prolonged-incubation-acute-neuronal-tissue-for-electrophysiology

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Western Sydney University

The mammalian cortical layer I is a convergence site for axons of sub- and intracortical origin, and the apical dendritic tufts of pyramidal neurons. A prominent feature of layer I is an extensive plexus of inhibitory axons, which originate from stellate cells in all cortical laminae. The role of this inhibitory projection in the activity of cortical networks has yet to be determined. We investigated the degree to which inhibitory inputs within layer I affect the activity of the underlying cellular network. Field potentials (FPs) were recorded in layer II/III. Focal application of the GABAA blocker picrotoxin in layer I above the recording pipette or the removal of layer I resulted in larger FP amplitudes for stimulations at control-equal intensities. When inhibition was partially blocked, the removal of layer I caused a significant reduction in the threshold stimulus intensity required for generating epileptiform events, and a rise in the propagation velocity of these events. Immunocytochemistry for chemical markers of interneurons proved that the inhibitory input to layer I is predominantly somatostatin immunoreactive (SM-ir), such that layer I contains approximately one-third of all SM-ir axons in the cortex. Calretinin-immunoreactive axons were also present in layer I at a lower density. We conclude that the impact of layer I on the cortical cellular network includes a significant inhibitory component. This inhibition confers a moderate restraining influence, and its removal increases the excitability of cortical circuits, but not sufficiently to induce epileptic phenomena.

Inhibitory effect of mouse neocortex layer I on the underlying cellular network.

Nitric oxide (NO), a cellular signaling molecule, is produced in the brain by both neurons and astrocytes. While neurons are capable of rapid release of small amounts of NO serving as neurotransmitter, astrocytic NO production has been demonstrated mainly as a slow reaction to various stress stimuli. Little is known about the role of astrocyte-produced NO. Using the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and acute slices from mouse brain, we distinguished neurons from astrocytes based on their different fluorescence kinetics and pattern, cellular morphology, electrophysiology, and responses to selective nitric oxide synthase (NOS) inhibitors. Typically, astrocytic fluorescence followed neuronal fluorescence with a delay of 1-2 min and was dependent on the inducible NOS isoform (iNOS) activity. Western blot analysis established the presence of functional iNOS in the neocortex. An assay for cell death revealed that most DAF-2DA-positive neurons, but not astrocytes, were damaged. Whole cell recordings from astrocytes confirmed that these cells maintained their membrane potential and passive properties during illumination and afterward. Induction of excitotoxicity by brief application of glutamate triggered an immediate and intense astrocytic response, while high-frequency electrical stimulation failed to do so. The present study demonstrates, for the first time, rapid and massive iNOS-dependent NO production by astrocytes in situ, which appears to be triggered by acute neuronal death. These data may bear important implications for our theoretical understanding and practical management of acute brain insults.

Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices.

It has been well accepted that glial cells in the central nervous system (CNS) produce nitric oxide (NO) through the induction of a nitric oxide synthase isoform (NOS2) only in response to various insults. Recently we described rapid astroglial, NOS2-dependent, NO production in the neocortex of healthy mice on a time scale relevant to neuronal activity. To explore a possible role for astroglial NOS2 in normal brain function we investigated a NOS2 knockout mouse (B6;129P2-Nos2(tm1Lau)/J, Jackson Laboratory). Previous studies of this mouse strain revealed mainly altered immune responses, but no compensatory pathways and no CNS abnormalities have been reported.

Enhanced astrocytic nitric oxide production and neuronal modifications in the neocortex of a NOS2 mutant mouse.

Nitric oxide (NO) has been recognized as an atypical neuronal messenger affecting synaptic transmission, but its cellular source has remained unresolved as the neuronal NO synthase isoform (nNOS) in brain areas such as the neocortex is expressed only by a small subset of inhibitory neurons. The involvement of the glial NOS isoform (iNOS) in modulating neuronal activity has been largely ignored because it has been accepted that this enzyme is regulated by gene induction following detrimental stimuli. Using acute brain slices from mouse neocortex and electrophysiology, we found that selective inhibition of iNOS reduced both spontaneous and evoked synaptic release. Moreover, iNOS inhibition partially prevented and reversed the potentiation of excitatory synapses in layer 2/3 pyramidal neurons. NOS enzymatic assay confirmed a small but reliable Ca(2+)-independent activity fraction, consistent with the existence of functioning iNOS in the tissue. Together these data point to astrocytes as a source for the nitrosative regulation of synaptic release in the neocortex.

Astrocytic iNOS-dependent enhancement of synaptic release in mouse neocortex.

&#x4EE5;&#x8272;&#x5217;&#x519C;&#x6C11;&#x51FA;&#x53E3; 25 &#x4E07;&#x5428;&#x7684;&#x9A6C;&#x94C3;&#x85AF;&#x5757;&#x830E;&#x6BCF;&#x5E74; &asymp; 40 &#x9879;&#xFF0C;&#x4E07;&#x5428;&#xFF0C;&#x5176;&#x4E2D;&#x65E9;&#xFF0C;&#x6536;&#x83B7;&#x524D;&#x76AE;&#x80A4;&#x96C6;&#x3002;&#x8FD1;&#x5E74;&#x6765;&#xFF0C;&#x6709;&#x65E9;&#x671F;&#x6536;&#x83B7;&#x7684;&#x9A6C;&#x94C3;&#x85AF;&#x5757;&#x830E; (&#39;&#x5C3C;&#x53E4;&#x62C9;&#39;) &#x88C5;&#x5728;&#x5927;&#x888B;&#xFF0C;&#x4EE5;&#x4FDD;&#x7559;&#x6C34;&#x5206;&#x7684;&#x6CE5;&#x70AD;&#x4E0A;&#x7684;&#x9ED1;&#x76AE;&#x80A4;&#x6591;&#x70B9;&#x7684;&#x53D1;&#x751F;&#x7387;&#x589E;&#x52A0;&#x3002;&#x5B58;&#x50A8;&#x548C;&#x6D77;&#x5916;&#x8FD0;&#x8F93;&#x671F;&#x95F4;&#x4E0D;&#x89C4;&#x5219;&#x574F;&#x6B7B;&#x70B9;&#x5F62;&#x5F0F;&#x3002;&#x8868;&#x5F81;&#x7684;&#x75C7;&#x72B6;&#x53D1;&#x5C55;&#x6240;&#x9700;&#x7684;&#x6761;&#x4EF6;&#x8868;&#x793A;&#x888B;&#x5305;&#x88C5;&#x540E;&#x7684;&#x6E29;&#x5EA6;&#x662F; 11 &#x5230; 13 &deg; C &#x548C;&#x5B83;&#x5230;&#x8FBE;&#x76EE;&#x6807; &#xFF08;8 &deg; C&#xFF09; &#x6E29;&#x5EA6;&#x53EA;&#x6709; 25 &#x5929;&#x91C7;&#x540E;&#x3002;&#x8FD9;&#x4E2A;&#x6E29;&#x5EA6;&#x7684;&#x7F13;&#x6162;&#x964D;&#x4F4E;&#x53EF;&#x80FD;&#x4F1A;&#x63A8;&#x52A8;&#x5EFA;&#x7ACB;&#x75C5;&#x539F;&#x611F;&#x67D3;&#x3002;&#x4ECE;&#x5178;&#x578B;&#x75C5;&#x53D8;&#x682A;&#x88AB;&#x786E;&#x5B9A;&#x4E3A;&#x4E1D;&#x6838;&#x83CC;&#x548C; Koch &#x7684;&#x5047;&#x8BBE;&#x4E0E; 25 &#x682A;&#x5B8C;&#x6210;&#x4E86;&#x4EBA;&#x5DE5;&#x63A5;&#x79CD;&#x6F14;&#x51FA; 13 &#x81F3; 14 &deg; c&#x3002;&#x7CFB;&#x7EDF;&#x53D1;&#x80B2;&#x5206;&#x6790;&#xFF0C;&#x4F7F;&#x7528;&#x5185;&#x90E8;&#x7684; rDNA &#x57FA;&#x56E0;&#xFF0C;&#x5206;&#x914D;&#x7ED9;&#x7B2C;&#x4E09;&#x7EC4;&#x543B;&#x5408;&#x7684;&#x67AF;&#x4E09;&#x682A;&#x8F6C;&#x5F55;&#x95F4;&#x9694;&#x5E8F;&#x5217; &#xFF08;ITS1 &#x548C; ITS2&#xFF09;&#x3002;&#x8FD9;&#x4E9B;&#x94A2;&#x7B4B;&#x67AF;&#x75C5;&#x83CC;&#x7684;&#x83CC;&#x4E1D;&#x7684;&#x4F24;&#x5458;&#x5757;&#x830E;&#x63A5;&#x79CD;&#x5BFC;&#x81F4;&#x53D7;&#x611F;&#x67D3;&#x5730;&#x533A; oversuberization &#x54CD;&#x5E94;&#x3002;&#x6C34;&#x7A3B;&#x7EB9;&#x67AF;&#x75C5;&#x83CC; R.Rh17 &#x9694;&#x79BB; suberin &#x751F;&#x7269;&#x5408;&#x6210;&#x76F8;&#x5173;&#x57FA;&#x56E0; StKCS6 &#x548C; CYP86A33 &#x7684;&#x8868;&#x8FBE;&#x589E;&#x52A0; 6.8-&#x548C; 3.4-fold&#xFF0C;&#x5206;&#x522B;&#xFF0C;24 h &#x4E0E;&#x521B;&#x4F24;&#x8BF1;&#x5BFC;&#x6813;&#xFF0C;&#x8868;&#x8FBE; 48 h postinoculation&#xFF0C;postinoculation&#xFF0C;&#x5176;&#x6B21;&#x662F;&#x5728; POP_A &#x4E2D;&#x589E;&#x52A0;&#x4E86;&#x79D1;&#x7814;&#xFF0C;&#x76F8;&#x5173;&#x57FA;&#x56E0;&#x76F8;&#x6BD4; noninoculated &#x7684;&#x5757;&#x830E;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x91C7;&#x540E;&#x6697;&#x6591;&#x75C5;&#x662F;&#x94A2;&#x7B4B;&#x6C34;&#x7A3B;&#x7EB9;&#x67AF;&#x75C5;&#x83CC;&#x7684;&#x5757;&#x830E;&#x76AE;&#x80A4;&#x96C6;&#x524D;&#x53D1;&#x751F;&#x7684; AG 3 &#x611F;&#x67D3; oversuberization &#x54CD;&#x5E94;&#x3002;

&#x9A6C;&#x94C3;&#x85AF;&#x5757;&#x830E;&#x91C7;&#x540E;&#x9ED1;&#x76AE;&#x80A4;&#x6591;&#x70B9;&#x662F;&#x5BF9;&#x4E1D;&#x6838;&#x83CC;&#x6C34;&#x7A3B;&#x7EB9;&#x67AF;&#x75C5;&#x83CC;&#x611F;&#x67D3; oversuberization &#x56DE;&#x5E94;&#x3002;

Israeli farmers export 250,000 tons of potato tubers annually, â‰ˆ40,000 tons of which are harvested early, before skin set. In recent years, there has been an increase in the occurrence of dark skin spots on early-harvested potato tubers ('Nicola') packed in large bags containing peat to retain moisture. The irregular necrotic spots form during storage and overseas transport. Characterization of the conditions required for symptom development indicated that bag temperature after packing is 11 to 13Â°C and it reaches the target temperature (8Â°C) only 25 days postharvest. This slow decrease in temperature may promote the establishment of pathogen infection. Isolates from typical lesions were identified as Rhizoctonia spp., and Koch's postulates were completed with 25 isolates by artificial inoculation performed at 13 to 14Â°C. Phylogenetic analysis, using the internal transcribed spacer sequences (ITS1 and ITS2) of rDNA genes, assigned three isolates to anastomosis group 3 of Rhizoctonia solani. Inoculation of wounded tubers with mycelium of these R. solani isolates resulted in an oversuberization response in the infected area. With isolate Rh17 of R. solani, expression of the suberin biosynthesis-related genes StKCS6 and CYP86A33 increased 6.8- and 3.4-fold, respectively, 24 h postinoculation, followed by a 2.9-fold increase in POP_A, a gene associated with wound-induced suberization, expression 48 h postinoculation, compared with the noninoculated tubers. We suggest that postharvest dark spot disease is an oversuberization response to R. solani of AG-3 infection that occurs prior to tuber skin set.

Postharvest dark skin spots in potato tubers are an oversuberization response to Rhizoctonia solani infection.

Les agriculteurs isra&#xE9;liens d&#x26;#39;exporter 250.000 tonnes de tubercules de pommes de terre chaque ann&#xE9;e, &#x2248; 40.000 tonnes qui sont r&#xE9;colt&#xE9;es de bonne heure, avant ensemble la peau. Ces derni&#xE8;res ann&#xE9;es, il ya eu une augmentation de l&#x26;#39;apparition de taches sombres sur la peau au d&#xE9;but des tubercules r&#xE9;colt&#xE9;s (&#x26;#39;Nicola&#x26;#39;) emball&#xE9;es dans de grands sacs contenant de la tourbe pour retenir l&#x26;#39;humidit&#xE9;. Les taches irr&#xE9;guli&#xE8;res n&#xE9;crotiques former pendant le transport de stockage et &#xE0; l&#x26;#39;&#xE9;tranger. Caract&#xE9;risation des conditions requises pour le d&#xE9;veloppement des sympt&#xF4;mes ont indiqu&#xE9; que la temp&#xE9;rature sac apr&#xE8;s l&#x26;#39;emballage est de 11 &#xE0; 13 &#xB0; C et il atteint la temp&#xE9;rature cible (8 &#xB0; C) apr&#xE8;s la r&#xE9;colte seulement 25 jours. Cette diminution lente de la temp&#xE9;rature peut favoriser la cr&#xE9;ation d&#x26;#39;une infection pathog&#xE8;ne. Les isolats provenant des l&#xE9;sions typiques ont &#xE9;t&#xE9; identifi&#xE9;s comme Rhizoctonia spp., Et les postulats de Koch ont &#xE9;t&#xE9; r&#xE9;alis&#xE9;es avec 25 isolats par inoculation artificielle effectu&#xE9;e &#xE0; 13 &#xE0; 14 &#xB0; C. L&#x26;#39;analyse phylog&#xE9;n&#xE9;tique, en utilisant les s&#xE9;quences espaceur interne transcrit (ITS1 et ITS2) de g&#xE8;nes ADNr, affect&#xE9;s &#xE0; trois isolats anastomose groupe 3 de Rhizoctonia solani. L&#x26;#39;inoculation de tubercules bless&#xE9;s avec du myc&#xE9;lium de ces isolats R. solani a entra&#xEE;n&#xE9; une r&#xE9;ponse oversuberization dans la zone infect&#xE9;e. Avec isoler RH17 de R. solani, l&#x26;#39;expression des sub&#xE9;rine biosynth&#xE8;se des g&#xE8;nes li&#xE9;s &#xE0; StKCS6 et CYP86A33 ont augment&#xE9; de 6,8-et 3,4 fois, respectivement, 24 h apr&#xE8;s l&#x26;#39;inoculation, suivie d&#x26;#39;une augmentation de 2,9 fois en POP_A, un g&#xE8;ne associ&#xE9; &#xE0; la blessure provoqu&#xE9;e par sub&#xE9;risation, l&#x26;#39;expression 48 h apr&#xE8;s, par rapport aux tubercules non inocul&#xE9;s. Nous sugg&#xE9;rons que la maladie post-r&#xE9;colte tache sombre est une r&#xE9;ponse oversuberization au R. solani AG-3 de l&#x26;#39;infection qui se produit avant ensemble peau du tubercule.

Post-r&#xE9;colte Spots peau fonc&#xE9;e dans les tubercules de pommes de terre sont une r&#xE9;ponse Oversuberization &#xE0; Rhizoctonia solani infection

Israelische Landwirte exportieren 250.000 Tonnen Kartoffeln Knollen j&auml;hrlich, &asymp;40, 000, die Tonnen davon vor Haut fr&uuml;h geerntet werden. In den letzten Jahren gab es eine Zunahme f&uuml;r das Auftreten von dunklen Haut Flecken auf fr&uuml;hen geerntete Kartoffel-Knollen (&#39;Nicola&#39;) verpackt in gro&szlig;en Taschen mit Torf, um Feuchtigkeit zu behalten. Unregelm&auml;&szlig;ige nekrotische Flecken Form w&auml;hrend der Lagerung und &Uuml;bersee-Transport. Charakterisierung von Voraussetzungen f&uuml;r eine Symptom-Entwicklung darauf hingewiesen, dass die Tasche nach der Verpackung betr&auml;gt 11 bis 13 &deg; C und sie erreicht eine Temperatur von 8 &deg; C nur 25 Tage Postharvest. Diese langsamen R&uuml;ckgang der Temperatur kann die Einrichtung von Pathogen-Infektion f&ouml;rdern. Isolate aus typischen L&auml;sionen waren als Rhizoctonia spp. und Kochs Postulate wurden abgeschlossen mit 25 Isolaten von k&uuml;nstlicher Inokulation durchgef&uuml;hrt bei 13 bis 14 &deg; C. Phylogenetische Analysen der internen transkribiert Spacer-Sequenzen (ITS1 und ITS2) der rDNA-Gene, Anastomose Gruppe 3 von Thanatephorus cucumeris drei Isolaten zugewiesen. Impfung von Verwundeten Knollen mit Myzel diese R. Solani Isolate ergab eine Oversuberization Antwort im Seuchengebiet. Mit Eingreiftruppe Rh17 von R. Solani, Ausdruck der Suberin Biosynthese-bezogenen Gene StKCS6 und CYP86A33 erh&ouml;ht 6,8 - und 3.4-fold, bzw. 24 h postinoculation, gefolgt von einem 2.9-fold Anstieg der POP_A, ein Gen mit Wunde-induzierte Suberization, Ausdruck 48 h Postinoculation, verbunden mit der noninoculated Knollen verglichen. Wir empfehlen, dass postharvest dunklen Ort Krankheit eine Oversuberization Antwort an R. Solani AG-3-Infektion, die vor der Knolle Haut Satz auftritt.

Postharvest dunkle Haut Spots in Kartoffel-Knollen sind eine Oversuberization Antwort zu Thanatephorus cucumeris Infektion.

Gli agricoltori israeliani esportare 250.000 tonnellate di tuberi di patata annualmente, &asymp;40, 000 tonnellate che vengono raccolte all&#39;inizio, prima set di pelle. Negli ultimi anni, c&#39;&egrave; stato un aumento dell&#39;insorgenza di macchie cutanee scure sui tuberi di patata precoce raccolte (&#39;Nicola&#39;) in grandi sacchi contenenti torba per trattenere l&#39;umidit&agrave;. La forma di macchie necrotiche irregolari durante lo stoccaggio e il trasporto all&#39;estero. Caratterizzazione delle condizioni necessarie per lo sviluppo del sintomo indicato che borsa dopo imballaggio &egrave; 11-13 &deg; C e raggiunge il postharvest destinazione (8 &deg; C) temperatura solo 25 giorni. Questa lenta diminuzione della temperatura pu&ograve; promuovere l&#39;istituzione di infezione del patogeno. Isolati da lesioni tipiche sono stati identificati come Rhizoctonia spp., e postulati di Koch sono stati completati con 25 isolati da inoculazione artificiale eseguito a 13-14 &deg; C. Analisi filogenetica, utilizzando l&#39;interno trascritto sequenze distanziale (ITS1 e ITS2) dei geni rDNA, assegnate tre isolati a gruppi di anastomosi 3 di Rhizoctonia solani. Inoculazione di tuberi feriti con micelio di questi isolati di r. solani ha provocato una risposta oversuberization in zona infetta. Con isoli Rh17 di R. solani, aumentata espressione di Suberina biosintesi relativi geni StKCS6 e CYP86A33 6,8 - e 3.4-fold, rispettivamente, 24 h postinoculation, seguita da un aumento del 2.9-fold in POP_A, un gene associato ferita indotta da suberization, postinoculation 48 h espressione, confrontati con i tuberi noninoculated. Suggeriamo che venga malattia macchia scura &egrave; una risposta oversuberization a r. solani AG-3 infezione che si verifica prima del set di pelle del tubero.

Macchie della pelle scura venga nei tuberi di patata sono una risposta oversuberization all&#39;infezione di Rhizoctonia solani.

&#x30A4;&#x30B9;&#x30E9;&#x30A8;&#x30EB;&#x306E;&#x8FB2;&#x6C11;&#x306F;&#x3001;&#x2248;4&#x4E07;&#x30C8;&#x30F3;&#x30B9;&#x30AD;&#x30F3;&#x30BB;&#x30C3;&#x30C8;&#x3059;&#x308B;&#x524D;&#x306B;&#x3001;&#x65E9;&#x671F;&#x306B;&#x53CE;&#x7A6B;&#x3055;&#x308C;&#x3001;&#x305D;&#x306E;&#x3046;&#x3061;&#x6BCE;&#x5E74;&#x30B8;&#x30E3;&#x30AC;&#x30A4;&#x30E2;25&#x4E07;&#x30C8;&#x30F3;&#x3092;&#x30A8;&#x30AF;&#x30B9;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x8FD1;&#x5E74;&#x3067;&#x306F;&#x3001;&#x6CE5;&#x70AD;&#x304C;&#x6C34;&#x5206;&#x3092;&#x4FDD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x542B;&#x3093;&#x3067;&#x3044;&#x308B;&#x5927;&#x304D;&#x306A;&#x888B;&#x306B;&#x8A70;&#x3081;&#x3001;&#x65E9;&#x671F;&#x53CE;&#x7A6B;&#x30B8;&#x30E3;&#x30AC;&#x30A4;&#x30E2;&#x306E;&#x584A;&#x830E;&#xFF08; &#x26;#39;&#x30CB;&#x30B3;&#x30E9;&#xB7;&#x26;#39;&#xFF09;&#x4E0A;&#x306B;&#x6D45;&#x9ED2;&#x3044;&#x808C;&#xB7;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306E;&#x767A;&#x751F;&#x304C;&#x5897;&#x52A0;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x4E0D;&#x898F;&#x5247;&#x306A;&#x58CA;&#x6B7B;&#x6591;&#x306F;&#x3001;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B8;&#x304A;&#x3088;&#x3073;&#x6D77;&#x5916;&#x8F38;&#x9001;&#x4E2D;&#x306B;&#x5F62;&#x6210;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x75C7;&#x72B6;&#x306E;&#x958B;&#x767A;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x6761;&#x4EF6;&#x306E;&#x7279;&#x6027;&#x306F;&#x3001;&#x68B1;&#x5305;&#x5F8C;&#x306E;&#x888B;&#x306E;&#x6E29;&#x5EA6;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x305F;11&#x304B;&#x3089;13&#xB0;C&#x3001;&#x305D;&#x308C;&#x304C;&#x76EE;&#x6A19;&#x6E29;&#x5EA6;&#xFF08;8&#x2103;&#xFF09;&#x3067;25&#x65E5;&#x53CE;&#x7A6B;&#x5F8C;&#x306B;&#x5230;&#x9054;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x6E29;&#x5EA6;&#x304C;&#x9045;&#x3044;&#x6E1B;&#x5C11;&#x306F;&#x75C5;&#x539F;&#x4F53;&#x611F;&#x67D3;&#x306E;&#x78BA;&#x7ACB;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x5178;&#x578B;&#x7684;&#x306A;&#x75C5;&#x5909;&#x304B;&#x3089;&#x306E;&#x5206;&#x96E2;&#x682A;&#x306F;&#x3002;&#x30EA;&#x30BE;&#x30AF;&#x30C8;&#x30CB;&#x30A2;&#x5C5E;&#x3068;&#x3057;&#x3066;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x3001;&#x30B3;&#x30C3;&#x30DB;&#x306E;&#x4EEE;&#x8AAC;13&#x304B;&#x3089;14&#x3067;&#x884C;&#x308F;&#x4EBA;&#x5DE5;&#x63A5;&#x7A2E;&#xB0;C&#x3067;25&#x83CC;&#x682A;&#x3067;&#x5B8C;&#x6210;&#x3057;&#x305F;&#x305F;rDNA&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5185;&#x90E8;&#x8EE2;&#x5199;&#x30B9;&#x30DA;&#x30FC;&#x30B5;&#x30FC;&#x914D;&#x5217;&#xFF08;ITS1&#x3068;ITS2&#xFF09;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x7CFB;&#x7D71;&#x89E3;&#x6790;&#x306F;&#x3001;&#x30EA;&#x30BE;&#x30AF;&#x30C8;&#x30CB;&#x30A2;&#xB7;&#x30BD;&#x30E9;&#x30CB;&#x306E;&#x543B;&#x5408;&#x30B0;&#x30EB;&#x30FC;&#x30D7;3&#x306B;3&#x3064;&#x306E;&#x5206;&#x96E2;&#x682A;&#x304C;&#x5272;&#x308A;&#x5F53;&#x3066;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;R. solani&#x306E;&#x5206;&#x96E2;&#x682A;&#x306E;&#x83CC;&#x7CF8;&#x4F53;&#x3068;&#x8CA0;&#x50B7;&#x3057;&#x305F;&#x584A;&#x830E;&#x306E;&#x63A5;&#x7A2E;&#x306F;&#x3001;&#x611F;&#x67D3;&#x5730;&#x57DF;&#x3067;oversuberization&#x5FDC;&#x7B54;&#x306B;&#x306A;&#x308A;&#x307E;&#x3057;&#x305F;&#x3002; R. solani&#x306E;Rh17&#x3092;&#x5206;&#x96E2;&#x3059;&#x308B;&#x3068;&#x3001;&#x30B9;&#x30D9;&#x30EA;&#x30F3;&#x751F;&#x5408;&#x6210;&#x95A2;&#x9023;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;StKCS6&#x3068;CYP86A33&#x306F;POP_A&#x306E;2.9&#x500D;&#x306B;&#x5897;&#x52A0;&#x3057;&#x3001;&#x7D9A;&#x3044;&#x3066;H&#x63A5;&#x7A2E;&#x5F8C;&#x3001;&#x5275;&#x50B7;&#x8A98;&#x767A;&#x6027;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;24&#x3001;6.8&#x3001;3.4&#x500D;&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x5897;&#x52A0;&#x3057;noninoculated&#x584A;&#x830E;&#x3068;&#x6BD4;&#x3079;&#x3066;&#x30B3;&#x30EB;&#x30AF;&#x5316;&#x306F;&#x3001;&#x5F0F;48&#x6642;&#x9593;&#x63A5;&#x7A2E;&#x5F8C;&#x3001;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x53CE;&#x7A6B;&#x5F8C;&#x306E;&#x30C0;&#x30FC;&#x30AF;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x75C5;&#x306F;&#x584A;&#x830E;&#x30B9;&#x30AD;&#x30F3;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x524D;&#x306B;&#x767A;&#x751F;&#x3057;&#x305F;AG-3&#x611F;&#x67D3;&#x75C7;&#x306E;R. solani&#x306E;&#x306B;oversuberization&#x306E;&#x5FDC;&#x7B54;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

&#x30B8;&#x30E3;&#x30AC;&#x30A4;&#x30E2;&#x584A;&#x830E;&#x306E;&#x53CE;&#x7A6B;&#x5F8C;&#x306E;&#x30C0;&#x30FC;&#x30AF;&#x30B9;&#x30AD;&#x30F3;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306F;&#x7D0B;&#x67AF;&#x75C5;&#x83CC;&#x611F;&#x67D3;&#x306B;&#x5BFE;&#x3059;&#x308B;Oversuberization&#x306E;&#x5FDC;&#x7B54;&#x3067;&#x3042;&#x308B;

&#xC774;&#xC2A4;&#xB77C;&#xC5D8; &#xB18D;&#xBD80; &#xAC10;&#xC790; tubers &#xC5F0;&#xAC04; &asymp;40, 000 &#xD1A4;&#xC758; &#xC2A4;&#xD0A8; &#xC124;&#xC815; &#xD558;&#xAE30; &#xC804;&#xC5D0; &#xC77C;&#xCC0D;, &#xC218;&#xD655;&#xC758; 250, 000 &#xD1A4; &#xC218;&#xCD9C;. &#xCD5C;&#xADFC; &#xBA87; &#xB144; &#xB3D9;&#xC548;, &#xCD08;&#xAE30; &#xC218;&#xD655; &#xAC10;&#xC790; tubers (&#39;&#xB2C8;&#xCF5C;&#xB77C;&#39;) &#xC218; &#xBD84;&#xC744; &#xC720;&#xC9C0; &#xD1A0; &#xD0C4;&#xC744; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xD070; &#xAC00;&#xBC29;&#xC5D0; &#xD3EC;&#xC7A5;&#xC5D0; &#xC5B4;&#xB450;&#xC6B4; &#xD53C;&#xBD80; &#xBC18;&#xC810;&#xC758; &#xBC1C;&#xC0DD;&#xC5D0;&#xC11C; &#xC99D;&#xAC00; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xC800;&#xC7A5; &#xBC0F; &#xD574;&#xC678; &#xC804;&#xC1A1; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xBD88;&#xADDC;&#xCE59; &#xD55C; &#xAD34; &#xC0AC; &#xC131; &#xBC18;&#xC810; &#xD615;&#xD0DC;. &#xC99D;&#xC0C1; &#xAC1C;&#xBC1C;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xC870;&#xAC74; &#xBD84;&#xD3EC;&#xC758; &#xD45C;&#xC2DC; &#xD3EC;&#xC7A5; &#xD6C4; &#xAC00;&#xBC29; &#xC628;&#xB3C4; 11 ~ 13 &deg; C &#xC774;&#xBA70; &#xB300;&#xC0C1; (8 &deg; C) &#xC628;&#xB3C4; 25 &#xC77C; postharvest&#xC5D0; &#xB3C4;&#xB2EC;. &#xC628;&#xB3C4;&#xC5D0;&#xC11C; &#xB290;&#xB9B0; &#xAC10;&#xC18C;&#xAC00; &#xBCD1;&#xC6D0; &#xCCB4; &#xAC10;&#xC5FC;&#xC758; &#xC124;&#xB9BD;&#xC744; &#xD64D;&#xBCF4;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC804;&#xD615;&#xC801;&#xC778; &#xBCD1; &#xBCC0;&#xC5D0;&#xC11C; &#xACA9;&#xB9AC; Rhizoctonia &#xC885;&#xC73C;&#xB85C; &#xD655;&#xC778; &#xB41C; &#xACE0; &#xCF54;&#xD06C;&#xC758; &#xC5EC;&#xAE34;&#xB2E4; 13 ~ 14 &deg; C&#xC5D0;&#xC11C; &#xC218;&#xD589; &#xD558;&#xB294; &#xC778;&#xACF5; &#xC811;&#xC885; &#xD558; &#xC5EC; 25 &#xACA9;&#xB9AC;&#xC640; &#xD568;&#xAED8; &#xC644;&#xB8CC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. Phylogenetic &#xBD84;&#xC11D;, &#xB0B4;&#xBD80;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBB38; &#xD569; &#xADF8;&#xB8F9; 3 Rhizoctonia solani&#xC758; &#xC138; &#xAC00;&#xC9C0; &#xACA9;&#xB9AC; &#xD560;&#xB2F9; rDNA &#xC720;&#xC804;&#xC790;&#xC758; &#xACF5;&#xBC31; &#xC2DC;&#xD000;&#xC2A4; (its1&#xC640; ITS2) &#xBCF5;&#xC0AC;&#xD560;. &#xC774;&#xB7EC;&#xD55C; R. solani &#xACA9;&#xB9AC;&#xC758; &#xC0AC;&#xCCB4;&#xC640; &#xD568;&#xAED8; &#xBD80; &#xC0C1;&#xB2F9;&#xD55C; tubers&#xC758; &#xC811;&#xC885; &#xAC10;&#xC5FC;&#xB41C; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C; oversuberization &#xC751;&#xB2F5; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. &#xACA9;&#xB9AC; Rh17 R. solani &#xD568;&#xAED8; suberin &#xC0DD; &#xD569;&#xC131; &#xAD00;&#xB828; &#xC720;&#xC804;&#xC790; StKCS6 &#xBC0F; cyp86a33&#xC758; &#xD45C;&#xD604;&#xC744; &#xC99D;&#xAC00; 6.8-3.4-fold, &#xAC01;&#xAC01; 24 h &#xC720;&#xC804;&#xC790; &#xAD00;&#xB828; &#xB41C; &#xC0C1;&#xCC98; &#xC720;&#xBC1C; suberization, &#xC2DD; 48 h postinoculation, postinoculation, POP_A 2.9-fold &#xC99D;&#xAC00; &#xB4A4;&#xC774;&#xC5B4;, noninoculated tubers&#xC640; &#xBE44;&#xAD50;. Postharvest &#xB2E4;&#xD06C; &#xC2A4;&#xD31F; &#xBCD1;&#xC740; &#xACB0; &#xC808; &#xD53C;&#xBD80; &#xC138;&#xD2B8; &#xC804;&#xC5D0; &#xBC1C;&#xC0DD; &#xD558;&#xB294; AG 3 &#xAC10;&#xC5FC;&#xC758; R. solani&#xC5D0; oversuberization &#xC751;&#xB2F5; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xAC10;&#xC790; &#xAD34; &#xACBD;&#xC5D0;&#xC11C; postharvest &#xC5B4;&#xB450;&#xC6B4; &#xD53C;&#xBD80; &#xBC18;&#xC810; Rhizoctonia solani &#xAC10;&#xC5FC;&#xC5D0; &#xB300; &#xD55C; oversuberization &#xC751;&#xB2F5;&#xC740;.

Los agricultores israel&#xED;es exportar 250.000 toneladas de tub&#xE9;rculos de papa al a&#xF1;o, &#x2248; 40.000 toneladas de las cuales se cosechan temprano, antes de conjunto la piel. En los &#xFA;ltimos a&#xF1;os, ha habido un aumento en la aparici&#xF3;n de manchas oscuras en la piel temprana cosechados tub&#xE9;rculos de patata, &#x26;#39;Nicola&#x26;#39;) envasados &#x200B;&#x200B;en bolsas grandes que contienen turba para retener la humedad. Las manchas necr&#xF3;ticas irregulares se forman durante el almacenamiento, transporte y en el extranjero. Caracterizaci&#xF3;n de las condiciones necesarias para el desarrollo de s&#xED;ntomas indica que la temperatura de la bolsa despu&#xE9;s de embalaje es de 11 a 13 &#xB0; C y alcanza la temperatura deseada (8 &#xB0; C), s&#xF3;lo el 25 despu&#xE9;s de la cosecha d&#xED;as. Este descenso lento de la temperatura puede promover el establecimiento de la infecci&#xF3;n por pat&#xF3;genos. Los aislados de las lesiones t&#xED;picas fueron identificados como Rhizoctonia spp., Y los postulados de Koch se completaron con 25 aislamientos mediante la inoculaci&#xF3;n artificial realizada a los 13 a&#xF1;os a 14 &#xB0; C. El an&#xE1;lisis filogen&#xE9;tico, utilizando las secuencias de espaciadores internos transcritos (ITS1 e ITS2) de los genes rDNA, asignados a la anastomosis tres aislamientos del grupo 3 de Rhizoctonia solani. La inoculaci&#xF3;n de tub&#xE9;rculos heridos con micelio de R. solani de estos aislamientos resultaron en una respuesta oversuberization en la zona infectada. Con aislar Rh17 de R. solani, la expresi&#xF3;n de los relacionados con la bios&#xED;ntesis de suberina-genes StKCS6 y CYP86A33 aumento de 6,8 y 3,4 veces, respectivamente, 24 horas despu&#xE9;s de la inoculaci&#xF3;n, seguido por un aumento de 2,9 veces en POP_A, un gen asociado con la herida inducida por suberizaci&#xF3;n, la expresi&#xF3;n de 48 horas despu&#xE9;s de la inoculaci&#xF3;n, en comparaci&#xF3;n con los tub&#xE9;rculos inoculados. Le sugerimos que despu&#xE9;s de la cosecha enfermedad de la mancha oscura es una respuesta oversuberization a R. solani AG-3 de la infecci&#xF3;n que se produce antes de la serie del tub&#xE9;rculo de piel.

Postcosecha puntos oscuros de la piel en tub&#xE9;rculos de patata son una respuesta Oversuberization a Rhizoctonia solani infecci&#xF3;n

We have synthesized in a 6-nitrodibenzofuranyl (NDBF) derivative of inositol-1,4,5-trisphosphate (IP(3)) for efficient two-photon uncaging in living cells. As its hexakis acetoxymethyl ester, this caged compound may be applied at low concentration to the extracellular milieu to load the intact astrocytes in acutely isolated brain slices from the mouse cortex. Two-photon irradiation of single astrocytes evoked intracellular calcium signals that required 10% of the energy dosage compared to nitroveratyl (NV)-IP(3). Since NDBF-IP(3) has a 5-fold higher quantum yield than NV-IP(3), these data imply that photolysis of the new NDBF caged compound mobilized intracellular calcium about twice as efficiently as the NV cage.

Synthesis and characterization of cell-permeant 6-nitrodibenzofuranyl-caged IP3.

Deciphering the role of GABAergic neurons in large neuronal networks such as the neocortex forms a particularly complex task as they comprise a highly diverse population. The neuronal isoform of the enzyme nitric oxide synthase (nNOS) is expressed in the neocortex by specific subsets of GABAergic neurons. These neurons can be identified in live brain slices by the nitric oxide (NO) fluorescent indicator diaminofluorescein-2 diacetate (DAF-2DA). However, this indicator was found to be highly toxic to the stained neurons. We used this feature to induce acute phototoxic damage to NO-producing neurons in cortical slices, and measured subsequent alterations in parameters of cellular and network activity. Neocortical slices were briefly incubated in DAF-2DA and then illuminated through the 4× objective. Histochemistry for NADPH-diaphorase (NADPH-d), a marker for nNOS activity, revealed elimination of staining in the illuminated areas following treatment. Whole cell recordings from several neuronal types before, during, and after illumination confirmed the selective damage to non-fast-spiking (FS) interneurons. Treated slices displayed mild disinhibition. The reversal potential of compound synaptic events on pyramidal neurons became more positive, and their decay time constant was elongated, substantiating the removal of an inhibitory conductance. The horizontal decay of local field potentials (LFPs) was significantly reduced at distances of 300-400 μm from the stimulation, but not when inhibition was non-selectively weakened with the GABA(A) blocker picrotoxin. Finally, whereas the depression of LFPs along short trains of 40 Hz stimuli was linearly reduced with distance or initial amplitude in control slices, this ordered relationship was disrupted in DAF-treated slices. These results reveal that NO-producing interneurons in the neocortex convey lateral inhibition to neighboring columns, and shape the spatiotemporal dynamics of the network's activity.

Spatiotemporal alterations of cortical network activity by selective loss of NOS-expressing interneurons.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

The advent of large scale neural computational platforms has highlighted the lack of algorithms for synthesis of neural structures to perform predefined cognitive tasks. The Neural Engineering Framework (NEF) offers one such synthesis, but it is most effective for a spike rate representation of neural information, and it requires a large number of neurons to implement simple functions. We describe a neural network synthesis method that generates synaptic connectivity for neurons which process time-encoded neural signals, and which makes very sparse use of neurons. The method allows the user to specify-arbitrarily-neuronal characteristics such as axonal and dendritic delays, and synaptic transfer functions, and then solves for the optimal input-output relationship using computed dendritic weights. The method may be used for batch or online learning and has an extremely fast optimization process. We demonstrate its use in generating a network to recognize speech which is sparsely encoded as spike times.

Synthesis of neural networks for spatio-temporal spike pattern recognition and processing.

We measured the action potential backpropagation delays in apical dendrites of layer V pyramidal neurons of the somatosensory cortex under different stimulation regimes that exclude synaptic involvement. These delays showed robust features and did not correlate to either transient change in the stimulus strength or low frequency stimulation of suprathreshold membrane oscillations. However, our results indicate that backpropagation delays correlate with high frequency (>10 Hz) stimulation of membrane oscillations, and that persistent suprathreshold sinusoidal stimulation injected directly into the soma results in an increase of the backpropagation delay, suggesting an intrinsic adaptation of the backpropagating action potential (bAP), which does not involve any synaptic modifications. Moreover, the calcium chelator BAPTA eliminated the alterations in the backpropagation delays, strengthening the hypothesis that increased calcium concentration in the dendrites modulates dendritic excitability and can impact the backpropagation velocity. These results emphasize the impact of dendritic excitability on bAP velocity along the dendritic tree, which affects the precision of the bAP arrival at the synapse during specific stimulus regimes, and is capable of shifting the extent and polarity of synaptic strength during suprathreshold synaptic processes such as spike time-dependent plasticity.

The adaptation of spike backpropagation delays in cortical neurons.

The lifespan of an acute brain slice is approximately 6-12 hours, limiting potential experimentation time. We have designed a new recovery incubation system capable of extending their lifespan to more than 36 hours. This system controls the temperature of the incubated artificial cerebral spinal fluid (aCSF) while continuously passing the fluid through a UVC filtration system and simultaneously monitoring temperature and pH. The combination of controlled temperature and UVC filtering maintains bacteria levels in the lag phase and leads to the dramatic extension of the brain slice lifespan. Brain slice viability was validated through electrophysiological recordings as well as live/dead cell assays. This system benefits researchers by monitoring incubation conditions and standardizing this artificial environment. It further provides viable tissue for two experimental days, reducing the time spent preparing brain slices and the number of animals required for research.

Extending the viability of acute brain slices.

In vitro brain slice preparations are instrumental in developing our understanding of the nervous system. However, the current lifespan of an acute brain slice is limited to approximately 6-12 hours. This reduces potential experimentation time and leads to considerable waste of neural tissue. We have designed, developed and tested a novel incubation system capable of extending the lifespan of these brain slices. This is done by controlling the temperature and pH of the artificial cerebral spinal fluid in which the slices are incubated while continuously passing the fluid through a UVC filtration system. This system is capable of maintaining extremely low bacterial levels and significantly extending the brain slice lifespan to at least 24 hours. Brain slice viability was validated through electrophysiological recordings as well as live/dead cell assays.

Braincubator: an incubation system to extend brain slice lifespan for use in neurophysiology.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 μM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and β) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurons leading to progressive paralysis and death. Using transcranial magnetic stimulation (TMS) and nerve excitability tests, several clinical studies have identified that cortical and peripheral hyperexcitability are among the earliest pathologies observed in ALS patients. The changes in the electrophysiological properties of motor neurons have been identified in both sporadic and familial ALS patients, despite the diverse etiology of the disease. The mechanisms behind the change in neuronal signalling are not well understood, though current findings implicate intrinsic changes in motor neurons and dysfunction of cells critical in regulating motor neuronal excitability, such as astrocytes and interneurons. Alterations in ion channel expression and/or function in motor neurons has been associated with changes in cortical and peripheral nerve excitability. In addition to these intrinsic changes in motor neurons, inhibitory signalling through GABAergic interneurons is also impaired in ALS, likely contributing to increased neuronal excitability. Astrocytes have also recently been implicated in increasing neuronal excitability in ALS by failing to adequately regulate glutamate levels and extracellular K(+) concentration at the synaptic cleft. As hyperexcitability is a common and early feature of ALS, it offers a therapeutic and diagnostic target. Thus, understanding the underlying pathways and mechanisms leading to hyperexcitability in ALS offers crucial insight for future development of ALS treatments.

Yossi Buskila

The MARCS Institute,
School of Medicine

Western Sydney University

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Yossi Buskila

The MARCS Institute,School of Medicine

Western Sydney University

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

The MARCS Institute,
School of Medicine