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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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GFP expression in Pv-cre mice injected with AAV-LS1L-GFP is stable for months.(a) Pv-cre mice (top row; n = 3 mice for each time point) and wildtype control mice (middle row; n = 3 mice for each time point) were injected with AAV-LS1L-GFP and were perfused at 6,15,30, and 60 days post-injection (dpi). See Figure S3 for 60 dpi. All epi-fluorescence images were acquired with a CCD camera using the same acquisition parameters. The maximum exposure time that did not lead to saturation of signal was used (75 ms), and the same look-up table was applied to all of the images shown. GFP expression levels increased with time in Pv-cre mice. Under these conditions, sparse weak signals were detected in some wildtype control mice (blue arrowheads); scale bar, 100 µm. Bottom row, quantification of GFP intensity at somata of labeled neurons plotted for wildtype control mice (blue) and Pv-cre mice (black). Average number of GFP+ cells counted per animal in Pv-cre injected animals was (6,15,30 dpi): 110+/−34, 215.3+/−34, 195.3+/−26 cells. (b) Whole cell current clamp recording of an AAV-LS1L-GFP-transfected neuron from a 2.5 month-old Pv-cre mouse at 20 dpi. Left, DIC image of recorded cell; inset shows GFP fluorescence; scale bar, 10 µm. Right, example trace of spikes in response to a 250 pA current step; scale bar, 10 mV, 25 ms.
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pone-0002005-g002: GFP expression in Pv-cre mice injected with AAV-LS1L-GFP is stable for months.(a) Pv-cre mice (top row; n = 3 mice for each time point) and wildtype control mice (middle row; n = 3 mice for each time point) were injected with AAV-LS1L-GFP and were perfused at 6,15,30, and 60 days post-injection (dpi). See Figure S3 for 60 dpi. All epi-fluorescence images were acquired with a CCD camera using the same acquisition parameters. The maximum exposure time that did not lead to saturation of signal was used (75 ms), and the same look-up table was applied to all of the images shown. GFP expression levels increased with time in Pv-cre mice. Under these conditions, sparse weak signals were detected in some wildtype control mice (blue arrowheads); scale bar, 100 µm. Bottom row, quantification of GFP intensity at somata of labeled neurons plotted for wildtype control mice (blue) and Pv-cre mice (black). Average number of GFP+ cells counted per animal in Pv-cre injected animals was (6,15,30 dpi): 110+/−34, 215.3+/−34, 195.3+/−26 cells. (b) Whole cell current clamp recording of an AAV-LS1L-GFP-transfected neuron from a 2.5 month-old Pv-cre mouse at 20 dpi. Left, DIC image of recorded cell; inset shows GFP fluorescence; scale bar, 10 µm. Right, example trace of spikes in response to a 250 pA current step; scale bar, 10 mV, 25 ms.

Mentions: In the neocortex, PV is expressed in a prominent class of GABAergic inhibitory interneurons, the basket interneuron, which are fast spiking and innervate the soma and proximal dendrites of pyramidal neurons. In the neocortex of Pv-cre mice injected with AAV-LS1L-GFP (single-tract injections, see Methods for details), GFP expression was almost exclusively restricted to PV+ interneurons (Fig. 1d). In a total volume of 1.46 mm3 examined from 4 animals, 97% of the GFP-expressing neurons (n = 416 GFP+ neurons) were also PV immuno-positive, indicating a high degree of cell-type specificity. In the same volume, a total of 479 PV+ neurons were identified, indicating an 86% average labeling efficiency by AAV-LS1L-GFP. High-level GFP expression allowed bright and spectacular labeling of exuberant basket cell axon arbors (Fig. 1d; Fig. 2a), including their highly characteristic local branches and perisomatic boutons around pyramidal cell somata (Fig. 1d; Fig. 3e). The LS2L gave a very similar labeling pattern (Fig. 1c and Fig. S1).


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)

GFP expression in Pv-cre mice injected with AAV-LS1L-GFP is stable for months.(a) Pv-cre mice (top row; n = 3 mice for each time point) and wildtype control mice (middle row; n = 3 mice for each time point) were injected with AAV-LS1L-GFP and were perfused at 6,15,30, and 60 days post-injection (dpi). See Figure S3 for 60 dpi. All epi-fluorescence images were acquired with a CCD camera using the same acquisition parameters. The maximum exposure time that did not lead to saturation of signal was used (75 ms), and the same look-up table was applied to all of the images shown. GFP expression levels increased with time in Pv-cre mice. Under these conditions, sparse weak signals were detected in some wildtype control mice (blue arrowheads); scale bar, 100 µm. Bottom row, quantification of GFP intensity at somata of labeled neurons plotted for wildtype control mice (blue) and Pv-cre mice (black). Average number of GFP+ cells counted per animal in Pv-cre injected animals was (6,15,30 dpi): 110+/−34, 215.3+/−34, 195.3+/−26 cells. (b) Whole cell current clamp recording of an AAV-LS1L-GFP-transfected neuron from a 2.5 month-old Pv-cre mouse at 20 dpi. Left, DIC image of recorded cell; inset shows GFP fluorescence; scale bar, 10 µm. Right, example trace of spikes in response to a 250 pA current step; scale bar, 10 mV, 25 ms.
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pone-0002005-g002: GFP expression in Pv-cre mice injected with AAV-LS1L-GFP is stable for months.(a) Pv-cre mice (top row; n = 3 mice for each time point) and wildtype control mice (middle row; n = 3 mice for each time point) were injected with AAV-LS1L-GFP and were perfused at 6,15,30, and 60 days post-injection (dpi). See Figure S3 for 60 dpi. All epi-fluorescence images were acquired with a CCD camera using the same acquisition parameters. The maximum exposure time that did not lead to saturation of signal was used (75 ms), and the same look-up table was applied to all of the images shown. GFP expression levels increased with time in Pv-cre mice. Under these conditions, sparse weak signals were detected in some wildtype control mice (blue arrowheads); scale bar, 100 µm. Bottom row, quantification of GFP intensity at somata of labeled neurons plotted for wildtype control mice (blue) and Pv-cre mice (black). Average number of GFP+ cells counted per animal in Pv-cre injected animals was (6,15,30 dpi): 110+/−34, 215.3+/−34, 195.3+/−26 cells. (b) Whole cell current clamp recording of an AAV-LS1L-GFP-transfected neuron from a 2.5 month-old Pv-cre mouse at 20 dpi. Left, DIC image of recorded cell; inset shows GFP fluorescence; scale bar, 10 µm. Right, example trace of spikes in response to a 250 pA current step; scale bar, 10 mV, 25 ms.
Mentions: In the neocortex, PV is expressed in a prominent class of GABAergic inhibitory interneurons, the basket interneuron, which are fast spiking and innervate the soma and proximal dendrites of pyramidal neurons. In the neocortex of Pv-cre mice injected with AAV-LS1L-GFP (single-tract injections, see Methods for details), GFP expression was almost exclusively restricted to PV+ interneurons (Fig. 1d). In a total volume of 1.46 mm3 examined from 4 animals, 97% of the GFP-expressing neurons (n = 416 GFP+ neurons) were also PV immuno-positive, indicating a high degree of cell-type specificity. In the same volume, a total of 479 PV+ neurons were identified, indicating an 86% average labeling efficiency by AAV-LS1L-GFP. High-level GFP expression allowed bright and spectacular labeling of exuberant basket cell axon arbors (Fig. 1d; Fig. 2a), including their highly characteristic local branches and perisomatic boutons around pyramidal cell somata (Fig. 1d; Fig. 3e). The LS2L gave a very similar labeling pattern (Fig. 1c and Fig. S1).

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