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Hirokazu Hirai
Gunma University Graduate School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0721-4293
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GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood-brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.
Keywords
Interneuron, inhibitory neuron, Chandelier cell, GAD65, Dlx, GABA, Promoter, AAV, AAV-PHP.B
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigures.pdf
Supplementary Figure 1. Cells expressing GFP by the mGAD65 (delE1) were co-immunolabeled for GABA. A mouse received an intravenous infusion of AAV-PHP.eB expressing GABA by the mGAD65 (delE1). Three weeks after the injection, sagittal brain sections were produced and double-immunolabeled for GFP and GABA. Note that all GFP-expressing cells expressed GABA, suggesting that the mGAD65 (delE1) serves as an inhibitory neuron-specific promoter. Scale bar; 50 μm. Supplementary Figure 2. Significant labeling of chandelier-cell cartridges by the mGAD65 promoter over the mDlx enhancer. (A) A viral mixture (equal viral titer) comprising of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter and that expressing tdTomato under the control of the mDlx enhancer was intravenously injected to adult mice. (B) A representative fluorescent image of the chandelier-cell cartridge double-labeled with GFP and tdTomato. Brain sections were immunostained 3 weeks after the viral injection for ankyrin-G (blue). (C) Graph showing percentage of GFP-labeled chandelier-cell cartridges co-labeled with tdTomato and vice versa. To determine the ratio, we identified GFP-labeled cartridges along with the AIS by the GFP channel and determined whether those were co-labeled with tdTomato by the RFP channel (magenta box) (39 GFP-labeled cartridges from 4 mice were examined, in which 14 cartridges were modestly co-labeled with tdTomato) (magenta box). The green box was obtained by the opposite procedure. Namely, tdTomato-labeled cartridges were identified by the RFP channel and it was determined if they were co-labeled with GFP by the GFP channel (22 tdTomato-labeled cartridges from 4 mice were examined, in which 16 cartridges were robustly co-labeled with tdTomato). See the Materials & Methods section for more details. Floating bars indicate upper, median, and lower quartiles (boxes) and value from each mouse (dots). p = 0.0041 by unpaired t-test. Scale bar; 5 μm. Supplementary Figure 3. Transduction of Golgi cells by the mGAD65 promoter. (A) Diagram illustrating the experimental procedure. Mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, the mice were sacrificed, and the cerebellar sections were immunostained for mGluR2, a marker for Golgi cells. (B) A native GFP (green) fluorescent image of the cerebellar cortex immunolabeled for mGluR2 (magenta). (C) Magnification of the box in A. Arrowheads indicate GFP-expressing Golgi cells, which were proved by mGluR2 immunolabeling. Scale bars; 50 μm (B) and 20 μm (C). IHC; immunohistochemistry. Supplementary Figure 4. Cerebellar interneuron-specific transduction by direct parenchymal injection of AAV-PHP.B carrying the mGAD65 promoter. (A) Schematic showing the experimental procedure. (B) Immunohistochemistry of the cerebellar slice from mice 3 weeks after the viral injection. Cerebellar sections were immunostained for calbindin (magenta), a marker for Purkinje cell. Scale bar; 50 μm. IHC; immunohistochemistry. Supplementary Figure 5. Transduction of GABAergic neurons in various brain regions by the mGAD65 promoter. Adult VGAT-tdTomato mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, mice were sacrificed. Confocal microscopy showed overall co-labeling of tdTomato-expressing cells with GFP in various brain regions such as the striatum (caudate-putamen), the hippocampus (CA1), the anterior interposed nucleus (IntA), the superior vestibular nucleus (SuVe), and the retina. Scale bars; 50 μm (right) and 20 μm (left).
- SupplementaryTableRevise12272020.pdf
Supplementary Table. Antibodies used for immunohistochemistry.
- SupplementaryMovie.mp4
Supplementary Movie. Spontaneous Ca2+ activity in G-CaMP-expressing GABAergic neurons in an acute brain slice of the motor cortex using the mGAD65 promoter. Another example of spontaneous Ca2+ signals observed by confocal live Ca2+ imaging in the motor cortex of VGAT-tdTomato mice expressing G-CaMP7.09 using our AAV-mediated GABAergic neuron-selective gene delivery method. Still image of tdTomato signals in the same field of view as G-CaMP ones is presented in a right panel in magenta. The movie plays 20 times faster than real time.
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See the published version of this preprint at Molecular Brain.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Hirokazu Hirai
Gunma University Graduate School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0721-4293
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
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Potential risks to human health
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Molecular Brain.
Version 3
Posted 01 Feb, 2021
No community comments so far
Reviewer #1 agreed
On 27 Jan, 2021
Editor assigned
On 26 Jan, 2021
Reviewers invited
Invitations sent on 26 Jan, 2021
Submission checks complete
On 26 Jan, 2021
Editor invited
On 26 Jan, 2021
No comments provided
Review #3 received
Received 13 Jan, 2021
Editorial decision: Minor revision
On 13 Jan, 2021
Review #1 received
Received 04 Jan, 2021
Reviewer #3 agreed
On 30 Dec, 2020
Reviewer #2 agreed
On 29 Dec, 2020
Review #2 received
Received 29 Dec, 2020
Editor assigned
On 28 Dec, 2020
Reviewers invited
Invitations sent on 28 Dec, 2020
Reviewer #1 agreed
On 28 Dec, 2020
Submission checks complete
On 28 Dec, 2020
Editor invited
On 28 Dec, 2020
No comments provided
Review #2 received
Received 08 Sep, 2020
Editorial decision: Major revision
On 08 Sep, 2020
Review #3 received
Received 07 Sep, 2020
Review #1 received
Received 06 Sep, 2020
Reviewer #3 agreed
On 26 Aug, 2020
Reviewer #2 agreed
On 25 Aug, 2020
Reviewers invited
Invitations sent on 23 Aug, 2020
Reviewer #1 agreed
On 23 Aug, 2020
Editor invited
On 13 Aug, 2020
Editor assigned
On 13 Aug, 2020
Submission checks complete
On 12 Aug, 2020
First submitted
On 07 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Molecular Brain.
GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood-brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigures.pdf
Supplementary Figure 1. Cells expressing GFP by the mGAD65 (delE1) were co-immunolabeled for GABA. A mouse received an intravenous infusion of AAV-PHP.eB expressing GABA by the mGAD65 (delE1). Three weeks after the injection, sagittal brain sections were produced and double-immunolabeled for GFP and GABA. Note that all GFP-expressing cells expressed GABA, suggesting that the mGAD65 (delE1) serves as an inhibitory neuron-specific promoter. Scale bar; 50 μm. Supplementary Figure 2. Significant labeling of chandelier-cell cartridges by the mGAD65 promoter over the mDlx enhancer. (A) A viral mixture (equal viral titer) comprising of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter and that expressing tdTomato under the control of the mDlx enhancer was intravenously injected to adult mice. (B) A representative fluorescent image of the chandelier-cell cartridge double-labeled with GFP and tdTomato. Brain sections were immunostained 3 weeks after the viral injection for ankyrin-G (blue). (C) Graph showing percentage of GFP-labeled chandelier-cell cartridges co-labeled with tdTomato and vice versa. To determine the ratio, we identified GFP-labeled cartridges along with the AIS by the GFP channel and determined whether those were co-labeled with tdTomato by the RFP channel (magenta box) (39 GFP-labeled cartridges from 4 mice were examined, in which 14 cartridges were modestly co-labeled with tdTomato) (magenta box). The green box was obtained by the opposite procedure. Namely, tdTomato-labeled cartridges were identified by the RFP channel and it was determined if they were co-labeled with GFP by the GFP channel (22 tdTomato-labeled cartridges from 4 mice were examined, in which 16 cartridges were robustly co-labeled with tdTomato). See the Materials & Methods section for more details. Floating bars indicate upper, median, and lower quartiles (boxes) and value from each mouse (dots). p = 0.0041 by unpaired t-test. Scale bar; 5 μm. Supplementary Figure 3. Transduction of Golgi cells by the mGAD65 promoter. (A) Diagram illustrating the experimental procedure. Mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, the mice were sacrificed, and the cerebellar sections were immunostained for mGluR2, a marker for Golgi cells. (B) A native GFP (green) fluorescent image of the cerebellar cortex immunolabeled for mGluR2 (magenta). (C) Magnification of the box in A. Arrowheads indicate GFP-expressing Golgi cells, which were proved by mGluR2 immunolabeling. Scale bars; 50 μm (B) and 20 μm (C). IHC; immunohistochemistry. Supplementary Figure 4. Cerebellar interneuron-specific transduction by direct parenchymal injection of AAV-PHP.B carrying the mGAD65 promoter. (A) Schematic showing the experimental procedure. (B) Immunohistochemistry of the cerebellar slice from mice 3 weeks after the viral injection. Cerebellar sections were immunostained for calbindin (magenta), a marker for Purkinje cell. Scale bar; 50 μm. IHC; immunohistochemistry. Supplementary Figure 5. Transduction of GABAergic neurons in various brain regions by the mGAD65 promoter. Adult VGAT-tdTomato mice received intravenous infusions of AAV-PHP.B expressing GFP under the control of the mGAD65 promoter. Three weeks after the viral injection, mice were sacrificed. Confocal microscopy showed overall co-labeling of tdTomato-expressing cells with GFP in various brain regions such as the striatum (caudate-putamen), the hippocampus (CA1), the anterior interposed nucleus (IntA), the superior vestibular nucleus (SuVe), and the retina. Scale bars; 50 μm (right) and 20 μm (left).
- SupplementaryTableRevise12272020.pdf
Supplementary Table. Antibodies used for immunohistochemistry.
- SupplementaryMovie.mp4
Supplementary Movie. Spontaneous Ca2+ activity in G-CaMP-expressing GABAergic neurons in an acute brain slice of the motor cortex using the mGAD65 promoter. Another example of spontaneous Ca2+ signals observed by confocal live Ca2+ imaging in the motor cortex of VGAT-tdTomato mice expressing G-CaMP7.09 using our AAV-mediated GABAergic neuron-selective gene delivery method. Still image of tdTomato signals in the same field of view as G-CaMP ones is presented in a right panel in magenta. The movie plays 20 times faster than real time.
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