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High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

Kuhlman SJ, Huang ZJ - PLoS ONE (2008)

Bottom Line: The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv) fast-spiking GABAergic interneuron, was monitored over the course of a week.We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days.Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs) that express GFP, dsRedExpress, or channelrhodopsin (ChR2) upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv) fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

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Related in: MedlinePlus

Cre-activated AAV vectors confer cell-type specific and high-level gene expression in brains of Cre knockin mice.(a) Injection of a generic AAV-GFP virus into neocortex of a Pv-cre mouse labeled neurons non-specifically. Pyramids can be recognized by the presence of thick apical dendrites (black arrowheads) and projecting axons (white arrows). (b) Injection of the AAV-lox-STOP-lox (LS1L)-GFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. The STOP cassette is shown in gray in the schematic and is described in detail in Methods. Δβgeo-3xpA, a modified beta-galactosidae/neomycin STOP cassette with 3 poly-adenylation sites. (c) Injection of the AAV-LS2L-RFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. A different version of STOP cassette is used here, Neo-2xpA, a neomycin STOP cassette with 2 poly-adenylation sites. PCMV, CMV promoter and β-globin intron; scale bar, 250 µm for a–c. (d) Co-localization (white arrowheads) of GFP and parvalbumin (PV) in neocortical basket interneurons in neocortex of a Pv-cre mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Far right, high resolution image of basket cell axons with “basket-like” terminal branches and boutons (yellow arrowheads) around pyramidal cell somata (labeled with NeuN immunofluorescence) characteristic to PV+ interneurons; scale bar, 5 µm. (e) Co-localization of GFP and LacZ in neocortical pyramidal neurons in a Emx-cre-nlsLacZ mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Note that not all LacZ+ pyramidal neurons were positive for GFP. Far right, spines (blue arrowheads) along a pyramidal cell apical dendrite; scale bar, 5 µm. (f) Quantification of GFP fluorescence intensity at the soma normalized to intensity of GFP-labeled pyramidal soma in Thy1-GFP mice; n = 15 cells for each group. The intensity range for both Thy1-GFP and AAV-labeled somata was large, only the brightest cells were selected for this particular comparison (see Methods for details). Thy1-GFP: 1+/−0.08; AAV-LS1L-GFP::Pv-cre: 1.5+/−0.1; AAV-LS1L-GFP:: Emx-cre 1.3+/−0.2). All AAV-injected tissue was fixed 12–15 days post injection of 1.5–3 month-old mice.
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pone-0002005-g001: Cre-activated AAV vectors confer cell-type specific and high-level gene expression in brains of Cre knockin mice.(a) Injection of a generic AAV-GFP virus into neocortex of a Pv-cre mouse labeled neurons non-specifically. Pyramids can be recognized by the presence of thick apical dendrites (black arrowheads) and projecting axons (white arrows). (b) Injection of the AAV-lox-STOP-lox (LS1L)-GFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. The STOP cassette is shown in gray in the schematic and is described in detail in Methods. Δβgeo-3xpA, a modified beta-galactosidae/neomycin STOP cassette with 3 poly-adenylation sites. (c) Injection of the AAV-LS2L-RFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. A different version of STOP cassette is used here, Neo-2xpA, a neomycin STOP cassette with 2 poly-adenylation sites. PCMV, CMV promoter and β-globin intron; scale bar, 250 µm for a–c. (d) Co-localization (white arrowheads) of GFP and parvalbumin (PV) in neocortical basket interneurons in neocortex of a Pv-cre mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Far right, high resolution image of basket cell axons with “basket-like” terminal branches and boutons (yellow arrowheads) around pyramidal cell somata (labeled with NeuN immunofluorescence) characteristic to PV+ interneurons; scale bar, 5 µm. (e) Co-localization of GFP and LacZ in neocortical pyramidal neurons in a Emx-cre-nlsLacZ mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Note that not all LacZ+ pyramidal neurons were positive for GFP. Far right, spines (blue arrowheads) along a pyramidal cell apical dendrite; scale bar, 5 µm. (f) Quantification of GFP fluorescence intensity at the soma normalized to intensity of GFP-labeled pyramidal soma in Thy1-GFP mice; n = 15 cells for each group. The intensity range for both Thy1-GFP and AAV-labeled somata was large, only the brightest cells were selected for this particular comparison (see Methods for details). Thy1-GFP: 1+/−0.08; AAV-LS1L-GFP::Pv-cre: 1.5+/−0.1; AAV-LS1L-GFP:: Emx-cre 1.3+/−0.2). All AAV-injected tissue was fixed 12–15 days post injection of 1.5–3 month-old mice.

Mentions: We chose to explore adeno-associated virus (AAV) because it is considered to be non-pathogenic and non-toxic to neurons [18]–[21], and easy to engineer. Here we show that a generic serotype 2/9 AAV-GFP virus efficiently transfected neurons in the neocortex and drove green fluorescent protein (GFP) expression in both glutamatergic pyramidal neurons and GABAergic interneurons by a strong and ubiquitous CMV promoter, PCMV (Fig. 1a, and data not shown). To render GFP expression conditional upon Cre-mediated recombination, we added a loxP-STOP-loxP (LSL) cassette between the CMV promoter and either GFP or red fluorescent protein (RFP) cDNA (Fig. 1b,c). STOP is a transcriptional and translational stop cassette containing multiple poly-adenylation signals. We constructed two different versions of STOP (see Methods for details): Δβgeo-3xpA (S1) is a shortened version of a beta-galactosidae/neomycin STOP cassette containing 3 poly-adenylation sites with proven functionality [22], and Neo-2xpA (S2) is a neomycin cDNA followed by 2 poly-adenylation sites. We then tested these “conditional AAVs” (AAV-LS1L-GFP; AAV-LS2L-RFP) using two different Cre knockin drivers, the Pv-cre mouse, which expresses Cre in parvalbumin-containing (PV+) GABAergic neurons [23], and the Emx-cre mouse, which expresses Cre in glutamatergic neurons in the forebrain [24]. Both versions of the LSL performed well.


High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

Kuhlman SJ, Huang ZJ - PLoS ONE (2008)

Cre-activated AAV vectors confer cell-type specific and high-level gene expression in brains of Cre knockin mice.(a) Injection of a generic AAV-GFP virus into neocortex of a Pv-cre mouse labeled neurons non-specifically. Pyramids can be recognized by the presence of thick apical dendrites (black arrowheads) and projecting axons (white arrows). (b) Injection of the AAV-lox-STOP-lox (LS1L)-GFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. The STOP cassette is shown in gray in the schematic and is described in detail in Methods. Δβgeo-3xpA, a modified beta-galactosidae/neomycin STOP cassette with 3 poly-adenylation sites. (c) Injection of the AAV-LS2L-RFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. A different version of STOP cassette is used here, Neo-2xpA, a neomycin STOP cassette with 2 poly-adenylation sites. PCMV, CMV promoter and β-globin intron; scale bar, 250 µm for a–c. (d) Co-localization (white arrowheads) of GFP and parvalbumin (PV) in neocortical basket interneurons in neocortex of a Pv-cre mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Far right, high resolution image of basket cell axons with “basket-like” terminal branches and boutons (yellow arrowheads) around pyramidal cell somata (labeled with NeuN immunofluorescence) characteristic to PV+ interneurons; scale bar, 5 µm. (e) Co-localization of GFP and LacZ in neocortical pyramidal neurons in a Emx-cre-nlsLacZ mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Note that not all LacZ+ pyramidal neurons were positive for GFP. Far right, spines (blue arrowheads) along a pyramidal cell apical dendrite; scale bar, 5 µm. (f) Quantification of GFP fluorescence intensity at the soma normalized to intensity of GFP-labeled pyramidal soma in Thy1-GFP mice; n = 15 cells for each group. The intensity range for both Thy1-GFP and AAV-labeled somata was large, only the brightest cells were selected for this particular comparison (see Methods for details). Thy1-GFP: 1+/−0.08; AAV-LS1L-GFP::Pv-cre: 1.5+/−0.1; AAV-LS1L-GFP:: Emx-cre 1.3+/−0.2). All AAV-injected tissue was fixed 12–15 days post injection of 1.5–3 month-old mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289876&req=5

pone-0002005-g001: Cre-activated AAV vectors confer cell-type specific and high-level gene expression in brains of Cre knockin mice.(a) Injection of a generic AAV-GFP virus into neocortex of a Pv-cre mouse labeled neurons non-specifically. Pyramids can be recognized by the presence of thick apical dendrites (black arrowheads) and projecting axons (white arrows). (b) Injection of the AAV-lox-STOP-lox (LS1L)-GFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. The STOP cassette is shown in gray in the schematic and is described in detail in Methods. Δβgeo-3xpA, a modified beta-galactosidae/neomycin STOP cassette with 3 poly-adenylation sites. (c) Injection of the AAV-LS2L-RFP virus into the neocortex of a Pv-cre mouse specifically labeled neurons expressing Cre-recombinase. A different version of STOP cassette is used here, Neo-2xpA, a neomycin STOP cassette with 2 poly-adenylation sites. PCMV, CMV promoter and β-globin intron; scale bar, 250 µm for a–c. (d) Co-localization (white arrowheads) of GFP and parvalbumin (PV) in neocortical basket interneurons in neocortex of a Pv-cre mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Far right, high resolution image of basket cell axons with “basket-like” terminal branches and boutons (yellow arrowheads) around pyramidal cell somata (labeled with NeuN immunofluorescence) characteristic to PV+ interneurons; scale bar, 5 µm. (e) Co-localization of GFP and LacZ in neocortical pyramidal neurons in a Emx-cre-nlsLacZ mouse injected with AAV-LS1L-GFP; scale bar, 20 µm. Note that not all LacZ+ pyramidal neurons were positive for GFP. Far right, spines (blue arrowheads) along a pyramidal cell apical dendrite; scale bar, 5 µm. (f) Quantification of GFP fluorescence intensity at the soma normalized to intensity of GFP-labeled pyramidal soma in Thy1-GFP mice; n = 15 cells for each group. The intensity range for both Thy1-GFP and AAV-labeled somata was large, only the brightest cells were selected for this particular comparison (see Methods for details). Thy1-GFP: 1+/−0.08; AAV-LS1L-GFP::Pv-cre: 1.5+/−0.1; AAV-LS1L-GFP:: Emx-cre 1.3+/−0.2). All AAV-injected tissue was fixed 12–15 days post injection of 1.5–3 month-old mice.
Mentions: We chose to explore adeno-associated virus (AAV) because it is considered to be non-pathogenic and non-toxic to neurons [18]–[21], and easy to engineer. Here we show that a generic serotype 2/9 AAV-GFP virus efficiently transfected neurons in the neocortex and drove green fluorescent protein (GFP) expression in both glutamatergic pyramidal neurons and GABAergic interneurons by a strong and ubiquitous CMV promoter, PCMV (Fig. 1a, and data not shown). To render GFP expression conditional upon Cre-mediated recombination, we added a loxP-STOP-loxP (LSL) cassette between the CMV promoter and either GFP or red fluorescent protein (RFP) cDNA (Fig. 1b,c). STOP is a transcriptional and translational stop cassette containing multiple poly-adenylation signals. We constructed two different versions of STOP (see Methods for details): Δβgeo-3xpA (S1) is a shortened version of a beta-galactosidae/neomycin STOP cassette containing 3 poly-adenylation sites with proven functionality [22], and Neo-2xpA (S2) is a neomycin cDNA followed by 2 poly-adenylation sites. We then tested these “conditional AAVs” (AAV-LS1L-GFP; AAV-LS2L-RFP) using two different Cre knockin drivers, the Pv-cre mouse, which expresses Cre in parvalbumin-containing (PV+) GABAergic neurons [23], and the Emx-cre mouse, which expresses Cre in glutamatergic neurons in the forebrain [24]. Both versions of the LSL performed well.

Bottom Line: The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv) fast-spiking GABAergic interneuron, was monitored over the course of a week.We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days.Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs) that express GFP, dsRedExpress, or channelrhodopsin (ChR2) upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv) fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

Show MeSH
Related in: MedlinePlus