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Neurogliaform cortical interneurons derive from cells in the preoptic area

Research Article
Cite this article as: eLife 2018;7:e32017 doi: 10.7554/eLife.32017
7 figures, 1 table and 2 additional files

Figures

A fraction of 5-HT3AR-expressing interneurons (INs) originates from Hmx3-derived cells in the preoptic area (POA) and expresses transcription factors related to the caudal ganglionic eminence (CGE).

(A) At E14.5, tdTOM specifically labels cells expressing Hmx3 (Hmx3; tdTOM+). Htr3a-GFP+ INs co-label with tdTOM in a rostral region of the POA (dashed lines; high magnified images) located ventrally to the anterior commissure (AC). (B) At more caudal levels, Hmx3; tdTOM+/Htr3a-GFP+ embryonic cells appear to further migrate caudally (arrowhead) towards the CGE. High magnified images show Hmx3; tdTOM+/Htr3a-GFP+ cells entering the ventral CGE (dashed lines). (C) More than 80% of Hmx3; tdTOM+ cells co-label with Htr3a-GFP (and conversely) in the overlap zone (orange dashed line) of the POA domain defined by Hmx3; tdTOM recombination (white dashed line). (D) In situ hybridization showing that almost all Hmx3; tdTOM+/Htr3a-GFP+ INs in the POA express the Htr3a mRNA (arrowheads; yellow outline), whereas Hmx3; tdTOM+ cells do not (empty arrowheads; cyan outline). (E) In the overlap zone of the POA, IHC reveals that Hmx3; tdTOM+/Htr3a-GFP+ embryonic cells express (arrowheads) the CGE-enriched transcription factors PROX1 (left) and NR2F2 but not SP8 (right). (F) By contrast, the vast majority of Hmx3; tdTOM+/Htr3a-GFP+ INs do not express NKX2.1 (arrowheads). dTh: dorsal thalamus, GP: globus pallidus, LGE: lateral ganglionic eminence, LV: lateral ventricle, MGE: medial ganglionic eminence. Scale bars: 300 µm in A, B: low magnified images; 100 µm in C, D: low magnified images; 50 µm in A, B: high magnified images; 25 µm in E, F; 10 µm in D: high magnified images.

https://doi.org/10.7554/eLife.32017.003
Figure 1—source data 1

Detailed counts of cells quantified in Figure 1 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.004
Figure 2 with 3 supplements
During the postnatal period, Hmx3-derived cells from the preoptic area (Gelman et al., 2009) constitute a small but persistent fraction of 5-HT3AR-expressing interneurons (INs) (Lee et al., 2010) in superficial cortical layers.

(A–B) Hmx3; tdTOM+/Htr3a-GFP+ cells represent a fraction of Htr3a-GFP+ INs that increases along postnatal ages P5 (left), P9 (middle) and P21 (right). Note that double-labeled cells (open arrowheads) are mainly located in superficial cortical layers. (C) In situ hybridization showing that, at P21, Htr3a mRNA is found in cortical Hmx3; tdTOM+/Htr3a-GFP+ INs (arrowheads) as in Htr3a-GFP+ INs (open arrowheads). (D–E) Hmx3; tdTOM+/Htr3a-GFP+ cells largely express the neuronal marker NeuN (arrowheads) whereas those negative for Htr3a-GFP only rarely do (arrows, open arrowhead). Scale bars: 100 µm in A, C: low magnified images; 25 µm in C: high magnified images, D.

https://doi.org/10.7554/eLife.32017.005
Figure 2—source data 1

Detailed counts of cells quantified in Figure 2 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.010
Figure 2—figure supplement 1
Rostro-caudal distribution of Hmx3; tdTOM+/Htr3a-GFP+ cells at P21.

(A) Hmx3; tdTOM+/Htr3a-GFP+ cells are observed in a variety of cortical areas as well as other brain regions including the hippocampus (Hip) and the amygdala (Am) (B) High magnified images showing Hmx3; tdTOM+/Htr3a-GFP+ cells in various brain regions. AH: amygdalo-hippocampal area, Am-a: amygdaloid areas, AuC: auditory cortex, bsc: brachius of the superior colliculus, CC: corpus callosum, CPu: caudate putamen (striatum), DLG: dorso-lateral geniculate nucleus, GP: globus pallidus, Hy: hypothalamus, lot: lateral olfactory tract, LV: lateral ventricle, MC: motor cortex, MeAm: medial amygdala, mfb: medial forebrain boundle, ml: medial lemniscus, MGN: medial geniculate nucleus, mOFC: medial orbitofrontal cortex, mPFC: medial prefrontal cortex, opt: optic tract, PG: progeniculate nucleus, Pir: piriform cortex, PM: pre-mamillary nuclei, PoN: preoptic nucleus, RCh: retrochiasmatic area, RM: retro-mamillary nuclei, SCh: suprachiasmatic nucleus, SSC: somatosensory cortex, SuC: superior colliculus (optic nerve layer and superficial grey layer), VC: visual cortex, WM: white matter. Scale bars: 5 mm in A; 50 µm in B.

https://doi.org/10.7554/eLife.32017.006
Figure 2—figure supplement 2
At postnatal ages, Hmx3; tdTOM+/Htr3a-GFP+ cells are rarely observed in the preoptic nuclei (PoN), the subpallial brain region corresponding to the embryonic preoptic area.

(A–B) Hmx3; tdTOM+ cells negative for Htr3a-GFP are present in PoN at P5 (A), and P21 (B) where rare Hmx3; tdTOM+/Htr3a-GFP+ were found (high magnified images). AC: anterior commissure. Scale bars: 300 µm in A, B: low magnified images; 50 µm in A, B: high magnified images.

https://doi.org/10.7554/eLife.32017.007
Figure 2—figure supplement 3
Hmx3; tdTOM+ cells negative for Htr3a-GFP often express glial markers.

(A) The fraction of Hmx3; tdTOM+ cells negative for Htr3a-GFP is relatively stable across postnatal ages. (B–D) Hmx3; tdTOM+ cells negative for Htr3a-GFP express the astrocytic markers GFAP (B; arrows), S100β (C; arrow) and the oligodendrocytic transcription factor SOX10 (D; arrows). In contrast, Hmx3; tdTOM+/Htr3a-GFP+ cells are negative for these markers (B-D; arrowheads). Scale bar: 50 µm in B, C, D.

https://doi.org/10.7554/eLife.32017.008
Figure 2—figure supplement 3—source data 1

Detailed counts of cells quantified in Figure 2—figure supplement 3 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.009
Figure 3 with 1 supplement
5-HT3AR-expressing interneurons (INs) largely originate from Hmx3+ but not Dbx1+ cells.

(A–C) A consistent fraction of cortical Htr3a-GFP+ INs co-labels with Hmx3; tdTOM (A, arrowheads) at P5 and P21, whereas only a minimal fraction does with Dbx1; tdTOM (B, arrowhead). (D) Dbx1; tdTOM+ INs express the MGE-enriched TF SOX6 (arrowhead) but not the CGE-enriched TF PROX1 (open arrowheads). Only a minimal fraction of Dbx1; tdTOM+ co-labelled for Htr3a-GFP, among which the majority were PROX1+. (E–F) In situ hybridization showing that Dbx1; tdTOM+ INs express the transcript for the MGE-enriched TF Lhx6 (E, arrowheads) whereas Htr3a-GFP+ INs do not (E, open arrowheads). In contrast, Dbx1; tdTOM+ INs do not express the Htr3a transcript (F, arrowheads) whereas Htr3a-GFP+ INs do (F, open arrowheads). Scale bars: 100 µm in A, B, E, F: low magnified images; 50 µm in A, B, D, E, F: high magnified images.

https://doi.org/10.7554/eLife.32017.011
Figure 3—source data 1

Detailed counts of cells quantified in Figure 3 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.013
Figure 3—figure supplement 1
Interneurons (INs) derived from Dbx.1+ cells express MGE-enriched markers.

(A–B) Dbx1; tdTOM+ cells negative for Htr3a-GFP express the neurochemical markers somatostatin (SST) (A, arrowheads) and parvalbumin (PV) (B, arrowheads). WM: white matter. Scale bars: 100 µm in A, B: low magnified images; 50 µm in A, B: high magnified images.

https://doi.org/10.7554/eLife.32017.012
Figure 4 with 1 supplement
Hmx3; tdTOM+/Htr3a-GFP+ cortical interneurons (INs) express markers related to the CGE but not to the MGE.

(A–B) Hmx3; tdTOM+/Htr3a-GFP+ INs express the CGE-enriched TF PROX1 (A; arrowheads) but not the MGE-related TF SOX6 (A; open arrowheads) similarly to Htr3a-GFP+ INs that do not derive from Hmx3+ cells (B, right graph). (C–D) Hmx3; tdTOM+/Htr3a-GFP+ cells express the CGE-enriched TFs NR2F2 (arrowheads) and (open arrowheads)/or SP8 (arrow). Htr3a-GFP+ derived from Hmx3+ cells are biased towards NR2F2 expression, in comparison to Htr3a-GFP+ INs that do not co-label with Hmx3; tdTOM (D, blue bars). Scale bars: 100 µm in A, C: low magnified images; 50 µm in A, C: high magnified images.

https://doi.org/10.7554/eLife.32017.014
Figure 4—source data 1

Detailed counts of cells quantified in Figure 4 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.016
Figure 4—figure supplement 1
Hmx3; tdTOM+/Htr3a-GFP+ cortical interneurons (INs) display a combinatorial expression of NR2F2 and SP8 that is biased toward NR2F2.

(A) Hmx3; tdTOM+/Htr3a-GFP+ INs (A; arrowheads) express significantly (one-way ANOVA; *p<0.05) more NR2F2 alone and less SP8 alone as compared to Htr3a-GFP+ INs negative for Hmx3; tdTOM (A; open arrowheads). Scale bar: 100 µm in A.

https://doi.org/10.7554/eLife.32017.015
Figure 5 with 1 supplement
Hmx3; tdTOM+/Htr3a-GFP+ cortical interneurons (INs) express reelin and NPY but not VIP.

(A–F) Hmx3; tdTOM+/Htr3a-GFP+ INs are stained with the neurochemical markers reelin (A, arrowheads) and NPY (B, arrowheads) as well as Htr3a-GFP+ cells negative for Hmx3; tdTOM (open arrowheads). Note that reelin-positive Hmx3; tdTOM+/Htr3a-GFP+ INs are preferentially found in L1-3 (C) whereas NPY-expressing Hmx3; tdTOM+/Htr3a-GFP+ INs are mainly found in L2-6 (E). Hmx3; tdTOM+/Htr3a-GFP+ INs account for more than one third of all reelin-positive Htr3a-GFP+ (D) and of all NPY-positive Htr3a-GFP+ cells (F). (G–H) Hmx3; tdTOM+/Htr3a-GFP+ INs mainly express reelin (G, arrowheads) or reelin and NPY (G, open arrowheads) but to a smaller extend only NPY (G, arrow). (I) Hmx3; tdTOM+/Htr3a-GFP+ INs do not express VIP (arrowheads), whereas Htr3a-GFP+ INs negative for Hmx3; tdTOM do (open arrowheads). WM: white matter. Scale bars: 100 µm in A, B: low magnified images; 50 µm in A, B: high magnified images, G, I.

https://doi.org/10.7554/eLife.32017.017
Figure 5—source data 1

Detailed counts of cells quantified in Figure 5 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.019
Figure 5—figure supplement 1
Hmx3; tdTOM+/Htr3a-GFP+ do not express MGE-enriched markers.

(A–B) Hmx3; tdTOM+/Htr3a-GFP+ cells do not express the neurochemical markers somatostatin (SST) (A, arrowheads) and parvalbumin (PV) (B, arrowheads). WM: white matter. Scale bars: 100 µm in A, B: low magnified images; 50 µm in A, B: high magnified images.

https://doi.org/10.7554/eLife.32017.018
Figure 6 with 4 supplements
Hmx3; tdTOM+/Htr3a-GFP+ cortical interneurons (INs) in layer 1(L1) display the molecular, morphological and electrophysiological features of neurogliaform cells (NGCs).

(A) RNAscope multiplex fluorescent hybridization for tdTOM, GFP and Car4 transcripts on P30 brains showing that L1 Hmx3; tdTOM+/Htr3a-GFP+ INs express Car4 at significantly higher levels (orange outline) as compared to Htr3a-GFP+ INs negative for tdTOM (green outline) (***p<0.0001; Mann-Whitney test). (B) Example of a L1 Hmx3; tdTOM+/Htr3a-GFP+ cell (arrowhead) or Htr3a-GFP+ INs negative for tdTOM (open arrowheads) checked before patching (left image). Example of a patched Hmx3; tdTOM+/Htr3a-GFP+ IN (middle and right images, arrowhead). (C) Illustrative reconstruction of a Hmx3; tdTOM+/Htr3a-GFP+ IN in L1 displaying the characteristic morphology of an elongated NGC with dense axonal ramifications restricted to L1. (D) Illustrative reconstruction of a Htr3a-GFP+ IN negative for tdTOM in L1 displaying the characteristic morphology of single bouquet-like cell (SBC) with axonal ramifications extending deep into L5. (E) Illustrative traces from recorded Hmx3; tdTOM+/Htr3a-GFP+ INs (orange) and Htr3a-GFP+ INs negative for tdTOM (green), showing the first action potentials (APs) at rheobase and trains of APs at higher current injections. (F) Superimposed single AP traces at rheobase of all Hmx3; tdTOM+/Htr3a-GFP+ INs (orange) and Htr3a-GFP+ INs negative for tdTOM (green). Thick traces correspond to type 1A and type 2A examples in E. (G) Same traces as in (F), aligned to the AP, with a lower time scale. Thin lines are individual cell traces and thick lines are trace averages. The average traces on the right are aligned to the threshold potential (Vthr). (H) Plots of AP peak amplitude (Peak; ***p<0.0001; unpaired t-test), after hyperpolarization potential amplitude (AHP; ***p<0.0001; unpaired t-test) and membrane resistance (Rm; *p=0.0318; Mann-Whitney test) showing significant differences between the two cell types. (I) Absolute linear weights assigned by the classification model trained on all cells with standardized electrophysiological properties. (J) Prediction probabilities estimated by the classifier on the cell left out in the leave-one-out-cross-validation (LOOCV) loop. Cells are ordered on the x-axis by origin and prediction value, and the color code reflect their origin. Cells above the probability threshold 0.5 are more likely to be Hmx3-derived according to the model. Scale bars: 10 µm A; 20 µm in B; 100 µm C, D.

https://doi.org/10.7554/eLife.32017.020
Figure 6—source data 1

Detailed counts of cells expressing Car4 quantified in Figure 6 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.025
Figure 6—source data 2

Electrophysiological properties of cells quantified in Figure 6.

https://doi.org/10.7554/eLife.32017.026
Figure 6—figure supplement 1
Hmx3; tdTOM+/Htr3a-GFP+ interneurons (INs) in layer 1 (L1) display the characteristic morphology of elongated neurogliaform cells (eNGCs).

Illustrative images confirming location of the cells, morphological reconstructions and electrophysiological traces of Hmx3; tdTOM+/Htr3a-GFP+ INs in L1. All cells have the morphology of eNGCs with dense axonal ramifications mostly restricted to layer 1. One cell (*) was excluded from the analysis of electrophysiological properties due to the presence of two peaks at the first 5 pA increment showing an action potential, but still its biocytin filling was good enough for morphological tracing (bottom right). Scale bars: 100 µm in all brightfield images and reconstructions.

https://doi.org/10.7554/eLife.32017.021
Figure 6—figure supplement 2
Prediction model parameters and electrophysiological features of Hmx3; tdTOM+/Htr3a-GFP+ interneurons (INs) in layer 1.

(A) Density plots of the electrophysiological values as well as of the LOOCV prediction probabilities for Hmx3-derived cells and non Hmx3-derived cells. (B) Receiver operating characteristic (ROC) curve for each electrophysiological property and on prediction probabilities obtained by LOOCV. (C) Significant difference is found between Hmx3-derived cells and non Hmx3-derived cells for threshold potential (Vthr; **p=0.0097; unpaired t-test) but not for delay to the first action potential (Delay; p=0.55; Mann-Whitney test) and resting potential (Vrest; p=0.68; Mann-Whitney test). Afterdepolarization potential (ADP) is0 for all Hmx3-derived cells.

https://doi.org/10.7554/eLife.32017.022
Figure 6—figure supplement 3
Hmx3; tdTOM+/Htr3a-GFP+ interneurons (INs) in cortical layers 2–6 (L2-6) display molecular and electrophysiological properties of NGCs.

(A, B) RNAscope multiplex fluorescent hybridization for tdTOM, GFP and Car4 transcripts on P30 brains showing that Hmx3; tdTOM+/Htr3a-GFP+ INs (red outline) express Car4 at significantly higher levels in L2–4 (A, ***p<0.0001; Mann-Whitney test) and L5/6 (B, ***p<0.0001; Mann-Whitney test) as compared to Htr3a-GFP+ INs negative for tdTOM (green outline). (C) Superimposed single AP traces at rheobase of Hmx3; tdTOM+/Htr3a-GFP+ INs recorded in L1 (orange) and in L2–6 (red). Hmx3; tdTOM+/Htr3a-GFP+ INs from L2–6 exhibit more late-spiking profile. (D) Same traces as in (C), aligned to the AP, in a lower time scale. Thin lines are individual cell traces and thick lines are trace averages. The average traces on the right are aligned to the threshold potential (Vthr). (E) As compared to L1 cells, Hmx3; tdTOM+/Htr3a-GFP+ INs in L2–6 display significant differences in afterhyperpolarization amplitude (AHP; ***p=0.0001; Mann-Whitney test), delay to the first action potential (Delay; *p=0.0202; unpaired t-test) and Vthr (*p=0.0002; unpaired t-test). No significant differences were found between Hmx3; tdTOM+/Htr3a-GFP+ INs in L2–6 and L1 for peak amplitude (Peak; p<0.1058; unpaired t-test), membrane resistance (Rm; p=0.5011; unpaired t-test) and resting potential (Vrest; p=0.7056; unpaired t-test), Scale bars: 10 µm in all.

https://doi.org/10.7554/eLife.32017.023
Figure 6—figure Supplement 3—source data 1

Detailed counts of cells expressing Car4 quantified in Figure 6—figure supplement 3 in the different experimental conditions.

https://doi.org/10.7554/eLife.32017.027
Figure 6—figure Supplement 3—source data 2

Electrophysiological properties of cells quantified in Figure 6—figure supplement 3.

https://doi.org/10.7554/eLife.32017.028
Figure 6—figure supplement 4
Hmx3; tdTOM+/Htr3a-GFP+ interneurons (INs) in cortical layers 2–6 (L2-6) display the characteristic morphology of NGCs.

Illustrative images confirming location of the cells, morphological reconstructions and electrophysiological traces of Hmx3; tdTOM+/Htr3a-GFP+ INs in L2–6. All cells have the morphology of NGCs with dense axonal ramifications extending throughout layers. Two cells (*) were excluded from the analysis of electrophysiological properties due to high series resistance, but still their biocytin filling was good enough for morphological tracing (left, upper two panels). Scale bars: 100 µm in all.

https://doi.org/10.7554/eLife.32017.024
Developmental origin of cardinal classes of cortical interneurons.

The Martinotti somatostatin (SST) cell, the parvalbumin (PV) basket cell and the PV+ chandelier cell originate from NKX2.1+ progenitors of the medial ganglionic eminence (MGE, blue) and rely on the transcription factors (TFs) LHX6 and SOX6. The Htr3a-GFP/reelin (RELN) single-bouquet cell (SBC), the Htr3a-GFP/vasointestinal peptide (VIP)/cholecystokinin (CCK) basket cell and the Htr3a-GFP/VIP/calretinin (CR) bipolar cell are derived from cells located in the caudal ganglionic eminence (CGE, green) that express the TFs PROX1, NR2F2 and SP8. The RELN/Car4/Htr3a-GFP neurogliaform cell (NGC) is specifically derived from Hmx3+ cells located in the preoptic area (POA, orange) and express the TFs PROX1 and NR2F2.

https://doi.org/10.7554/eLife.32017.029

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus Musculus)Tg(Htr3a-EGFP)DH30Gsat (referred as Htr3a-GFP)GENSAT ConsortiumMGI:3846657Maintained on a C57Bl/6 background
Strain, strain background (Mus Musculus)B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (referred as R26R-tdTOMfl/fl)The Jackson LaboratoryMGI:104735Maintained on a C57Bl/6 background
Strain, strain background (Mus Musculus)Tg(Hmx3-icre)1Kessprovided by Oscar MarinMGI:5566775Maintained on a C57Bl/6 background
Strain, strain background (Mus Musculus)Dbx1tm2(cre)Apie (referred as Dbx1-Cre)provided by Alessandra PieraniMGI:3757955Maintained on a C57Bl/6 background
AntibodyAnti-GFAP, rabbit polyclonalAbcam, United Kingdomab7260(1:2000)
AntibodyAnti-GFP, rabbit polyclonalMillipore, GermanyAB3080(1:500)
AntibodyAnti-GFP, goat polyclonalAbcamab5450(1:2000)
AntibodyAnti-NeuN (clone A60), mouse monoclonalMilliporeMAB377(1:500)
AntibodyAnti-NKX2.1 (H-190), rabbit polyclonalSanta Cruz Biotechnology, Dallas, TXsc-13040(1:100)
AntibodyAnti-NPY, rabbit polyconalAbcamab10980(1:500)
AntibodyAnti-NR2F2, rabbit polyclonalAbcamab42672(1:500) antigen retrieval (Citrate buffer pH 6.0; 85°C; 20 min)
AntibodyAnti-PROX1, goat polyclonalR&D System,Minneapolis, MNAF2727(1:250)
AntibodyAnti-PV, mouse monoclonalSwant, SwitzerlandPV235(1:2000)
AntibodyAnti-Reelin, mouse monoclonalAbcamab78540(1:500)
AntibodyAnti-S100β, rabbit polyclonalAbcamab41548(1:2000)
AntibodyAnti-SST, rat monoclonalMilliporeMAB354(1:500)
AntibodyAnti-SOX6, rabbit polyclonalAbcamab30455(1:500)
AntibodyAnti-SOX10 (N-20), goat polyclonalSanta Cruz Biotechnologysc-17343(1:100)
AntibodyAnti-SP8 (C-18), goat polyclonalSanta Cruz Biotechnologysc-104661(1:50) antigen retrieval (Citrate buffer pH 6.0; 85°C; 20 min)
AntibodyAnti-tdTOM, goat polyclonalSicgen, PortugalAB8181-200(1:500)
AntibodyAnti-VIP, rabbit polyclonalAbcamab22736(1:500) ASCF perfusion; 2 hr PFA 4% postfixation
AntibodyDonkey anti-rabbit Alexa
Fluor488
Invitrogen, Carlsbad, CAA21206(1:500)
AntibodyDonkey anti-goat Alexa
Fluor488
InvitrogenA11055(1:500)
AntibodyDonkey anti-goat Alexa
Fluor405
Abcamab175664(1:500)
AntibodyDonkey anti-rabbit Alexa
Fluor405
Abcamab175651(1:500)
AntibodyDonkey anti-rabbit Alexa Fluor647InvitrogenA31573(1:500)
AntibodyDonkey anti-rat Alexa Fluor647InvitrogenA21247(1:500)
AntibodyDonkey anti-mouse Alexa Fluor647InvitrogenA31571(1:500)
AntibodyDonkey anti-goat Alexa Fluor647InvitrogenA21447(1:500)
AntibodyStreptavidin, Alexa Fluor647-conjugatedThermoFisher/InvitrogenS21374(1:500)
AntibodyAnti-Digoxigenin-AP, Fab fragmentRoche, Switzerland11082736103(1:2000)
Recombinant DNA reagentHtr3a plasmid probeGift from Dr. B. EmeritNALinearization: HindIII-HF; antisense synthesis: T7; concentration 1 µg
Recombinant DNA reagentLhx6 plasmid probeGift from Dr. M. DenaxaNALinearization: Not1; antisense synthesis: T3; concentration 1 µg
Recombinant DNA reagentRNAScope Probe-tdTomato-C2Affimetrix, Santa Clara, CA317041-C2
Recombinant DNA reagentRNAScope Probe-EGFPAffimetrix400281
Recombinant DNA reagentRNAScope Probe-Car4-C3Affimetrix468421-C3
Sequence-based reagentGenotyping PCR primer forB6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JMicrosynth, Switzerland
(desalted;100 µM stock)
WT-F (oIMR9020): AAG GGA GCT GCA GTG GAG TAhttps://www2.jax.org/protocolsdb/f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:29436,007909
Sequence-based reagentGenotyping PCR primer forB6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JMicrosynth (desalted;100 µM stock)WT-R (oIMR9021): CCG AAA ATC TGT GGG AAG TC
Sequence-based reagentGenotyping PCR primer forB6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JMicrosynth (desalted;100 µM stock)Mut-R (oIMR9103): GGC ATT AAA GCA GCG TAT CC
Sequence-based reagentGenotyping PCR primer forB6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JMicrosynth (desalted;100 µM stock)Mut-F (oIMR9105): CTG TTC CTG TAC GGC ATG G
Sequence-based reagentGenotyping PCR primer for Tg(Htr3a-EGFP)DH30GsatMicrosynth (desalted;100 µM stock)Com-F (273): GCA AGA TGT GAC CAA GCC ACC TAT TThttp://www.med.unc.edu/mmrrc/resources/genotyping-protocols/mmrrc-273
Sequence-based reagentGenotyping PCR primer for Tg(Htr3a-EGFP)DH30GsatMicrosynth (desalted; 100 µM stock)WT-R: CAG CCC TCA GCC CTT TGA GAC TTA AG
Sequence-based reagentGenotyping PCR primer for Tg(Htr3a-EGFP)DH30GsatMicrosynth (desalted; 100 µM stock)Mut-R: TGA ACT TGT GGC CGT TTA CGT CG
Sequence-based reagentGenotyping PCR primer forTg(Hmx3-icre)1KessMicrosynth (desalted;100 µM stock)Mut-F: CTC TGA CAG ATG CCA GGA CA
Sequence-based reagentGenotyping PCR primer forTg(Hmx3-icre)1KessMicrosynth (desalted;100 µM stock)Mut-R: TCT CTG CCC AGA GTC ATC CT
Sequence-based reagentGenotyping PCR primer forDbx1tm2(cre)ApieMicrosynth (desalted;100 µM stock)WT-F (1307): GCA AGG AAA TGT CTC TGG GAChttps://www.infrafrontier.eu/sites/infrafrontier.eu/files/upload/public/pdf/genotype_protocols/EM01924_geno.pdf
Sequence-based reagentGenotyping PCR primer forDbx1tm2(cre)ApieMicrosynth (desalted;100 µM stock)WT-R (1115): GAG GAT GAG GAA ATC ACG GTG
Sequence-based reagentGenotyping PCR primer forDbx1tm2(cre)ApieMicrosynth (desalted;100 µM stock)Mut-F (cre83): GTC CAA TTT ACT GAC CGT ACA CC
Sequence-based reagentGenotyping PCR primer forDbx1tm2(cre)ApieMicrosynth (desalted;100 µM stock)Mut-R (cre85): GTT ATT CGG ATC ATC AGC TAC ACC
Commercial assay or kitVECTASTAIN Elite ABC-HRP KitVectorLab, Burlingame, CAPK-6100Manufacturer's protocol
Commercial assay or kitDAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3’-diaminobenzidineVectorLabSK-4100Manufacturer's protocol
Commercial assay or kitRNAscope Fluorescence Multiplex Reagent KitAdvanced Cell Diagnostics, Newark, CA320850Manufacturer's protocol (fresh frozen tissue)
Chemical compound, drugNε-(+)-Biotinyl-L-lysine (biocytin)Sigma Aldrich, GermanyB4261Used at 8.1 mM
Chemical compound, drugFast Red tabletsKem-En-Tech, Denmark4210Manufacturer's protocol
Chemical compound, drugHoechst 33258Sigma Aldrich23491-45-4(1:10000)
Chemical compound, drugThiopental Inresa 0.5 gInresa Arzneimittel GmbH, GermanyUsed at 50 mg/kg
Software, algorithmMicrosoft Office 2017 (Excel, Word)© 2017 Microsoft, Redmond, WAv.16.9.1Manuscript editing
Software, algorithmAdobe Suit CC (Photoshop, Illustrator, Acrobat)Adobe Systems, San José, CAv.22.0.1Image treatment, figure editing
Software, algorithmGraphPad PrismGraphPad software, Inc., La Jolla, CAv.7.0Statistics, graph editing
Software, algorithmFijidoi:10.1038/nmeth.2019v.2.0.0Image editing, manual counting
Software, algorithmEndNote XThomson Reuters, Canadav.7.7.1Reference editing, bibliography
Software, algorithmNeurolucida v.11.02.1Microbrightfield, MBF Bioscience, Williston, VTv.11.02.1Neuron reconstruction
Software, algorithmNeurolucida ExplorerMicrobrightfield, MBF Biosciencev.11.02.1Morphological reconstruction editing
Software, algorithmMatLabMathWorks, Natick, MAElectrophysiological recordings measurment/editing
Software, algorithmEphus (MATLAB-based)doi: 10.3389/fncir.2010.00100v. 2.1.0Electrophysiological recordings data aquisition
Software, algorithmClampfitMolecular Devices, San José, CAv. 10.1.0.10Electrophysiological recordings offline analysis
Software, algorithmR programming languagewww.R-project.orgv. 3.4.0Statistics
Software, algorithmR package bmrmdoi:10.1038/ncomms14219v. 3.5Prediction model
Software, algorithmNLMorpholoyConverterhttp://neuronland.org/NLMorphologyConverter/NLMorphologyConverter.htmlv. 0.8.1Morphological reconstruction editing
Software, algorithmR package NeuroAnatomy Toolboxdoi: 10.1016/j.neuron.2016.06.012v. 1.8.12.9000Morphological reconstruction editing

Additional files

Supplementary file 1

Detailed description of the technical (brains) and biological (cells) replicates used in the different experiments.

https://doi.org/10.7554/eLife.32017.030
Transparent reporting form
https://doi.org/10.7554/eLife.32017.031

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