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><tbody><tr><td colspan="4">1. Culture Media (50 ml)</td></tr><tr><td>Neurobasal&amp;nbsp;</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 colspan="4">2. washing media (HBSS, 100 ml)</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red&amp;nbsp;</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 colspan="4">3. HEPES buffered saline&amp;nbsp; (HBS)&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">4. HBS Cocktail for live imaging pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td>tetrodotoxin&amp;nbsp;</td><td>Tocris</td><td>1078</td><td>2 &amp;micro;M</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>10 &amp;micro;M</td></tr><tr><td>D-AP-5</td><td>Tocris</td><td>105</td><td>50 &amp;micro;M</td></tr><tr><td>CNQX</td><td>Tocris</td><td>1045</td><td>20 &amp;micro;M</td></tr><tr><td>LY341495</td><td>Tocris</td><td>1209</td><td>10 &amp;micro;M</td></tr><tr><td colspan="4">5. Calcium-free&amp;nbsp; HBS&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">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&amp;nbsp;</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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9.0K 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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation von Neuronen aus Nager Hirnschnitten zu Study Synaptic Transmission and Image präsynaptischen

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Spectral konfokale Bildgebung von fluoreszenzmarkierten Nikotinrezeptoren in Knock-in Mäusen mit chronischer Gabe von Nikotin

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Längs-Zwei-Photon-Bildgebung des Dorsal Hippocampal CA1 in lebenden Mäusen

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Verhalten

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Sondieren Nikotinsäure Acetylcholin-Rezeptor-Funktion in Maus Gehirnscheiben per Laser Flash Photolyse von Photoactivatable Nikotin

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Kraniotomie-Verfahren zur Visualisierung neuronaler Aktivitäten im Hippocampus von sich benehmenden Mäusen

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Im lebenden Organismus Calcium-Bildgebung neuronaler Ensembles in Netzwerken primärer sensorischer Neurone in intakten dorsalen Wurzelganglien

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Source: Zhong, C., et al. <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

Co-Kultivierung des ventralen Hippocampus und des Nucleus accumbens

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Co-culture and Interaction Studies

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

Bildgebung der Nikotin-induzierten Kalzium-Signalübertragung entlang ventraler Hippocampus-Axone in synaptischen Co-Kulturen

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

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