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Lentiviral expression of GAD6(7) and CCK promoter-driven opsins to target interneurons in vitro and in vivo

Ritter, LM; Macdonald, DC; Ritter, G; Escors, D; Chiara, F; Cariboni, A; Schorge, S; ... Collins, M; + view all (2016) Lentiviral expression of GAD6(7) and CCK promoter-driven opsins to target interneurons in vitro and in vivo. Journal of Gene Medicine , 18 (1-3) pp. 27-37. 10.1002/jgm.2873. Green open access

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Abstract

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.

Type: Article
Title: Lentiviral expression of GAD6(7) and CCK promoter-driven opsins to target interneurons in vitro and in vivo
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/jgm.2873
Publisher version: http://doi.org/10.1002/jgm.2873
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: adenoassocated virus (AAV), gene ‐ delivery, microscopy, neuroscience, retroviruses, virology ‐ lentiviruses
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10061437
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