dopamine-release-at-individual-presynaptic-terminals-visualized-with

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Alterations in the cytosolic pool directly affect neurotransmitter synthesis and release and are suggested to be key factors in various neurodegenerative disorders. Although this cytosolic pool is the most metabolically active, it is miniscule compared with the amount of vesicular transmitter and has never been quantified separately. Here, we introduce intracellular patch electrochemistry (IPE), a technique that for the first time provides direct measurements of cytosolic oxidizable molecules in single mammalian cells. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it preferentially measures catecholamines. In cultured chromaffin cells, the total cytosolic catechol concentration was 50-500 microm, of which approximately 10% were catecholamines. Reserpine, a vesicular monoamine transporter inhibitor, had no effect on the catecholamine pool but increased total catechols by fourfold to fivefold. Combined with pargyline, a monoamine oxidase inhibitor, reserpine increased catecholamine levels in the cytosol by approximately sixfold. Amphetamine induced a transient approximately fivefold accumulation of cytosolic catecholamines and a slow increase of total catechols. In cells incubated with 3,4-dihydroxy-L-phenylalanine (L-DOPA), catecholamines increased by approximately 2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels <or=10 min after L-DOPA withdrawal, whereas total catechols remained approximately twofold elevated even after a 1.5 hr incubation in L-DOPA-free media. Our data indicate that cytosolic catecholamines are strictly maintained at a defined level, and drug-induced increases in their concentrations lead to the accumulation of other catecholamine derivatives, such as DOPAC and 3,4-dihydroxyphenylethyleneglycol. These derivatives reside in the cytosol for hours after treatment and may be an underlying cause of drug-related cytotoxicity.

Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.

Intrazellul&#xE4;re Patch Elektrochemie: Regulation der cytosolischen Katecholamine in chromaffinen Zellen.

A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.

Dopamine neurons release transmitter via a flickering fusion pore.

Dopamin-Neuronen Freisetzung Sender &#xFC;ber ein Flackern Fusionspore.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

SNAREs can promote complete fusion and hemifusion as alternative outcomes.

SNAREs k&#xF6;nnen vollst&#xE4;ndige Fusion und hemifusion als Alternative Ergebnisse zu f&#xF6;rdern.

Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.

Analysis of exocytotic events recorded by amperometry.

Die Analyse der Exozytoseereignisse durch Amperometrie aufgezeichnet.

Dysregulation of dopamine homeostasis and elevation of the cytosolic level of the transmitter have been suggested to underlie the vulnerability of catecholaminergic neurons in Parkinson's disease. Because several known mutations in alpha-synuclein or overexpression of the wild-type (WT) protein causes familial forms of Parkinson's disease, we investigated possible links between alpha-synuclein pathogenesis and dopamine homeostasis. Chromaffin cells isolated from transgenic mice that overexpress A30P alpha-synuclein displayed significantly increased cytosolic catecholamine levels as measured by intracellular patch electrochemistry, whereas cells overexpressing the WT protein and those from knock-out animals were not different from controls. Likewise, catechol concentrations were higher in L-DOPA-treated PC12 cells overexpressing A30P or A53T compared with those expressing WT alpha-synuclein, although the ability of cells to maintain a low cytosolic dopamine level after L-DOPA challenge was markedly inhibited by either protein. We also found that incubation with low-micromolar concentrations of WT, A30P, or A53T alpha-synuclein inhibited ATP-dependent maintenance of pH gradients in isolated chromaffin vesicles and that the WT protein was significantly less potent in inducing the proton leakage. In summary, we demonstrate that overexpression of different types of alpha-synuclein disrupts vesicular pH and leads to a marked increase in the levels of cytosolic catechol species, an effect that may in turn trigger cellular oxyradical damage. Although multiple molecular mechanisms may be responsible for the perturbation of cytosolic catecholamine homeostasis, this study provides critical evidence about how alpha-synuclein might exert its cytotoxicity and selectively damage catecholaminergic cells.

Alpha-synuclein overexpression increases cytosolic catecholamine concentration.

Dysregulation der Dopamin-Hom&ouml;ostase und Erh&ouml;hung der zellul&auml;ren Ebene des Senders haben wurden vorgeschlagen, um die Verwundbarkeit der Catecholaminergic Neuronen bei Parkinson-Krankheit zugrunde liegen. Da mehrere bekannte Mutationen in Alpha-Synuclein oder eine &Uuml;berexpression des Wildtyp (WT) Proteins bewirkt famili&auml;re Formen der Parkinson-Krankheit, untersuchten wir m&ouml;gliche Verbindungen zwischen Alpha-Synuclein-Pathogenese und Dopamin-Hom&ouml;ostase. Chromaffin Zellen isoliert von transgenen M&auml;usen, die A30P Alpha-Synuclein deutlich angezeigt &uuml;berexprimiert h&ouml;here Zytosolischer Katecholamine intrazellul&auml;ren Patch Elektrochemie gemessen, w&auml;hrend Zellen &uuml;berexprimierenden das WT-Protein und die von Knock-out-Tiere waren nicht anders als Steuerelemente. Ebenso waren Brenzcatechin Konzentrationen h&ouml;her L-DOPA behandelten PC12-Zellen, die overexpressing A30P oder A53T mit jenen Ausdr&uuml;cken WT Alpha-Synuclein, verglichen, obwohl die F&auml;higkeit der Zellen, die eine niedrige Zytosolische Dopamin nach DOPA Challenge aufrechtzuerhalten durch entweder Protein deutlich gehemmt wurde. Wir fanden auch, dass Inkubation mit niedrig-micromolar Konzentrationen von WT, A30P oder A53T Alpha-Synuclein ATP-abh&auml;ngige Wartung pH-Gradienten in isolierten chromaffin Vesicles gehemmt und das WT-Protein wesentlich weniger potenten in induzierende Proton Leckage war. Zusammenfassend l&auml;sst sich sagen zeigen wir, dass eine &Uuml;berexpression von verschiedenen Arten von Alpha-Synuclein vesikul&auml;ren pH-Wert st&ouml;rt und f&uuml;hrt zu einem deutlichen Anstieg der Ebenen Zytosolische Brenzcatechin Arten, ein Effekt, der im Mai verwandeln Ausl&ouml;ser zellul&auml;ren Oxyradical Schaden. Obwohl mehrere Molekulare Mechanismen f&uuml;r die St&ouml;rung der Hom&ouml;ostase Zytosolischer Katecholamine verantwortlich sein k&ouml;nnen, bietet diese Studie kritische Beweise dar&uuml;ber, wie Alpha-Synuclein aus&uuml;ben, die Zytotoxizit&auml;t und selektiv Catecholaminergic Zellen besch&auml;digen k&ouml;nnte.

Alpha-Synuclein &Uuml;berexpression erh&ouml;ht Zytosolischer Katecholamine Konzentration.

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy.

Ver&auml;nderte Abbau von Alpha-Synuclein (Alpha-Syn) hat bei der Entstehung der Parkinson-Krankheit (PD) verwickelt. Wir haben gezeigt, dass die Alpha-Syn &uuml;ber Chaperon-vermittelte Autophagy (CMA), einen selektiven lysosomale Mechanismus zur Verschlechterung der cytosolic Proteinen beeintr&auml;chtigt werden kann. Pathogene Mutanten von Alpha-Syn blockieren lysosomale Translokation, eigene Abbau zusammen mit anderen Substraten CMA zu beeintr&auml;chtigen. W&auml;hrend pathogene Alpha-Syn-Mutationen sind selten, Alpha-Syn erf&auml;hrt posttranslationale Modifikationen, seine Anh&auml;ufung in Zytosolische Aggregate in den meisten Formen der PD Using Maus ventralen medialen Neuron Kulturen, SH-SY5Y Zellen in Kultur und isoliert Maus Lysosomen zugrunde liegen k&ouml;nnen, wir haben festgestellt, dass die meisten dieser posttranslationale Modifikationen von Alpha-Syn Abbau dieses Proteins von CMA beeintr&auml;chtigen aber haben keinen Einfluss auf die Verschlechterung der anderen Substraten. Dopamin-modifizierte Alpha-Syn, ist nicht nur schlecht vom CMA abgebaut aber auch Bl&ouml;cke Verschlechterung der andere Substrate sich diese Option. Mit zunehmender Verstopfung von CMA zellul&auml;re Anf&auml;lligkeit gegen&uuml;ber Stressoren schlagen wir Dopamin-induzierte autophagic Hemmung die selektive Degeneration der dopaminergen Neuronen PD erkl&auml;ren konnte.

Dopamin-modifizierte Alpha-Synuclein blockiert Chaperon-vermittelte Autophagy in Verbindung.

Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides valuable data about molecular steps involved in stimulation-dependent transmitter release. The sensitivity of amperometry permits the detection of the monoamines released from individual vesicles, giving information about intracellular transmitter homeostasis (synthesis, degradation, reuptake, etc.) and about the release probability (vesicle availability and sensitivity of fusion machinery to Ca(+2)). Furthermore, temporal resolution of amperometry allows for the observation of the real-time kinetics of monoamine release during exocytosis. Here, we provide algorithms for computerized analysis of the amperometric signal with emphasis on routines that confer consistent determination of spike parameters on recordings with different sampling rates, signal-to-noise levels, spike durations, and surrounding signal.

Analysis of single-vesicle exocytotic events recorded by amperometry.

Analyse der Form der amperometrisch-Spikes, die stammen aus der Oxidation der Monoamin-Molek&uuml;le w&auml;hrend der Fusion der einzelnen sekretorischen Vesikeln freigesetzt liefert wertvolle Daten &uuml;ber molekulare Schritte zur Anregung-abh&auml;ngige Sender Version. Die Empfindlichkeit des Amperometry erm&ouml;glicht das Erkennen von der Monoamine ver&ouml;ffentlicht von einzelnen Bl&auml;schen, geben Informationen &uuml;ber intrazellul&auml;re Sender Hom&ouml;ostase (Synthese, Abbau, Reuptake usw.) und die Freigabe-Wahrscheinlichkeit (Vesikel Verf&uuml;gbarkeit und Empfindlichkeit des Fusion-Maschinen zu Ca(+2)). Dar&uuml;ber hinaus kann zeitlicher Aufl&ouml;sung der Amperometry f&uuml;r die Beobachtung in Echtzeit Kinetics Monoamin-Version w&auml;hrend der Exozytose. Hier bieten wir Algorithmen f&uuml;r computergest&uuml;tzte Analyse der amperometrisch Signal mit Schwerpunkt auf Routinen, die konsistente Ermittlung Spitze Parameter bei Aufnahmen mit unterschiedlichen Abtastraten, Signal-Rausch-Ebenen, Spike-Dauer zu verleihen, und umliegenden Signal.

Analyse von Einzel-Vesikel exocytotic Ereignisse von Amperometry aufgenommen.

Acute exposure to amphetamines (AMPHs) collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreasing the quantal size of catecholamine release during fusion events. AMPH and methamphetamine (METH), however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term METH exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute METH effects, exposure to the drug for 6-48 h at 10 microM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5-10 microM levels, prolonged METH increased the quantal size and reinstated exocytotic catecholamine release, although very high (> 100 microM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at approximately 37 degrees C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including AMPHs, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, AMPHs and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification.

Secretory vesicle rebound hyperacidification and increased quantal size resulting from prolonged methamphetamine exposure.

Akute Gef&auml;hrdung durch Amphetamine (AMPHs) reduziert sekretorischen Vesikel pH-Wert Farbverl&auml;ufe, die erh&ouml;ht Zytosolischer Katecholamine w&auml;hrend die quantal Gr&ouml;&szlig;e der Katecholamin-Freisetzung bei Fusion Events zu verringern. Ladeeffekt und Methamphetamin (METH), werden jedoch im Gewebe &uuml;ber langer Dauer beibehalten. Wir haben optische und Elektron mikroskopische Sonden zur Messung der Auswirkungen einer langfristigen METH auf sekretorischen Vesikel pH, und Amperometry und intrazellul&auml;ren Patch Elektrochemie, die Auswirkungen auf Neurosecretion und Zytosolischer Katecholamine in kultivierten Ratte chromaffin Zellen zu beobachten. Im Gegensatz zu Akutwirkungen METH produziert Gef&auml;hrdung durch die Droge f&uuml;r 6-48 h bei 10 MicroM und h&ouml;heren Konzentrationen eine Konzentration abh&auml;ngige R&uuml;ckprall-Hyperacidification von sekretorischen Vesikeln. Auf 5-10 MicroM Ebenen, anhaltende METH quantal vergr&ouml;&szlig;ert und wieder exocytotic Katecholamin-Version eingesetzt, obwohl sehr hoch (&gt; 100 MicroM) Ebenen des Medikaments, Erholung Hyperacidification, produzieren weiterhin nicht quantal Gr&ouml;&szlig;e zu erh&ouml;hen. Sekretorischen Vesikel Erholung Hyperacidification temperaturabh&auml;ngig optimale Resonanz bei ca. 37 Grad C war, wurde nicht von der Transkription-Inhibitor blockiert Puromycin, und scheint eine allgemeine kompensatorische Reaktion auf l&auml;ngerer Exposition mit Membranophilic schwache Basen, einschlie&szlig;lich AMPHs, Methylphenidat, Kokain und Ammoniak. Also unter bestimmten Bedingungen einer l&auml;ngeren Einwirkung, k&ouml;nnen AMPHs und andere schwachen Basen verbessern, anstatt zum Abbau, der vesikul&auml;ren Freisetzung von Katecholaminen &uuml;ber eine kompensatorische Antwort Vesikel Versauerung f&uuml;hrt.

Sekretorischen Vesikel Zugstufe Hyperacidification und erh&ouml;hte quantal Gr&ouml;&szlig;e aufgrund l&auml;ngerer Methamphetamin.

The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DA(cyt)) in cultured midbrain neurons, we confirm that elevated DA(cyt) and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DA(cyt) provide neuroprotection. L-DOPA increased DA(cyt) in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DA(cyt) was not altered by alpha-synuclein deletion, although dopaminergic neurons lacking alpha-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DA(cyt), and alpha-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons.

Die Grundlage f&uuml;r die selektive Tod von spezifischen neuronalen Populationen in Neurodegenerative Krankheiten bleibt unklar. Parkinson-Krankheit (PD) ist ein Synucleinopathy zeichnet sich durch eine bevorzugte Verlust der dopaminergen Neuronen in der Substantia Nigra (SN), w&auml;hrend die Neuronen des ventralen Haubengebiet (VTA) erspart werden. Mit intrazellul&auml;ren Patch Elektrochemie Zytosolische Dopamin direkt messen (DA(cyt)) in kultivierten Mittelhirn Nervenzellen, wir best&auml;tigen, dass erh&ouml;hte DA(cyt) und seine Metaboliten neurotoxische sind und genetische und pharmakologische Interventionen, die DA(cyt) verringern Nervenschutz bieten. DOPA erh&ouml;hte DA(cyt) im SN Neuronen Ebenen 2 und 3 8-fach h&ouml;her als im VTA Neuronen, eine Antwort auf Dihydropyridin-Sensitive Ca2 + Kan&auml;le, was erh&ouml;hte Anf&auml;lligkeit von SN-Neuronen auf L-DOPA-induzierte Neurotoxizit&auml;t angewiesen. Da(cyt) wurde von Alpha-Synuclein l&ouml;schen, nicht ge&auml;ndert, obwohl dopaminergen Neuronen fehlt Alpha-Synuclein zu L-DOPA-induzierte Zelltod resistent waren. Somit kann eine Interaktion zwischen Ca2 +, DA(cyt) und Alpha-Synuclein die Empf&auml;nglichkeit von SN Neuronen in PD, schl&auml;gt mehrere therapeutische Ziele zugrunde liegen.

Interplay zwischen Zytosolischer Dopamin, Kalzium und Alpha-Synuclein Ursachen selektive Tod von Neuronen der Substantia Nigra.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals.

Das Nervensystem &uuml;bertr&auml;gt Signale zwischen Neuronen &uuml;ber Neurotransmitter-Freisetzung w&auml;hrend synaptische Vesikel Verschmelzung. Um Neurotransmitter Aufnahme zu beobachten und Pressemitteilung vom einzelnen pr&auml;synaptischen Klemmen direkt, gestalteten wir fluoreszierende falsche Neurotransmitter als Substrate f&uuml;r die synaptische Vesikel Monoamin-Transporter. Verwenden diese Pr&uuml;fpunkte auf Bild-Dopamin-Release im Striatum, haben wir mehrere Beobachtungen, die synaptische Plastizit&auml;t. Wir fanden, dass der Anteil der synaptische Vesikel, die Freigabe Neurotransmitter pro Impuls die Impuls-Frequenz abh&auml;ngig war. Kinetisch unterschiedliche &quot;Reservieren&quot; synaptische Vesikel Einwohner wurde unter diesen Versuchsbedingungen nicht beobachtet. Eine frequenzabh&auml;ngige Heterogenit&auml;t der pr&auml;synaptischen Terminals wurde aufgedeckt, dass D2-Dopamin-Rezeptoren, angibt, einen Mechanismus f&uuml;r die frequenzabh&auml;ngige Kodierung der pr&auml;synaptischen Auswahl teilweise abh&auml;ngig war.

Article	Year
Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 12843288	2003
Dopamine neurons release transmitter via a flickering fusion pore. Nature neuroscience\| PubMed ID: 14990933	2004
SNAREs can promote complete fusion and hemifusion as alternative outcomes. The Journal of cell biology\| PubMed ID: 16027221	2005
Analysis of exocytotic events recorded by amperometry. Nature methods\| PubMed ID: 16118635	2005
Alpha-synuclein overexpression increases cytosolic catecholamine concentration. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16957086	2006
Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy. The Journal of clinical investigation\| PubMed ID: 18172548	2008
Analysis of single-vesicle exocytotic events recorded by amperometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 18369956	2008
Secretory vesicle rebound hyperacidification and increased quantal size resulting from prolonged methamphetamine exposure. Journal of neurochemistry\| PubMed ID: 19014382	2008
Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron\| PubMed ID: 19409267	2009
Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals. Science (New York, N.Y.)\| PubMed ID: 19423778	2009

Eugene V. Mosharov

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