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 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>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
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		<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>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
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		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
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			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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		<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>
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		<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>
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		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<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>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
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		<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.

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

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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

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.

Neuromodulazione e mitocondriale di trasporto: Imaging Live in neuroni dell&#39;ippocampo oltre durate a lungo

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

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Neuroscienze

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.

Imaging in diretta degli assoni spinali dopo rizotomia

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.

Preparazione di acuta fette zona subventricolare for Imaging di calcio

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.

Analizzando lo sviluppo delle cellule di Schwann Murine lungo gli assoni in crescita

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.

Fosfato di calcio trasfezione di primaria neuroni dell&#39;ippocampo

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.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

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.

Isolamento dei gangli di radice dorsale e colture primarie per lo studio del rilascio di neurotrasmettitore

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 Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina 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 ...

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.

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

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Immagini dal vivo di

Isolamento dei gangli di radice dorsale e colture primarie per lo studio del rilascio di neurotrasmettitore

Ex Vivo Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina in Drosophila

Microscopia spettrale riflettometrica su assoni Myelinated In Situ