We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

Alle Tierversuche wurden in Übereinstimmung mit den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren durchgeführt (NIH Publications No. 80-23, überarbeitet 2012) und Studien wurden von Institutional Animal Care genehmigt und Verwenden für Forschung Ausschüsse at Stony Brook University (# 1618 und # 1792). 1. vHipp-NACC Synaptic Co-Kulturen <ol><li> Sacrifice Mäuse (postnatalen Tag 0-3, aus Wildtyp (WT) oder α7 nAChR transgene Mauslinie) mit CO 2. Enthaupten jeder Welpe und speichern Sie das Heck in ein 1,5-ml-Zentrifugenröhrchen für post facto Genotypisierung. Führen Sie folgende Schritte in einem sterilisierten Kapuze. </li><li> Entfernen Sie die Oberseite des Schädels mit Schere und Pinzette, um das gesamte Gehirn aus. Beginnend an der Verbindungsstelle, wo die zerebralen Kortex Teil voneinander kaudal, erhalten einen Frontalschnitt, der die hinteren Kortex einschließlich der vHipp 5,21 unter einem Binokular enthält. </li><li> Sezieren vHipp Gewebestreifen, die die CA1-Region subiculum gemäß einem entwickelnden Mausgehirnatlas 22 (Figur 1A). Übertragung auf eine 35 mm Kulturschale mit kaltem Kulturmedien (4 ° C), in kleine dünne Scheiben (ca. 100 &amp; mgr; m × 100 &amp; mgr; m microslices) geschnitten. </li><li> Pre-inkubiere 12 mm Poly-D-Lysin / Laminin beschichteten Deckgläschen Inneren 24-Well-Gewebekulturplatten mit Kulturmedien (~ 500 &amp; mgr; l) für mindestens 30 min im Brutschrank bei 37 ° C. Entfernen Sie das Medium vor dem Ausplattieren microslices. </li><li> Platte mit einer feuerpolierten Pasteur-Pipette in der Mitte des Deckglases mit einer minimalen Menge an Medium (~ 50 &amp; mgr; l, enthaltend ~ 20 Stücke microslices), um die Befestigung der Explantate erleichtern. Platte vHipp microslices aus einer tierischen Ursprung (entweder der + / + oder - / - Genotyp der transgenen α7 Linie) für jedes Deckglas. </li><li> Nachdem die Explantate auf das Deckglas absetzen, sanft fügen Sie zusätzliche Medien (~ 100 &amp; mgr; l) von the Wand, so dass der Pegel des Mediums hoch genug ist, um die Explantate am Umfang vollständig abdecken, nicht aber die in der Mitte des Deckglases. </li><li> Nach einer O / N Inkubation in 37 ° C, 5% CO 2 Inkubator, zerstreuen NACC Neuronen von embryonalen Tagen 18 bis postnatalen Tag 1 WT-Mäusen und zu den Explantaten. <ol><li> Sezieren NACC Gewebe gemäß einer entwickelnden Mausgehirnatlas 20 (1B). </li><li> In kleine Stücke geschnitten (ca. 500 &amp; mgr; m × 500 &amp; mgr; m microslices) aufnehmen und in einem 15-ml-Tube. </li><li> Behandlung mit 0,25% Trypsin (2 ml) für 15 min bei 37 ° C. Dreimal mit kaltem Waschmedien (5 ml, 4 ° C), gefolgt von einer Wäsche in kaltem Kulturmedium (5 ml, 4 ° C) Waschgewebebrocken. Erlauben Suspension für 5 Minuten in jedem Waschschritt ausruhen und dann abgießen Stand vorsichtig. </li><li> Dissoziieren Zellen durch vorsichtiges Zerreiben in 2 ml Kulturmedium mit einem leicht feuerpolierten Pasteur-Pipette. </li><li> Transfer Zellsuspension in einen anderen 15 ml-Tube und Zentrifugation bei 2.000 x rpm für 5 min. Entfernen Sie den Überstand und resuspendieren Zellen in Kulturmedium bei 1 ml pro 6 Welpen. Zerstreuen Zellen durch vorsichtiges Pipettieren in Medien mehrmals mit einer feuerpolierten Pasteur-Pipette. </li><li> Zugabe von 0,25 ml dispergiert NACC-Zellen zu jedem Deckglas mit vHipp Explantate. </li><li> Aufrechtzuerhalten, die Kulturen in einer befeuchteten 37 ° C, 5% CO 2 -Inkubator. Zeigen Kulturplatten auf gedämpft steriler Gaze-Pads, um mechanisch zu stabilisieren und zu pflegen Feuchtigkeit, Synapsenbildung zu erleichtern. </li></ol></li></ol> 2. Immunocytochemistry <ol><li> Fix Kulturen (5 - 7 Tage die in vitro) in 4% Paraformaldehyd / 4% Saccharose / PBS (~ 500 &amp; mgr; l pro Deckglas, 20 min, RT). </li><li> Permeabilisieren Kulturen mit 0,25% Triton X-100 / PBS (~ 500 &amp; mgr; l pro Deckglas, 5 min, RT). </li><li> Block Kulturen mit 10% normalem Eselserum in PBS (~ 500 &amp; mgr; l pro Deckglas, 30 min, RT) vor Antibody Färbung. Inkubieren mit primären Antikörpern O / N bei 4 ° C. Verwenden Sie die folgenden primären Antikörpern: anti nAChRs (α 4 -ECD 1: 500; anti-α-Untereinheiten 5, 1: 500), anti-vesikulären Glutamattransporter 1 (1: 250), anti-GAD65 (1: 100). </li><li> Waschen Sie primäre Antikörper mit PBS (3 mal / ~ 500 ul pro Deckglas, 5 min) und dann inkubieren Kulturen in Sekundärantikörper Alexa 488 konjugierten (~ 500 &amp; mgr; l pro Deckglas, 1: 500) oder Alexa 594 (~ 500 &amp; mgr; l pro Deckglas , 1: 500) für 1 h bei 37 ° C. </li><li> Vor der Fixierung für Etikettieroberfläche α7 * nAChRs für 45 min bei 37 ° C: Kulturen in αBgTx Alexa 594 konjugiert inkubieren (1000 in Kulturmedien ~ 500 ul pro Deckglas 1). </li><li> Halterung und Dichtung Deckgläser. </li><li> Machen Sie Bilder mit einem Fluoreszenzmikroskop mit Plan-Apochromat Objektive (20x mit 0,8 NA oder 63X Öl mit 1,4 NA) ausgestattet und einer CCD-Kamera. </li></ol> 3. FM1-43 Based Vesicular Fusion <ol><li> Pflegen Kulturen (5 - 7 Tage die in vitro) in einem bildgebenden Kammer, montieren Sie die Kammer auf Spinning Platten konfokalen Mikroskop. Kontinuierlich durchströmen (1 ml / min) mit HBS-Cocktail bei RT. </li><li> Stoppen Perfusion und Last Kulturen mit 10 &amp; mgr; M in 56 mM FM1-43 K + ACSF (~ 500 &amp; mgr; l) für 90 sec (Färbung), waschen externen Farbstoff entfernt 15 Minuten mit Ca 2+ frei HBS, enthaltend ADVASEP-7 (0,1 mm) membrangebundene FM1-43 abzufangen. </li><li> Stimulieren die Kulturen mit 56 mM K + ACSF (~ 500 &amp; mgr; l) ohne FM1-43 für 120 sec (Entfärben), laden Kulturen mit FM1-43 unter den gleichen Bedingungen und nachwaschen mit Ca 2+ frei HBS, enthaltend ADVASEP-7 15 min. </li><li> Erwerben FM1-43 Fluoreszenzbilder mit sich drehenden Scheibe konfokalen Mikroskop mit einem Plan-Apochromat Objektiv (60X Wasser mit 1,4 NA, Anregung 488 nm, Emission 530 nm) und die Aufnahmen mit einer CCD-Kamera ausgestattet. </li><li> Sammeln Basis FM1-43 Fluoreszenzbilder (alle 2 s 1 min) Als Pre-Nikotin-Steuerung; gelten Nikotin (1 uM) durch schnelle Durchblutung (2 ml / min) für 1 min, und dann waschen Nikotin mit HBS-Cocktail. Halten Sie Capture-Zeitraffer-Bilder vor, während und nach dem Nikotin-Anwendung alle 2 Sek 5 Min. </li><li> Quantifizieren FM1-43 Fluoreszenzintensität vor (F-Färbung) und nach (F Entfärben) Nikotin-Anwendung an jedem synaptischen bouton. </li><li> Berechnen Gesamtmenge an freisetzbarem FM1-43 Fluoreszenz entlang vHipp Axone durch Quantifizierung der Differenz FM1-43 Fluoreszenzintensität vor und nach dem Nikotin-Anwendung (&amp; Dgr; F = F Färbung -F Entfärbung). Analyse Fraktion von Fluoreszenzintensitätsabnahme nach Nikotin mit der folgenden Gleichung: F = &amp; Dgr; F% Abnahme / F-Färbung. </li></ol> 4. Calcium Imaging <ol><li> (- 7 Tage in vitro 5) schnell mit normalen HBS, dann laden Kulturen mit 5 &amp; mgr; M Fluo-4 Ca 2+ Anzeige (Kulturen SpülenAM-Ester) und 0,02% Pluronic F-127 in HBS (2 ml) für 30 min bei 37 ° C und 5% CO 2. </li><li> Waschen Sie Fluo-4-Lösung mit HBS (3-mal / 5 min). Zurück Kulturen Inkubator (37 ° C / 5% CO 2) für mindestens 30 min. </li><li> Aufrechtzuerhalten Kulturen in derselben Abbildungskammer und gleichen Bedingungen wie in FM1-43 basierend Vesikelfusion Abschnitt beschrieben (siehe Schritt 3.1). </li><li> Sammeln Fluo-4 Fluoreszenzbilder der axonalen Projektionen von den vHipp Mikro-Scheiben mit einem Plan-Apochromat Objektiv (60X Wasser mit 1,4 NA, Anregung 488 nm, Emission 530 nm) und einer CCD-Kamera. </li><li> Sammeln Basis Fluo-4 Fluoreszenzbilder (alle 10 s für 2 min) als vorge Nikotin Steuer gelten Nikotin (1 uM) durch rasche Perfusion (2 ml / min) für 1 min, und dann waschen Nikotin mit HBS-Cocktail. Halten Sie Capture-Zeitraffer-Bilder vor, während und nach dem Nikotin-Anwendung alle 10 Sek 30 Min. </li><li> Speichern Sie alle Bilder der Ausgangs Fluo-4 Fluoreszenz-Aufnahmen, Export als eine Reihe vonTIFF-Format Bilder für die weitere Analyse. </li><li> Sammeln Sie die integrierte Intensität der Fluo-4 Fluoreszenz entlang vHipp Axone vor und nach Nikotin-Anwendung. </li><li> Normalisieren integrierten Fluoreszenzintensität mit der folgenden Gleichung: &amp; Dgr; F / F 0 = (FF 0) / F 0 ist, wobei F 0 die hintergrundkorrigierten vorge Nikotin integrierten Fluoreszenzintensität und F ist die integrierte Fluoreszenzintensität entlang vHipp Axone zu jedem Zeitpunkt nach Nikotin-Anwendung. Zu analysieren und zu plotten normalisierte integrierte Fluoreszenzintensität. </li></ol>

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Das Co-Kulturpräparat beschrieben re-capitulates ventralen Hippocampus-accumbens-Schaltungen in vitro. Diese Zubereitung ermöglicht relativ einfache und sichere Überprüfung der räumlichen und zeitlichen Verläufe von denen die Aktivierung des präsynaptischen nAChRs ELICIT verbesserte glutamaterge Transmission 5, 21. Co-Kulturen werden als das Wachstum der verschiedenen spezifischen Zelltypen in eine Schüssel, die physiologischen Bedingungen in vitro zur Ve...

Cholinergen Modulation Schaltung Erregbarkeit trägt zur grundlegenden Aspekte des Erkennens und verändert cholinergen Modulation ist eine Funktion von neurodegenerativer und neuropsychiatrischen Störungen, einschließlich Alzheimer-Krankheit, Parkinson-Krankheit, Schizophrenie und Sucht 1-4. Eine etablierte Mechanismus der cholinergen Erleichterung der synaptischen Übertragung im ZNS ist über eine direkte Aktivierung von nAChR in präsynaptischen Websites lokalisiert. Aktivierung dieser präsynaptischen Rezeptoren führt zur intrazellulären Ca 2+ ([Ca 2+] i) in präsynaptischen Terminals erhöht - sowohl direkt, aufgrund der relativ hohen Calcium Leitfähigkeit bestimmter nAChR-Subtypen, und indirekt über die intrazellulären Signalkaskaden 5, wodurch die Freisetzung von Neurotransmittern. In der Tat hat die Aktivierung der präsynaptischen nAChRs mit Veränderungen in der Freisetzung von einer Vielzahl von Neurotransmittern wie Glutamat, GABA, Acetylcholin, in Verbindung gebracht worden einnd Dopamin 6-10. Obwohl dieser Prozess wurde indirekt mit elektrophysiologischen Methoden an verschiedenen Synapsen untersucht, optische Reportern von [Ca 2+] i und synaptischen Vesikel-Recycling ermöglichen mehr direkte und zeitlich präzise Messung der präsynaptischen Phänomene. Präsynaptischen Lokalisation nAChRs überzeugend mit direktem Immunogold-Markierung von nAChR in der elektronenmikroskopische (EM) Ebene 11,12 demonstriert. Mehrere andere Verfahren wurden ebenfalls verwendet, um die Lokalisierung nAChR indirekt zu adressieren, einschließlich Erfassen Standorte nAChRs subunit- fluoreszierendes Protein-Chimären in kultivierten Neuronen 13,14, elektrophysiologischen Aufzeichnung von nAChR Ströme in synaptische Anschlüssen 15,16, Überwachung Nikotin induzierte Veränderungen von [Ca 2+] i in synaptischen Nervenendigungen durch Live Cell Imaging 17 und indirekte Überwachung der Freisetzung von Neurotransmittern im synaptischen Terminal durchLive Cell Imaging-Techniken mit Fluoreszenzindikatoren, einschließlich Exozytose synaptischer Vesikel durch styryl amphipathischen FM Farbstoffe (FM1-43 und FM4-64) und / oder synapto-pHluorin und durch spezifische fluoreszierende Neurotransmitter Reportern, wie CNiFERs für ACh und iGluSnFr für Glutamat angesehen 18-20. Insgesamt sind diese aktuelle Ansätze zur Identifizierung von präsynaptischen Lokalisierung nAChRs sind kompliziert und erfordern spezielle Systeme und Techniken, um eine zuverlässige Identifizierung und physiologische Überwachung der präsynaptischen Aktivität zu ermöglichen. Hier beschreiben wir Protokolle und Ausrüstung für die in vitro Co-Kultursystem eines ventralen Hippocampus (vHipp) - Nucleus accumbens (NAKR) Schaltung, die einen direkten Zugang zu identifizieren und zu analysieren, sowohl vor als auch postsynaptischen Komponenten der synaptischen Übertragung bietet. Wir zeigen Beispiele von präsynaptischen Lokalisierung nAChRs und Live Cell Imaging von nAChR vermittelte Ca 2+ Signalisierung und die Freisetzung von Neurotransmittern along vHipp Axone. Eine natürliche (und unkompliziert) Erweiterung der hier vorgestellten Protokoll ist die Herstellung von Pre- und Post-synaptische Kontakte von Nervenzellen aus unterschiedlichen Genotypen besteht. Auf diese Weise wird der Beitrag eines bestimmten Genprodukts an der Vor- und / oder postsynaptische Mechanismen der Modulation kann direkt bewertet werden.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

Die eingesetzte Zubereitung umfasst chimäre Gen Kokulturen vHipp-NACC Schaltungen in vitro. Projektionen aus vHipp microslices ausgeht, als präsynaptischen Axon Eingang können synaptische Kontakte mit postsynaptischen Ziele, die verteilt Neuronen aus NACC machen. Nikotin induzierte eine anhaltende (≥ 30 min) Erleichterung der glutamatergen Übertragung von NACC Neuronen durch vHipp innerviert Axone 21 und verlängert Calcium-Signal entlang vHipp Axone 5 über präsynaptischen α7 nACh...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Live-Imaging von Nikotin induzierte Calcium Signaling und Neurotransmitterausschüttung Zusammen Ventral Hippocampus Axone

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

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Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

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8.9K Aufrufe.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Serie: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation und Mitochondriale Transport: Live Imaging in Hippocampus-Neuronen über lange Zeiträume

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

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Neurowissenschaften

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Live-Imaging von Spinalganglien Axone nach Rhizotomie

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Vorbereitung der akuten Subventrikulärzone Slices für Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Analyse Murine Schwann Zell Entwicklung entlang wachsenden Axone

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calciumphosphat-Transfektion von primären Hippocampus-Neuronen

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live-Imaging von<em&gt; Drosophila</em&gt; Larven Neuroblasten

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal Root Ganglien Isolation und Primärkultur Neurotransmitter-Freisetzung zu studieren

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex-Vivo Calcium Imaging zur Visualisierung von Gehirn Reaktionen auf endokrine Signalisierung bei Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Live-Imaging von Nikotin induzierte Calcium Signaling und Neurotransmitterausschüttung Zusammen Ventral Hippocampus Axone

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