An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex
- 1Princeton Neuroscience Institute, Princeton University, Princeton, NJ, USA
- 2Department of Molecular Biology, Princeton University, Princeton, NJ, USA
- 3Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan
- 4School of Engineering, Brown University, Providence, RI, USA
- 5Max Planck Institute for Medical Research, Heidelberg, Germany
- 6NeuroCure Cluster of Excellence, Charité - Universitätsmedizin, Berlin, Germany
Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs) requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs) to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN) led to high levels of expression (50–100 μM) in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs) (≤10 μM), yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA), a second rAAV containing the bidirectional TET promoter (Ptetbi) could drive strong D3cpv expression in PCs (10–300 μM), enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.
Keywords: AAV, cerebellum, crus, FCIP, GECI, in vivo, TET, two-photon
Citation: Kuhn B, Ozden I, Lampi Y, Hasan MT and Wang SS-H (2012) An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex. Front. Neural Circuits 6:49. doi: 10.3389/fncir.2012.00049
Received: 10 March 2012; Accepted: 09 July 2012;
Published online: 31 July 2012.
Edited by:Edward M. Callaway, Salk Institute for Biological Studies, USA
Reviewed by:Michael Häusser, University College London, UK
Axel Nimmerjahn, Salk Institute for Biological Studies, USA
Copyright © 2012 Kuhn, Ozden, Lampi, Hasan and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
*Correspondence: Bernd Kuhn, Optical Neuroimaging Unit, Okinawa Institute of Science and Technology, 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan. e-mail: email@example.com
† These authors contributed equally to this work.