NIH R01MH084894 partecipato al finanziamento di questo studio. Vorremmo ringraziare il Dott. Yasmin Hurd e Scott Russo al Mount Sinai School of Medicine per la donazione di topi e l&#39;uso dei loro chirurgia e comportamento strutture durante le riprese di questo lavoro.

NOTA: Tutte le procedure per l&#39;allevamento degli animali e le cure sono state condotte secondo la norma istituzionale cura e l&#39;uso Comitato Animal (IACUC) di Icahn Scuola di Medicina Mount Sinai. Assicurarsi di utilizzare guanti sterili durante tutta la procedura. 1. droga e Virus Preparazione <ol><li> Drug Preparazione <ol><li> Preparare 15,0 ml di ketamina / xilazina anestetico sciogliendo 1,35 ml di 100 mg / ml ketamina e 0,75 ml di 20 mg / xilazina ml in 12,9 ml di 0,9% di soluzione salina. Mescolare bene la soluzione. </li></ol></li><li> Virus Preparazione <ol><li> Clonare il mGlu2 e mGlu2ΔTM4N costruisce in un vettore bicistronico virus herpes simplex (HSV) a seguito di protocolli standard precedentemente descritti 6. Pacchetto le particelle virali come descritto in precedenza 6,13,14. Sostituzione di residui Ala-677 4.40, Ala-681 4.44 e 4.48 Ala685 in mGlu2 per Ser686 4.40, PhE690 4.44 e Gly-694 4.48 in mGlu3 (HA-mGlu2ΔTM4N) sono state descritte in precedenza 6. NOTA: E &#39;stato precedentemente dimostrato che il chimerico costrutto HA-mGlu2ΔTM4N è espressa a livello della membrana plasmatica con G intatti proteina-dipendente di segnalazione 6. </li><li> Conservare vettori virali -80 ° C quando non in uso. Disgelo vettore virale su ghiaccio, e quindi un&#39;aliquota in 10 microlitri aliquote. Per la procedura chirurgica, tenere su ghiaccio. </li></ol></li></ol> 2. Chirurgia <ol><li> Preparazione Chirurgia <ol><li> Pesare il mouse e iniettare il mouse con la dose appropriata di cocktail di ketamina / xylazina (per i dettagli, vedere 1.1.1). </li><li> Controllare mouse per vedere se adeguatamente anestetizzato, spremere piede e la coda per la risposta del dolore, se non è possibile ottenere una risposta, il mouse è adeguatamente anestetizzato. </li><li> Radersi la testa del mouse dalla base del cranio alla punta del naso con tagliaunghie. Applicare il gel oftalmico per l&#39;AF del mouseterward per prevenire la cecità del mouse. </li><li> Caricare ogni siringa sul telaio stereotassico. Poi inclinare porzione perpendicolare di ciascun braccio del telaio stereotassico modo che siano 10 gradi dalla normale. Assicurarsi che i bracci sono inclinati, in modo tale che gli aghi sono di fronte all&#39;altra. </li><li> Pulire ogni siringa riempiendo l&#39;ago con il 70% di etanolo. Riempire l&#39;ago almeno tre volte per assicurare che la siringa è pulito. </li><li> Una volta che l&#39;ago è stato pulito, lavare l&#39;ago riempiendo l&#39;ago con doppia H 2 O. distillata Una volta lavata, riempire ogni ago con 1,3 ml di doppia H 2 O. distillata Ruotare lo stantuffo della siringa per rilasciare 0,3 ml di doppia H 2 O. distillata Se perle d&#39;acqua sulla punta dell&#39;ago, accuratamente spazzare via l&#39;acqua. Se non viene fuori niente della siringa, spingere lo stantuffo fino in fondo e poi ripetere la pulizia della siringa. </li><li> Dopo il riempimento con acqua, poi tirare la siringa riempire la siringa con0,5 ml di aria. </li><li> Una volta che l&#39;aria e l&#39;acqua sono nella siringa, riempire accuratamente la siringa con 1,3 ml di soluzione di virus. A questo punto assicurarsi che il volume totale nella siringa è di 2,8 microlitri. Ancora ruotare la punta della siringa per rilasciare 0,3 ml di virus. Se perle liquide alla punta dell&#39;ago, accuratamente spazzare via liquido. Se non viene fuori niente della siringa, spingere lo stantuffo fino in fondo e poi ripetere la pulizia della siringa. </li></ol></li><li> Chirurgia <ol><li> Collegare il mouse al telaio stereotassico, avendo cura di regolare il telaio stereotassico in modo che il cranio è di livello e piatta. Applicare povodine-iodio al cuoio capelluto esposto. Usando un bisturi, fare una incisione sagittale lungo la linea mediana del cranio all&#39;interno dell&#39;area rasata esposta. Quindi collegare i morsetti Buret alla pelle al sito di incisione per assicurarsi che il cranio rimane esposto. </li><li> Utilizzare H 2 O 2 al dissolversi periostio per esporre le suture dellacranio. Ora che il bregma e suture sono visibili, assicurarsi di regolare il telaio stereotassico per assicurarsi che il teschio sia a livello. </li><li> Allineare le punte degli aghi delle siringhe con bregma e registrare le coordinate del bregma. Calcolare le coordinate del dove gli aghi stanno per essere inserito. <ol><li> Per il piano Rostral-Cauldal (RC), aggiungere 1,6 mm ai registrate RC coordinate bregma (+1,6 da Bregma). Per il piano dorso-ventrale (DV), sottrarre 2,4 millimetri dalle coordinate bregma DV registrate (-2,4 da Bregma). </li><li> Infine, per il piano mediale laterale (ML), aggiungere 2,6 mm ai registrate ML coordinate bregma (+2,6 da Bregma). Per tutte le coordinate essere sicuri di registrare sia le coordinate sinistra e destra, come questo è un iniezione bilaterale. </li></ol></li><li> Portare gli aghi per le coordinate desiderate. Segnare i luoghi di cui gli aghi stanno per essere inserito e con un trapano, trapano aree contrassegnate. </li><li> Con una punta di cotone dell&#39;applicatore wipe via qualsiasi sangue o del midollo frammento di eccesso. </li><li> Portare gli aghi al cranio dove le punte degli aghi sono toccare la superficie del cervello. Quindi abbassare gli aghi per le coordinate desiderate lentamente abbassando. </li><li> Una volta che gli aghi sono sulle coordinate volute, iniettare lentamente il contenuto della siringa ruotandolo lo stantuffo dell&#39;ago 0,1 microlitri al minuto nel corso di 5 minuti (in totale 0,5 microlitri). </li><li> Dopo l&#39;iniezione è stata fatta lasciare la siringa nella corteccia per altri 5 minuti. </li></ol></li><li> Chiusura Up / Care <ol><li> Rimuovere gli aghi dalla corteccia del mouse costantemente e lentamente. Quindi rimuovere il mouse dal telaio stereotassico. </li><li> Applicare cianoacrilato (colla cutanea) per i lembi di pelle di base dall&#39;incisione e poi con una pinza afferrare i lembi di pelle e metterli insieme. </li><li> Lasciare che il cianoacrilato asciugare. Posizionare il mouse nella gabbia sopra una piastra elettrica (rilievo di riscaldamento è facoltativo se l&#39;surgSuite ical viene mantenuta sotto RT di 37 ° C - altrimenti non necessario). Assicurarsi di posizionare il mouse su un tovagliolo di carta per fare in modo che nel letto non aderisce al sito chirurgico. </li><li> A seconda della lunghezza della procedura chirurgica, assicurarsi che i topi è fuori di anestesia in 30 - 60 min dopo la procedura. Dopo l&#39;intervento chirurgico, l&#39;animale viene posto nella sua gabbia e monitorato fino diventa consapevole prima di tornare alla camera per recuperare. Non si usano analgesici post-operatorio, perché possono alterare i risultati dei nostri esperimenti, modificando alcune delle vie biochimiche del cervello. Tuttavia, gli animali vengono controllati fino a quando si riprendono dall&#39;anestesia il giorno della chirurgia, e post-operazione al giorno per segni di infezione e di valutazione del dolore / disagio. </li></ol></li></ol> Esperimento 3. Capo Twitch Risposta <ol><li> Impostare <ol><li> Effettuare tutti i test comportamentali 10:00-14:00, 2 - 3 giorni dopo stereotaCTIC iniezione di particelle virali. </li><li> Sciogliere (±) -1- (2,5-dimetossi-4-iodofenil) cloridrato -2-aminopropano (DOI) in una soluzione salina allo 0,9% e 2,0 mg / kg. Anche preparare una soluzione salina allo 0,9%. </li><li> Preparare una gabbia di casa (28 x 18 x 15 cm) senza biancheria da letto e l&#39;utilizzo di un tri-pod, regolare una macchina fotografica in modo che la vista della telecamera si trova direttamente sopra la gabbia casa. </li><li> Abituare i topi al ambiente per almeno 4 ore prima dell&#39;inizio dell&#39;esperimento. </li><li> Impostare una videocamera per registrare la contrazione testa. </li></ol></li><li> Sperimentare <ol><li> Posizionare la telecamera in modo che sia direttamente sopra una gabbia casa. Calibrare la telecamera in modo che l&#39;intera gabbia è nel campo visivo. </li><li> Pesare il mouse e iniettare il mouse intraperitoneale con la dose appropriata di 0,9% soluzione salina o DOI (0,01 ml / g). NOTA: Se un mouse pesa 25 grammi, somministrare la dose per un volume totale di 0,25 ml. </li><li> Mettete ogni mouse avanti nella loro gabbia casa FOR 10 min. Dopo 10 min, posto mouse al centro della gabbia casa vuota e convalidare che non ci sono punti ciechi nel campo visivo della telecamera. Premere RECORD sulla videocamera. Lascia la stanza. NOTA: i movimenti del mouse e le varie risposte comportamentali in esso (... La testa a contrazione, zero orecchio, ecc Si prega di fare riferimento alla tabella di dati supplementari 1 di Gonzalez-Maeso et al 2007 per la lista completa di risposte comportamentali indotte da DOI) 3, saranno registrate per 30 minuti. Pertanto, è importante non ci sono punti ciechi nel campo visivo registrata. </li><li> Dopo 30 min interrompere la registrazione sul mouse videocamera e posto di nuovo in gabbia casa originale. Ripetere questo processo per ogni mouse. </li></ol></li><li> Revisione <ol><li> Avere ogni revisione arbitro i nastri cieco alle condizioni sperimentali di mouse (es., Farmaci utilizzati durante la testa contrarsi esperimento o virus usato durante l&#39;iniezione intracranica)). registrare manualmente ogni testa contrazione througHout video. NOTA: Head-contrazione è definito come un movimento della testa scossa rapida condotta da un mouse (video supplementare). </li><li> Per ogni mouse, media la risposta HTR finale dai tre totali degli arbitri ciechi. Poi gruppo di questi valori di condizione sperimentale ed eseguire analisi statistiche (ad es., T-test o ANOVA). </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Insieme con i precedenti risultati in mGlu2-KO topi 5, i risultati con mGlu2 e mGlu2 / mGlu3 costrutti chimerici che non formano la 5-HT complesso recettoriale 2A -mGlu2 in cellule in coltura suggeriscono che la 2A 5-HT -mGlu2 eteromerico complesso recettoriale in topo corteccia frontale è necessario per indurre comportamenti testa contrazione da allucinogeni 5-HT 2A agonisti del recettore LSD-like. Una limitazione di questo metodo è che non misura prossimità molecolare a livello subcellulare in tessuto nativo. In aggiunta, ci sono vari punti critici da notare. Poiché i topi vengono iniettati con un vettore virale HSV, l&#39;intervallo di tempo che gli esperimenti da effettuare sono 2 - 4 giorni dopo l&#39;iniezione. La posizione e l&#39;espressione dei vettori virali devono essere verificate con immunofluorescenza del cervello sezionato non più di 4 giorni dopo l&#39;iniezione iniziale. I topi in cui le coordinate non corrispondono o non esprimere il vettore virale dovrebberoessere esclusi dai dati sperimentali in quanto non esprimono la mGlu2 o mGlu2ΔTM4N. Cura dopo l&#39;intervento chirurgico stereotassica è anche fondamentale, come la chiusura improprio del ferita alla testa può portare a infezioni che possono causare problemi in entrambe le esperimenti in vivo e la colorazione di immunofluorescenza. Infine, dopo l&#39;iniezione stereotassica qualsiasi paradigma comportamentale (. Campo aperto, alternando t-test, ecc) può essere utilizzato fintanto che è entro 2 - 4 dopo l&#39;iniezione di particelle virali. Anche in questo caso, le coordinate e l&#39;espressione dovrebbe essere confermata mediante immunofluorescenza. Il concetto che la funzione GPCR come omo e / o heteromers in cellule viventi è ormai ben consolidata 16-18. Tuttavia, nonostante alcuni progressi, sono necessari ulteriori studi per definire il ruolo preciso (s) di GPCR complessi heteromeric in modelli animali interi 19. Approcci, come BRET e / o immagini FRET per indagare proteina-proteina vicinanza fisica all&#39;interno di tessuti profondi di piccoli animalimodelli possono fornire i mezzi per migliorare la comprensione del ruolo funzionale di heteromers GPCR in soggetti 20 vivente. Usando l&#39;espressione HSV-mediata GPCR nativa o con proteine ​​transmembrana modificati, fornire un modello in vivo per valutare la funzione e l&#39;interazione di GPCR. Sono inoltre necessari ulteriori studi in modelli di roditori per esaminare la stabilità e il tempo di vita (formazione e la dissociazione) di heteromers GPCR 21-23, riarrangiamenti strutturali tra i loro componenti 24, e le potenzialità di accoppiamento proteina G dopo internalizzazione recettoriale 25,26. Dato il ruolo fondamentale dei GPCR in segnalazione cellulare e la funzione, sembra probabile che questa zona potrebbe portare a interessanti studi di base e traslazionale. Anche se ulteriori indagini è necessaria per caratterizzare quantitativamente la ultrastrutturali co-localizzazione di entrambi i recettori nel sistema nervoso centrale umano e del mouse, insieme a Stu precedentemuore che convincente dimostrano la elettrofisiologico, cellulare, e le risposte comportamentali indotte da allucinogeni in modelli murini sono intrinseci alla 5-HT 2A recettore-esprimendo neuroni piramidali corticali 3,27,28, i risultati ottenuti con il metodo di espressione HSV-mediata qui descritte suggeriscono che la formazione eteromerico tra 2A e mGlu2 recettori 5-HT nel topo corteccia frontale è necessario per il comportamento simil-psicotico testa a contrazione indotta dal allucinogeno del recettore 5-HT 2A agonisti DOI. Espressione di mGlu2 HSV-mediata, ma non mGlu2ΔTM4N, in frontali neuroni corticali di topi mGlu2-KO salva il comportamento testa contrazione indotta dal allucinogeno del recettore 5-HT 2A agonisti DOI. Questo strumento di traslazione potrebbe essere vantaggioso per gli studi preclinici per valutare fenotipi comportamentali di heteromers GPCR.

Allucinogeni, come l&#39;LSD, psilocibina e mescalina provocano significativi cambiamenti nella coscienza umana, cognizione ed emozione 7-9. L&#39;inattivazione della serotonina 5-HT 2A recettore segnalazioni da approcci sia genetici o farmacologici cause notevolmente attenuato le risposte comportamentali a allucinogeni in entrambi i modelli di roditori 3,10 e gli esseri umani 11. Anche se allucinogeni legano altri sottotipi di recettori 8, il recettore 5-HT 2A è considerato come necessario per l&#39;attività comportamentale unica di queste sostanze chimiche. Gruppo II recettori metabotropici del glutammato (es., MGlu2 e mGlu3) sono stati oggetto di considerevole attenzione per quanto riguarda il meccanismo molecolare di allucinogeni e il loro ruolo fondamentale sottostante psicosi 12. In precedenza, è stato dimostrato che topi con nessuna espressione di proteine ​​mGlu2 (topi mGlu2-KO) sono insensibili agli effetti cellulari e comportamentali di hallucinogens 5. È stato anche suggerito che la 5-HT 2A e recettori mGlu2 formano un complesso specifico eteromerico attraverso cui serotonina e glutammato ligandi modulano il modello dell&#39;accoppiamento proteina G nelle cellule viventi 1,2. Strutturalmente, transmembrana (TM) domini 4 e 5 di mGlu2 giocano un ruolo fondamentale nella formazione eteromerico con la 5-HT 2A recettore 5. Inoltre, ulteriori indagini hanno dimostrato che tre residui si trovano alla fine intracellulare di TM4 di mGlu2 sono necessari per formare il 5-HT 2A -mGlu2 recettore eterocomplesso nelle cellule viventi 6. Sulla base di questi risultati osservati in sistemi di espressione eterologa, qui si descrive l&#39;uso di espressione HSV-mediata di tipo selvaggio mGlu2 e mGlu2 / mGlu3 costrutti chimerici nella corteccia frontale di topi mGlu2-KO per verificare se la formazione eteromerico tra 5-HT 2A e mGlu2 è necessario per ilcomportamento testa contrazione indotta da allucinogeni 5-HT 2A agonisti del recettore.

<table border="0" cellpadding="0" cellspacing="0" width="2329">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

Precedenti risultati dimostrano che la testa a contrazione risposta comportamentale murino è affidabile e robusto suscitata da allucinogeni, ed è assente in 5-HT 2A topi -Ko 3. Inoltre, è stato dimostrato che la risposta testa contrazione provocata dai allucinogeni 5-HT 2A agonisti DOI e LSD era significativamente diminuita in mGlu2-KO topi 5. Tuttavia, anche se i risultati precedenti dimostrano che convincente 2A 5-HT e mGlu2 sono montati in un complesso eteromerico in vitro in cellule trasfettate 1,2,15 Novità, se questa disposizione strutturale si comporta come tale in topi vivi rimasto irrisolto. Per comprendere appieno il ruolo della 5-HT 2A -mGlu2 recettore eterocomplesso negli effetti psicoattivi simili indotte da allucinogeni 5-HT agonisti del recettore 2A, espressione di entrambi mGlu2 o mGlu2ΔTM4N nella corteccia frontale di topi mGlu2-KO di esaminare se questo maniplamento regola il comportamento. I topi ha ricevuto iniezioni intra-corticali frontali di bicistronico HSV che esprimono la proteina fluorescente verde (GFP) e sia mGlu2 o mGlu2ΔTM4N, o GFP da solo. In primo luogo, è stato confermato che il virus over-esprime mGlu2 o mGlu2ΔTM4N nella corteccia del mouse frontale (figure 1A e 1B). Come precedentemente dimostrato 5, comportamento testa a contrazione indotta da DOI era assente nei topi mGlu2-KO iniettati con il vettore vuoto HSV-GFP. In particolare, la risposta testa contrazione indotta dal allucinogeno 5-HT 2A agonisti DOI è stato salvato in topi mGlu2-KO over-esprimono mGlu2, ma non mGlu2ΔTM4N, nella corteccia frontale rispetto a quello osservato negli animali che esprimono GFP (Figura 1C). Insieme, questi risultati suggeriscono che il 5-HT 2A -mGlu2 complesso recettoriale nella corteccia frontale è fondamentale per la regolazione stati psicosi-like. Figura 1. Espressione di mGlu2 come recettore eterocomplesso. Espressione di mGlu2 come un recettore con 5-HT 2A è necessaria per il comportamento simil-psicotico indotto da droghe allucinogene. (A) Immagine rappresentativa della espressione del transgene HSV-mediato nella corteccia frontale. HSV-mGlu2, che esprime anche GFP, è stato iniettato in corteccia frontale, e l&#39;espressione della GFP è stato rivelato da immunocitochimica, barra di scala, di 200 um. (B) HSV-mediata espressione del transgene nel topo corteccia frontale di topi mGlu2-KO, e reattività anti-mGlu2 è stata misurata mediante Western Blotting. Specificità dell&#39;anticorpo primario contro il recettore mGlu2 è stata precedentemente confermata nei topi knockout 6. recettori metabotropici del glutammato sono GPCR che formano homodimers covalentemente legate. Abbiamo misurato immunoreattività di mGlu2 come monomero (100 kDa) 6. (C </strong&gt;) espressione virale-mediata di mGlu2, ma non mGlu2ΔTM4N, nella corteccia frontale di topi KO mGlu2- salva in modo significativo la risposta testa contrazione indotta dal allucinogeno 5-HT2A agonista DOI (n = 4 per gruppo). *** P &lt;0,001; ns, non significativo; test post hoc di Bonferroni di ANOVA. Le barre di errore rappresentano SEM figura è stata modificata da Moreno et al (2012) 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">Supplemental Video 1. Testa Twitch risposta. (Clic destro per il download).</a> Topi CD-1 WT sono stati iniettati con 2,0 mg / kg DOI e collocati in una gabbia (muro oscurato tra le due gabbie) per sollecitare la risposta testa-contrazione (comportamento manifesta dopo *).

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Questo articolo viene descritto come iniettare vettori virali nel mouse corteccia frontale per testare test comportamentali che richiedono la formazione eteromerico GPCR.

HSV-mediata Transgene Espressione di chimerici costrutti di studio comportamentale funzione di GPCR Heteromers nei topi

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.2K Views.  Icahn School of Medicine at Mount Sinai.  Questo articolo viene descritto come iniettare vettori virali nel mouse corteccia frontale per testare test comportamentali che richiedono la formazione eteromerico GPCR. 

Video: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un metodo generale per la valutazione di incubazione della brama di saccarosio in ratti

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Ricerca

Neuroscienze

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Applicazione locale di farmaci allo studio delle funzioni recettore nicotinico in fettine di cervello del mouse

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registrazione e analisi dei ritmi circadiani in Running-ruota Attività nei roditori

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Un test comportamentale semplice for Testing Funzione visiva in<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Valutazione della dendritiche arborizzazione nel giro dentato della Regione ippocampale nei topi

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

La quantificazione di filamentosa Actina (F-actina) Puncta nel ratto neuroni corticali

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

Un metodo fMRI a respirazione libera per studiare la funzione olfattiva umana

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Espressione del Transgene di mediata da Virus adeno-associato in neuroni geneticamente definiti del midollo spinale

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Il potere di Interstimulus intervallo per la valutazione di elaborazione temporale in roditori

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Etichettatura balistica dei neuroni piramidali nelle sezioni cerebrali e nella cultura cellulare primaria

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...