Name: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice (Video) | JoVE Journal
Uploaded: 2016-07-09T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Learn HSV-mediated gene transfer to study GPCR heteromer function in mouse behavioral neuroscience. This method examines 5-HT2A-mGlu2 receptor interactions using the head-twitch response assay.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

NIH R01MH084894 participated in the funding of this study. We would like to thank Drs. Yasmin Hurd and Scott Russo at Mount Sinai School of Medicine for the donation of mice and the use of their surgery and behavior facilities during the filming of this work.

NOTE: All procedures for animal breeding and cares were conducted according to the Institutional Animal Care and Use Committee&#160;(IACUC) regulation of Icahn School of Medicine at Mount Sinai. Be sure to use sterile gloves throughout the procedure.&#160;
1. Drug and Virus Preparation
<ol>
	<li>Drug Preparation
	<ol>
		<li>Prepare 15.0 ml ketamine/xylazine anesthetic by dissolving 1.35 ml of 100 mg/ml Ketamine and 0.75 ml of 20 mg/ml xylazine in 12.9 ml of 0.9% Saline solution. Thoroughly mix solution.</li>
	</ol>
	</li>
	<li>Virus Preparation
	<ol>
		<li>Clone the mGlu2 and mGlu2&#916;TM4N constructs into a bicistronic herpes simplex virus (HSV) vector following standard protocols previously described6. Package the viral particles as previously described6,13,14. Substitution of residues Ala-6774.40, Ala-6814.44 and Ala6854.48 in mGlu2 for Ser6864.40, Phe6904.44 and Gly-6944.48 in mGlu3 (HA-mGlu2&#916;TM4N) have been described previously6. 
		NOTE: It was previously demonstrated that the chimeric construct HA-mGlu2&#916;TM4N is expressed at the plasma membrane with intact G protein-dependent signaling6.</li>
		<li>Store viral vectors in -80 &#176;C when not in use. Thaw viral vector on ice, and then aliquot into 10 &#956;l aliquots. For the surgical procedure, keep on ice.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Surgery Preparation
	<ol>
		<li>Weigh mouse and inject mouse with appropriate dose of ketamine/xylazine cocktail (for details, see 1.1.1).</li>
		<li>Check mouse to see if properly anesthetized, squeeze foot and tail for pain response, if unable to elicit a response, the mouse is properly anesthetized.</li>
		<li>Shave mouse head from the base of the skull to the tip of the nose using clippers. Apply ophthalmic gel to the mouse afterward to prevent blindness of the mouse.</li>
		<li>Load each syringe onto the stereotaxic frame. Then tilt perpendicular portion of each arm of the stereotaxic frame so that they are 10 degrees away from the normal. Ensure that the arms are tilted, such that the needles are facing each other.</li>
		<li>Clean each syringe by filling the needle with 70% ethanol. Fill the needle at least three times to ensure that the syringe is clean.</li>
		<li>Once the needle has been cleaned, flush the needle by filling the needle with double distilled H2O. Once flushed, fill each needle with 1.3 &#181;l of double distilled H2O. Twist the plunger of the syringe to release 0.3 &#181;l of double distilled H2O. If water beads at the tip of the needle, carefully wipe away water. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
		<li>After filling with water, then pull up the syringe filling the syringe with 0.5 &#181;l of air.</li>
		<li>Once the air and water are in the syringe, carefully fill the syringe with 1.3 &#181;l of virus solution. At this point ensure that the total volume in the syringe is 2.8 &#181;l. Again twist the tip of the syringe to release 0.3 &#181;l of virus. If liquid beads at the tip of the needle, carefully wipe away liquid. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Attach the mouse to the stereotaxic frame, making sure to adjust the stereotaxic frame so that the skull is level and flat. Apply povodine-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 the buret clamps to the skin at the incision site to make sure that the skull remains exposed.</li>
		<li>Use H2O2 to dissolve away the periosteum to expose the sutures of the skull. Now that the bregma and sutures are visible, be sure to adjust the stereotaxic frame to make sure that the skull is level.</li>
		<li>Align the needle tips of the syringes with the bregma and record the coordinates of the bregma. Calculate the coordinates of the where the needles are going to be inserted.
		<ol>
			<li>For the Rostral-Cauldal (R-C) plane, add 1.6 mm to the recorded R-C bregma coordinates (+1.6 from Bregma). For the Dorsal-Ventral (D-V) plane, subtract 2.4 mm from the recorded D-V bregma coordinates (-2.4 from Bregma).</li>
			<li>Finally, for the Medial Lateral (M-L) plane, add 2.6 mm to the recorded M-L bregma coordinates (+2.6 from Bregma). For all coordinates be sure to record both left and right coordinates, as this is a bilateral injection.</li>
		</ol>
		</li>
		<li>Bring the needles to the desired coordinates. Mark the places of where the needles are going to be inserted and with a drill, drill the marked areas.</li>
		<li>With a cotton tip applicator wipe away any excess blood or bone fragment.</li>
		<li>Bring the needles to the skull where the tips of the needles are touching the surface of the brain. Then lower the needles to the desired coordinates slowly lowering them.</li>
		<li>Once the needles are at the desired coordinates, slowly inject the contents of the syringe by twisting the plunger of the needle 0.1 &#181;l per minute over the course of 5 min&#160;(in total 0.5 &#181;l).</li>
		<li>Once the injection has been made leave the syringe in cortex for another 5 min.</li>
	</ol>
	</li>
	<li>Closing Up/Care
	<ol>
		<li>Remove the needles from the mouse cortex steadily and slowly. Then remove the mouse from the stereotaxic frame.</li>
		<li>Apply cyanoacrylate (dermal adhesive) to the base flaps of skin from the incision and then with forceps grab the flaps of skin and place them together.</li>
		<li>Allow the cyanoacrylate to dry. Place the mouse in cage over a heating pad (heating pad is optional if the surgical suite is kept under RT of 37 &#176;C - otherwise not necessary). Be sure to place the mouse on a paper towel to make sure that bedding does not adhere to surgical site.</li>
		<li>Depending on the length of the surgical procedure, ensure that the mice is out of anesthesia within 30 - 60 min&#160;after procedure.&#160;Following surgery, the animal is placed in its own cage and monitored until it becomes conscious before returning to its room to recover. No analgesics are used post-operatively, because they may alter the results of our experiments by modifying some of the brain biochemical pathways. However, animals are monitored until they recover from anesthesia on the day of surgery, and daily post-operation for signs of infection and evaluation of pain/distress.</li>
	</ol>
	</li>
</ol>
3. Head Twitch Response Experiment
<ol>
	<li>Set-up
	<ol>
		<li>Carry out all behavioral testing between 10:00 AM and 2:00 PM, 2 - 3 days after stereotactic injection of viral particles.</li>
		<li>Dissolve (&#177;)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) into a 0.9% saline solution to 2.0 mg/kg. Also prepare a 0.9% saline solution.</li>
		<li>Prepare a home cage (28 x 18 x 15 cm) without any bedding and using a tri-pod, adjust a camera so that the view of the video camera is directly above the home cage.</li>
		<li>Habituate the mice to the room for at least 4 hr prior to the beginning of the experiment.</li>
		<li>Set up a camcorder to record the head twitch.</li>
	</ol>
	</li>
	<li>Experiment
	<ol>
		<li>Position the camera so that it is directly over a home cage. Calibrate the camera so that the entire home cage is in the field of view.</li>
		<li>Weigh mouse and inject mouse intraperitoneally with appropriate dose of either 0.9% saline or DOI (0.01 ml/g). NOTE: If a mouse weighs 25 grams, administer dose to a total volume of 0.25 ml.</li>
		<li>Place each mouse back in their home cage for 10 min. After 10 min, place mouse into the center of the empty home cage and validate that there are no blind spots in the field of view of the camera. Press record on the camcorder. Leave the room. 
		NOTE: Mouse movements and various behavioral responses therein (head twitch, ear scratch, etc. Please refer to supplemental data table 1 of Gonzalez-Maeso et al. 2007 for full list of behavioral responses induced by DOI.)3, will be recorded for 30 min. Therefore, it is important there are no blind spots in the field of view recorded.</li>
		<li>After 30 min&#160;stop recording on the camcorder and place mouse back in original home cage. Repeat this process for each mouse.</li>
	</ol>
	</li>
	<li>Review
	<ol>
		<li>Have each referee review the tapes blind to the experimental conditions of mouse (i.e., drugs used during head twitch experiment or virus used during intracranial injection)). Manually record every head twitch throughout the video. 
		NOTE: Head-twitch is defined as a rapid shaking head movement conducted by a mouse (supplemental video).</li>
		<li>For each mouse, average the final HTR response from the three totals of the blind referees. Then group these values by experimental condition and carry out statistical analysis (i.e., t-test or ANOVA).</li>
	</ol>
	</li>
</ol>

<ol><li>Fribourg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+the+Signaling+of+a+GPCR+Heteromeric+Complex+Reveals+a+Unifying+Mechanism+of+Action+of+Antipsychotic+Drugs%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Identification+of+a+serotonin%2Fglutamate+receptor+complex+implicated+in+psychosis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Hallucinogens+Recruit+Specific+Cortical+5-HT%282A%29+Receptor-Mediated+Signaling+Pathways+to+Affect+Behavior%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Transcriptome+fingerprints+distinguish+hallucinogenic+and+nonhallucinogenic+5-hydroxytryptamine+2A+receptor+agonist+effects+in+mouse+somatosensory+cortex%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Metabotropic+glutamate+mGlu2+receptor+is+necessary+for+the+pharmacological+and+behavioral+effects+induced+by+hallucinogenic+5-HT2A+receptor+agonists%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">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/pubmed?term=((Identification+of+Three+Residues+Essential+for+5-HT2A-mGlu2+Receptor+Heteromerization+and+its+Psychoactive+Behavioral+Function%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Serotonin+research%3A+contributions+to+understanding+psychoses%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((Hallucinogens%5BTitle%5D)+AND+%22Pharmacol+Ther%22%5BJournal%5D)">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/pubmed?term=((Animal+models+of+serotonergic+psychedelics%5BTitle%5D)+AND+%22ACS+Chem+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Role+of+5-HT2A+and+5-HT2C+receptors+in+the+stimulus+effects+of+hallucinogenic+drugs.+II%3A+Reassessment+of+LSD+false+positives%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">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/pubmed?term=((Psilocybin+induces+schizophrenia-like+psychosis+in+humans+via+a+serotonin-2+agonist+action%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">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/pubmed?term=((Group+II+metabotropic+glutamate+receptors+and+schizophrenia%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">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/pubmed?term=((HDAC2+regulates+atypical+antipsychotic+responses+through+the+modulation+of+mGlu2+promoter+activity%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Repressive+Epigenetic+Changes+at+the+mGlu2+Promoter+in+Frontal+Cortex+of+5-HT2A+Knockout+Mice%5BTitle%5D)+AND+%22Mol+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Crosstalk+between+GABAB+and+mGlu1a+receptors+reveals+new+insight+into+GPCR+signal+integration%5BTitle%5D)+AND+%22Embo+J%22%5BJournal%5D)">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/pubmed?term=((The+Prevalence%2C+Maintenance+and+Relevance+of+GPCR+Oligomerization%5BTitle%5D)+AND+%22Mol+Pharmacol%22%5BJournal%5D)">The Prevalence, Maintenance and Relevance of GPCR Oligomerization.</a> Mol Pharmacol. (84), Epub ahead of print 158-169 (2013).</li>
<li>Ferre, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G+protein-coupled+receptor+oligomerization+revisited%3A+functional+and+pharmacological+perspectives%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">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/pubmed?term=((GPCR+oligomers+in+pharmacology+and+signaling%5BTitle%5D)+AND+%22Mol+Brain%22%5BJournal%5D)">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/pubmed?term=((Family+a+GPCR+heteromers+in+animal+models%5BTitle%5D)+AND+%22Front+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Bioluminescence+resonance+energy+transfer+%28BRET%29+imaging+of+protein-protein+interactions+within+deep+tissues+of+living+subjects%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Instability+of+a+class+a+G+protein-coupled+receptor+oligomer+interface%5BTitle%5D)+AND+%22Mol+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Formation+and+dissociation+of+M1+muscarinic+receptor+dimers+seen+by+total+internal+reflection+fluorescence+imaging+of+single+molecules%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Sequential+inter-+and+intrasubunit+rearrangements+during+activation+of+dimeric+metabotropic+glutamate+receptor+1%5BTitle%5D)+AND+%22Sci+Signal%22%5BJournal%5D)">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/pubmed?term=((Conformational+biosensors+reveal+GPCR+signalling+from+endosomes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Signaling+by+internalized+G-protein-coupled+receptors%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex%3A+reversal+by+antipsychotic+drugs%5BTitle%5D)+AND+%22Biol+Psychiatry%22%5BJournal%5D)">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>Béïque, J. -C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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>

The authors have nothing to disclose.

Together with previous findings in mGlu2-KO mice5, the results with mGlu2 and mGlu2/mGlu3 chimeric constructs that do not form the 5-HT2A-mGlu2 receptor complex in cultured cells suggest that the 5-HT2A -mGlu2 heteromeric receptor complex in mouse frontal cortex is needed to induce head-twitch behavior by LSD-like hallucinogenic 5-HT2A receptor agonists. A limitation of this method is that it does not measure close molecular proximity at a subcellular level in native tissue. In...

Hallucinogens, such as LSD, psilocybin and mescaline cause significant changes in human consciousness, cognition and emotion7-9. Inactivation of serotonin 5-HT2A receptor signaling by either genetic or pharmacological approaches causes markedly attenuated behavioral responses to hallucinogens in both rodent models3,10 and humans11. Although hallucinogens bind other receptor subtypes8, the 5-HT2A receptor is considered as necessary for the unique behavioral activity of these chemicals.
Group II metabotropic glutamate receptors (i.e., mGlu2 and mGlu3) have been the target of considerable attention regarding the molecular mechanism of hallucinogens and their integral role underlying psychosis12. Previously, it has been demonstrated that mice with no expression of mGlu2 protein (mGlu2-KO mice) are insensitive to the cellular and behavioral effects of hallucinogens5. It has also been suggested that the 5-HT2A and the mGlu2 receptors form a specific heteromeric complex through which serotonin and glutamate ligands modulate the pattern of G protein coupling in living cells 1,2.
Structurally, transmembrane (TM) domains 4 and 5 of mGlu2 play a fundamental role in heteromeric formation with the 5-HT2A receptor5. Additionally, further investigation demonstrated that three residues located at the intracellular end of TM4 of mGlu2 are necessary to form the 5-HT2A-mGlu2 receptor heterocomplex in living cells6.
Based on these findings observed in heterologous expression systems, here we describe the use of HSV-mediated expression of wild-type mGlu2 and mGlu2/mGlu3 chimeric constructs in the frontal cortex of mGlu2-KO mice to test whether heteromeric formation between 5-HT2A and mGlu2 is necessary for the head-twitch behavior induced by hallucinogenic 5-HT2A receptor agonists.

<table><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></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><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% 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

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.

Previous findings demonstrate that the head-twitch murine behavioral response is reliably and robustly elicited by hallucinogens, and it is absent in 5-HT2A-KO mice3. Furthermore, it has been shown that the head-twitch response elicited by the hallucinogenic 5-HT2A agonists DOI and LSD was significantly decreased in mGlu2-KO mice5. However, although previous findings convincingly demonstrate that 5-HT2A and mGlu2 are assembled as a he...

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

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

We present a protocol to produce antigen-specific mouse T-cells using retroviral...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining to Visualize Murine Enteric Nervous System Development

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

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.

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

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

We present improved protocols for retroviral transduction of trafficking receptors and competitive homing to study receptor-mediated organ- and microenvironment-specific lymphocyte positioning. This method offers valuable insights into immune cell trafficking mechanisms and has potential applications in future basic and therapeutic research.

Efficient Retroviral Transduction and Competitive Homing for Investigating GPCR-Mediated T-Cell Localization in Diverse Tissue Microenvironments

Learn HSV-mediated gene transfer to study GPCR heteromer function in mouse behavioral neuroscience. This method examines 5-HT2A-mGlu2 receptor interactions using the head-twitch response assay.

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

Explore More Videos