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, 22 (3), 492-502

Intersectional Monosynaptic Tracing for Dissecting Subtype-Specific Organization of GABAergic Interneuron Inputs

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Intersectional Monosynaptic Tracing for Dissecting Subtype-Specific Organization of GABAergic Interneuron Inputs

Michael J Yetman et al. Nat Neurosci.

Abstract

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.

Figures

Figure 1.
Figure 1.
iMT of cortical IN subtypes that send direct inputs to defined PNs. (a) Canonical wiring diagram of three non-overlapping IN subtypes including PV-, SOM-, and VIP-INs. (b) Schematic of iMT principle. (c) Schematic of Cre/Flp-dependent RFP expression in dual reporter mice. See also Supplementary Fig. 1.
Figure 2.
Figure 2.
iMT specifically label PV-INs sending direct inputs to supragranular PNs. (a) Experimental design for iMT of PV-INs sending direct inputs to supragranular PNs. (b) Confocal projection image merging H2BYFP (yellow), CFP (cyan), and RFP (red) signals. CFP+/H2BYFP+, CFP+/H2BYFP-, and RFP+ neurons represent starter PNs, general input neurons, and putative input PV-INs, respectively. Scale bar, 500 μm. (c) Merged and single channel images from a single confocal optical section of a CFP+/H2BYFP+ starter PN surrounded by basket-like structures from RFP+ putative PV-INs. Scale bar, 10 μm. (d,e) Confocal projection images showing RFP+/PV+ and RFP+/PV- neurons (d) and RFP+/CFP+ and RFP+/CFP- neurons (e). Double and single positive neurons are indicated by closed and open arrowheads, respectively. Scale bar, 100 μm. (f) Percentage of RFP+/PV+ and RFP+/CFP+ neurons to total RFP+ neurons (n = 3 animals). (g) DIC and fluorescent images of an RFP+/CFP+ neuron in patch clamp configuration. Outline of putative PV-IN is shown in dotted white oval. Scale bar, 15 μm. (h) Representative voltage trace from an RFP+ putative PV-IN given a 150 pA current injection for 500 ms. (i) Sample AP trace showing definition of the resting membrane potential (RMP), AP threshold, and AP duration. Scale bar, 1 ms, 10 mV. (j) RMP and AP threshold for RFP+ neurons (n = 13 RFP+ neurons, 4 animals). (k) AP duration for RFP+ putative PV-INs (n = 13 RFP+ neurons, 4 animals). (l) Spikes elicited from RFP+ putative PV-INs during 500 ms current injections (n = 13 RFP+ neurons, 4 animals). Data are presented as mean ± SEM. All experiments were repeated independently three (b-f) and four (g-l) times, respectively, with similar results. See also Supplementary Fig. 1,3.
Figure 3.
Figure 3.
Supragranular PNs receive not only local but also translaminar inputs from PV-INs. (a) Experimental design for iMT of PV-INs sending direct inputs to supragranular PNs. (b-d) Confocal projection images of RFP+ PV-INs innervating supragranular PNs captured by iMT. Lower (b) and higher (L2/3 and L5 in c and d, respectively) magnification images of RFP+ input PV-INs. Scale bars, 200 μm (b), 50 μm (c and d). (e-h) Laminar distribution of CFP+/H2BYFP+ starter PNs (e), CFP+/H2BYFP- general input neurons (f), RFP+ input PV-INs (g), and RFP+ processes (h) (n = 5 animals). (i) Experimental design for iMT of PV-INs sending direct inputs to supragranular PNs using dual SypYFP reporter to visualize synaptic terminals. (j-l) iMT of presynaptic terminals from PV-INs innervating supragranular PNs. Lower (j and k) and higher (l) magnification images. Merged (j and l) and single channel (k) confocal projection images. HAH2B (white), RFP (red), and SypYFP (green). Right small panels in l show merged and single channel images from a single confocal optical section, which is enlarged from a boxed area in a left panel. RFP+/HAH2B+ starter PN is surrounded by SypYFP puncta. Scale bars, 200 μm (j and k), 50 μm (left panel in l), 10 μm (right panels in l). (m-o) Laminar distribution of RFP+/HAH2B+ starter PNs (m), RFP+/HAH2B- general input neurons (n), and SypYFP+ puncta (o) (n = 5 animals). Data are presented as mean ± SEM. All experiments were repeated independently five (b-h) or three (j-o) times with similar results. See also Supplementary Fig. 5,2b,c,8a,b. See Supplementary Table 4,5 for numerical values.
Figure 4.
Figure 4.
A single supragranular PN in the SSC receives inputs from PV-INs in multiple layers. (a) Experimental design for iMT of PV-INs sending direct inputs to a single supragranular PN. (b,c) iMT of input PV-INs innervating a single supragranular PN. Confocal projection image of HAH2B (yellow) and DAPI (blue) signals showing extremely sparse expression of HAH2B in supragranular PNs (b). Closed and open arrowheads represent an infected CFP+/HAH2B+ starter PN and a non-infected HAH2B+ PN, respectively. Small panels in b represent merged and single channel images from a single confocal optical section of a CFP+/HAH2B+ starter PN indicated by a closed arrowhead in left panel. Confocal projection images of RFP+ input PV-INs (red) that send inputs to a single CFP+/HAH2B+ PN shown in b, which are distributed in serial, anterior-to-posterior 60 μm sections (c). Scale bars, 100 μm (b, left panel, and c) and 10 μm (b, right panels). (d-h) 3-D reconstruction of IN-PN circuit modules, each of which contains a single starter PN (yellow sphere) and RFP+ input PV-INs (purple-white spheres). A-P positions of RFP+ input PV-INs relative to a starter PN are indicated by purple-to-white heatmap colors ranging from −98.5 to +116 μm. Scale bars, 100 μm. All experiments were repeated independently five times with similar results. See also Supplementary Fig. 4,6.
Figure 5.
Figure 5.
Granular/infragranular PNs receive local inputs from granular/infragranular PV-INs. (a) Experimental design for iMT of PV-INs sending direct inputs to granular/infragranular PNs. (b-d) iMT of PV-INs sending direct inputs to granular/infragranular PNs. Merged (b and c) and single channel (d) confocal projection images. DAPI (blue), H2BYFP (yellow), CFP (cyan), and RFP (red). CFP+/H2BYFP+, CFP+/H2BYFP-, and RFP+ neurons represent starter PNs, general input neurons, and input PV-INs, respectively. Scale bar, 200 μm. (e-h) Laminar distribution of CFP+/H2BYFP+ starter PNs (e), CFP+/H2BYFP- general input neurons (f), RFP+ input PV-INs (g), and RFP+ processes (h) (n = 3 animals). Data are presented as mean ± SEM. All experiments were repeated independently three times with similar results. See also Supplementary Fig. 2e,8c. See Supplementary Table 6,7 for numerical values.
Figure 6.
Figure 6.
Unique cellular/axonal organization of SOM-INs that innervate supragranular PNs in distinct cortical areas. (a) Experimental design for iMT of SOM-INs sending direct inputs to supragranular PNs. (b,c) Confocal projection images merging H2BYFP (yellow) and CFP (cyan) in the aSSC (b) and the MC (c). CFP+/H2BYFP+ and CFP+/H2BYFP- represent starter PNs and general input neurons, respectively. Scale bar, 200 μm. (d,e) Confocal projection images of RFP+ input SOM-INs (red) sending inputs to supragranular PNs in the aSSC (d) and the MC (e). Somata and axons in all layers (upper panels) and L1 axons (lower panels) of RFP+ input SOM-INs. Scale bars, 200 μm (upper panels), 20 μm (lower panels). (f-m) Laminar distribution of CFP+/H2BYFP+ starter PNs (f,h), CFP+/H2BYFP- general input neurons (g,i), RFP+ SOM-INs (j,l), and RFP+ processes (k,m) in aSSC (f,h,j,l) and MC (g,i,k,m), respectively (n = 5 animals). (n) Area occupied by L1 RFP+ processes normalized to the number of RFP+ somata in the aSSC and the MC (n = 5 animals). Data are presented as mean ± SEM. All experiments were repeated independently five times with similar results. See also Supplementary Fig. 2f,g,7a,8d,e,g,h,9. See Supplementary Table 3,8,9 for numerical values and statistics.
Figure 7.
Figure 7.
Cellular/axonal organization of VIP-INs that innervate supragranular PNs in the SSC. (a) Experimental design for iMT of VIP-INs sending direct inputs to supragranular PNs. (b) Confocal projection image merging H2BYFP (yellow) and CFP (cyan). CFP+/H2BYFP+ and CFP+/H2BYFP- represent starter PNs and general input neurons respectively. Scale bar, 200 μm. (c,d) iMT of VIP-INs sending direct inputs to supragranular PNs in the SSC. Confocal projection image showing RFP+ input VIP-INs (red) at lower magnification (c). Higher magnification images of individual RFP+ input VIP-INs with bipolar and multipolar morphologies in L2/3 and L4, respectively (d). Scale bars, 200 μm (c), 50 μm (d). (e,f) Merged confocal projection images of RFP+ basket-like axonal terminals from input VIP-INs (red) and NeuN or Gad67 (green) in e and f, respectively. Right small panels in e and f represent merged and single-channel images from a single confocal optical section, which is enlarged from a boxed area in left panels. Scale bars, 50 μm (left panels), 10 μm (right panels). (g-j) Laminar distribution of CFP+/H2BYFP+ starter PNs (g), CFP+/H2BYFP- general input neurons (h), RFP+ input VIP-INs (i), and RFP+ processes (j) (n = 5 animals). Data are presented as mean ± SEM. All experiments were repeated independently five times with similar results. See also Supplementary Fig. 2h,7b,c,8f. See Supplementary Table 10,11 for numerical values.

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