Limits...
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.

Show MeSH

Related in: MedlinePlus

Chronic 2-photon imaging of structural dynamics of basket interneurons in-vivo.(a) Example images of the morphology of an axon branch and boutons in which both were stable over the course of two days. (b) One-week imaging interval in which the axon branch and boutons were largely stable (yellow arrowheads), additions (blue arrowheads) and subtractions (red arrowheads) on the axon shaft were observed. Note, two far right boutons are not shown for day 7. (c) Proportion of axon bouton additions and subtractions (total number of boutons examined = 183, from three animals). (d) Example in which newly formed boutons persistented for at least two days (blue arrowheads). Scale bars: 5 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2289876&req=5

pone-0002005-g005: Chronic 2-photon imaging of structural dynamics of basket interneurons in-vivo.(a) Example images of the morphology of an axon branch and boutons in which both were stable over the course of two days. (b) One-week imaging interval in which the axon branch and boutons were largely stable (yellow arrowheads), additions (blue arrowheads) and subtractions (red arrowheads) on the axon shaft were observed. Note, two far right boutons are not shown for day 7. (c) Proportion of axon bouton additions and subtractions (total number of boutons examined = 183, from three animals). (d) Example in which newly formed boutons persistented for at least two days (blue arrowheads). Scale bars: 5 µm.

Mentions: A major advantage of sparse labeling is that it allows reliable and repeated imaging of dendritic and axonal structures of the same neuron [27]. In the GIN transgenic line [35], GFP expression level is sufficient for in vivo imaging of somatostatin-positive interneurons, but the dense labeling of overlapping dendritic arbors prevented tracking of dendritic branches longer than ∼40 microns and dense labeling of axonal structures made it difficult to locate the same region for repeated measures (S.K., unpublished data). In the neocortex of AAV-LS1L-GFP injected Pv-cre mice in areas of sparse labeling, we were able to repeatedly image long stretches of the same dendritic branch (not shown) and axonal structures (Fig. 5) over the course of weeks. Furthermore, dendritic branches were easily traced back to the soma (Fig. 4).


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)

Chronic 2-photon imaging of structural dynamics of basket interneurons in-vivo.(a) Example images of the morphology of an axon branch and boutons in which both were stable over the course of two days. (b) One-week imaging interval in which the axon branch and boutons were largely stable (yellow arrowheads), additions (blue arrowheads) and subtractions (red arrowheads) on the axon shaft were observed. Note, two far right boutons are not shown for day 7. (c) Proportion of axon bouton additions and subtractions (total number of boutons examined = 183, from three animals). (d) Example in which newly formed boutons persistented for at least two days (blue arrowheads). Scale bars: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002005-g005: Chronic 2-photon imaging of structural dynamics of basket interneurons in-vivo.(a) Example images of the morphology of an axon branch and boutons in which both were stable over the course of two days. (b) One-week imaging interval in which the axon branch and boutons were largely stable (yellow arrowheads), additions (blue arrowheads) and subtractions (red arrowheads) on the axon shaft were observed. Note, two far right boutons are not shown for day 7. (c) Proportion of axon bouton additions and subtractions (total number of boutons examined = 183, from three animals). (d) Example in which newly formed boutons persistented for at least two days (blue arrowheads). Scale bars: 5 µm.
Mentions: A major advantage of sparse labeling is that it allows reliable and repeated imaging of dendritic and axonal structures of the same neuron [27]. In the GIN transgenic line [35], GFP expression level is sufficient for in vivo imaging of somatostatin-positive interneurons, but the dense labeling of overlapping dendritic arbors prevented tracking of dendritic branches longer than ∼40 microns and dense labeling of axonal structures made it difficult to locate the same region for repeated measures (S.K., unpublished data). In the neocortex of AAV-LS1L-GFP injected Pv-cre mice in areas of sparse labeling, we were able to repeatedly image long stretches of the same dendritic branch (not shown) and axonal structures (Fig. 5) over the course of weeks. Furthermore, dendritic branches were easily traced back to the soma (Fig. 4).

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