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は、この研究の資金調達に参加しました。我々は、博士に感謝したいと思います。マウスの寄付と、この作品の撮影時に、その手術や行動施設の利用のためのマウントサイナイ医科大学でヤスミンHurd氏とスコット・ルッソ。

注：動物の飼育や心配事のためのすべての手順は、マウントサイナイ医科大学の施設内動物管理使用委員会（IACUC）規則に従って行いました。作業中は滅菌手袋を使用してください。 1.薬剤およびウイルスの準備<ol><li> 医薬品の準備 <ol><li> 0.9％の食塩水の12.9ミリリットルで100 mg / mlでケタミンおよび20mg / mlのキシラジンの0.75ミリリットルの1.35ミリリットルを溶解するこ?...

<ol>
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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. 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一緒にmGlu2-KOマウス5の前の所見と、培養細胞において5-HT 2A -mGlu2受容体複合体を形成しないmGlu2およびmGlu2 / mGlu3キメラ構築物を用いた結果は、5-HT 2A -mGlu2ヘテロマー受容体複合体ことを示唆していますマウスの前頭皮質は、LSDのような幻覚5-HT 2A受容体アゴニストによって頭部攣縮挙動を誘発するために必要とされます。この方法の制限は、それが天然の組織内...

そのようなLSD、サイロシビンやメスカリンなどの幻覚剤は、人間の意識、認知と感情7-9に大きな変化を引き起こします。セロトニンの不活性化は、遺伝的または薬理学的のいずれかの方法により、5-HT 2A受容体のシグナル伝達は、両方の齧歯類モデル3,10、およびヒト11で幻覚剤に著しく減衰行動反応を引き起こします。幻覚剤は、他の受容体サブタイプ8に結合するが、5-HT 2A受容体は、これらの化学物質の固有の行動活性に必要と考えられています。 グループII代謝型グルタミン酸受容体（ すなわち 、mGlu2およびmGlu3）は幻覚剤の分子機構に関するかなりの注目の対象と精神病12の基礎となる彼らの重要な役割となっています。これまでは、mGlu2タンパク質の発現なしでのマウス（mGlu2-KOマウス）はHALの細胞や行動への影響に鈍感であることが実証されていますlucinogens 5。また、5-HT 2AおよびmGlu2受容体は、セロトニンおよびグルタミン酸リガンドが生細胞1,2のGタンパク質結合のパターンを調節し、それを通して、特定のヘテロマー複合体を形成することが示唆されています。 構造的には、膜貫通（TM）ドメイン4およびmGlu2の5は、5-HT 2A受容体5とヘテロマー形成に重要な役割を果たしています。さらに、さらなる調査がmGlu2のTM4の細胞内の端部に位置する3つの残基が、細胞を6生体内5-HT 2A -mGlu2レセプターヘテロを形成するのに必要であることを実証しました。 異種発現系で観察されたこれらの知見に基づいて、ここでは、5-HT 2Aとの間のヘテロマー形成するかどうかをテストするために、野生型mGlu2およびmGlu2-KOマウスの前頭皮質におけるmGlu2 / mGlu3キメラ構築物のHSV媒介表現の使用を記載していますそして、mGlu2はのために必要です幻覚5-HT 2A受容体アゴニストによって誘導される頭部攣縮挙動。

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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>
</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.

以前の知見は、ヘッド単収縮マウスの行動応答を確実かつロバスト幻覚剤によって誘発され、それは、5-HT 2A -KOマウス3には存在しないことを示しています。また、DOIとLSD幻覚の5-HT 2Aアゴニストによって誘発される頭部攣縮応答が有意mGlu2-KOマウス5に減少したことが示されました。しかし、以前の知見は、説得力の5-HT 2Aおよびm...

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

この記事では、GPCRヘテロマーの形成を必要とする行動アッセイをテストするために、マウスの前頭皮質にウイルスベクターを注入する方法について説明します。

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

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

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神経科学

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A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

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

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

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

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