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 deltog i finansieringen af ​​denne undersøgelse. Vi vil gerne takke Drs. Yasmin Hurd og Scott Russo på Mount Sinai School of Medicine ved donation af mus og brugen af ​​deres kirurgi og adfærd faciliteter under indspilningen af ​​dette arbejde.

BEMÆRK: Alle procedurer for dyreavl og bekymringer blev udført i overensstemmelse med Institutional Animal Care og brug Udvalg (IACUC) regulering af Icahn School of Medicine på Mount Sinai. Sørg for at bruge sterile handsker under hele proceduren. 1. Drug og Virus Forberedelse <ol><li> Drug Preparation <ol><li> Forbered 15,0 ml ketamin / xylazin bedøvelsesmiddel ved opløsning 1,35 ml 100 mg / ml ketamin og 0,75 ml 20 mg / ml xylazin i 12,9 ml 0,9% saltvandsopløsning. Bland løsning. </l...

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Sammen med tidligere resultater i mGlu2-KO-mus 5, resultaterne med mGlu2 og mGlu2 / mGlu3 kimære konstruktioner, der ikke danner 5-HT 2A -mGlu2 receptorkomplekset i dyrkede celler tyder på, at 5-HT 2A -mGlu2 heteromere receptorkomplekset i muse frontale cortex er nødvendig for at inducere head-twitch adfærd ved LSD-lignende hallucinogene 5-HT 2A receptor-agonister. En begrænsning ved denne metode er, at den ikke måler tæt molekylære lighed på et subcellulært niveau i...

Hallucinogener, såsom LSD, psilocybin og mescalin medføre betydelige ændringer i den menneskelige bevidsthed, kognition og emotion 7-9. Inaktivering af serotonin 5-HT 2A receptor signalering ved enten genetiske eller farmakologiske metoder forårsager markant svækket adfærdsmæssige reaktioner på hallucinogener i begge gnavermodeller 3,10 og mennesker 11. Selvom hallucinogener binder andre receptorsubtyper 8 er 5-HT2A-receptoren betragtes som nødvendig for den unikke adfærdsmæssige aktivitet af disse kemikalier. Gruppe II metabotrope glutamatreceptorer (dvs.., MGlu2 og mGlu3) har været mål for betydelig opmærksomhed om den molekylære mekanisme af hallucinogener og deres integrerende rolle underliggende psykose 12. Tidligere er det blevet vist, at mus med ingen ekspression af mGlu2 protein (mGlu2-KO-mus) er ufølsomme over for de cellulære og adfærdsmæssige virkninger af hallucinogens fem. Det er også blevet foreslået, at 5-HT 2A og mGlu2 receptorer danner en specifik heteromerkompleks hvorigennem serotonin og glutamat ligander modulere mønster af G-protein kobling i levende celler 1,2. Strukturelt transmembrane (TM) domæner 4 og 5 i mGlu2 spiller en fundamental rolle i heteromere formation med 5-HT 2A receptor 5. Derudover yderligere undersøgelse viste, at tre rester placeret i den intracellulære ende af TM4 af mGlu2 er nødvendige for at danne 5-HT 2A -mGlu2 receptor heterokompleks i levende celler 6. Baseret på disse resultater observeret i heterologe ekspressionssystemer, Her beskrives anvendelsen af HSV-medieret ekspression af vildtype mGlu2 og mGlu2 / mGlu3 kimære konstruktioner i den frontale cortex hos mGlu2-KO-mus at teste, om heteromer dannelse mellem 5-HT 2A og mGlu2 er nødvendig forhead-twitch opførsel induceret af hallucinogene 5-HT 2A receptor-agonister.

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			<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>
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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.

Tidligere resultater viser, at head-spjæt murine adfærdsmæssig reaktion er pålideligt og robust fremkaldt af hallucinogener, og det er fraværende i 5-HT 2A -KO mus 3. Endvidere er det blevet vist, at hovedet-twitch respons fremkaldt af hallucinogene 5-HT2A agonister DOI og LSD blev signifikant reduceret i mGlu2-KO-mus 5. Men selv om tidligere resultater overbevisende, at 5-HT 2A og mGlu2 er samlet som en heteromerkompleks in...

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

Denne artikel beskriver, hvordan at injicere virale vektorer i musen frontale cortex at teste adfærdsmæssige analyser, der kræver GPCR heteromere formation.

HSV-medieret transgene ekspression af kimære konstruktioner at undersøge adfærdsmæssige funktion GPCR heteromerer i mus

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.

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

Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

En generel metode til evaluering Inkubation af Saccharose Savner hos rotter

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 ...

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 ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Juxtasomal biocytin Mærkning at studere strukturen-funktionen forholdet mellem individuelle corticalneuroner

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

En simpel Behavioral Assay for Testing Visual Funktion i<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

Vurdering af dendritiske arborization i gyrus dentatus i hippocampus-regionen i mus

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

Kvantificering af Filamentøs actin (F-actin) puncta i Rat kortikale neuroner

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

En fri vejrtrækning fMRI metode til at studere human olfactory funktion

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-associeret Virus-medieret transgen udtryk i genetisk definerede neuroner i rygmarven

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

Kraften i Interstimulus Interval for vurdering af tidsmæssige forarbejdes i Rodents

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

Ballistisk mærkning af pyramideformede neuroner i hjerneskiver og i primærcellekultur

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