Specific and comprehensive genetic targeting reveals brain-wide distribution and synaptic input patterns of GABAergic axo-axonic interneurons
- Cold Spring Harbor Laboratory, United States
- Program in Neurobiology, Stony Brook University, United States
- Department of Neurobiology, Duke University, United States
- Department of Neural and Behavioral Sciences, Penn State College of Medicine, United States
- Department of Biomedical Engineering, Duke University, United States
Figures
Figure 1 with 3 supplements
Developmental genetic intersectional targeting reveals brain-wide axo-axonic cell (AAC) distribution patterns.
Schematic of lineage (Nkx2.1) and marker (Unc5b) intersection for pan-AAC labeling. (a) Embryonic Nkx2.1 expression defines medial ganglionic eminence (MGE) interneuron identity while subsequent …
Figure 1—figure supplement 1
Dense labeling of axo-axonic cells (AACs) across brain regions in a Unc5bCreER; Nkx2.1Flp; Ai65 mouse.
(a–f) Additional representative coronal sections showing dense Ai65 expression patterns at specified anterior–posterior coordinates (from Bregma). Note labeling in cerebral cortex (a–f); hippocampus …
Figure 1—figure supplement 2
Additional sagittal sections of Unc5bCreER; Nkx2.1Flp; Ai65 mice highlighting dense putative AACs (pAACs) in medial, lateral, and ventral pallium-derived structures.
(a–c) A lateral sagittal section showing the lateral pallium (lPAL)-derived anterior insular (AI), claustrum (CLA), endopiriform (EP) (magnified in b), and medial pallium (mPAL)-derived hippocampal …
Figure 1—figure supplement 3
Intersectional targeting with PthlhFlp and Nkx2.1Cre, Nkx2.1CreER or VipCre to label specific subsets of axo-axonic cells (AACs).
(a) Schematic of intersectional strategy. (b–d) Tamoxifen induction of Nkx2.1CreER; PthlhFlp; Ai65 at E14.5 or E17.5 sparsely labels chandelier cells (ChCs) with diverse morphology in sensorimotor …
Figure 2 with 1 supplement
Characterization of chandelier cells (ChCs, i.e. cortical axo-axonic cells [AACs]) in cerebral cortex.
(a) Representative two-dimensional (2D) stereotactic plot of cortical ChC distribution in Unc5b; Nkx2.1. Dots represent individual red fluorescent (RFP) labeled cells. Anterior–posterior distance …
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Figure 2—source data 1
Mean, SEM and raw depths for data points in Figure 2b-e, m.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Anatomical diversity of chandelier cells (ChCs) across neocortical areas.
AnkyrinG or IκBα immunohistochemical labeling (green) of PyN axon initial segment (AIS) was used for validation of axo-axonic targeting. Examples are shown of characteristic upper L2 ChCs in motor (a…
Figure 3
Distribution and validation of axo-axonic cells (AACs) in hippocampus.
(a) Population labeling of AACs throughout hippocampal compartments by Unc5b; Nkx2.1 intersection. Note the highly stereotyped banded distribution of AACs in CA1–CA3, with somata in the stratum …
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Figure 3—source data 1
Mean and SEM for data points in Figure 3c.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig3-data1-v1.xlsx
Figure 4
Distribution of axo-axonic cells (AACs) across lateral pallium (lPAL)- and ventral pallium (vPAL)-derived structures.
(a) Dense-labeled RFP cells in claustrum, endopiriform nucleus, and insula. Note AACs in insula follow the banded laminar pattern of AACs similar to cortical upper layers and piriform, while those …
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Figure 4—source data 1
Mean and SEM for data points in Figure 4d, i, j.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig4-data1-v1.xlsx
Figure 5
Axo-axonic cells (AACs) in piriform cortex.
(a–c) Dense AAC labeling in piriform cortex, organized from anterior (a) to posterior (c) and overlaid with ARAv3 area outlines. Note the highly laminar distribution patterns. (d-j). Examples of …
Figure 6
Identification and distribution of novel axo-axonic cell (AAC) subpopulations in anterior olfactory nucleus (AON), taenia tecta (TT), and septum.
Dense population labeling in anterior AON and TT (a) and more posterior sections containing the TT and LS (b). Anteriorly, AACs are more concentrated in ventral TT while posteriorly in dorsal TT. …
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Figure 6—source data 1
Mean, SEM and percentages for data points in Figure 6h.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Intersectional tTA2 conversion enables viral access and input tracing to chandelier cells (ChCs) in sensorimotor cortices.
(a) Schematic of intersectional tTA2-conversion strategy. Expression of Nkx2.1Flp and subsequent Unc5bCreER or PvalbCre results in activation of tTA2 expression in axo-axonic cells (AACs). This …
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Figure 7—source data 1
Number of input cells, depth, mean, SEM and associated statistics for data points in Figure 7c-g.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Intersectional tTA2 conversion enables viral access and input tracing to chandelier cells (ChCs) and Pvalb cells.
(a) Schematic and procedure for synaptic input tracing from ChC and Pvalb interneurons. Top, driver and reporter lines to achieve tTA expression in ChC or Pvalb cells. Middle, schematic of AAV and …
Figure 8
Monosynaptic input tracing to AACsCA1 of hippocampus.
(a) Schematic of retrograde synaptic tracing from CA1 axo-axonic cells (AACs) with site of viral injection. (b) Starter AAV expression in CA1 (green). White arrowheads show soma position is largely …
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Figure 8—source data 1
Mean and SEM for data points in (Figure 8c, e, k).
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig8-data1-v1.xlsx
Figure 9
Monosynaptic input tracing to AACsBLA.
(a) Schematic of retrograde synaptic tracing from basolateral amygdala (BLA) axo-axonic cells (AACs) with site of viral injection. (b) Starter AAV distribution as indicated by white arrowheads, …
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Figure 9—source data 1
Mean and SEM for data points in Figure 9c, l-o.
- https://cdn.elifesciences.org/articles/93481/elife-93481-fig9-data1-v1.xlsx
Videos
Video 1
Whole-brain serial two-photon (STP) imaging and reconstruction of Unc5bCreER; Nkx2.1Flp; Ai65.
Serially reconstructed coronal images of representative dense-labeled Unc5bCreER; Nkx2.1Flp; Ai65 brain imaged by STP microscopy at 50 µm intervals. Scale bar, 500 µm.
Tables
Table 1
Brain-wide distribution pattern of putative axo-axonic cells (AACs).
| Developmental origin | Brain structure | Evolutionary term | Validation |
|---|---|---|---|
| Dorsal pallium | Neocortex all cortical areas entorhinal, perirhinal | Isocortex | Yes |
| Medial pallium | Hippocampal formation dentate, CA1, CA2, CA3 subiculum | Archicortex | Yes |
| Lateral pallium | Claustrum insular endopirifom dorsal | Not yet | |
| Ventral pallium | Amygdaloid complex
| Paleocortex | Yes |
| Septal neuroepithelium |
| Yes | |
| Hypothalamo-telencephalic prosomere h1/h2 | Hypothalamus
| Not yet |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-AnkryinG (mouse monoclonal) | Neuromab | Cat#: 75-146 | 1:500 |
| Antibody | Anti-ChAT (goat polyclonal) | Millipore | Cat#: AB144P | 1:300 |
| Antibody | Anti-EAAC1 (goat polyclonal) | Millipore | Cat#: AB1520 | 1:500 |
| Antibody | Anti-GABA (rabbit polyclonal) | Sigma | Cat#: A2052 | 1:500 |
| Antibody | Anti-GFP (chicken polyclonal) | Aves Labs | Cat#: 1020 | 1:1000 |
| Antibody | Anti-parvalbumin (mouse monoclonal) | Sigma | Cat#: P3088 | 1:1000 |
| Antibody | Anti-Phospho-IκBα (rabbit polyclonal) | Cell Signaling Technology | Cat#: 2859 | 1:500 |
| Antibody | Anti-RFP (rabbit polyclonal) | Rockland | Cat#: 600-401-379 | 1:1000 |
| Commercial assay or kit | HCR v3.0 | Molecular Instruments | ||
| Other | AAVDJ-TRE3g-EGFP | Vigene | Adeno-associated virus, titer: 5 × 1012 genome copies (gc)/ml | |
| Other | AAVDJ-TRE3g-TVA-oG-EGFP | Vigene | Adeno-associated virus, titer: 9.24 × 1012 genome copies (gc)/ml | |
| Other | G-Deleted Rabies dsRedXpress | Salk Institute | Rabies virus, titer: 5.0 × 107 colony-forming units (cfu)/ml | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | MATLAB | Mathworks | RRID:SCR_001622 | R2017b |
| Software, algorithm | Elastix | Image Sciences Institute | RRID:SCR_009619 | |
| Strain (Mus musculus) | Ai65 | The Jackson Laboratory | RRID:IMSR_JAX:021875 | |
| Strain (M. musculus) | C57BL/6J | The Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Strain (M. musculus) | dual-SynaptotagminEGFP-2A-tdTomato | The Jackson Laboratory | RRID:IMSR_JAX:030206 | |
| Strain (M. musculus) | dual-tTA2 | The Jackson Laboratory | RRID:IMSR_JAX:036304 | |
| Strain (M. musculus) | Nkx2.1Cre | The Jackson Laboratory | RRID:IMSR_JAX:008661 | |
| Strain (M. musculus) | Nkx2.1FlpO | The Jackson Laboratory | RRID:IMSR_JAX:028577 | |
| Strain (M. musculus) | PthlhFlpO | This paper | ||
| Strain (M. musculus) | PvalbCre | The Jackson Laboratory | RRID:IMSR_JAX:017320 | |
| Strain (M. musculus) | Unc5bCreER | Dudok et al., 2021 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/93481/elife-93481-mdarchecklist1-v1.docx
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Supplementary file 1
List of Allen Reference Area v3 (ARAv3) brain area labels and abbreviations.
- https://cdn.elifesciences.org/articles/93481/elife-93481-supp1-v1.xlsx
