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A viral strategy for targeting and manipulating interneurons across vertebrate species

A Corrigendum to this article was published on 01 July 2017

An Addendum to this article was published on 01 July 2017

This article has been updated

Abstract

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

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Figure 1: rAAV with mDlx enhancer restricts reporter expression to GABAergic interneurons.
Figure 2: rAAV-mDlx-Flex-GFP allows intersectional targeting of Cck-expressing interneurons.
Figure 3: rAAV-hDLX-Gq-DREADD allows chemogenetic modulation of interneuronal activity in mice.
Figure 4: rAAV-mDlx is selectively expressed within GABAergic interneurons in various non-genetic model organisms.
Figure 5: rAAV-Dlx restricts expression to interneurons derived from iPSCs and human embryonic stem cells.

Change history

  • 29 November 2016

    In the version of this article initially published, authors Joshua S. Grimley, Anne-Rachel Krostag and Ajamete Kaykas were missing. These authors have been inserted into the author list after Jianhua Chu; they are at the Allen Institute for Brain Science, Seattle, Washington, USA, and performed experiments related to hESCs. The error has been corrected in the HTML and PDF versions of the article.

  • 29 May 2017

    The authors wish to acknowledge a potentially relevant work that they were made aware of after publication: Lee, A.T., Vogt, D., Rubenstein, J.L. & Sohal, V.S. A class of GABAergic neurons in the prefrontal cortex sends long-range projections to the nucleus accumbens and elicits acute avoidance behavior. J. Neurosci. 34, 11519–11525, http://dx.doi.org/10.1523/JNEUROSCI.1157-14.2014 (2014). This work describes an approach for using Dlx1/2 enhancers to attempt to achieve selective expression in cortical GABAergic interneurons. Although thorough validation was not performed, the results are consistent with the possibility that other regulatory elements may be generalizable as an effective way of targeting specific cell types.

  • 01 July 2017

    Nat. Neurosci. 19, 1743–1749 (2016); published online 31 October 2016; corrected after print 29 November 2016 In the version of this article initially published, authors Joshua S. Grimley, Anne-Rachel Krostag and Ajamete Kaykas were missing. These authors have been inserted into the author list after Jianhua Chu; they are at the Allen Institute for Brain Science, Seattle, Washington, USA, and performed experiments related to hESCs.

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Acknowledgements

We thank S. Gerard, L. Sjulson and T. Petros for discussions and comments on the manuscript. Plasmids AAV-CaMKIIa-GCaMP6f-P2A-nls-dTomato and AAV-hSyn1-GCaMP6s-P2A-nls-dTomato were a gift from J. Ting (Allen Institute for Brain Science). Plasmid pNeuroD-Ires-GFP was a gift from F. Polleux (Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University Medical Center). Plasmid pGP-CMV-GCaMP6f was a gift from D. Kim (Janelia Research Campus, Howard Hughes Medical Institute). Plasmid pAAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP was a gift from K. Deisseroth (Department of Bioengineering, Stanford University). Plasmid pAAV-hSyn-DIO-hM3D(Gq)-mCherry was a gift from B. Roth (Department of Pharmacology, UNC Chapel Hill Medical School).This work was supported by grants from the National Institutes of Health (NIH): MH071679, NS08297, NS074972, MH095147, as well as support from the Simons Foundation (SFARI) (to G.F.); MH066912 (to S.S.A.); and EY022577 and MH063912 (to E.M.C.).

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Authors

Contributions

J.D. conceived and designed the viral constructs, designed and performed experiments, analyzed data, prepared figures and wrote the manuscript. Q.C., C.L. and R.L. performed experiments, analyzed data and prepared figures related to mouse primary cultures. R.T. performed the slice recording experiments, analyzed the data and prepared the associated figures and text. G.-A.S. and S.L.R. performed viral injections and IHC on mice. Q.X. and L.G. produced the viruses at NYUAD using plasmids conceived and generated at NYU. G.K. performed experiments related to zebra finches. A.L.J., G.B.S. and D.E.W. performed experiments related to ferrets. V.C.K. and T.M.M. performed experiments related to gerbils. J.H.R., M.C.A. and M.S.R. performed experiments related to marmosets. I.K. and T.R. performed experiments related to iPSCs. J.C., S.A., J.S.G., A.-R.K. and A.K. performed experiments related to hESCs, M.B. and J.S.D. performed experiments related to spinal cord. S.A.A., E.M.C., D.J.S., D.F., V.F., M.A.L., S.N., J.H.R., D.H.S., B.R. and G. Feng helped with the study design. G. Fishell helped with study design, manuscript and figure preparation and supervised the project. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Gord Fishell.

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Competing interests

The New York University Langone Medical Center has filed patent applications related to this work with J.D and G.F. listed as inventors.

Integrated supplementary information

Supplementary Figure 1 rAAV-mDlx-GCaMP6f allows calcium transients to be visualized in interneurons in vitro

(a) The indicated rAAV was added to primary cortical culture at DIV8 and analyzed at DIV19 by immunostaining using GFP and GAD67 antibodies and counterstained with Dapi. Scale bars represent 20μm. (b) Visualization of ΔF/F calcium current traces recorded from GCaMP6f-expressing cortical interneurons. Each trace corresponds to a single neuron.

Supplementary Figure 2 rAAV-mDlx-GFP is not selective for interneurons in the spinal cord

(a, b) P0 wildtype Swiss Webster mice (n=10) were injected with the indicated virus in the lumbar spinal cord and analyzed by immunostaining for GFP and PAX2 immunoreactivity after one weeks. Representative example of expression of the reporter GFP and Pax2 and corresponding quantifications. (Upper panel: 2.9 +/- 1.7%, n= 255 cells from 4 animals; lower panel: 12.2 +/- 1.1%, n= 1904 cells from 6 animals). Quantification are indicated in the text as mean ± s.e.m and are represented as box-and-whisker plot with upper and lower whiskers represent the maximum and minimum value respectively and the box represent upper, median and lower quartile. Dashed lines represent limits of the spinal cord. Scale bars represent 25μm (left merge) 100μm (right merge).

Supplementary Figure 3 rAAV-mDlx-Flex-GFP allows intersectional targeting of Cck-expressing interneurons

(a) Adult CCK-Cre mice (n=4) or wildtype C57Bl6 mice (n=2) were injected with the indicated virus in the somatosensory cortex (S1) and were analyzed for GFP expression. (b) Adult wildtype C57Bl6 mice (n=2) were injected bilaterally with rAAV-mDlx-Flex-GFP and analyzed by immunostaining for GAD67 immunoreactivity after 2 weeks. Note that no cells expressing could be observed (n=4 injection sites from 2 animals). Scale bars represent 100μm (a) or 10 μm (b).

Supplementary Figure 4 rAAV-hDlx-Gq-DREADD restricts reporter expression to the membrane of GABAergic interneurons

(a) Scheme of the rAAV-hDlx-Gq viral construct. (a-c) Adult Dlx6aCre::RCE (n=4) or (d) C57Bl6 mice (n=16) were injected with rAAV-hDlx-Flex-GFP in somatosensory cortex (S1) or hippocampus (CA1) and were analyzed by immunostaining for the indicated markers after 7 days. Representative example of expression of the reporter dTomato and the indicated marker and corresponding quantifications (S1: 91.9 +/- 0.7%, n= 2091 cells from 4 animals; CA1: 93.9 +/- 2.3%, n= 173 cells from 3 animals). Quantification are indicated in the text as mean ± s.e.m and are represented as box-and-whisker plot with upper and lower whiskers represent the maximum and minimum value respectively and the box represent upper, median and lower quartile. Dashed lines represent limits of the indicated anatomical structures. P – pyramidal layer. Scale bars represent 100μm (a), 25μm (c,d).

Supplementary Figure 5 rAAV-mDlx-GFP allows in vivo recording of interneurons in the HVC of zebra finches

Adult Zebra Finches (n=4) were injected with rAAV-mDlx-GFP in HVC and were either sectioned for electrophysiological recording after 2 weeks or a cranial window was placed above the injection site for two-photon imaging and in-vivo electrophysiological recording. (a, b) Representative examples of biocytin GFP positive cells recorded from acute slice showing typical neocortical interneuron morphology and corresponding membrane potential of cells in response to current pulses. (c) Representative examples of biocytin GFP positive recorded in vivo and membrane potential of the two cells marked with stars in response to current pulses. Dark gray: hyperpolarizing; Black trace: rheobase; light gray: -200pA / twice rheobase. Scale bars represent 20μm (a, b) or 10μm (c).

Supplementary Figure 6 rAAV-mDlx-ChR2-mCherry allows modulation of neuronal activity in gerbil auditory cortex

Adult gerbils were stereotactically injected with 50-100nl of rAAV-mDlx-ChR2-mCherry in auditory cortex (A1) and sectioned for optogenetics and electrophysiological recording after 5 weeks (n=6 animals). (a) Infrared and fluorescence images showing the cells targeted for electrophysiological recording. Dashed lines highlight the edges of the recording pipet. Note that the cell in (c) does not express mCherry but is surrounded by mCherry-expressing cells. (b) Left panel: Membrane potential of a mCherry+ cell in response current pulses corresponding to twice the rheobase. Baseline membrane potential is -60 to -70 mV. Right panel: intensity-dependent increase of firing upon 1ms pulse of blue light for both FS and LTS interneurons. (c) Left panel: Membrane potential of a cell mCherry- in response current pulses corresponding to twice the rheobase. Baseline membrane potential is -60 to -70 mV. Right panel: the firing of pyramidal neurons is interrupted by a 1ms pulse of blue light suggesting activation of interneurons produces such inhibitory postsynaptic response. Scale bars represent 5μm.

Supplementary Figure 7 rAAV-mDlx-GCaMP6f allows the visualization of calcium transients in interneurons in ferret visual cortex

Ferrets aged P27-P30 were injected with rAAV-mDlx-GCaMP6f in primary visual cortex and then imaged at P50 (n=4 animals). (a) rAAV-mDlx-GCaMP6f labeled large numbers of inhibitory neurons, allowing for in vivo two-photon calcium imaging of inhibitory neurons. (b) Representative recording of calcium fluorescence of individual neurons upon presentation of visual stimulus. Labeled neurons show robust calcium fluorescence changes to visual stimuli during functional imaging. Black traces represent mean over 8 trials and grey represent ± s.e.m. Blue bars above responses indicate period of stimulus presentation. PS: preferred stimulus. NPS: non preferred stimulus. Scale bar represents 100 μm.

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Dimidschstein, J., Chen, Q., Tremblay, R. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat Neurosci 19, 1743–1749 (2016). https://doi.org/10.1038/nn.4430

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