A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex

Abstract

A key obstacle to understanding neural circuits in the cerebral cortex is that of unraveling the diversity of GABAergic interneurons. This diversity poses general questions for neural circuit analysis: how are these interneuron cell types generated and assembled into stereotyped local circuits and how do they differentially contribute to circuit operations that underlie cortical functions ranging from perception to cognition? Using genetic engineering in mice, we have generated and characterized approximately 20 Cre and inducible CreER knockin driver lines that reliably target major classes and lineages of GABAergic neurons. More select populations are captured by intersection of Cre and Flp drivers. Genetic targeting allows reliable identification, monitoring, and manipulation of cortical GABAergic neurons, thereby enabling a systematic and comprehensive analysis from cell fate specification, migration, and connectivity, to their functions in network dynamics and behavior. As such, this approach will accelerate the study of GABAergic circuits throughout the mammalian brain.

Figure 1

Overall scheme on the genetic targeting of GABAergic neurons. (A) Top: Cortical GABAergic neurons in rodents are largely generated from the medial and caudal ganglionic eminence (MGE, CGE), and preoptic area (POA). Bottom, postmitotic GABAergic neurons undergo long distance migration (blue arrow) into the cortex and form circuits with glutamatergic pyramidal neurons that are generated in the dorsal ventricular zone and migrate radially into the cortex (red arrow). (B) Mature cortical GABA neurons can be parsed into several major classes according to their preferential axon innervation of specific cellular and subcellular targets; the expression of several molecular markers tend to correlate, although not exclusively, with these classes. (C) List of genes that were used for genetic targeting in this study and their approximate temporal expression pattern in developing neocortex. (D) Design of CreER and Cre gene targeting strategy and list of knockin lines. Stars (*) indicate lines with low efficiency CreER induction.

Figure 2

The Nkx2.1-CreER driver captures MGE progenitors. (A) A schematic of the ventricular (VZ) and subventricular (SVZ) zones of ganglionic eminence at mid-gestation and a summary of transcription factor expression in these regions. (B) GFP expression in an Nkx2.1-CreER::RCE-LoxP brain 1day after induced at E12. MGE progenitors and postmitotic GABA neurons are labeled by GFP; progenitors were colabeled by an NKX2.1 antibody (red) in VZ (arrow). Postmitotic neurons were migrating towards cortex and striatum (Str). (C) High dose tamoxifen induction at E12 labeled many MGE progenitors (colabeled by an NKX2.1 antibody in red) and postmitotic neurons. (D) Low dose tamoxifen induction at E12 labeled sparser MGE progenitors, which showed radial clone-like organization (arrows) in VZ (colabeled by an NKX2.1 antibody). (E) A schematic of NKX2.1 expression in the ventral portion of late embryonic SVZ. (F, G) E17 NKX2.1⁺ cells (red) in the ventral SVZ were colabeled by a BrdU antibody (green), suggesting that they were mitotic. Four pulses of BrdU were administered every 4 hours at E17. (H, I) MGE progenitors labeled at E12 (green) contributed to NKX2.1⁺ cells (red) in E17 SVZ. (J-L) NKX2.1 progenitors labeled at E13 gave rise to major types of cortical interneurons including PV (J) and SST (K) cells but not VIP cells (L) at P31. Scale bars: 300 μm in B, 100 μm in C, D, I, 50 μm in F, G, 500 μm in H, 50 μm in J-L, 25 μm in insets of J, K.

Figure 3

The Dlx1- and Dlx5-CreER drivers preferentially target different subsets of developing and mature cortical interneurons. (A-I) Migration and distribution patterns of interneuorns labeled by the Dlx1-CreER or Dlx5-CreER driver induced at E12. (A, B) At E13, both cohorts of interneuorns entered the cortex through the SVZ. (C-F) At E15, while the Dlx5 cohort migrated predominantly in MZ (D,F), the Dlx1 cohort migrated in both MZ and SVZ and had entered the cortical plate (C,E). (G-I) At P21, both cohorts settled in deep layers of the cortex but with slightly different laminar patterns. Quantification of laminar distribution of GFP positive neurons (schematic in the upper panel of I) in the somatosensory area of Dlx-1-CreER (G) and Dlx5-CreER (H) mice demonstrated that the fraction of Dlx5-CreER labeled neurons occupying the deeper part of layer 6 was higher than that of Dlx1-CreER labeled neurons (I). Results are expressed as the mean±SEM from 3 animals for each genotype. *p<0.01, t-test. (J-M) Distribution of interneuorns at P21 labeled by the Dlx1-CreER or Dlx5-CreER driver induced at E15. While the Dlx1 driver labeled a small subset, the Dlx5 labeled broad population of interneurons. (L-M) Distribution of interneuorns labeled by the Dlx1- and Dlx5-CreER drivers in adult cortex induced in adult. While the Dlx1 driver labeled a small subset, the Dlx5 labeled broad population of interneurons. Scale bars: 100 μm in A, B, E, F, L, M, 500 μm in C, D, 300 μm in G,H,J,K.

Figure 4

The Gad2-CreER driver. (A) Overview of a P28 brain sagital section with P20 tamoxifen induction. (B,C) GFP expression activated by Cre recombination was restricted to GABAergic neurons (Gad67, red) in cortex. (D-H). GFP labeled neurons in cortex include major populations of interneurons which are positive for parvalbumin (PV), somatostatin (SST), calretinin (CR), vasoactive intestinal peptide (VIP) or nNOS. See SpplFig.1 for recombination patterns of the Gad2-ires-Cre driver. Scale bars: 500 μm in A, 25 μm in B, C, 25 μm in D-H.

Figure 5

The SST-ires-Cre and SST-CreER drivers. (A) Distribution of SST interneurons in cortex and hippocampus in an adult SST-ire-Cre::RCE-LoxP mouse. Laminar organization in cortex and hippocampus are indicated. Note the prominent axon track in cortical layer 1 (arrow). In hippocampal CA1, O-LM cells (soma indicated by an arrowhead) innervate the distal dendrite of pyramidal cells. O-LM cell axons form a prominent and sharp band in the stratum lacunosum moleculare (arrow) (also see SpplFig.2). (B) A schematic of distal dendrite-targeting interneurons. (C, D) Low frequency recombination in the SST-CreER driver labeled single Martinotti cells (arrow heads) in layer 5 (C) and layer2 (D). Note the characteristic axon arborization in layer1 (arrows). The background green puncta were non-specific signals from GFP antibody staining which were commonly seen when brain sections contained very small number of GFP expressing cells. (E) A schematic showing the migration streams of SST neurons at ~E13. Marginal zone (MZ), cortical plate (CP), intermediate zone (IZ), subventricular zone (SVZ), and ventricular zone (VZ) are indicated. (F, G) At E13, the SST-ires-Cre driver labeled migrating SST neurons soon after they exited MGE. SST neurons entered the cortex mainly through MZ and SVZ. (H) A schematic of P0 cortex depicting SST neurons (green) migrating and entering cortical plate. Pyramidal neurons are in light yellow and radial glia in gray. (I) At P0, while some SST neurons were still migrating in MZ and IZ, a significant portion had entered the cortical plate. Many SST neurons showed vertically oriented neurites, and their layer1 axons (an arrow) became discernable. (J) A schematic of P5 cortex depicting SST neurons (green) which are settling into appropriate cortical layers. (K) At P5, SST neurons were settling into appropriate cortical layers, and their layer1 axons (an arrow) were already prominent. Scale bars: 500 μm in A, 100 μm in C, D, 500 μm in F, 200 μm in G, 100 μm in I, K. stratum oriens (s.o), stratum pyramidale (s.p), stratum radiatum (s.r), stratum lacunosum moleculare (s.lm), dentate gyrus (DG).

Figure 6

Intersectional strategy captures CCK positive GABA interneurons. (A, B) Schematics depict CCK and PV basket interneurons that target pyramidal cell (Py) soma and proximal dendrites (A). CCK interneuron axon terminals show distinct molecular profiles compared to those of PV interneurons, including prominent expression of the CB1 receptor and signaling through distinct postsynaptic GABA_A receptors (B). (C) Intersectional strategy using CCK-ires-Cre, Dlx5/6-Flp, and a dual reporter (RCE-dual) to label CCK GABAergic interneurons. (D) GFP reporter expression in the cortex and hippocampus in a CCK-ires-Cre::RCE-LoxP mouse. Note the broad expression presumably including both pyramidal neurons and GABAergic neurons. (E) GFP reporter expression in the cortex and hippocampus in a Dlx5/6-Flp::RCE-Frt mouse. Most of the GABAergic neurons are labeled with GFP. (F) CCK positive GABAergic interneurons in cortex and hippocampus are targeted by the intersectional strategy. Note the dense GABAergic axons (an arrow) in the pyramidal cell layer (stratum pyramidale) in hippocampal CA1. Laminar organization in cortex and hippocampus are as indicated. (G) In hippocampal dentate gyrus, CCK axon terminals (green) segregated from PV axon terminals (red). (H) CCK GABAregic neurons (star) and axons (green) in neocortex. PV axons were labeled with an antibody (red), and all cell somata were labeled with TOTO-3 (blue). Inset: CCK axon terminals (white arrowheads) segregated from PV axon terminals (yellow arrowheads) around the same cell soma. Scale bars: 300 μm in D-F, 25 μm in G, H, 5 μm in inset of H.

Figure 7

The VIP-ires-Cre driver. (A) Distribution of VIP interneurons in an adult VIP-ires-cre::Ai9 mouse cortex and hippocampus. Tdtomato signals in A-C were psudocolored green. Laminar organization in cortex and hippocampus are as indicated. Note the prominent vertical orientation of neurites in cortex. In hippocampal CA1, the axons of VIP neurons segregated into distinct lamina in stratum oriens and stratum pyramidale (arrows). (B,C) Higher magnification view of cortical VIP interneurons. A stacked images is available as sppl movie 2. Note that VIP interneurons were completely segregated from PV interneurons (red) in (C). (D) VIP interneurons in a P2 VIP-ires-cre::RCE-LoxP mouse cortex. Young VIP interneurons migrated up into cortical plate from the subventricular zone with strikingly homogeneous and vertically oriented neurites. Inset shows higher magnification view of the boxed region. (E) A schematic depicts VIP neurons in P2 cortex. Pyramidal neurons and radial glias are drawn in light yellow and gray, respectively. (F) Cortical VIP interneurons (green traces) showed distinct intrinsic properties compared with PV (red traces) and SST interneurons (blue trances), including membrane resistance (Rm), action potential (AP) threshold, half width, amplitude, maximum firing frequency, and adaptation. Scale bars: 400 μm in A, 100 μm in B, 300 μm in D. See SpplFig5 for expression pattern in other brain regions.

Figure 8

The nNOS-CreER driver (A) A schematic of nNOS GABAergic neurons depicting the neurogliaform (NGFC) cells and long-range projection neurons (LPN) that extend axons across cortical areas and to the contralateral cortex. (B) Sagittal view of cortex and hippocampus from a P29 nNOS-CreER;Ai9 mouse induced from P21. TdTomato-labeled nNOS neurons were psudocolored green. Cortical nNOS neurons were distributed in all layers and extend highly profuse axons (arrows) throughout the cortex. In hippocampal CA1, NGFC cells elaborate extremely dense axons in stratum oriens and stratum radiatum but were nearly absent in stratum lacunosum moleculare and stratum pyramidale. In the dentate gyrus (DG), some nNOS neurons were located in the area of subgranular zone and hilus. (C, D) Confocal images of superficial (C) and deep (D) cortical layers. nNOS neuron dendrites were indicated by double arrows and axons by arrows. Note the extremely dense axon networks. (E) Confocal image of NGFCs in hippocampal CA1. Note the often vertically oriented dendrites (double arrows) and the extremely dense thin axons (arrows). (F, G) High magnification confocal images of nNOS axons (arrows) in cortical layer 3 (F) and hippocampal CA1 (G). Movie files from the stacked images of F and G are available as sppl movie 3 and 4, respectively. (f.g) Single optical sections in boxed areas from F and G show that nNOS axonal boutons (green) contain GAD65 (red), a GABAergic presynaptic marker. Note the small and closely spaced GAD65 boutons (arrowheads) along green axons. (H, I) Non-overlapping distribution of nNOS and PV axon terminals. In neocortex (H), pyramidal neuron somata (stars) were surrounded by PV axon terminals (red) but were not approached by nNOS axon terminals (green). Inset: higher magnification view of boxed region; white arrowheads indicate nNOS axon terminals, and yellow arrowheads indicate PV axon terminals. In hippocampal CA1 (I), PV basket cell axon terminals heavily innervated the perisomatic regions of pyramidal neurons (stars in stratum pyramidale), whereas nNOS terminals predominantly concentrated around pyramidal cell dendrites in stratum oriens and stratum radiatum. Inset: higher magnification view of the boxed region; white arrowheads indicate nNOS axon terminals, and yellow arrowheads indicate PV axon terminals. Scale bars: 300 μm in B, 50 μm in C-E, 25 μm in F-I, 5 μm in insets of H, I, 5 μm in f1, f2, g1, g2. See SpplFig6 for expression pattern in other brain regions.