Name: hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice
Uploaded: 2016-07-09T13:00:00
Description: 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

NIH R01MH084894 a participé au financement de cette étude. Nous tenons à remercier les Drs. Yasmin Hurd et Scott Russo au Mount Sinai School of Medicine pour le don de la souris et l&#39;utilisation de leurs installations de chirurgie et de comportement pendant le tournage de ce travail.

NOTE: Toutes les procédures pour l&#39;élevage et soins des animaux ont été menées conformément à la réglementation du Comité des soins et l&#39;utilisation des animaux institutionnels (IACUC) de Mount Sinai School of Medicine. Assurez-vous d&#39;utiliser des gants stériles pendant toute la procédure. 1. drogues et Virus Préparation <ol><li> Préparation des médicaments <ol><li> Préparer 15,0 ml de kétamine / xylazine anesthésique en dissolvant 1,35 ml de 100 m...

<ol>
	<li>Fribourg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+Signaling+of+a+GPCR+Heteromeric+Complex+Reveals+a+Unifying+Mechanism+of+Action+of+Antipsychotic+Drugs.">Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs.</a> Cell. 147 (5), 1011-1023 (2011).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+serotonin/glutamate+receptor+complex+implicated+in+psychosis.">Identification of a serotonin/glutamate receptor complex implicated in psychosis.</a> Nature. 452 (7183), 93-97 (2008).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens+Recruit+Specific+Cortical+5-HT(2A)+Receptor-Mediated+Signaling+Pathways+to+Affect+Behavior.">Hallucinogens Recruit Specific Cortical 5-HT(2A) Receptor-Mediated Signaling Pathways to Affect Behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Gonzalez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+fingerprints+distinguish+hallucinogenic+and+nonhallucinogenic+5-hydroxytryptamine+2A+receptor+agonist+effects+in+mouse+somatosensory+cortex.">Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.</a> J Neurosci. 23 (26), 8836-8843 (2003).</li><li>Moreno, J. L., Holloway, T., Albizu, L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+mGlu2+receptor+is+necessary+for+the+pharmacological+and+behavioral+effects+induced+by+hallucinogenic+5-HT2A+receptor+agonists.">Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.</a> Neurosci Lett. 493 (3), 76-79 (2011).</li><li>Moreno, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Three+Residues+Essential+for+5-HT2A-mGlu2+Receptor+Heteromerization+and+its+Psychoactive+Behavioral+Function.">Identification of Three Residues Essential for 5-HT2A-mGlu2 Receptor Heteromerization and its Psychoactive Behavioral Function.</a> J Biol Chem. 287, 44301-44319 (2012).</li><li>Geyer, M. A., Vollenweider, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+research:+contributions+to+understanding+psychoses.">Serotonin research: contributions to understanding psychoses.</a> Trends Pharmacol Sci. 29 (9), 445-453 (2008).</li><li>Nichols, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens.">Hallucinogens.</a> Pharmacol Ther. 101 (2), 131-181 (2004).</li><li>Hanks, J. B., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+serotonergic+psychedelics.">Animal models of serotonergic psychedelics.</a> ACS Chem Neurosci. 4 (1), 33-42 (2013).</li><li>Fiorella, D., Rabin, R. A., Winter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+5-HT2A+and+5-HT2C+receptors+in+the+stimulus+effects+of+hallucinogenic+drugs.+II:+Reassessment+of+LSD+false+positives.">Role of 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. II: Reassessment of LSD false positives.</a> Psychopharmacology (Berl). 121 (3), 357-363 (1995).</li><li>Vollenweider, F. X., Vollenweider-Scherpenhuyzen, M. F., Babler, A., Vogel, H., Hell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psilocybin+induces+schizophrenia-like+psychosis+in+humans+via+a+serotonin-2+agonist+action.">Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action.</a> Neuroreport. 9 (17), 3897-3902 (1998).</li><li>Moreno, J. L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+II+metabotropic+glutamate+receptors+and+schizophrenia.">Group II metabotropic glutamate receptors and schizophrenia.</a> Cell Mol Life Sci. 66 (23), 3777-3785 (2009).</li><li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC2+regulates+atypical+antipsychotic+responses+through+the+modulation+of+mGlu2+promoter+activity.">HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.</a> Nat Neurosci. 15 (9), 1245-1254 (2012).</li><li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repressive+Epigenetic+Changes+at+the+mGlu2+Promoter+in+Frontal+Cortex+of+5-HT2A+Knockout+Mice.">Repressive Epigenetic Changes at the mGlu2 Promoter in Frontal Cortex of 5-HT2A Knockout Mice.</a> Mol Pharmacol. 83 (6), 1166-1175 (2013).</li><li>Rives, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+GABAB+and+mGlu1a+receptors+reveals+new+insight+into+GPCR+signal+integration.">Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration.</a> Embo J. 28 (15), 2195-2208 (2009).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prevalence,+Maintenance+and+Relevance+of+GPCR+Oligomerization.">The Prevalence, Maintenance and Relevance of GPCR Oligomerization.</a> Mol Pharmacol. (84), 158-169 (2013).</li><li>Ferre, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptor+oligomerization+revisited:+functional+and+pharmacological+perspectives.">G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives.</a> Pharmacol Rev. 66 (2), 413-434 (2014).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+oligomers+in+pharmacology+and+signaling.">GPCR oligomers in pharmacology and signaling.</a> Mol Brain. 4 (1), 20 (2011).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Family+a+GPCR+heteromers+in+animal+models.">Family a GPCR heteromers in animal models.</a> Front Pharmacol. 5, 226 (2014).</li><li>Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+resonance+energy+transfer+(BRET)+imaging+of+protein-protein+interactions+within+deep+tissues+of+living+subjects.">Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (29), 12060-12065 (2011).</li><li>Calebiro, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization.">Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.</a> Proc Natl Acad Sci U S A. 110 (2), 743-748 (2013).</li><li>Fonseca, J. M., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+a+class+a+G+protein-coupled+receptor+oligomer+interface.">Instability of a class a G protein-coupled receptor oligomer interface.</a> Mol Pharmacol. 75 (6), 1296-1299 (2009).</li><li>Hern, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+dissociation+of+M1+muscarinic+receptor+dimers+seen+by+total+internal+reflection+fluorescence+imaging+of+single+molecules.">Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.</a> Proc Natl Acad Sci U S A. 107 (6), 2693-2698 (2010).</li><li>Hlavackova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+inter-+and+intrasubunit+rearrangements+during+activation+of+dimeric+metabotropic+glutamate+receptor+1.">Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1.</a> Sci Signal. 5 (237), 59 (2012).</li><li>Irannejad, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+biosensors+reveal+GPCR+signalling+from+endosomes.">Conformational biosensors reveal GPCR signalling from endosomes.</a> Nature. 495 (7442), 534-538 (2013).</li><li>Calebiro, D., Nikolaev, V. O., Persani, L., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+internalized+G-protein-coupled+receptors.">Signaling by internalized G-protein-coupled receptors.</a> Trends Pharmacol Sci. 31 (5), 221-228 (2010).</li><li>Celada, P., Puig, M. V., Diaz-Mataix, L., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex:+reversal+by+antipsychotic+drugs.">The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs.</a> Biol Psychiatry. 64 (5), 392-400 (2008).</li><li>B&#233;&#239;que, J. -. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).</li></ol>

Ainsi que les résultats précédents en mGlu2-KO 5 souris, les résultats obtenus avec mGlu2 et mGlu2 / mGlu3 constructions chimères qui ne forment pas le complexe récepteur 2A -mGlu2 5-HT dans des cellules en culture suggèrent que la 5-HT 2A -mGlu2 complexe récepteur hétéromère en souris cortex frontal est nécessaire pour induire un comportement tête twitch par hallucinogènes 5-HT des agonistes des récepteurs 2A LSD-like. Une limitation de cette méthode est qu&#3...

Hallucinogènes, comme le LSD, la psilocybine et la mescaline provoquent des changements importants dans la conscience humaine, la cognition et l&#39; émotion 7-9. L&#39; inactivation de la sérotonine 5-HT signalisation du récepteur 2A par des approches génétiques ou pharmacologiques soit provoque nettement atténué les réactions comportementales aux hallucinogènes dans les deux modèles de rongeurs 3,10 et humains 11. Bien que les hallucinogènes se lient d&#39; autres sous - types de récepteurs 8, le récepteur 5-HT2A est considéré comme nécessaire à l&#39;activité comportementale unique de ces produits chimiques. Groupe II récepteurs de glutamate métabotropique (ie., MGlu2 et mGlu3) ont été la cible d&#39; une attention considérable en ce qui concerne le mécanisme moléculaire d&#39;hallucinogènes et de leur rôle essentiel qui sous - tend la psychose 12. Auparavant, il a été démontré que des souris sans expression de la protéine mGlu2 (souris mGlu2-KO) sont insensibles aux effets cellulaires et comportementaux hallucinogens 5. Il a également été suggéré que la 5-HT 2A et les récepteurs de mGlu2 forment un complexe hétéromère spécifique à travers lequel la sérotonine et de glutamate de ligands modulent le motif de G couplage des protéines dans les cellules vivantes 1,2. Structurellement, transmembranaires (TM) , les domaines 4 et 5 de mGlu2 jouent un rôle fondamental dans la formation hétéromère avec le récepteur 5-HT 2A 5. En outre, une investigation a montré que trois résidus situés à l&#39;extrémité intracellulaire de TM4 de mGlu2 sont nécessaires pour former la 5-HT 2A -mGlu2 hétérocomplexe de récepteur dans des cellules vivant à 6. Sur la base de ces résultats observés dans les systèmes d&#39;expression hétérologues, ici , nous décrivons l&#39;utilisation de l&#39; expression HSV-médiée de type sauvage mGlu2 et mGlu2 / mGlu3 constructions chimériques dans le cortex frontal des souris mGlu2-KO pour vérifier si la formation hétéromère entre 5-HT 2A et mGlu2 est nécessaire pour lacomportement tête contraction induite par hallucinogènes 5-HT des agonistes des récepteurs de 2A.

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	<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.

Des résultats antérieurs démontrent que la tête-twitch réponse comportementale murin est fiable et robuste provoquée par les hallucinogènes, et il est absent dans 5-HT 2A -kō souris 3. En outre, il a été démontré que la réponse de la tête contraction provoquée par les hallucinogènes 5-HT 2A agonistes DOI et le LSD a été significativement diminuée chez les souris KO mGlu2-5. Cependant, bien que les résultats antérieurs démo...

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

Cet article décrit comment injecter des vecteurs viraux dans le cortex frontal de la souris pour tester tests comportementaux qui nécessitent la formation hétéromère GPCR.

HSV-Mediated Expression du transgène de chimériques Constructs pour étudier la fonction Behavioral des GPCR hétéromères chez la souris

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

The overall goal of this method is to observe how virus-mediated gene transfer affects behavioral phenotypes that require heterodimerization of G-protein coupled receptors in mouse models. This method can help answer key questions in the field of neuro and psycho pharmacology. Specifically how g protein coupled receptors interact with each other at a molecular level.By using this method we can understand how this interaction affects behavior in models of psychiatric disorders, such as schizophrenia and depression. Using this method we are going to specifically look at seratonin 2a and metabotropic glutamate receptor 2, both of which have been shown to be implicated in the mechanism of action of drugs used to treat patients with schizophrenia. Begin by properly anesthetizing the mouse according to approved methods.Ensure that a surgical plane of anesthesia has been obtained by performing a toe pinch and noting the absence of a response. Then apply ophthalmic gel to protect the eyes. Attach the mouse to the stereotactic frame, making sure to adjust the tilt of the head so that the skull is level and flat.Apply povidone iodine to the exposed scalp. Using a scalpel make a sagittal incision along the midline of the skull within the exposed shaved area. Then attach pureit clamps to the skin at the incision site to make sure that the skull remains exposed.Apply hydrogen peroxide to dissolve away the periosteum, and expose the sutures of the skull. Now that the bregma and sutures are visible, be sure to adjust the stereotactic frame to make sure that the skull is level. Align the needle tips of the syringes with bregma and record the coordinates of bregma.To bilaterally inject the frontal cortex, add 1.6 mm to the recorded rostral caudal bregma coordinate, and 2.6 mm to the recorded medial lateral bregma coordinate. Mark the injection sites on the skull and then drill small holes at the injection sites. With a cotton tip applicator, wipe away any excess blood or bone fragments.Bring the needle to the surface of the brain, and lower to the depth of the dorsal ventral coordinate. Then slowly inject the contents of the syringe by twisting the plunger of the needle to administer 0.1 microliters per minute for five minutes, for a 0.5 microliter injection. Allow the syringe to remain in place for five minutes.After the allotted time, remove the animal from the stereotaxis, close the incision, and place on a warming pad. Administer post operative analgesia according to your approved animal protocol. Perform behavioral testing two to three days after stereotactic injection of viral particles.Habituate the mice to the room for at least four hours prior to the beginning of the experiment. Begin by dissolving DOI into a 0.9 percent saline solution to two milligrams per kilogram. Prepare a home cage without any bedding and using a tripod adjust a camera so that it is directly over the home cage.Calibrate the camera so that the entire home cage is in the field of view. Weigh the mouse and inject intraperitoneally with the appropriate dose of either 0.9 percent saline or DOI. Place the mouse back in the home cage for ten minutes.After ten minutes, place the mouse into the center of the empty home cage, and ensure that there are no blind spots in the field of view of the camera. Press record on the camcorder and leave the room. After 30 minutes, stop the recording and place the mouse back in the original home cage.Repeat the behavioral test on the next mouse. Have the referee review the videos blind to the experimental conditions. Manually record every head twitch throughout the video.A head twitch is defined as a rapid shaking head movement conducted by a mouse. Process the data as outlined in the written portion of the protocol. Shown here is a representative image of HSV mediated transgene expression in the frontal cortex.HSV mGlu2 GFP was injected into the frontal cortex and GFP expression was revealed by immunocytochemistry. This scale bar represents 200 microns. This image shows a western blot of HSV mediated transgene expression and anti-mGlu2 reactivity in the frontal cortex of mGlu2 knockout mice.Here we measured immunoreactivity of mGlu2 as a monomer. This histogram shows that viral mediated expression of mGlu2, but not of chimeric mGlu2 in the frontal cortex of mGlu2 knockout mice significantly rescues the head twitch response induced by the hallucinogenic seratonin 2a receptor agonist DOI. The most critical steps with this experiment are the timing between the viral injections, the head twitch response, and when the mice are sacrificed.Any delay can alter the results of the experiment or introduce ambiguity into the data. Once mastered, the mouse surgery will take approximately 30 minutes to inject the virus. The head twitch experiment should also take approximately 35 minutes per mouse.Staggering surgeries, or operating on more than one mouse at a time is recommended. This way one can maintain a timeline that allows the head twitch experiments to be done in the three to four days after surgery which is the peak expression time of the viral construct. Thanks for watching and good luck with your experiment.

Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

Une méthode générale pour l&#39;évaluation d&#39;incubation du désir saccharose chez les 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 ...

Recherche

Neurosciences

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

Application locale de médicaments pour l&#39;étude des fonctions récepteur de l&#39;acétylcholine nicotinique dans des tranches de cerveau de souris

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A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Un test comportemental simple pour tester la fonction visuelle dans<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

Évaluation de l&#39;arborisation dendritique dans le gyrus denté de la région de l&#39;hippocampe chez la souris

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

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

Quantification des filamenteuses Actine (F-actine) punctas chez le rat neurones corticaux

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

Une méthode FMRI à respiration libre pour étudier la fonction olfactive humaine

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

Adeno-associated Virus-mediated Transgene Expression dans les neurones de la moelle épinière génétiquement définies

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

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification de la Dopamine D1-Alpha du récepteur au sein de rongeurs noyau Accumbens par une ARN innovantes In Situ la technologie de détection

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

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

La puissance de l’intervalle Interstimulus pour l’évaluation du traitement temporel chez les rongeurs

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

Étiquetage balistique des neurones pyramidaux dans les tranches de cerveau et dans la culture cellulaire primaire

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