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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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Functional demonstration that the Cre-activated AAV vector is cell-type specific.Pv-cre mice were injected with AAV-LS2L-ChR2mCherry and acute slices for electrophysiological recording were prepared. (a–c) Light stimulation directly elicited depolarizing responses in ChR2-positive neurons. DIC and fluorescent images of a ChR2-positive cell targeted for recording (a); scale bar: 10 µm. Spike responses to light stimulation of increasing duration (7, 14, and 500 ms as indicated by blue line) were recorded in cell-attached mode (b); scale bars: 40 pA, 25 ms. ChR2-mediated current was maintained in the presence of TTX. (c); scale bar: 50 pA, 10 ms. (d) Electrical field stimulation evoked both inhibitory post-synaptic current (upper trace, holding potential set to the reversal potential for AMPA/NMDA-mediated current, 0 mV) and excitatory post-synaptic current (lower trace, holding potential set to the reversal potential for GABA-mediated current, −55 mV), recorded in a pyramidal cell in whole-cell voltage-clamp mode; scale bar: 40 pA, 20 ms. (e) Light stimulation specifically evoked inhibitory post-synaptic current (upper trace), no excitatory post-synaptic current was detected (lower trace); scale bar: 20 pA, 20 ms. Inset, both inhibitory and excitatory spontaneous synaptic events were detected using the same recording conditions; scale bar: 20 pA, 50 ms.
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pone-0002005-g003: Functional demonstration that the Cre-activated AAV vector is cell-type specific.Pv-cre mice were injected with AAV-LS2L-ChR2mCherry and acute slices for electrophysiological recording were prepared. (a–c) Light stimulation directly elicited depolarizing responses in ChR2-positive neurons. DIC and fluorescent images of a ChR2-positive cell targeted for recording (a); scale bar: 10 µm. Spike responses to light stimulation of increasing duration (7, 14, and 500 ms as indicated by blue line) were recorded in cell-attached mode (b); scale bars: 40 pA, 25 ms. ChR2-mediated current was maintained in the presence of TTX. (c); scale bar: 50 pA, 10 ms. (d) Electrical field stimulation evoked both inhibitory post-synaptic current (upper trace, holding potential set to the reversal potential for AMPA/NMDA-mediated current, 0 mV) and excitatory post-synaptic current (lower trace, holding potential set to the reversal potential for GABA-mediated current, −55 mV), recorded in a pyramidal cell in whole-cell voltage-clamp mode; scale bar: 40 pA, 20 ms. (e) Light stimulation specifically evoked inhibitory post-synaptic current (upper trace), no excitatory post-synaptic current was detected (lower trace); scale bar: 20 pA, 20 ms. Inset, both inhibitory and excitatory spontaneous synaptic events were detected using the same recording conditions; scale bar: 20 pA, 50 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)

Functional demonstration that the Cre-activated AAV vector is cell-type specific.Pv-cre mice were injected with AAV-LS2L-ChR2mCherry and acute slices for electrophysiological recording were prepared. (a–c) Light stimulation directly elicited depolarizing responses in ChR2-positive neurons. DIC and fluorescent images of a ChR2-positive cell targeted for recording (a); scale bar: 10 µm. Spike responses to light stimulation of increasing duration (7, 14, and 500 ms as indicated by blue line) were recorded in cell-attached mode (b); scale bars: 40 pA, 25 ms. ChR2-mediated current was maintained in the presence of TTX. (c); scale bar: 50 pA, 10 ms. (d) Electrical field stimulation evoked both inhibitory post-synaptic current (upper trace, holding potential set to the reversal potential for AMPA/NMDA-mediated current, 0 mV) and excitatory post-synaptic current (lower trace, holding potential set to the reversal potential for GABA-mediated current, −55 mV), recorded in a pyramidal cell in whole-cell voltage-clamp mode; scale bar: 40 pA, 20 ms. (e) Light stimulation specifically evoked inhibitory post-synaptic current (upper trace), no excitatory post-synaptic current was detected (lower trace); scale bar: 20 pA, 20 ms. Inset, both inhibitory and excitatory spontaneous synaptic events were detected using the same recording conditions; scale bar: 20 pA, 50 ms.
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pone-0002005-g003: Functional demonstration that the Cre-activated AAV vector is cell-type specific.Pv-cre mice were injected with AAV-LS2L-ChR2mCherry and acute slices for electrophysiological recording were prepared. (a–c) Light stimulation directly elicited depolarizing responses in ChR2-positive neurons. DIC and fluorescent images of a ChR2-positive cell targeted for recording (a); scale bar: 10 µm. Spike responses to light stimulation of increasing duration (7, 14, and 500 ms as indicated by blue line) were recorded in cell-attached mode (b); scale bars: 40 pA, 25 ms. ChR2-mediated current was maintained in the presence of TTX. (c); scale bar: 50 pA, 10 ms. (d) Electrical field stimulation evoked both inhibitory post-synaptic current (upper trace, holding potential set to the reversal potential for AMPA/NMDA-mediated current, 0 mV) and excitatory post-synaptic current (lower trace, holding potential set to the reversal potential for GABA-mediated current, −55 mV), recorded in a pyramidal cell in whole-cell voltage-clamp mode; scale bar: 40 pA, 20 ms. (e) Light stimulation specifically evoked inhibitory post-synaptic current (upper trace), no excitatory post-synaptic current was detected (lower trace); scale bar: 20 pA, 20 ms. Inset, both inhibitory and excitatory spontaneous synaptic events were detected using the same recording conditions; scale bar: 20 pA, 50 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