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Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.

Gompf HS, Budygin EA, Fuller PM, Bass CE - Front Behav Neurosci (2015)

Bottom Line: TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection).IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted).Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Division of Sleep Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center Boston, MA, USA.

ABSTRACT
Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

No MeSH data available.


Related in: MedlinePlus

Adeno-associated virus (AAV) transgenes used in this study. Schematic maps of AAV vector constructs. (A) Effector gene constructs, ChR2 and hM3Dq and the promoters used to drive high neuronal expression rates, EF1-α and hSyn, respectively. Note that both effector molecule sequences are in the DIO position. (B) Maps of the three vector constructs used to drive expression of Cre-recombinase. Sizes are approximate and demonstrate that the TH promoter is much larger than either EF1-α or CMV. (C) Unlocking of the hM3Dq construct from the DIO orientation to the sense orientation following Cre-mediated recombination.
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Figure 1: Adeno-associated virus (AAV) transgenes used in this study. Schematic maps of AAV vector constructs. (A) Effector gene constructs, ChR2 and hM3Dq and the promoters used to drive high neuronal expression rates, EF1-α and hSyn, respectively. Note that both effector molecule sequences are in the DIO position. (B) Maps of the three vector constructs used to drive expression of Cre-recombinase. Sizes are approximate and demonstrate that the TH promoter is much larger than either EF1-α or CMV. (C) Unlocking of the hM3Dq construct from the DIO orientation to the sense orientation following Cre-mediated recombination.

Mentions: Here we present a new combinatorial dual AAV vector approach to achieve specificity while maintaining high expression levels in wildtype animals, namely rats. The first AAV incorporates the gene of interest in a double-inverted open (DIO) reading frame, (Figure 1A) driven by a strong neuron specific or generalized promoter (human synapsin, hSyn or elongation factor-1 alpha, EF1α, respectively). In the absence of Cre the gene is retained in an inverse, non-sense orientation. The second AAV delivers Cre-recombinase under control of a subtype specific promoter (1b). We hypothesize that only small amounts of Cre are needed to reorient the transgene for expression, thus a significantly weaker, but neuronal subtype-specific promoter—in this case tyrosine hydroxylase (Oh et al., 2009)—can drive sufficient Cre expression. In combination, these AAVs enable expression of a Cre-dependent transgene in a cell-type (TH+) specific pattern (1c). This approach then allows the flexibility of using viral vector constructs expressing different effectors (e.g., excitatory or inhibitory channelrhodopsins, DREADDS and other genes of interest) with the same promoter-Cre AAV (e.g., TH-Cre-AAV), or limiting a transgene to different neuronal subpopulations utilizing a toolbox of verified promoter-Cre constructs. For example, a single EF1α-DIO-gene of interest can be combined with a Cre virus driven by promoters that restrict to glutamatergic neurons in the targeted brain region (e.g., the CamK2α promoter in the hippocampus, forebrain or amygdala; Minichiello et al., 1999). Alternatively, GABAergic neurons may possibly be targeted with a vesicular GABA transporter (VGAT) or GAD67 promoter (Figure 2).


Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.

Gompf HS, Budygin EA, Fuller PM, Bass CE - Front Behav Neurosci (2015)

Adeno-associated virus (AAV) transgenes used in this study. Schematic maps of AAV vector constructs. (A) Effector gene constructs, ChR2 and hM3Dq and the promoters used to drive high neuronal expression rates, EF1-α and hSyn, respectively. Note that both effector molecule sequences are in the DIO position. (B) Maps of the three vector constructs used to drive expression of Cre-recombinase. Sizes are approximate and demonstrate that the TH promoter is much larger than either EF1-α or CMV. (C) Unlocking of the hM3Dq construct from the DIO orientation to the sense orientation following Cre-mediated recombination.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488755&req=5

Figure 1: Adeno-associated virus (AAV) transgenes used in this study. Schematic maps of AAV vector constructs. (A) Effector gene constructs, ChR2 and hM3Dq and the promoters used to drive high neuronal expression rates, EF1-α and hSyn, respectively. Note that both effector molecule sequences are in the DIO position. (B) Maps of the three vector constructs used to drive expression of Cre-recombinase. Sizes are approximate and demonstrate that the TH promoter is much larger than either EF1-α or CMV. (C) Unlocking of the hM3Dq construct from the DIO orientation to the sense orientation following Cre-mediated recombination.
Mentions: Here we present a new combinatorial dual AAV vector approach to achieve specificity while maintaining high expression levels in wildtype animals, namely rats. The first AAV incorporates the gene of interest in a double-inverted open (DIO) reading frame, (Figure 1A) driven by a strong neuron specific or generalized promoter (human synapsin, hSyn or elongation factor-1 alpha, EF1α, respectively). In the absence of Cre the gene is retained in an inverse, non-sense orientation. The second AAV delivers Cre-recombinase under control of a subtype specific promoter (1b). We hypothesize that only small amounts of Cre are needed to reorient the transgene for expression, thus a significantly weaker, but neuronal subtype-specific promoter—in this case tyrosine hydroxylase (Oh et al., 2009)—can drive sufficient Cre expression. In combination, these AAVs enable expression of a Cre-dependent transgene in a cell-type (TH+) specific pattern (1c). This approach then allows the flexibility of using viral vector constructs expressing different effectors (e.g., excitatory or inhibitory channelrhodopsins, DREADDS and other genes of interest) with the same promoter-Cre AAV (e.g., TH-Cre-AAV), or limiting a transgene to different neuronal subpopulations utilizing a toolbox of verified promoter-Cre constructs. For example, a single EF1α-DIO-gene of interest can be combined with a Cre virus driven by promoters that restrict to glutamatergic neurons in the targeted brain region (e.g., the CamK2α promoter in the hippocampus, forebrain or amygdala; Minichiello et al., 1999). Alternatively, GABAergic neurons may possibly be targeted with a vesicular GABA transporter (VGAT) or GAD67 promoter (Figure 2).

Bottom Line: TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection).IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted).Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Division of Sleep Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center Boston, MA, USA.

ABSTRACT
Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

No MeSH data available.


Related in: MedlinePlus