#x3001; NMDA &#x523A;&#x6FC0;&#x8BF1;&#x5BFC;&#x7EBF;&#x7C92;&#x4F53;&#xFF0C;&#x5305;&#x62EC;&#x5F3A;&#x9499;&#x9AD8;&#x7A0B;&#x3001; &#x819C;&#x6F5C;&#x5728;&#x7684;&#x53BB;&#x6781;&#x5316;&#x548C;&#x53EF;&#x53D8;&#x80BF;&#x80C0;&#x7684;&#x620F;&#x5267;&#x5316;&#xFF0C;&#x4F46;&#x4E3B;&#x8981;&#x662F;&#x53EF;&#x9006;&#x7684;&#x53D8;&#x5316;&#x3002;&#x9AD8;&#x7A0B;&#x77E9;&#x9635; Ca &#x4E2D;&#x5927;&#x7EA6;&#x4E09;&#x5206;&#x4E4B;&#x4E00;&#x7684;&#x5F3A;&#x70C8;&#x80BF;&#x4E86;&#x8D77;&#x6765;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x4EE5;&#x53CA;&#x81A8;&#x80C0;&#x65F6; Ca2 + &#x8FDB;&#x5165;&#x88AB;&#x5E9F;&#x9664;&#xFF0C;&#x6CA1;&#x6709;&#x8BF4;&#x660E; Ca &#x91CD;&#x8F7D;&#x7684;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;NMDA &#x66DD;&#x5149;&#x540E;&#x4E0D;&#x4E45;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c&#x3001; &#x7EBF;&#x7C92;&#x4F53;&#xFF0C;&#x901A;&#x5E38;&#x672C;&#x5730;&#x5316;&#x6210;&#x4E3A;&#x5DE5;&#x519C;&#x4E1A;&#x5206;&#x5E03;&#x5728;&#x7EC6;&#x80DE;&#x8D28;&#x7684;&#x4E25;&#x91CD;&#x80BF;&#x7EBF;&#x7C92;&#x4F53;&#x5916;&#x819C;&#x7834;&#x88C2; &#xFF1B; &#x5916;&#x89C2;&#x4E0E;&#x91CD;&#x5408;&#x5728;&#x8FD9;&#x4E9B;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x5B8C;&#x6574;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#xFF0C;&#x6697;&#x793A;&#x5B83;&#x88AB;&#x91CA;&#x653E;&#x4E3B;&#x8981;&#x4ECE;&#x635F;&#x574F;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x4E2D;&#x4FDD;&#x7559;&#x4E86;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c&#x3002;&#x4E0E;&#x7EBF;&#x7C92;&#x4F53; Ca &#x91CD;&#x8F7D;&#x7684;&#x4F5C;&#x7528;&#x76F8;&#x4E00;&#x81F4;&#xFF0C;&#x7FB0;&#x57FA;&#x6C30;&#x5316;&#x7269; p-(&#x4E09;&#x6C1F;&#x7532;&#x6C27;&#x57FA;) &#x82EF;&#x8159;&#x51CF;&#x5C11; Ca &#x79EF;&#x7D2F;&#x3001; &#x963B;&#x6B62;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c &#x91CA;&#x653E;&#xFF0C;&#x795E;&#x7ECF;&#x4FDD;&#x62A4;&#x4F5C;&#x7528;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x652F;&#x6301;&#x5EF6;&#x8FDF;&#x5174;&#x594B;&#x6BD2;&#x6027;&#x6B7B;&#x4EA1;&#x5305;&#x62EC;&#x4ECE;&#x4E9A;&#x7FA4;&#x7684;&#x9499;&#x8D85;&#x8F7D;&#x7684;&#x7EBF;&#x7C92;&#x4F53; apoptogen &#x91CA;&#x653E;&#x673A;&#x5236;&#xFF0C;&#x800C;&#x5176;&#x4ED6;&#x7684;&#x3001; &#x672A;&#x635F;&#x574F;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x4FDD;&#x6301;&#x6B63;&#x5E38;&#x529F;&#x80FD;&#x3002;

&#x5174;&#x594B;&#x6BD2;&#x6027;&#x9499;&#x8D85;&#x8F7D;&#x7684;&#x4E9A;&#x7FA4;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x89E6;&#x53D1;&#x5668;&#x5EF6;&#x8FDF;&#x6D77;&#x9A6C;&#x795E;&#x7ECF;&#x5143;&#x6B7B;&#x4EA1;&#x3002;

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons. Beyond transient elevation of cytosolic calcium and perturbation of Na+/K+ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca2+ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function.

Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.

Dans les neurones, excitotoxique stimulation induit une surcharge de calcium mitochondrial et la lib&eacute;ration des prot&eacute;ines pro-apoptotiques, quels d&eacute;clencheurs retard&eacute; la mort cellulaire. Les m&eacute;canismes de lib&eacute;ration apoptogen, cependant, demeurent controvers&eacute;es. Pour caract&eacute;riser le lien entre la vuln&eacute;rabilit&eacute; de charge et cellules de calcium mitochondrial et de v&eacute;rifier l&#39;hypoth&egrave;se que seulement une sous-population de mitochondries endommag&eacute;es par calcium surcharge communiqu&eacute;s d&#39;apoptogens, nous avons mesur&eacute; directement la concentration de Ca total (gratuit plus d&eacute;pendant) dans les mitochondries et surveill&eacute; en parall&egrave;les changements structurels et la localisation subcellulaire de pro-apoptotic cytochrome c apr&egrave;s la surstimulation de NMDA dans les neurones hippocampiques cultiv&eacute;s. Au-del&agrave; une &eacute;l&eacute;vation transitoire du calcium cytosolique et la perturbation de la Na + / K + l&#39;hom&eacute;ostasie, stimulation de NMDA induit des changements dramatiques, mais surtout r&eacute;versibles, dans les mitochondries, &eacute;l&eacute;vation de calcium forte d&eacute;polarisation de la membrane potentiels et gonflement variable. &Eacute;l&eacute;vation de matrice Ca dans l&#39;environ le tiers des mitochondries qui &eacute;taient fortement gonfl&eacute;s, ainsi que l&#39;absence de gonflement lors de l&#39;entr&eacute;e de Ca2 + a &eacute;t&eacute; abolie, indiquent un r&ocirc;le essentiel pour la surcharge de Ca. Peu de temps apr&egrave;s l&#39;exposition NMDA, cytochrome c, normalement localis&eacute;e dans les mitochondries, est devenu diffus&eacute;ment distribu&eacute; dans le cytoplasme, qui co&iuml;ncide avec l&#39;apparition des mitochondries fortement gonfl&eacute;s avec rupture des membranes externes ; dans ces conditions, le cytochrome c a &eacute;t&eacute; retenu dans les mitochondries intactes, ce qui implique qu&#39;il a &eacute;t&eacute; lib&eacute;r&eacute; principalement des mitochondries endommag&eacute;es. Coh&eacute;rente avec le r&ocirc;le de la surcharge de Ca mitochondrial, carbonyl cyanure p-(carbonylcyanure) phenylhydrazone diminue l&#39;accumulation de Ca, a emp&ecirc;ch&eacute; la lib&eacute;ration du cytochrome c et &eacute;tait neuroprotecteurs. Ces r&eacute;sultats supportent un m&eacute;canisme dans lequel la mort excitotoxique retard&eacute;e implique apoptogen lib&eacute;ration d&#39;une sous-population de mitochondries surcharg&eacute; de calcium, tandis que les autres, intact mitochondrie maintenir la fonction normale.

Surcharge calcique excitotoxique dans une sous-population de d&eacute;clencheurs de mitochondries retard&eacute; la mort dans les neurones de l&#39;hippocampe.

Exzitotoxische Calcium-&#xDC;berladung in einer Subpopulation von Mitochondrien ausl&#xF6;st verz&#xF6;gerten Tod in Hippocampus-Neuronen.

Nei neuroni, excitotoxic stimolazione induce sovraccarico di calcio mitocondriale e il rilascio di pro-apoptotic proteine, quali trigger ritardato morte cellulare. I precisi meccanismi di rilascio apoptogen, tuttavia, rimangono controversi. A caratterizzare il collegamento tra vulnerabilit&agrave; delle cellule e carico di calcio mitocondriale e per testare l&#39;ipotesi che solo una sottopopolazione di mitocondri danneggiati da calcio overload comunicati apoptogens, abbiamo misurato direttamente le concentrazioni del Ca totale (gratuito pi&ugrave; vincolato) nei mitocondri individuali e monitorati in paralleli cambiamenti strutturali e la localizzazione sottocellulare di pro-apoptotic citocromo c dopo sovrastimolazione NMDA in neuroni ippocampali colti. Di l&agrave; di elevazione transitoria di calcio citosolico e perturbazione di Na + K + omeostasi, stimolazione del NMDA indotta da cambiamenti drammatici, ma soprattutto reversibile, nei mitocondri, tra cui calcio forte elevazione, depolarizzazione potenziale di membrana e gonfiore variabile. Elevazione della matrice Ca nel circa un terzo dei mitocondri che erano fortemente gonfio, come pure l&#39;assenza di gonfiore quando Ca2 + voce venne abolita, indicano un ruolo essenziale per sovraccarico di Ca. Poco dopo l&#39;esposizione NMDA, citocromo c, normalmente localizzato ai mitocondri, divenne diffusamente distribuiti nel citoplasma, coincida con la comparsa dei mitocondri gravemente gonfiati con rottura delle membrane esterne; in queste condizioni, il citocromo c &egrave; stato mantenuto nei mitocondri intatti, implicando che &egrave; stato rilasciato principalmente dai mitocondri danneggiati. Coerente con il ruolo del sovraccarico di Ca mitocondriale, Carbonil cianuro-p (trifluorometossi) phenylhydrazone diminuito accumulo di Ca, ha impedito la versione del citocromo c e fu neuroprotective. Questi risultati supportano un meccanismo in cui morte excitotoxic ritardato comporta rilascio di apoptogen da una sottopopolazione di mitocondri sovraccarico di calcio, mentre altri, integri mitocondri mantengono la normale funzione.

Sovraccarico di calcio excitotoxic in una sottopopolazione di trigger mitocondri ritardata morte in neuroni ippocampali.

&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E; excitotoxic &#x523A;&#x6FC0;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x904E;&#x8CA0;&#x8377;&#x3068;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x8A98;&#x5C0E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3001;&#x7D30;&#x80DE;&#x6B7B;&#x306E;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x306E;&#x9045;&#x5EF6;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3092;&#x8A98;&#x767A;&#x3057;&#x307E;&#x3059;&#x3002;Apoptogen &#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x306E;&#x6B63;&#x78BA;&#x306A;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x3001;&#x305F;&#x3060;&#x3057;&#x3001;&#x7269;&#x8B70;&#x3092;&#x91B8;&#x3059;&#x6B8B;&#x308A;&#x307E;&#x3059;&#x3002;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x8CA0;&#x8377;&#x304A;&#x3088;&#x3073;&#x30BB;&#x30EB;&#x306E;&#x8106;&#x5F31;&#x6027;&#x306F;&#x3001;&#x9593;&#x306E;&#x9023;&#x643A;&#x3092;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x308B;&#x3068;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x4E9C;&#x7A2E;&#x3060;&#x3051;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x904E;&#x8CA0;&#x8377;&#x306E;&#x30EA;&#x30EA;&#x30FC;&#x30B9; apoptogens &#x306B;&#x3088;&#x3063;&#x3066;&#x640D;&#x50B7;&#x3053;&#x3068;&#x4EEE;&#x8AAC;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x304C;&#x500B;&#x3005; &#x306E;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x5408;&#x8A08;&#x306E; Ca &#xFF08;&#x7121;&#x6599;&#x30D7;&#x30E9;&#x30B9; &#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x305F;&#xFF09; &#x306E;&#x6FC3;&#x5EA6;&#x3092;&#x76F4;&#x63A5;&#x6E2C;&#x5B9A;&#x3057;&#x3001;&#x4E26;&#x5217;&#x69CB;&#x9020;&#x5909;&#x5316;&#x3068;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x8A98;&#x5C0E;&#x30B7;&#x30C8;&#x30AF;&#x30ED;&#x30E0; c &#x306E;&#x7D30;&#x80DE;&#x5185;&#x5C40;&#x5728;&#x5316;&#x57F9;&#x990A;&#x6D77;&#x99AC;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; NMDA &#x904E;&#x5270;&#x306A;&#x523A;&#x6FC0;&#x5F8C;&#x306E;&#x76E3;&#x8996;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x3068;&#x6442;&#x52D5;&#x306B;&#x5BFE;&#x3059;&#x308B; Na &#x306E;&#x4E0A;&#x6607;&#x904E;&#x6E21;&#x3092;&#x8D85;&#x3048;&#x3066; c++/cli K + &#x6052;&#x5E38;&#x6027;&#x3001;NMDA &#x523A;&#x6FC0;&#x5F37;&#x3044;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6A19;&#x9AD8;&#x3001;&#x819C;&#x96FB;&#x4F4D;&#x8131;&#x5206;&#x6975;&#x3068;&#x53EF;&#x5909;&#x306E;&#x816B;&#x308C;&#x3092;&#x542B;&#x3080;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x5287;&#x7684;&#x306A;&#x3057;&#x304B;&#x3057;&#x4E3B;&#x306B;&#x53EF;&#x9006;&#x5909;&#x5316;&#x3002;&#x6A19;&#x9AD8;&#x306B;&#x304A;&#x3051;&#x308B; ca 2 + &#x30A8;&#x30F3;&#x30C8;&#x30EA;&#x3092;&#x5EC3;&#x6B62;&#x3057;&#x305F;&#x3068;&#x304D;&#x306F;&#x3001;&#x816B;&#x308C;&#x306E;&#x6709;&#x7121;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x7D04; 1/3 &#x5F37;&#x304C;&#x816B;&#x308C;&#x3066;&#x3044;&#x305F;&#x3001;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9; Ca &#x306E; Ca &#x306E;&#x30AA;&#x30FC;&#x30D0;&#x30FC; &#x30ED;&#x30FC;&#x30C9;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;NMDA &#x66B4;&#x9732;&#x5F8C;&#x307E;&#x3082;&#x306A;&#x304F;&#x30B7;&#x30C8;&#x30AF;&#x30ED;&#x30E0; c&#x3001;&#x901A;&#x5E38;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306B;&#x5C40;&#x5728;&#x306F;&#x3073;&#x307E;&#x3093;&#x6027;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x306E;&#x6DF1;&#x523B;&#x306A;&#x816B;&#x308C;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x5916;&#x819C;&#x306E;&#x7834;&#x88C2;&#x306E;&#x5916;&#x89B3;&#x3068;&#x4E00;&#x81F4;&#x3059;&#x308B;&#x5206;&#x6563;&#x306B;&#x306A;&#x3063;&#x305F;;&#x3053;&#x308C;&#x3089;&#x306E;&#x6761;&#x4EF6;&#x4E0B;&#x3067;&#x306F;&#x3001;&#x30B7;&#x30C8;&#x30AF;&#x30ED;&#x30E0; c &#x306F;&#x305D;&#x308C;&#x304B;&#x3089;&#x4E3B;&#x306B;&#x7834;&#x640D;&#x3057;&#x305F;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x304C;&#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3055;&#x308C;&#x305F;&#x3053;&#x3068;&#x3092;&#x610F;&#x5473;&#x3057;&#x3066;&#x3001;&#x7121;&#x50B7;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x4E2D;&#x4FDD;&#x6301;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x30AA;&#x30FC;&#x30D0;&#x30FC; &#x30ED;&#x30FC;&#x30C9; Ca &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x5F79;&#x5272;&#x306F;&#x3001;&#x30AB;&#x30EB;&#x30DC;&#x30CB;&#x30EB; &#x30B7;&#x30A2;&#x30F3; p - &#xFF08;&#x30D5;&#x30C3;&#x7D20;&#x7CFB;&#x4F4E;&#x5206;&#x5B50;) phenylhydrazone Ca &#x306E;&#x84C4;&#x7A4D;&#x3092;&#x6E1B;&#x5C11;&#x3001;&#x30B7;&#x30C8;&#x30AF;&#x30ED;&#x30E0; c &#x306E;&#x653E;&#x51FA;&#x3092;&#x9632;&#x6B62;&#x3001;&#x795E;&#x7D4C;&#x4FDD;&#x8B77;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x4ED6;&#x3001;&#x7121;&#x50B7;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x304C;&#x901A;&#x5E38;&#x6A5F;&#x80FD;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x306E;&#x306B;&#x5BFE;&#x3057;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x304B;&#x3089;&#x9045;&#x5EF6; excitotoxic &#x6B7B; apoptogen &#x30EA;&#x30EA;&#x30FC;&#x30B9; &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x304C;&#x30AA;&#x30FC;&#x30D0;&#x30FC; &#x30ED;&#x30FC;&#x30C9; &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x4E9C;&#x7A2E;&#x304B;&#x3089;&#x3092;&#x542B;&#x3080;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;

Excitotoxic &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x904E;&#x8CA0;&#x8377;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2; &#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x306E;&#x4E9C;&#x96C6;&#x56E3;&#x306B;&#x6D77;&#x99AC;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x6B7B;&#x9045;&#x5EF6;&#x3002;

&#xB274;&#xB7F0;&#xC5D0;&#xC11C; excitotoxic &#xC790;&#xADF9; &#xC720;&#xB3C4; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xCE7C;&#xC298; &#xACFC;&#xBD80;&#xD558; &#xBC0F; &#xD504;&#xB85C;-apoptotic &#xB2E8;&#xBC31;&#xC9C8;, &#xC5B4;&#xB5A4; &#xD2B8;&#xB9AC;&#xAC70; &#xC9C0;&#xC5F0; &#xC138;&#xD3EC; &#xC8FD;&#xC74C;&#xC758; &#xB9B4;&#xB9AC;&#xC2A4; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, apoptogen &#xC790;&#xB8CC;&#xC758; &#xC815;&#xD655;&#xD55C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xB17C;&#xB780;&#xC774; &#xB0A8;&#xC544; &#xC788;&#xB2E4;. &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xCE7C;&#xC298; &#xBD80;&#xD558; &#xBC0F; &#xC140; &#xCDE8;&#xC57D;&#xC810; &#xAC04;&#xC758; &#xC5F0;&#xACC4; &#xD2B9;&#xC131;&#xD654; &#xACE0; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xB9CC; subpopulation &#xCE7C;&#xC298; &#xC624;&#xBC84; &#xB85C;&#xB4DC; &#xBC84;&#xC804; apoptogens&#xC5D0; &#xC758;&#xD574; &#xC190;&#xC0C1; &#xB41C; &#xAC00;&#xC124;&#xC744; &#xD14C;&#xC2A4;&#xD2B8; &#xD558; &#xC6B0;&#xB9AC; &#xAC1C;&#xBCC4; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC5D0;&#xC11C; &#xC804;&#xCCB4; Ca (&#xBB34;&#xB8CC; &#xD50C;&#xB7EC;&#xC2A4; &#xBC14;&#xC6B4;&#xB4DC;)&#xC758; &#xB18D;&#xB3C4; &#xC9C1;&#xC811; &#xCE21;&#xC815; &#xD558; &#xACE0; &#xBCD1;&#xB82C; &#xAD6C;&#xC870; &#xBCC0;&#xACBD; &#xBC0F; &#xAD50;&#xC591;&#xB41C; hippocampal &#xC2E0;&#xACBD;&#xC5D0; &#xC788;&#xB294; NMDA overstimulation &#xD6C4; &#xD504;&#xB85C; apoptotic &#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c&#xC758; subcellular &#xC9C0;&#xC5ED;&#xD654; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;. &#xACFC;&#xB3C4; &#xD55C; &#xC0C1;&#xC2B9; cytosolic &#xCE7C;&#xC298;&#xACFC; &#xB098;&#xC758; &#xC12D; &#xB3D9;&#xC744; &#xB118;&#xC5B4; + K + &#xD56D;&#xC0C1;&#xC131;, NMDA &#xC790;&#xADF9; &#xC720;&#xBC1C; &#xAC15;&#xB825;&#xD55C; &#xCE7C;&#xC298; &#xC0C1;&#xC2B9;, &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xC7A0;&#xC7AC; depolarization &#xAC00;&#xBCC0; &#xBD93;&#xAE30; &#xB4F1; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC5D0;&#xC11C; &#xADF9;&#xC801;&#xC73C;&#xB85C;, &#xD558;&#xC9C0;&#xB9CC; &#xC8FC;&#xB85C; &#xAC00;&#xC5ED; &#xBCC0;&#xD654;. &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4; Ca&#xB294; &#xC57D; 1 / 3&#xC740; &#xAC15;&#xB825; &#xD558; &#xAC8C; &#xBD80; &#xC5B4;, &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; Ca2 + &#xD56D;&#xBAA9; &#xD3D0;&#xC9C0; &#xB418;&#xC5C8;&#xB2E4; &#xB54C; &#xBD93;&#xAE30;&#xC758; &#xBD80;&#xC7AC;&#xC5D0;&#xC11C; &#xC0C1;&#xC2B9; &#xCE98;&#xB9AC;&#xD3EC;&#xB2C8;&#xC544; &#xC624;&#xBC84; &#xB85C;&#xB4DC;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. NMDA &#xB178;&#xCD9C; &#xD6C4; &#xACE7; &#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c, &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;, &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xC9C0;&#xC5ED;&#xD654; &#xB41C; &#xBE0C;&#xB7EC;&#xC2DC;&#xAC00; &#xD30C;&#xC5F4;&#xB41C; &#xC678;&#xBD80; &#xBA64;&#xBE0C;&#xB808;&#xC778;;&#xACFC; &#xC2EC;&#xAC01; &#xD558; &#xAC8C; &#xBD80; &#xC5B4; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC758; &#xBAA8;&#xC591;&#xC744; &#xC77C;&#xCE58; &#xC138;&#xD3EC;&#xC9C8;&#xC5D0; &#xBD84;&#xC0B0; &#xB418;&#xC5C8;&#xB2E4; &#xC774;&#xB7EC;&#xD55C; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c&#xB294; &#xC190;&#xC0C1; &#xB41C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC5D0;&#xC11C; &#xC8FC;&#xB85C; &#xB9B4;&#xB9AC;&#xC2A4; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xB73B; &#xADF8;&#xB300;&#xB85C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC5D0;&#xC11C; &#xC720;&#xC9C0; &#xB418;&#xC5C8;&#xB2E4;. &#xC624;&#xBC84; &#xB85C;&#xB4DC; Ca &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC758; &#xC5ED;&#xD560;&#xC640; &#xC77C;&#xCE58;, &#xCE74;&#xBCF4;&#xB2D0;&#xAE30; &#xCCAD;&#xC0B0;&#xAC00; &#xB9AC; p-(trifluoromethoxy) phenylhydrazone Ca &#xCD95;&#xC801; &#xAC10;&#xC18C;, &#xBC29;&#xD574; &#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c &#xCD9C;&#xC2DC; &#xD558; &#xACE0; &#xC2E0;&#xACBD; &#xBCF4;&#xD638; &#xD588;&#xB2E4;. &#xC774; &#xACB0;&#xACFC; &#xB2E4;&#xB978;, &#xC190;&#xC0C1; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xAE30;&#xB2A5;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xB294; &#xBC18;&#xBA74; &#xC9C0;&#xC5F0;&#xB41C; excitotoxic &#xC8FD;&#xC74C; &#xCE7C;&#xC298; &#xC624;&#xBC84; &#xB85C;&#xB4DC; &#xB41C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;, subpopulation&#xC5D0;&#xC11C; apoptogen &#xB9B4;&#xB9AC;&#xC2A4;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

&#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xD2B8;&#xB9AC;&#xAC70; subpopulation&#xC5D0; Excitotoxic &#xCE7C;&#xC298; &#xACFC;&#xBD80;&#xD558; hippocampal &#xB274;&#xB7F0;&#xC5D0;&#xC11C; &#xC8FD;&#xC74C;&#xC744; &#xC9C0;&#xC5F0;.

En las neuronas, excitatoria est&iacute;mulo induce la sobrecarga de calcio mitocondrial y la liberaci&oacute;n de prote&iacute;nas pro-apopt&oacute;ticas, qu&eacute; disparadores retrasa la muerte celular. Sin embargo, los mecanismos precisos de liberaci&oacute;n apoptogen, siguen siendo pol&eacute;micos. Para caracterizar la vinculaci&oacute;n de la vulnerabilidad de carga y de la c&eacute;lula de calcio mitocondrial y para probar la hip&oacute;tesis que s&oacute;lo una subpoblaci&oacute;n de mitocondrias da&ntilde;adas por sobrecarga de calcio comunicados apoptogens, hemos medido directamente las concentraciones de Ca total (libre m&aacute;s consolidado) en las mitocondrias individuales y monitoreados en paralelos cambios estructurales y la localizaci&oacute;n subcelular de pro-apopt&oacute;tica citocromo c despu&eacute;s de estimulaci&oacute;n NMDA en las neuronas de hipocampales cultivadas. M&aacute;s all&aacute; de la elevaci&oacute;n transitoria de calcio citos&oacute;lico y perturbaci&oacute;n de Na + / K + homeostasis, estimulaci&oacute;n NMDA inducida por cambios dram&aacute;ticos, pero principalmente reversibles, en las mitocondrias, incluyendo la elevaci&oacute;n de calcio fuerte, despolarizaci&oacute;n potencial de membrana e inflamaci&oacute;n variable. Elevaci&oacute;n de la matriz Ca en el aproximadamente un tercio de las mitocondrias que fueron fuertemente hinchados, as&iacute; como la ausencia de inflamaci&oacute;n cuando se aboli&oacute; la entrada de Ca2 +, indican un papel esencial para la sobrecarga de Ca. Poco despu&eacute;s de la exposici&oacute;n NMDA, citocromo c, normalmente localizado en las mitocondrias, se convirti&oacute; en distribuidas difusamente en el citoplasma, coincidente con la aparici&oacute;n de mitocondrias severamente inflamadas con rotura de membranas externa; en estas condiciones, el citocromo c se mantuvo en mitocondria intacto, lo que implica que fue lanzado principalmente de mitocondrias da&ntilde;adas. Consistente con el papel de la sobrecarga de Ca mitocondrial, cianuro de carbonilo p-(trifluoromethoxy) phenylhydrazone disminuida acumulaci&oacute;n de Ca, impidi&oacute; la liberaci&oacute;n del citocromo c y fue neuroprotector. Estos resultados apoyan un mecanismo en que retrasada excitatoria muerte implica apoptogen lanzamiento de una subpoblaci&oacute;n de mitocondrias de sobrecarga de calcio, mientras que las mitocondrias, flamante mantienen la funci&oacute;n normal.

Sobrecarga de calcio excitatoria en una subpoblaci&oacute;n de disparadores de mitocondrias hab&iacute;a retrasado muerte de neuronas hipocampales.

Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention.

In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.

Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons.

Overactivation of NMDA receptors (NMDARs) is a critical early step in glutamate-evoked excitotoxic injury of CNS neurons. Distinct NMDAR-coupled pathways specified by, for example, receptor location or subunit composition seem to govern glutamate-induced excitotoxic death, but there is much uncertainty concerning the underlying mechanisms of pathway selection. Here we ask whether, and if so how, route-specific vulnerability is coupled to Ca(2+) overload and mitochondrial dysfunction, which is also a known, central component of exitotoxic injury. In cultured hippocampal neurons, overactivation of only extrasynaptic NMDARs resulted in Ca(2+) entry strong enough to promote Ca(2+) overload, which subsequently leads to mitochondrial dysfunction and cell death. Receptor composition per se appears not to be a primary factor for specifying signal coupling, as NR2B inhibition abolished Ca(2+) loading and was protective only in predominantly NR2B-expressing young neurons. In older neurons expressing comparable levels of NR2A- and NR2B-containing NMDARs, amelioration of Ca(2+) overload required the inhibition of extrasynaptic receptors containing both NR2 subunits. Prosurvival synaptic stimuli also evoked Ca(2+) entry through both N2A- and NR2B-containing NMDARs, but, in contrast to excitotoxic activation of extrasynaptic NMDARs, produced only low-amplitude cytoplasmic Ca(2+) spikes and modest, nondamaging mitochondrial Ca(2+) accumulation. The results--showing that the various routes of excitotoxic Ca(2+) entry converge on a common pathway involving Ca(2+) overload-induced mitochondrial dysfunction--reconcile and unify many aspects of the "route-specific" and "calcium load-dependent" views of exitotoxic injury.

Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity.

Hippocampal CA1 pyramidal neurons are selectively vulnerable to ischemia, while adjacent CA3 neurons are relatively resistant. Although glutamate receptor-mediated mitochondrial Ca(2+) overload and dysfunction is a major component of ischemia-induced neuronal death, no direct relationship between selective neuronal vulnerability and mitochondrial dysfunction has been demonstrated in intact brain preparations. Here, we show that in organotypic slice cultures NMDA induces much larger Ca(2+) elevations in vulnerable CA1 neurons than in resistant CA3. Consequently, CA1 mitochondria exhibit stronger calcium accumulation, more extensive swelling and damage, stronger depolarization of their membrane potential, and a significant increase in ROS generation. NMDA-induced Ca(2+) and ROS elevations were abolished in Ca(2+)-free medium or by NMDAR antagonists, but not by zinc chelation. We conclude that Ca(2)(+) overload-dependent mitochondrial dysfunction is a determining factor in the selective vulnerability of CA1 neurons.

Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons.

Calcium is an extraordinarily versatile signaling ion, encoding cellular responses to a wide variety of external stimuli. In neurons, mitochondria can accumulate enormous amounts of calcium, with the consequence that mitochondrial calcium uptake, sequestration and release play pivotal roles in orchestrating calcium-dependent responses as diverse as gene transcription and cell death. In this review, we consider the basic chemistry of calcium as a 'sticky' cation, which leads to extremely high bound/free ratios, and discuss areas of current interest or controversy. Topics addressed include methodologies for measuring local intracellular calcium, mitochondrial calcium buffering and loading capacity, mitochondrially directed spatial calcium gradients, and the role of calcium overload-dependent mitochondrial dysfunction in glutamate-evoked excitotoxic injury and neurodegeneration. Finally, we consider the relationship between delayed calcium de-regulation, the mitochondrial permeability transition and the generation of reactive oxygen species, and propose a unified view of the 'source specificity' and 'calcium overload' models of N-methyl-d-aspartate (NMDA) receptor-dependent excitotoxicity. Non-NMDA receptor mechanisms of excitotoxicity are discussed briefly.

Calcium-dependent mitochondrial function and dysfunction in neurons.

Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD.

The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder.

Glutamate excitotoxicity, a major component of many neurodegenerative disorders, is characterized by excessive calcium influx selectively through NMDARs. However, there is a substantial uncertainty concerning why other known routes of significant calcium entry, in particular, VGCCs, are not similarly toxic. Here, we report that in the majority of neurons in rat hippocampal and cortical cultures, maximal L-type VGCC activation induces much lower calcium loading than toxic NMDAR activation. Consequently, few depolarization-activated neurons exhibit calcium deregulation and cell death. Activation of alternative routes of calcium entry induced neuronal death in proportion to the degree of calcium loading. In a small subset of neurons, depolarization evoked stronger calcium elevations, approaching those induced by toxic NMDA. These neurons were characterized by elevated expression of VGCCs and enhanced voltage-gated calcium currents, mitochondrial dysfunction and cell death. Preventing VGCC-dependent mitochondrial calcium loading resulted in stronger cytoplasmic calcium elevations, whereas inhibiting mitochondrial calcium clearance accelerated mitochondrial depolarization. Both observations further implicate mitochondrial dysfunction in VGCC-mediated cell death. Results indicate that neuronal vulnerability tracks the extent of calcium loading but does not appear to depend explicitly on the route of calcium entry.

Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury.

Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons - normal (2 mM) calcium plus elevated (> 100 μM) exogenous zinc - mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium-containing media. This contrasts with the lack of any mitochondrial response in zinc-containing but calcium-free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore-induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium- and zinc-containing media, depolarization-induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium. Zinc and calcium are both implicated in ischemic injury. While calcium toxicity is mediated by mitochondrial dysfunction, mechanisms of zinc toxicity are unclear. Here we show that under conditions mimicking ischemia in hippocampal neurons, glutamate-induced mitochondrial dysfunction - swelling (red mitochondrion), depolarization and increased ROS generation - is triggered only by calcium, while zinc mainly facilitates calcium-dependent mitochondrial damage.

Natalia B. Pivovarova

Laboratory of Neurobiology,
National Institute of Neurological Disorders and Stroke,
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke

National Institutes of Health

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

Natalia B. Pivovarova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Neurobiology,National Institute of Neurological Disorders and Stroke,Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke

National Institutes of Health

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

Laboratory of Neurobiology,
National Institute of Neurological Disorders and Stroke,
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke