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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

Dual LC injection of TH-Cre-AAV and hM3Dq-AAV. (A) hM3Dq expression is limited to LC neurons. (B–D) Following CNO injections, c-Fos expression markedly increased in the LC (B) as well as the entire neocortex, including the ACC (C), which was not evident in LC or cortex of animals lacking hM3Dq receptors (D). (E) CNO induced wakefulness for most of the 6 h period following injection (blue line) compared to the normal amount of wakefulness observed following saline injection (red line). (F) Power spectral analysis revealed normal wake EEG signatures following CNO injection compared to saline (blue and red lines, respectively).
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Figure 6: Dual LC injection of TH-Cre-AAV and hM3Dq-AAV. (A) hM3Dq expression is limited to LC neurons. (B–D) Following CNO injections, c-Fos expression markedly increased in the LC (B) as well as the entire neocortex, including the ACC (C), which was not evident in LC or cortex of animals lacking hM3Dq receptors (D). (E) CNO induced wakefulness for most of the 6 h period following injection (blue line) compared to the normal amount of wakefulness observed following saline injection (red line). (F) Power spectral analysis revealed normal wake EEG signatures following CNO injection compared to saline (blue and red lines, respectively).

Mentions: After animals were anesthetized with chloral hydrate and injections were performed as above, the skulls were exposed. Four EEG screw electrodes were implanted into the frontal (two) and parietal bones (two) of each side of the skull, and two flexible EMG wire electrodes were placed into the neck muscles. The free ends of the leads were fitted into a socket that was attached to the skull with dental cement. At least 2 weeks after surgery, the sockets were connected via flexible recording cables and a commutator to an amplifier (A-M Systems model 3600, Carlsborg, WA, USA) and computer, and signals were digitized using a Dell PC running the Sleep Sign recording system (Kissei Comtec, Irvine, CA, USA). The EEG/EMG was recorded at the end of the second week after surgery, for 48 h. Injections of CNO (0.3 mg/kg) or saline were performed at ZT 5, 24 h into this continuous recording period in a cross-over design (animals received either saline or CNO first), and injections were at least 1 week apart from one another to allow sufficient time for CNO washout and recovery. The cages were placed in such a way that animals receiving the same treatment were located next to one another so that the CNO-injected rat did not keep the saline-injected rat awake. The only exception to this were the sham virus, CNO injected animals in Figure 7D.


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)

Dual LC injection of TH-Cre-AAV and hM3Dq-AAV. (A) hM3Dq expression is limited to LC neurons. (B–D) Following CNO injections, c-Fos expression markedly increased in the LC (B) as well as the entire neocortex, including the ACC (C), which was not evident in LC or cortex of animals lacking hM3Dq receptors (D). (E) CNO induced wakefulness for most of the 6 h period following injection (blue line) compared to the normal amount of wakefulness observed following saline injection (red line). (F) Power spectral analysis revealed normal wake EEG signatures following CNO injection compared to saline (blue and red lines, respectively).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Dual LC injection of TH-Cre-AAV and hM3Dq-AAV. (A) hM3Dq expression is limited to LC neurons. (B–D) Following CNO injections, c-Fos expression markedly increased in the LC (B) as well as the entire neocortex, including the ACC (C), which was not evident in LC or cortex of animals lacking hM3Dq receptors (D). (E) CNO induced wakefulness for most of the 6 h period following injection (blue line) compared to the normal amount of wakefulness observed following saline injection (red line). (F) Power spectral analysis revealed normal wake EEG signatures following CNO injection compared to saline (blue and red lines, respectively).
Mentions: After animals were anesthetized with chloral hydrate and injections were performed as above, the skulls were exposed. Four EEG screw electrodes were implanted into the frontal (two) and parietal bones (two) of each side of the skull, and two flexible EMG wire electrodes were placed into the neck muscles. The free ends of the leads were fitted into a socket that was attached to the skull with dental cement. At least 2 weeks after surgery, the sockets were connected via flexible recording cables and a commutator to an amplifier (A-M Systems model 3600, Carlsborg, WA, USA) and computer, and signals were digitized using a Dell PC running the Sleep Sign recording system (Kissei Comtec, Irvine, CA, USA). The EEG/EMG was recorded at the end of the second week after surgery, for 48 h. Injections of CNO (0.3 mg/kg) or saline were performed at ZT 5, 24 h into this continuous recording period in a cross-over design (animals received either saline or CNO first), and injections were at least 1 week apart from one another to allow sufficient time for CNO washout and recovery. The cages were placed in such a way that animals receiving the same treatment were located next to one another so that the CNO-injected rat did not keep the saline-injected rat awake. The only exception to this were the sham virus, CNO injected animals in Figure 7D.

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