Lentiviral expression of GAD67 and CCK promoter-driven opsins to target interneuronsin vitroandin vivo
| Autor: | Stephanie Schorge, Douglas C. MacDonald, David Escors, Dimitri M. Kullmann, Mary Collins, Francesca Chiara, Laura Mantoan Ritter, Georg Ritter, Anna Cariboni |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Opsin digestive oral and skin physiology Glutamate decarboxylase Channelrhodopsin Biology Optogenetics Molecular biology Viral vector 03 medical and health sciences Transduction (genetics) 030104 developmental biology 0302 clinical medicine medicine.anatomical_structure nervous system In vivo Drug Discovery Genetics medicine Molecular Medicine Pyramidal cell Molecular Biology hormones hormone substitutes and hormone antagonists 030217 neurology & neurosurgery Genetics (clinical) |
| Zdroj: | The Journal of Gene Medicine. 18:27-37 |
| ISSN: | 1099-498X |
| DOI: | 10.1002/jgm.2873 |
| Popis: | Background: The ability to manipulate the activity of interneurons with optogenetic tools offers the possibility of interfering with diseases caused by altered neuronal inhibition and synchrony, including epilepsy and schizophrenia. To develop vectors for therapeutic approaches, targeting optogenetic constructs to interneurons is therefore a key requirement. We investigated whether the interneuron‐specific promoters glutamic acid decarboxylase (GAD)67 and cholecystokinin (CCK) allowed targeted lentiviral delivery of opsins to interneurons as a whole, or specifically CCK+ interneurons. Methods: We generated lentiviral (LV) plasmids encoding channelrhodopsin (ChR2) and halorhodopsin (NpHR) tagged with fluorophores and driven by GAD67 or CCK promoters. Adeno‐associated virus (AAV) and LV vectors carrying opsins driven by pyramidal cell promoters were used as controls. We transduced neuronal cultures and rodent brain in vivo, immunostained specimens 6–8 weeks after in vivo injection and 7–14 days after in vitro transduction, and evaluated volume and specificity of expression by confocal microscopy. Results: In vitro, 90% (19/21) of LV‐CCK‐NpHR2.0‐EYFP expressing neurons were CCK+. In vivo, LV‐GAD67‐ChR2‐mCherry was expressed in 2.6% (5/193), LV‐GAD67‐NpHR2.0‐EYFP in approximately 15% (43/279) and LV‐CCK‐NpHR2.0‐EYFP in 47% (9/19) of hippocampal GABA+ interneurons. GAD67 vectors expressed in larger volumes than CCK‐driven constructs. AAV vector controls achieved the largest expression volumes. Conclusions: LV‐CCK‐NpHR2.0‐EYFP may be useful for targeting CCK+ interneurons in culture. GAD67/CCK‐driven lentiviral constructs are expressed in vivo, although expression is not specific for interneurons. Overall, expression levels are low compared to opsins driven by pyramidal cell promoters. A better understanding of GAD67 and CCK promoter structure or alternative techniques is required to reliably target opsins to interneurons using viral vectors. |
| Databáze: | OpenAIRE |
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