NOTE: All procedures for animal breeding and cares were conducted according to the Institutional Animal Care and Use Committee (IACUC) regulation of Icahn School of Medicine at Mount Sinai. Be sure to use sterile gloves throughout the procedure.
1. Drug and Virus Preparation
2. Surgery
3. Head Twitch Response Experiment
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 heteromeric complex in vitro in transfected cells1,2,15, whether this structural arrangement behaves as such in living mice remained unsolved. To fully understand the role of the 5-HT2A-mGlu2 receptor heterocomplex in the psychoactive-like effects induced by hallucinogenic 5-HT2A receptor agonists, expression of either mGlu2 or mGlu2ΔTM4N in frontal cortex of mGlu2-KO mice to examine whether this manipulation regulates behavior.
Mice received intra-frontal cortical injections of bicistronic HSV expressing green fluorescent protein (GFP) and either mGlu2 or mGlu2ΔTM4N, or GFP alone. First, it was confirmed that the virus over-expresses mGlu2 or mGlu2ΔTM4N in mouse frontal cortex (Figures 1A and 1B). As previously demonstrated5, head-twitch behavior induced by DOI was absent in mGlu2-KO mice injected with the empty vector HSV-GFP. Notably, the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI was rescued in mGlu2-KO mice over-expressing mGlu2, but not mGlu2ΔTM4N, in frontal cortex as compared to that seen in animals expressing GFP (Figure 1C). Together, these findings suggest that the 5-HT2A-mGlu2 receptor complex in frontal cortex is critical for regulating psychosis-like states.
Figure 1. Expression of mGlu2 as a Receptor Heterocomplex. Expression of mGlu2 as a receptor with 5-HT2A is necessary for psychosis-like behavior induced by hallucinogenic drugs. (A) Representative image of HSV-mediated transgene expression in frontal cortex. HSV-mGlu2, which also expresses GFP, was injected into frontal cortex, and GFP expression was revealed by immunocytochemistry, scale bar, 200-um. (B) HSV-mediated transgene expression in mouse frontal cortex of mGlu2-KO mice, and anti-mGlu2 reactivity was measured by Western Blotting. Specificity of the primary antibody against the mGlu2 receptor has previously been confirmed in knockout mice6. Metabotropic glutamate receptors are GPCRs that form covalently linked homodimers. We measured immunoreactivity of mGlu2 as a monomer (100 kDa)6. (C) Viral-mediated expression of mGlu2, but not mGlu2ΔTM4N, in frontal cortex of mGlu2- KO mice significantly rescues the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI (n = 4 per group). ***p <0.001; n.s, not significant; Bonferroni's post hoc test of one-way ANOVA. Error bars represent S.E.M. Figure was modified from Moreno et al (2012)6. Please click here to view a larger version of this figure.
Supplemental Video 1. Head Twitch Response. (Right click to download). CD-1 WT mice were injected with 2.0 mg/kg DOI and placed in a cage (wall blacked out between the two cages) to elicit head-twitch response (behavior elicited after *).
mGlu2 bicitronic herpes simplex virus (HSV) vector | MIT Core | 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) | |
mGlu2ΔTM4N bicitronic herpes simplex virus (HSV) vector | MIT Core | 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) | |
GFP bicitronic herpes simplex virus (HSV) vector | MIT Core | 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) | |
xylazine | Lloyd | List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10 | 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 |
ketamine | Vedco | KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06 | 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 |
ophthalmic gel | Fisher Scientific | NC0550805 | |
burret clips | Fisher Scientific | NC9268369 | |
Feather surgical blade | Fisher Scientific | NC9032736 | |
Hydrogen Peroxide | Fisher Scientific | 19-898-919 | |
Hamilton syringe | Fisher Scientific | 14815203 | |
Hamilton™ Small Hub Removable Needles (33 Ga) | Fisher Scientific | 14816206 | |
Cordless Micro Drill | Fisher Scientific | NC9089241 | |
Dermabond Dermal Adhesive | Fisher Scientific | NC0690470 | |
(±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) | Sigma-Aldrich | 42203-78-1 | Dissolved in .9% saline solution to the concentration of 2.0 mg/kg |
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 mGlu2ΔTM4N (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.
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 mGlu2ΔTM4N (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.
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 mGlu2ΔTM4N (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.