Developmental genetic intersectional targeting reveals brain-wide AAC distribution patterns.

a) Schematic of lineage (Nkx2.1) and marker (Unc5b) intersection for pan-AAC labeling. Embryonic Nkx2.1 expression defines MGE interneuron identity while subsequent postnatal Unc5b expression restricts interneurons to AACs. Height of green or gray shade represents relative expression levels.

b) Configuration of triple allele intersectional labeling, combining Unc5b-CreER and Nkx2.1-Flp drivers and the Cre-AND-Flp Ai65 reporter. Embryonic Flp expression removes the frt-flanked STOP cassette and postnatal CreER induction removes the loxp-flanked STOP, thereby activating constitutive tdTomato expression.

c) Schematic showing that MGE-derived AACs migrate and populate all four pallial-derived brain structures depicted in color: medial pallium (red), dorsal pallium (green), lateral pallium (violet and blue), and ventral pallium (yellow and orange). Adapted from Puelles, 2017.

d) Representative midsagittal section showing AAC labeling in cerebral cortex, hippocampal formation (HPF), and olfactory centers such as piriform cortex (PIR) and anterior olfactory nucleus (AON). Note sparse labeling in lateral hypothalamus (LH) and striatum (str).

e-g) Representative coronal sections at specified anterior-posterior coordinates (from Bregma) showing dense AAC labelling in cerebral cortex (e-g), HPF (f-g), claustrum (CLA, e), endopiriform (EP, f,-g), taenia tecta (TTd, e), lateral septum (LS, e), basolateral amygdala (BLA, f-g), cortical amygdala (COA, f-g), medial amygdala (MeA, f), and hypothalamus (HYP, g). (Scale bars, 500µm.) Abbreviations for anatomical structures are listed in Appendix. All images showing Ai65-tdTomato were immunostained for signal amplification.

Brain-wide distribution pattern of putative AACs

Characterization of chandelier cells (ChCs, i.e. cortical AACs) in cerebral cortex.

(a) Representative 2D stereotactic plot of cortical ChC distribution in Unc5b; Nkx2.1. Dots represents individual RFP-labelled cells. Anterior-posterior distance from Bregma (vertical scale on right).

(b) Normalized ChC cell density (cells/um3) following registration to ARAv3 isocortical areas. For b and c, data are mean ± SEM.

(c) Comparative bar plot showing relative proportion in each cortical layer in sensorimotor cortices.

(d) Violin plots of ChC cell density proportion along pia-to-white matter cortical depth in each sensorimotor cortical areas. Median is used for each violin plot.

(e) Scatterplot of ChC distribution along pia-to-white matter depth (vertical axis) and anterior-posterior distance from Bregma (horizontal axis, in mm); red dots represent individual cells. While a vast majority (∼80%) of ChCs occupy a band near the layer I/II border, ChCs in other layers account for ∼20% of the total cortical population and vary in depth distribution by anterior-posterior cortical location.

(f) Representative ChC laminar distribution pattern, showing largely separated supragranular (top inset) and infragranular (bottom inset) laminar subtypes. (Scale bars, 200µm and insets, 100 µm.)

(g-i) Multiple ChC laminar subtypes revealed by single-cell labeling using low-dose tamoxifen induction.

These include supragranular subtypes (g), infragranular and inverted subtypes (h), and translaminar L2/3 subtypes that extend long vertical axons (white arrowheads) to deep layers (i). White arrowheads highlight axonal plexi distant from soma in translaminar types. (Scale bars, 65µm.)

(j) Example of regional ChC morphological diversity in auditory cortex, with relatively high proportion of translaminar subtypes. (Scale bar, 200µm.)

(k-n) ChC axon terminal (i.e. “cartridge”) labeling and mapping using intersectional synaptophysin-EGFP/cytoplasmic-tdtomato reporter mouse line.

(k) Diagram of intersectional genetic labeling of ChC cartridges (EGFP) and soma (tdTomato).

(l) Pixel classifier trained to segment EGFP-expressing ChC cartridges in cortex and CA1 hippocampus. White arrowheads indicate detected EGFP-labelled cartridges. (Scale bars, 100µm).

(m) Density of ChC cartridges (cartridges/um3) registered to ARAv3

(n) Representative relative depth plots (from pia) of individual detected cartridges at a single AP coordinate for each cortical area. Refer to Appendix for ARAv3 area label abbreviations. Note reduced deep layer cartridges in FRP, RSP, and V1.

Distribution and validation of 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 pyramidale and vertically oriented dendrites extending basally to the stratum oriens and apically to the striatum lacunosum-moleculare. AACs are much sparser in dentate gyrus, with individual cells having elaborate and extended horizontal axon arbor. Scale bars, 200µm and 50µm for high-magnification inset.

(b) AACs in ventral CA1 have overall similar morphology as in dorsal CA1, though with longer basal-apical dendrites matching the ventral CA1 anatomy.

(c) Normalized AAC cell density (cells/um3) across hippocampal compartments. CA2 has 3-4-fold higher AAC cell density compared to CA1 and CA3 and 12-fold higher compared to DG.

(d-i) Immunohistochemistry validation with the AIS markers AnkG or IκBα (green) and sparse-labelled RFP cells confirm axo-axonic targeting by the intersectional strategy. Examples of hippocampal AACs in CA1 (e, g), CA2 (d) and CA3 (d, f, h). In contrast, AACs in DG have much wider axon arbor in the stratum granulosum, potentially innervating many more granule cell AIS compared to in other hippocampal compartments (i). High-magnification inset: white arrowheads indicate segments of IκBα and RFP apposition. (Scale bars, 50µm and 20µm for high-magnification inset.) For b, data are mean ± SEM.

Distribution of AACs across lPAL- and vPAL-derived structures

(a) Dense labelled 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 in claustrum and endopiriform are more dispersed in distribution.

(b-c) Sparse labeling of single cell morphologies in endopiriform nucleus (b) and claustrum (c) showing elaborate dendritic and axonal branching structure. High-magnification insets: white arrowheads indicate segments of AnkG and RFP apposition. Scale bars, 50µm and 5µm for single cell examples and insets, respectively.

(d) Normalized AAC cell density (cells/um3) across lateral pallial structures. pAACs are approximately five-fold more prevalent in dorsal EP compared to ventral EP. Data are mean ± SEM.

(e) Dense labelling throughout vPall-derived amygdalar nuclei and piriform cortex. Note there is a degree of compartmentalization, with fewer cells in the ventral-most portions of EPd and the ventral BMA. The laminar pattern of AAC distribution in piriform and Piriform-amygdalar area (PAA) is similar to other three-layered allocortices.

(f-h) Immunohistochemistry of single cell labeling shows the multipolar morphologies characteristic of amygdalar AACs. High-magnification inset: white arrowheads indicate segments of AnkG and RFP apposition. (Scale bars, 500 µm for grayscale panels, 50µm and 5µm for single cell examples and insets.)

(i-j) Normalized AAC cell density (cells/um3) across cortical subplate (i) and striatum-like (j) amygdala. Data are mean ± SEM.

All images showing Ai65-tdTomato were immunostained for signal amplification, except for panels a and e which were native fluorescence.

AACs in piriform cortex.

(a-c) Dense AAC labelling 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 single piriform AACs co-stained with AnkG (green). Note the relatively similar morphologies with only a few apical dendrites extending to the pial surface (d-g, i-j). An example of a deeper layer AAC at the border of piriform and EPv (h). White arrowheads in high-magnification insets indicate segments of overlap for RFP-labelled AAC and AnkG-labelled AIS. (Scale bars, 500µm for grayscale images, 50µm for confocal, 10µm for high-magnification insets.)

Identification and distribution of novel AAC subpopulations in anterior olfactory nucleus (AON), taenia tecta (TT), and septum.

(a-b) 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. Within LS, AACs tend to occupy the medial most portions along the midline.

(c-g, i-k) Single cell (confocal) labeling of AACs in anterior olfactory nucleus (c-d), taenia tecta (e-g), and lateral septum (i-k). Note that despite areal variations, individual AACs preserve their stereotyped morphological characteristics (apical-oriented dendrite and basal-oriented axon arbor) that conform to laminar patterns of targets in each anatomical area. High-magnification insets: white arrowheads indicate segments of AnkG and RFP apposition. (Scale bars, 200 µm for grayscale panels, 50µm and 5µm for single cell examples and insets.)

(h) Normalized cell density (cells/um3) for AACs in AON, TT, and lateral septum. Within lateral septum, the majority of AACs are located in rostral compartments (LSr). Data are mean ± SEM.

Intersectional tTA2 conversion enables viral access and input tracing to ChCs in sensorimotor cortices.

(a) Schematic of intersectional tTA2-conversion strategy. Expression of Nkx2.1-Flp and subsequent Unc5b-CreER or PV-Cre results in activation of tTA2 expression in AACs. This provides a genetic handle for versatile viral targeting and manipulation.

(b) Cortical injection of TRE3G-promoter AAV vectors enables specific targeting of ChCs (green, left). A similar backbone was used to construct a TRE3G rabies starter AAV for monosynaptic input tracing, which contains both TVA and optimized rabies glycoprotein (right). Compared to TRE3G-EGFP, the TRE3G starter AAV has weaker EGFP labeling of neurites. (Scale bar, 200µm.)

(c) Linear regression fit of inputs cells/starter cells. Each point represents one animal, n= 18, r = 0.8874, slope = 135.51.

(d) Relative pia-to-white matter depth distribution of starter cells (expressing both AAV starter and rabies). ChC starter cell tended to cluster closer to the pia while PV cells were more broadly distributed throughout the cortical upper layers, reflecting the differences in the distribution of the two subtypes.

(e-g) Synaptic input source to ChCs in MOs, MOp, and SSp, with PV cells as comparison. Only long-range inputs (i.e. outside the ARAv3 area injected) are plotted.

Left: representative coronal sections from three indicated cortical areas overlaid with macroscale view of rabies tract labeling (in red, gray is autofluorescence) to each subpopulation of ChC.

Middle: Histogram comparing percentage of inputs from each structure innervating the indicated cortical subpopulation of ChCs (dark gray) and PV (light gray), sorted from largest input source to smallest. Inset: Representative top-down map of all inputs to AACs from a single animal.

Right: Representative sections highlighting select sources of rabies-labelled inputs to each ChC population, overlaid with ARAv3 boundaries. (Scale bars, 200µm.) For e-g, data are mean ± SEM, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicates P ≥ 0.05 (two-way ANOVA with post hoc Bonferroni correction); for scatterplot in d, median is plotted. Homotypic projections to injection site from contralateral hemisphere are denoted by (#). All images showing EGFP were immunostained for signal amplification, while rabies-RFP shown in panels e-g were native fluorescence.

For each MOs, MOp and SSp, N=6 mice for (3 Unc5b; Nkx2.1, 3 PV; Nkx2.1; total N=18 mice)

Monosynaptic input tracing to AACsCA1 of hippocampus.

(a) Schematic of retrograde synaptic tracing from CA1 AACs with site of viral injection.

(b) Starter AAV expression in CA1 (green). White arrowheads show soma position is largely confined to the pyramidal cell layer. (Scale bar, 50µm.)

(c) Comparative input distribution from ipsilateral (right) and contralateral (left) hippocampal compartments.

(d) Mesoscale rabies labeling of presynaptic input cells (red) to AACsCA1

(e) Histogram of percentage of total inputs from each structure, sorted from largest to smallest. Inset: Summary schematic of input sources to AACsCA1. AACsCA1receive strong innervation from entorhinal, subiculum, and contralateral hippocampus, with more minor innervation from thalamus, medial septum, diagonal band nucleus, and various cortical areas.

(f-j) Immunohistochemistry and HCR RNA-FISH marker analysis of input cells in medial septum (MS) and nucleus of the diagonal band (NDB), indicated by color-coded labels. Examples of co-labeled cells are shown following HCR RNA-FISH for the inhibitory marker vGAT (g) and immunohistochemistry for the excitatory marker EAAC1 (h), PV (i) and ChAT (j). White arrowheads indicate co-labelled cells for each marker. (Scale bars, 50µm and 20µm for high-magnification insets.)

(k) Quantification of marker colocalization with rabies labeling in MS and NDB.

(l-p) Representative coronal sections highlighting select sources of rabies-labelled inputs to AACsCA1, overlaid with ARAv3 atlas boundaries. Inputs within hippocampus were mostly from within CA1 both ipsilateral (l) and contralateral (m) to the injection site. Input cells were also found in adjacent cortical areas more posterior such as entorhinal cortex (ENT), auditory cortex (AUD), and temporal association area (TEa) (n-p). Thalamic inputs were largely confined to the anterior group of the dorsal thalamus (ATN) (o). (Scale bars, 200µm.)

For c, e, and k, data are mean ± SEM from N=4 mice. Homotypic projections to injection site from contralateral hemisphere are denoted by (#CA1) in e. EGFP in panel b and rabies-RFP in panels f-j were immunostained for signal amplification, while rabies-RFP shown in panels l-p were native fluorescence.

Monosynaptic input tracing to AACsBLA.

(a) Schematic of retrograde synaptic tracing from BLA AACs with site of viral injection.

(b) Starter AAV distribution as indicated by white arrowheads, overlaid with ARAv3 atlas. (Scale bar, 200µm.)

(c) Left: Mesoscale rabies labeling of presynaptic input cells (red) to AACsBLA. Right: Histogram of percentage of total inputs from each structure, sorted form largest to smallest. Inset: Summary schematic synaptic input source to AACsBLA. AACsBLA are innervated by diverse sets of inputs, with strongest innervation from entorhinal cortical-hippocampal network (EC-HPC), piriform areas (PIR), cortical subplate (CTXsp), and minor subcortical and cortical inputs.

(d-j) Representative coronal sections of rabies-labelled inputs (grey) across input structures, overlaid with ARAv3 boundaries. (Scale bars, 200µm.) Inputs to AACsBLA were found in amygdalar and cortical-amygdalar areas (d-e), piriform (e), midline group of the dorsal thalamus (f), substantia innominata (g), posterior hippocampus and cortex (h) and the ventral subiculum (i). Examples of dense input cells in medial (MEZ) and lateral (LZ) hypothalamus overlaid with excitatory vGLUT2 HCR RNA-FISH (j).

(k-o) Immunohistochemistry and RNA-FISH marker analysis of input cells in striatum/CP, GPe, EP, and hypothalamus. VGAT FISH or GABA IHC were used for identifying GABAergic input cells (l-n) while VGLUT2 FISH was used an excitatory marker (o) as indicated. White arrowheads indicate co-labelled cells. (Scale bars, 50µm and 20µm for high-magnification insets.) Right: Quantification of marker colocalization with rabies labeling for each corresponding area. Inputs to AACsBLA from CP, GPe, and EP were inhibitory, while inputs from hypothalamus were excitatory. Data are mean ± SEM from N=3 mice.

Rabies-RFP in panels j-o were immunostained for signal amplification, while EGFP in panel b and rabies-RFP shown in panels d-i were native fluorescence.

Dense labelling of AACs across brain regions in a Unc5b-CreER;Nkx2.1-Flp;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 (e-f); olfactory centers, including piriform, anterior olfactory nucleus, and tenia tecta dorsal (PIR, AON, TTd) (a-b); agranular insula, claustrum, and endopiriform nucleus (AI, CLA, EP) (b-c); amygdaloid complex and extended amygdala (COA, BLA, MeA) (d-f); Bed nuclei of the stria terminalis (BST) (d); and hypothalamus (HYP) (e-f). (Scale bars, 500µm.)

(g-j) Ai65-labelled pAACs in the bed nuclei of the stria terminalis (BST) are largely confined to the principal nucleus (BSTpr), with pAACs clustering more densely in anterior BSTpr (g-h) compared to more posterior segments (i-j).

(k-n) Ai65-labelled pAACs in the hypothalamus are densest in the ventral-medially area of the arcuate nucleus (ARH), with highest density in caudal portions (k-m) and more posteriorly in the paraventricular hypothalamic nuclei (PVP) (n). pAACs are also present sparsely in the dorsal-medial nucleus of the hypothalamus (DMH) (k-m).

Additional sagittal sections of Unc5b-CreER; Nkx2.1-Flp; Ai65 mice highlighting dense pAACs in medial, lateral, and ventral pallium-derived structures.

(a-c) A lateral sagittal section showing the lPAL-derived anterior insular (AI), claustrum (CLA), endopiriform (EP) (magnified in b) and mPAL-derived hippocampal formation (magnified in c). Note the single-banded orientation of hippocampal (c) and insular (b) AACs that is absent in more inferior lPAL structures (b).

(d-e), A more medial sagittal section showing dorsal and ventral hippocampus and retrohippocampal region (RHP) (d), retrohippocampus, and visual and retrosplenial cortex (e). Compare the dual-banded AAC distribution in upper and deeper cortical layers to the single-banded pattern in hippocampus. Also note dense labeling in amygdaloid complex and extended amygdala (BLA, COA, and MeA) (a, d). (Scale bars, 500µm.)

Intersectional targeting with Pthlh-Flp and Nkx2.1-Cre, Nkx2.1-CreER or VIP-Cre to label specific subsets of AACs.

(a) Schematic of intersectional strategy.

(b-d) Tamoxifen induction of Nkx2.1-CreER; Pthlh-Flp; Ai65 at E14.5 or E17.5 sparsely labels ChCs with diverse morphology in sensorimotor cortices. Arrowheads highlight a translaminar AAC type in layer 2, which extends axon arbors into deep layers. (Scale bars, 65µm.).

(e-i) Coronal hemisections from Nkx2.1-Cre; Pthlh-Flp; Ai65 at specified anterior-posterior coordinates (mm from Bregma) showing pattern of dense labeling. Note labeling in cerebral cortex (e-i); hippocampus (h-i); olfactory centers, including piriform, anterior olfactory nucleus, and tenia tecta dorsal (e-h); agranular insula, claustrum, and endopiriform nucleus (AI, CLA, EP) (f); amygdaloid complex and extended amygdala (COA, BLA, MeA) (h-i); and hypothalamus (HYP) (i). Labeling by Nkx2.1-Cre; Pthlh-Flp is sparser in AON and anterior TTd and absent in BST compared to Unc5b-CreER; Nkx2.1-Flp (Fig1). Note non-AAC labeling of layer 4 interneurons in somatosensory cortex (g-h) and striatum (CP, f-h). The absence of projection fibers in SNr (i) suggests these striatal cells are not medium spiny neurons and likely striatal interneurons (Scale bars, 500µm.).

(j-l) Coronal hemisections from Nkx2.1-CreER; Pthlh-Flp; Ai65 following a single tamoxifen dose at embryonic day 18.5 (E18.5) shows exclusive specificity for individual ChCs across neocortex (Scale bars, 500µm.)

(m) Intersectional strategy of Pthlh and VIP using VIP-Cre;Pthlh-Flp; Ai65 mice.

(n-p) Dense labeling of cortical and hippocampal interneurons with predominantly bipolar morphology (putative interneuron-selective cells) in cortical layers II/III and deep layers (Scale bars, 500µm.)

Anatomical diversity of ChCs across neocortical areas.

AnkyrinG or IκBα immunohistochemical labeling (green) of PyN AIS was used for validation of axo-axonic targeting. Examples are shown of characteristic upper L2 ChCs in motor (a), sensory (b, d,j), cingulate (f, k), and insular cortex (i). Examples of deeper L5 or L6 ChCs in somatosensory (c, e, g) and temporal association area (TEA) (h). Note the clustering of a distinct morphological subtypes in ventromedial infralimbic cortex (l). High-magnification inset (g): white arrowheads indicate segments of overlap between AnkG and RFP. Scale bars, 50µm and 5µm for high-magnification insets.

Intersectional tTA2 conversion enables viral access and input tracing to ChCs and PV cells.

(a) Schematic and procedure for synaptic input tracing from ChC and PV interneurons. Top, driver and reporter lines to achieve tTA expression in ChC or PV cells. Middle, schematic of AAV and EnvA pseudo-typed rabies viral vectors. Bottom, timeline of tamoxifen and viral vector for retrograde synaptic tracing.

(b) Bulk injection of TRE3G-EGFP in Unc5b-CreER; Nkx2.1-Flp; LSL-FSF-tTA2 results in efficient and specific labeling of ChCs, identifiable by laminar position and morphological features (inset). (Scale bars, 200µm and 100µm for high-magnification inset.)

(c) Starter AAV expression is comparable to TRE3G-EGFP with high specificity for ChCs in Unc5b; Nkx2.1, albeit with weaker EGFP neurite expression. (Scale bars, 100µm and 20µm for high-magnification insets.)

(d, e) Example of starter cells (arrowheads) co-expressing EGFP (AAV vector) and RFP (rabies vector) in Unc5b/Nkx2.1 (d) and PV/Nkx2.1 (e) intersections. (Scale bars, 100µm and 20µm for high-magnification insets.) All images showing EGFP were immunostained for signal amplification, while rabies-RFP shown in panels d-e were native fluorescence.