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.

Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio (NIH Publications No. 80-23, riveduta 2012) e gli studi sono stati approvati dalla Institutional Animal Care e Uso delle commissioni di ricerca alla Stony Brook University (# 1618 e # 1792). 1. vHipp-NACC sinaptiche co-culture <ol><li> Topi Sacrifice (postnatale giorno 0-3, da wild-type (WT) o nAChR α7 linea di topo transgenico) con emissioni di CO 2. Decapitare ogni cucciolo e salvare la coda in una provetta da 1,5 ml centrifugare per post facto genotipizzazione. Effettuare le seguenti operazioni in una cappa sterile. </li><li> Togliere la parte superiore del cranio con le forbici e pinze per esporre tutto il cervello. Partendo dalla congiunzione dove le cortecce cerebrali parte una dall&#39;altra caudalmente, ottenere una sezione coronale che contiene le cortecce posteriori che includono la vHipp 5,21 sotto un microscopio da dissezione. </li><li> Sezionare vStrisce di tessuto Hipp contenenti regione CA1-subiculum secondo un atlante sviluppo cerebrale del mouse 22 (Figura 1A). Trasferimento a un piatto di coltura 35 millimetri contenente terreni di coltura freddo (4 ° C), tagliato a fettine sottili (circa 100 micron × 100 micron microslices). </li><li> Pre-incubazione 12 millimetri poli-D-lisina / vetrini laminina rivestite all&#39;interno di 24 pozzetti di coltura tissutale con terreni di coltura (~ 500 microlitri) per almeno 30 minuti in incubatrice a 37 ° C. Rimuovere i supporti prima di placcatura microslices. </li><li> Piatto con una pipetta Pasteur lucidati a fuoco al centro del vetrino con minima quantità di supporti (~ 50 microlitri, contenente ~ 20 pezzi di microslices) per facilitare il fissaggio degli espianti. Piatto microslices vHipp provenienti da un singolo animale (sia il + / + o il - / - genotipo della linea α7 transgenica) su ogni vetrino. </li><li> Dopo gli espianti depositano sul vetrino, aggiungere delicatamente multimediale aggiuntivo (~ 100 ml) di the parete in modo che il livello dei media è sufficientemente alto per coprire completamente le espianti sulla periferia, ma non quelli al centro del vetrino. </li><li> Dopo un / N incubazione O a 37 ° C, 5% CO 2 incubatore, disperdere i neuroni NACC da giorni embrionali 18 di post-natale giorno 1 topi WT e aggiungere gli espianti. <ol><li> Sezionare tessuti NACC secondo un sviluppo atlante cervello di topo 20 (Figura 1B). </li><li> Tagliare a piccoli pezzi (circa 500 micron × 500 micron microslices) e trasferimento in una provetta da 15 ml. </li><li> Trattare con 0,25% tripsina (2 ml) per 15 min a 37 ° C. Lavare pezzi di tessuto tre volte con mezzi di lavaggio a freddo (5 ml, 4 ° C) seguito da un lavaggio in mezzi di coltura fredda (5 ml, 4 ° C). Lasciare riposare la sospensione per 5 minuti in ogni fase di lavaggio, e poi versare con cautela il surnatante. </li><li> Separa celle di dolce triturazione in 2 ml di coltura con un leggermente incendio lucidato pipetta Pasteur. </li><li> Transsospensione cellulare fer ad un&#39;altra provetta da 15 ml e centrifugare a 2.000 x rpm per 5 min. Rimuovere il surnatante e sospendere nuovamente le cellule in coltura a 1 ml per 6 cuccioli. Disperdere le cellule pipettando delicatamente in mezzi più volte con un fuoco lucidato pipetta Pasteur. </li><li> Aggiungere 0,25 ml di cellule NACC dispersi ad ogni vetrino con espianti vHipp. </li><li> Mantenere le culture in un umidificata 37 ° C, 5% CO 2 incubatore. Mettere piastre di coltura su inumidite garze sterili per stabilizzare meccanicamente e mantenere l&#39;umidità, favorendo la formazione di sinapsi. </li></ol></li></ol> 2. Immunocitochimica <ol><li> Fissare culture (5-7 giorni in vitro) nel 4% paraformaldeide / 4% di saccarosio / PBS (~ 500 microlitri per vetrino, 20 min, RT). </li><li> Culture permeabilize con 0,25% Triton X-100 / PBS (~ 500 microlitri per vetrino, 5 min, RT). </li><li> Culture di blocco con il 10% di siero asino normale in PBS (~ 500 microlitri per vetrino, 30 min, RT) prima gli anticorpiy colorazione. Incubare con anticorpi primari O / N a 4 ° C. Utilizzare i seguenti anticorpi primari: nAChRs anti- (α 4 -ECD 1: 500; anti-α 5 subunità, 1: 500), anti-vescicolare trasportatore del glutammato 1 (1: 250), anti-GAD65 (1: 100). </li><li> Lavare anticorpi primari con PBS (3 volte / ~ 500 microlitri per vetrino, 5 min) e poi incubare culture anticorpi secondari coniugati Alexa 488 (~ 500 ml per vetrino, 1: 500) o Alexa 594 (~ 500 microlitri per coprioggetto , 1: 500) per 1 ora a 37 ° C. </li><li> Incubare culture in αBgTx coniugati Alexa 594 (~ 500 ml per vetrino, 1: 1.000 in coltura) per 45 minuti a 37 ° C prima della fissazione per l&#39;etichettatura superficie α7 * nAChR. </li><li> Inserire e tenuta coprioggetto. </li><li> Catturare le immagini con un microscopio a fluorescenza dotato di obiettivi Plan-Apocromatici (20X con 0,8 NA o 63X olio con 1.4 NA) e una fotocamera CCD. </li></ol> 3. FM1-43 Based vescicolare Fusion <ol><li> Mantenere culture (5-7 giorni in vitro) in un camera di imaging, montare la camera su disco rotante microscopio confocale. Continuously profumato (1 ml / min) con HBS cocktail RT. </li><li> Smettere di culture di perfusione e di carico con 10 micron FM1-43 in 56 mm K + ACSF (~ 500 microlitri) per 90 secondi (colorazione), lavare tintura esterno via per 15 minuti con Ca2 + HBS gratuito contenente ADVASEP-7 (0,1 mm) per pulire FM1-43 legato alla membrana. </li><li> Stimolare le culture con 56 mm K + ACSF (~ 500 microlitri) senza FM1-43 per 120 sec (decolorazione), ricaricare le culture con FM1-43 utilizzando le stesse condizioni, e lavare di nuovo con Ca2 + HBS gratuito contenente ADVASEP-7 per 15 min. </li><li> Acquisire FM1-43 immagini a fluorescenza con disco rotante microscopio confocale equipaggiato con un obiettivo Plan-Apochromat (60X acqua con 1.4 NA, di eccitazione 488 nm, emissione 530 nm) e catturare immagini con una telecamera CCD. </li><li> Raccogliere immagini FM1-43 fluorescenza di base (ogni 2 s per 1 min) Il controllo pre-nicotina; applicare nicotina (1 mM) di perfusione rapida (2 ml / min) per 1 min, e poi lavare la nicotina con HBS cocktail. Tenere time-lapse immagini cattura prima, durante e dopo l&#39;applicazione di nicotina ogni 2 secondi per 5 min. </li><li> Quantificare FM1-43 intensità di fluorescenza prima (F colorazione) e dopo (F decolorante) applicazione nicotina ad ogni bouton sinaptica. </li><li> Calcola importo complessivo di rilasciabile fluorescenza FM1-43 lungo gli assoni vHipp quantificando la differenza di FM1-43 fluorescenza prima e dopo l&#39;applicazione della nicotina (Af = F colorazione -F decolorazione). Analizzare frazione della diminuzione dell&#39;intensità di fluorescenza dopo nicotina con la seguente equazione: diminuzione F% = Af / F colorazione. </li></ol> 4. Imaging di calcio <ol><li> Sciacquare culture (5-7 giorni in vitro) rapidamente con normale HBS, quindi caricare le culture con 5 Indicatore mM Fluo-4 Ca 2+ (AM estere) e 0,02% Pluronic F-127 in HBS (2 ml) per 30 min a 37 ° C e 5% CO 2. </li><li> Lavare Fluo-4 soluzione HBS (3 volte / 5 min). Culture Ritorno a incubatore (37 ° C / 5% di CO 2) per almeno 30 minuti. </li><li> Mantenere le culture nella stessa camera di imaging e stesse condizioni descritte nella sezione FM1-43 fusione vescicolare base (vedi punto 3.1). </li><li> Raccogliere le immagini Fluo-4 di fluorescenza di proiezioni assonale dai vHipp micro-fette con un obiettivo Plan-Apochromat (60X acqua con 1.4 NA, di eccitazione 488 nm, emissione 530 nm) e una fotocamera CCD. </li><li> Raccogliere basali Fluo-4 fluorescenza (ogni 10 sec per 2 min) il controllo pre-nicotina, applicare nicotina (1 micron) di perfusione rapido (2 ml / min) per 1 min, e poi lavare la nicotina con HBS cocktail. Tenere time-lapse immagini cattura prima, durante e dopo l&#39;applicazione di nicotina ogni 10 sec per 30 min. </li><li> Salvare tutti i fotogrammi dei fluo-4 fluorescenza prime, l&#39;esportazione come una serie diLe immagini in formato TIFF per ulteriori analisi. </li><li> Raccogliere l&#39;intensità integrata di fluo-4 fluorescenza lungo gli assoni vHipp prima e dopo l&#39;applicazione di nicotina. </li><li> Normalizzare intensità di fluorescenza integrato con la seguente equazione: Af / F 0 = (0 FF) / F 0, dove F 0 è il pre-nicotina intensità di fluorescenza integrato fondo corretto e F è l&#39;intensità di fluorescenza integrato lungo gli assoni vHipp ad ogni tempo dopo l&#39;applicazione di nicotina. Analizzare e tracciare normalizzato intensità di fluorescenza integrato. </li></ol>

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La preparazione co-coltura descritto ri-capitola ventrale circuiti dell&#39;ippocampo-accumbens in vitro. Questo esame permette di preparazione relativamente semplice e affidabile dei profili spazio-temporali per cui l&#39;attivazione di nAChR pre-sinaptici suscitare esaltata trasmissione glutamatergica 5, 21. Co-colture sono definite come la crescita di diversi tipi cellulari specifici in un piatto che può fornire condizioni fisiologiche in vitro per dimostrare...

Modulazione colinergica circuito eccitabilità contribuisce ad aspetti fondamentali della cognizione, e modulazione colinergici alterato è una caratteristica di malattie neurodegenerative e neuropsichiatrici tra cui il morbo di Alzheimer, il morbo di Parkinson, la schizofrenia e la dipendenza 1-4. Un meccanismo stabilito di facilitazione colinergici della trasmissione sinaptica nel sistema nervoso centrale è attraverso l&#39;attivazione diretta di nAChR localizzati nei siti pre-sinaptici. L&#39;attivazione di questi recettori presinaptici porta ad un aumento intracellulare Ca 2+ ([Ca 2+] i) nei terminali presinaptici - sia direttamente, a causa della relativamente alta conduttanza di calcio di alcuni sottotipi di nAChR, e indirettamente, tramite intracellulari cascate di segnalazione 5, rafforzando in tal modo il rilascio di neurotrasmettitore. Infatti, l&#39;attivazione del nAChR pre-sinaptici è stato collegato con i cambiamenti nel rilascio di un&#39;ampia varietà di neurotrasmettitori compresi glutammato, GABA, ACh, unnd dopamina 6-10. Sebbene questo processo è stato studiato indirettamente utilizzando metodi elettrofisiologici a vari sinapsi, reporter ottiche di [Ca 2+] i e riciclo delle vescicole sinaptiche permettono misura più diretta e temporalmente precisa dei fenomeni pre-sinaptici. Localizzazione pre-sinaptica dei nAChR è stata dimostrata in modo convincente con etichettatura immuno-oro diretto della nAChR al microscopio elettronico (EM) il livello 11,12. Diverse altre tecniche sono state utilizzate anche per affrontare la localizzazione nAChR indirettamente, anche rilevando posizioni di nAChRs subunit- fluorescenti chimere proteine ​​in neuroni in coltura 13,14, registrazione elettrofisiologica delle correnti nAChR nei terminali sinaptici 15,16, il monitoraggio nicotina indotto cambiamenti in [Ca 2 +] i in terminazioni nervose sinaptiche di immagini dal vivo delle cellule 17, e monitoraggio indiretto del rilascio di neurotrasmettitore al terminale sinaptico datecniche di imaging cellulare dal vivo con indicatori fluorescenti, tra cui esocitosi delle vescicole sinaptiche visti da stirilici coloranti anfipatiche FM (FM1-43 e FM4-64) e / o Synapto-pHluorin e da specifici neurotrasmettitori reporter fluorescenti, come CNiFERs per ACh e iGluSnFr per il glutammato 18-20. Nel complesso, questi approcci attuali per identificare la localizzazione pre-sinaptica di nAChR sono complicate e richiedono sistemi e tecniche speciali per permettere l&#39;identificazione affidabile e monitoraggio fisiologico di attività pre-sinaptica. Qui si descrive protocolli e attrezzature per un sistema di co-coltura in vitro di un ippocampo ventrale (vHipp) - nucleus accumbens (NACC) circuito che fornisce accesso diretto a identificare e analizzare i due componenti pre e post-sinaptici della trasmissione sinaptica. Mostriamo esempi di localizzazione pre-sinaptica di nAChR e imaging cellulare dal vivo di nAChR mediata Ca 2+ di segnalazione e di rilascio dei neurotrasmettitori aloassoni ng vHipp. Una naturale estensione (e semplice) del protocollo presentato qui è la preparazione di contatti pre e post-sinaptiche composti da neuroni provenienti da diversi genotipi. In questo modo il contributo di un particolare prodotto genico al pre e / o meccanismi di post-sinaptici di modulazione può essere valutata direttamente.

<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.

La preparazione impiegato consiste gene chimerico co-colture di circuiti vHipp-NACC in vitro. Le proiezioni provenienti da microslices vHipp, come input assonale pre-sinaptica, possono prendere contatti sinaptici con obiettivi post-sinaptici, i neuroni dispersi da NACC. La nicotina ha indotto una sostenuta (≥ 30 min) la facilitazione della trasmissione glutammatergica dai neuroni NACC innervati dal vHipp assoni 21 e prolungata di calcio di segnalazione lungo gli assoni vHipp 5 via α7 pr...

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.

Immagini dal vivo di nicotina indotta segnalazione Calcio e neurotrasmettitori uscita Lungo ventrali Hippocampal assoni

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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8.9K Views.  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.

Video: 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 di neuroni da fettine di cervello di roditore studio di trasmissione sinaptica e Immagine terminali presinaptici

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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Neuroscienze

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 Imaging di fluorescenza confocale tag recettori nicotinici a Knock-in topi con la somministrazione cronica alla nicotina

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.

Imaging longitudinale a due fotoni dell'ippocampo dorsale CA1 in topi vivi

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

Comportamento

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.

Funzione del recettore nicotinico dell'acetilcolina in fettine di cervello del Mouse tramite Laser fotolisi di nicotina fuse di sondaggio

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.

Procedura craniotomia per la visualizzazione delle attività neuronali nell'ippocampo dei topi che si comportano

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.

In vivo Imaging del calcio di insiemi neuronali in reti di neuroni sensoriali primari nei gangli della radice dorsale intatti

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-coltura dell'ippocampo ventrale e del nucleo 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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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.

Imaging della segnalazione del calcio indotta dalla nicotina lungo gli assoni dell'ippocampo ventrale in co-colture sinaptiche

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

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