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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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In-vivo 2-photon imaging of GFP-labeled basket interneurons in Pv-cre mice.(a) Low-magnification projection of a z-series starting at ∼20 µm below the pia surface to a depth of ∼120 µm, from a densely labeled area. The asterisks indicate the tops of two cell bodies. Numerous dendritic branches are indicated by blue arrows. (b–c) Higher magnification z-projections of regions highlighted in (a) at two different depths: 85–90 µm (b) and 65–75 µm (c) below the pia. Note the smooth, aspiny dendrites (blue arrows), and dense cluster of boutons of varying size (yellow arrowheads). (d) Projection of a z-series 60–170 µm below the pia from a sparsely labeled area, showing an isolated GFP-labeled basket interneuron. Dendrites (blue arrows) could be traced back to soma, and axonal boutons (yellow arrowheads) appear as a cloudy signal at this magnification. (e–f) Examples of axon morphology magnified from areas indicated by gray boxes in (d). (e) shows an axonal basket-like structure (asterisk), and (f) shows a well-isolated axon branch (yellow arrow) with distinct boutons (yellow arrowheads). (g–h) dendrite structures from areas indicated in (d). Dendrites of the cell were largely aspiny (g), though occasionally small protrusions were visible on some dendritic branches (h). Scale bars a,d: 20 µm; b–c, e–h: 5 µm.
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pone-0002005-g004: In-vivo 2-photon imaging of GFP-labeled basket interneurons in Pv-cre mice.(a) Low-magnification projection of a z-series starting at ∼20 µm below the pia surface to a depth of ∼120 µm, from a densely labeled area. The asterisks indicate the tops of two cell bodies. Numerous dendritic branches are indicated by blue arrows. (b–c) Higher magnification z-projections of regions highlighted in (a) at two different depths: 85–90 µm (b) and 65–75 µm (c) below the pia. Note the smooth, aspiny dendrites (blue arrows), and dense cluster of boutons of varying size (yellow arrowheads). (d) Projection of a z-series 60–170 µm below the pia from a sparsely labeled area, showing an isolated GFP-labeled basket interneuron. Dendrites (blue arrows) could be traced back to soma, and axonal boutons (yellow arrowheads) appear as a cloudy signal at this magnification. (e–f) Examples of axon morphology magnified from areas indicated by gray boxes in (d). (e) shows an axonal basket-like structure (asterisk), and (f) shows a well-isolated axon branch (yellow arrow) with distinct boutons (yellow arrowheads). (g–h) dendrite structures from areas indicated in (d). Dendrites of the cell were largely aspiny (g), though occasionally small protrusions were visible on some dendritic branches (h). Scale bars a,d: 20 µm; b–c, e–h: 5 µm.

Mentions: We performed in-vivo, long-term imaging of basket interneurons by two-photon microscopy in the neocortex of Pv-cre mice (n = 3) injected with AAV-LS1L-GFP. Mice were implanted with glass windows directly over the cortex. GFP expression was bright enough to allow reliable imaging of dendrites, axons, and putative presynaptic boutons visualized as distinct swellings along the axon (Fig. 4). In areas of dense labeling, plexuses of basket cell dendrites and axons intertwined, and clusters of “basket-like” boutons were routinely observed (Fig. 4a–c, see Movie S1 and Movie S2 to view high-magnification z-series stacks). At the edge of the injection site in all 3 experimental animals, where there was sparse labeling, dendrites of individual cells could be easily traced back to the soma (Fig. 4d, see Movie S3 to view 3-D rotation). We found that dendrites were primarily aspiny and smooth (Fig. 4b,c,g), although in some cases dendritic protrusions were apparent (Fig. 4h, see Movie S4 to view z-series stack). The density and size of dendritic protrusions seen here is similar to previous characterizations of cortical fast-spiking basket cells [34]. Well-isolated axon branches and boutons were frequently visible (Fig. 4e,f).


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)

In-vivo 2-photon imaging of GFP-labeled basket interneurons in Pv-cre mice.(a) Low-magnification projection of a z-series starting at ∼20 µm below the pia surface to a depth of ∼120 µm, from a densely labeled area. The asterisks indicate the tops of two cell bodies. Numerous dendritic branches are indicated by blue arrows. (b–c) Higher magnification z-projections of regions highlighted in (a) at two different depths: 85–90 µm (b) and 65–75 µm (c) below the pia. Note the smooth, aspiny dendrites (blue arrows), and dense cluster of boutons of varying size (yellow arrowheads). (d) Projection of a z-series 60–170 µm below the pia from a sparsely labeled area, showing an isolated GFP-labeled basket interneuron. Dendrites (blue arrows) could be traced back to soma, and axonal boutons (yellow arrowheads) appear as a cloudy signal at this magnification. (e–f) Examples of axon morphology magnified from areas indicated by gray boxes in (d). (e) shows an axonal basket-like structure (asterisk), and (f) shows a well-isolated axon branch (yellow arrow) with distinct boutons (yellow arrowheads). (g–h) dendrite structures from areas indicated in (d). Dendrites of the cell were largely aspiny (g), though occasionally small protrusions were visible on some dendritic branches (h). Scale bars a,d: 20 µm; b–c, e–h: 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2289876&req=5

pone-0002005-g004: In-vivo 2-photon imaging of GFP-labeled basket interneurons in Pv-cre mice.(a) Low-magnification projection of a z-series starting at ∼20 µm below the pia surface to a depth of ∼120 µm, from a densely labeled area. The asterisks indicate the tops of two cell bodies. Numerous dendritic branches are indicated by blue arrows. (b–c) Higher magnification z-projections of regions highlighted in (a) at two different depths: 85–90 µm (b) and 65–75 µm (c) below the pia. Note the smooth, aspiny dendrites (blue arrows), and dense cluster of boutons of varying size (yellow arrowheads). (d) Projection of a z-series 60–170 µm below the pia from a sparsely labeled area, showing an isolated GFP-labeled basket interneuron. Dendrites (blue arrows) could be traced back to soma, and axonal boutons (yellow arrowheads) appear as a cloudy signal at this magnification. (e–f) Examples of axon morphology magnified from areas indicated by gray boxes in (d). (e) shows an axonal basket-like structure (asterisk), and (f) shows a well-isolated axon branch (yellow arrow) with distinct boutons (yellow arrowheads). (g–h) dendrite structures from areas indicated in (d). Dendrites of the cell were largely aspiny (g), though occasionally small protrusions were visible on some dendritic branches (h). Scale bars a,d: 20 µm; b–c, e–h: 5 µm.
Mentions: We performed in-vivo, long-term imaging of basket interneurons by two-photon microscopy in the neocortex of Pv-cre mice (n = 3) injected with AAV-LS1L-GFP. Mice were implanted with glass windows directly over the cortex. GFP expression was bright enough to allow reliable imaging of dendrites, axons, and putative presynaptic boutons visualized as distinct swellings along the axon (Fig. 4). In areas of dense labeling, plexuses of basket cell dendrites and axons intertwined, and clusters of “basket-like” boutons were routinely observed (Fig. 4a–c, see Movie S1 and Movie S2 to view high-magnification z-series stacks). At the edge of the injection site in all 3 experimental animals, where there was sparse labeling, dendrites of individual cells could be easily traced back to the soma (Fig. 4d, see Movie S3 to view 3-D rotation). We found that dendrites were primarily aspiny and smooth (Fig. 4b,c,g), although in some cases dendritic protrusions were apparent (Fig. 4h, see Movie S4 to view z-series stack). The density and size of dendritic protrusions seen here is similar to previous characterizations of cortical fast-spiking basket cells [34]. Well-isolated axon branches and boutons were frequently visible (Fig. 4e,f).

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