Animal procedures were conducted in strict compliance with approved institutional protocols and in accordance with the provisions for animal care and use described in the European Communities Council directive of 24 November 1986 (86-16-09/EEC). The day of vaginal plug detection was counted as embryonic day (E) E 0.5. Two transgenic mouse lines expressing the enhanced GFP under the control of the 5-HT3A
:GFP) obtained by using modified BACs were used: The first one has been generated in H. Monyer's laboratory (Inta et al. 2008
), was maintained under the C57/Bl6 background, and was mainly used to assess the feasibility of the project. The second transgenic mouse line Tg(Htr3a-GFP)1Gsat was provided by the GENSAT Consortium (Rockeffeler University-GENSAT Consortium; Heintz 2004
) and was maintained under the Swiss background. Both strains gave identical expression patterns in cortical areas and match mRNAs expression. Genitors were polymerase chain reaction (PCR) genotyped for GFP insertion using the primers (from 5′ to 3′): ATGGTGAGCAAGGGCGAGGAGCT and GCCGAGAGTGATCCCGGCGGCGGT. Embryos were phenotyped by macroscopic observation under fluorescent optics. Characterization of juvenile 5-HT3A
-expressing interneurons was performed using the Tg(Htr3a-GFP) mouse line provided by GENSAT. Both the GENSAT and Hannah Monyer's mouse lines were used for analysis of embryonic GFP expression and grafting experiments and gave similar results.
Radioactive In Situ Hybridization
cRNA probe corresponded to the full-length domain of the protein. The plasmid was linearized with Bam
H1 for antisense RNA synthesis by T7 polymerase and with Eco
RI for sense RNA synthesis by T3 polymerase. The Dlx2
cRNA probe (Hin
d3 linearization, T7 polymerization) was also used. The transcription was carried out using the Promega kit, and probes were labeled with 35
S-UTP (>1000 Ci/mmol; Amersham). Hybridization was performed on fresh frozen brain sections (15 μm thick) as previously described (Fontaine and Changeux 1989
). Slides were dipped in photographic emulsion (NTB2, Kodak) and exposed for about 5–10 days. Emulsions were then developed, and sections were Nissl counterstained (0.25 % thionin solution).
The laminar density of cells expressing the 5-HT3A mRNA was estimated at P25. Quantifications of 5-HT3A+ cells were performed at the level of the primary somatosensory cortex, in 500-μm-wide cortical strips (data are expressed as percentage). Three adjacent sections of at least 5 independent preparations were used.
Neuronal populations expressing 5-HT3A:GFP were analyzed at embryonic (E13/E13.5 [n = 8], E14.5 [n = 12], E15.5 [n = 8], E16.5 [n = 12], E17.5 [n = 10], and E18.5 [n = 10]), and postnatal stages (P0 [n = 14], P1 [n = 8], P3 [n = 10], P5 [n = 6], P15–P16 [n = 10], and P25 [n = 14]). Embryos collected by cesarean section after cervical dislocation of the dam were placed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 (PFA). Embryos were cryoprotected, embedded into gelatine (7%)–sucrose (10%), frozen into isopentane (−40°C) and sectioned coronally (20 μm) with a cryostat. Postnatal animals were deeply anesthetized with an intraperitoneal (IP) injection of pentobarbital (150 mg/kg body weight) and perfused transcardially with 4% PFA. Postnatal brains were cryoprotected in 30% sucrose and cut on a freezing microtome (35 μm). For immunofluorescence, sections were incubated overnight at 4 °C with the following antibodies diluted in saline PB (PBS): rabbit anti-CR (1:8000, Swant), rabbit anti-Parv (1:1000; Swant), rabbit anti-SOM (1:500; a kind gift of Dr Epelbaum), rabbit anti-NPY (neuropeptide Y) (1:500, Amersham), rabbit anti-VIP (1:800, Incstar), rabbit anti-Nr2F2 (1:200; Acris GmbH), rabbit anti-GABA (1:5000; Sigma), rabbit anti-GFP (1:1000, Molecular Probes), mouse anti-tuj1 (1:2000, Babco), or mouse anti-Parv (1:200, Sigma). After washing in PBS, sections were incubated with Cy3-conjugated goat antirabbit or/and antimouse antibodies (1:200; Jackson Laboratory). Sections were rinsed in PB, mounted in Vectashield (Vector) containing 4′,6′-diamidino-2-phénylindole (Dapi) and were observed with a fluorescent microscope (Leica, DMR). Images were acquired with a Coolsnap camera (Photometrics, Tucson, AZ) and analyzed with the Metamorph software (Molecular Devices, Foster City, CA).
The laminar density of cells expressing GFP was estimated at P25. Quantifications of GFP:5-HT3A+ cells were performed at the level of the primary somatosensory cortex, in 500-μm-wide cortical strips (data are expressed as percentage). Three adjacent sections of at least 5 independent preparations were used.
The proportion of green fluorescent cells labeled for GABA at E14.5 (n = 9) in the low intermediate zone (LIZ) was estimated at 2 different levels, 1 rostral including MGE and 1 caudal including CGE. For each case, data obtained from 3 adjacent sections were averaged.
Preparation of Juvenile Brain Slices and Electrophysiological Recordings of 5-HT3A-Expressing Cells
5-HT3A:GFP transgenic mice (postnatal days 14–18) were decapitated, brains were quickly removed and placed into cold (~4 °C) artificial cerebrospinal fluid (ACSF) containing (in mM): 110 choline chloride, 11.6 Na-ascorbate, 7 MgCl2, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2, continuously bubbled with 95%O2–5%CO2. Coronal brain slices (300 μm thick) containing the somatosensory cortex were cut with a vibratome (VT1200S; Leica, Nussloch, Germany), and transferred to a holding chamber containing ACSF (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 20 glucose, and 1 kynurenic acid (nonspecific glutamate receptor antagonist, Sigma), constantly oxygenated (95% O2/5% CO2) and held at room temperature.
Individual slices were placed in a submerged recording chamber and perfused (1–2 mL/min) with oxygenated ACSF (in the absence of kynurenic acid). Patch micropipettes pulled from borosilicate glass capillaries (3–5 MΩ) were filled with 8 μL of autoclaved reverse transcription polymerase chain reaction (RT-PCR) internal solution (in mM): 144 K-gluconate, 3 MgCl2, 0.5 ethylene glycol tetraacetic acid, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2, 285/295 mOsm), and 3 mg/mL biocytin for intracellular labeling. Neurons were visualized in the slice using infrared transmitted light with Dodt gradient contrast optics or epifluorescence illumination, using a Zeiss (Axioskop 2FS) microscope equipped with a ×40 water-immersion objective. Images were captured with CoolSnap HQ2 CCD camera (Photometrics) controlled by Image-Pro 5.1 software (Media Cybernetics Inc., San Diego, CA). Just before breaking the seal, GFP expression in the targeted cell was rechecked by fluorescence detection. Whole-cell recordings in current-clamp mode were performed at room temperature using a patch-clamp amplifier (Multiclamp 200B, Molecular Devices). Data were filtered at 5 kHz and digitized at 50 kHz using an acquisition board (Digidata 1322A, Molecular Devices) attached to a computer running pCLAMP 9.2 software package (Molecular Devices). All membrane potentials were not corrected for liquid junction potential.
For each recorded neuron, 28 electrophysiological parameters were quantified using custom written routines running within IgorPro (Wavemetrics). The detailed procedures are described in the Supplementary Methods S1
Single-Cell Reverse Transcription Polymerase Chain Reaction (scRT-PCR)
At the end of the recording, the cell's cytoplasm was aspirated into the recording pipette while maintaining the tight seal. Then, the pipette was removed delicately to allow outside-out patch formation. Next the content of the pipette was expelled into a test tube, and reverse transcription (RT) was performed in a final volume of 10 μL as described previously (Lambolez et al. 1992
). The scRT-PCR protocol was designed to detect simultaneously the expression of the 2 isoforms of glutamic acid decarboxylase (GAD65
), 3 genes encoding for calcium-binding proteins: calbindin D28k (CB), CR, and Parv, 3 neuropeptides NPY, SOM, and VIP, 2 transcription factors (Lhx6 and Nr2F2), and the protein reelin implicated in neuronal migration and morphology (Chameau et al. 2009
). The next 2 steps of PCR were performed essentially as described previously (Ruano et al. 1995
). The cDNAs present in 10 μL of the RT reaction were first simultaneously amplified by using all of the primer pairs described in Supplementary Table S1
(for each primer pair, the sense and antisense primers were positioned on 2 different exons). GoTaq polymerase (2.5 U; Promega, Madison, United States) and 20 pmol of each primer were added to the buffer supplied by the manufacturer (final volume, 100 μL), and 21 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 35 s) of PCR were run. Second rounds of amplification were performed using 2 μL of the first PCR product as template. In this second round, each cDNA was amplified individually with a second primer pair internal to the pair used in the first PCR, excepted for Nr2F2
(nested primers, see Supplementary Table S1
). Thirty-five PCR cycles were performed as described earlier (Cauli et al. 1997
). Then, 10 μL of each individual PCR product were run on a 2% agarose gel using 100-bp ladders (Promega) as molecular weight marker and stained with ethidium bromide. All the transcripts were detected from 500 pg of neocortical RNA using this protocol (data not shown). The sizes of the PCR-generated fragments were as predicted by the mRNA sequences (see Supplementary Table S1
Intracellular Labeling and Morphological Reconstructions
Slices containing recorded neurons filled with biocytin were fixed overnight at 4 °C in 4% PFA. The morphology of the recorded neurons was investigated by histochemical labeling of intracellular biocytin with diaminobenzidine (DAB) by using the ABC elite kit (Vector Laboratories, Burlingame, CA). After blocking endogenous peroxidase with 0.3% H2O2 in PB 0,1 M for 15–30 min, slices were rinsed in PB (4× 10 min), permeabilized in 2% Triton X-100 in PBS for 1 h, and incubated in AB diluted 1:200 in PBS and 1% Triton X-100 for 2 h. Slices were then washed in PBS (6× 10 min). For the visualization of the stain, the sections were incubated with the DAB reagent for ABC (elite kit, Vector) containing 0.05% DAB and 0.01% H2O2 in PBS. The reaction was monitored under a dissecting microscope and stopped by rinsing in PBS (4× 10 min) when the cell body and dendritic processes were clearly visible. The slices were then mounted in 50%PBS–50%glycerol, coverslipped, and sealed with nail polish. Biocytin-filled neurons were visualized, traced, and digitally reconstructed using the Neurolucida software (MicroBrightField, Bioscience Europe, Magdeburg, Germany) with a ×100 oil-immersion objective (Leica). The morphological parameters, quantified as mean ± standard deviation (SD) were compared for significance using Student's test. The average tortuosity of the neurons was calculated as the ratio between the distance along a process over the straight line distance, the smallest tortuosity possible being 1 for a straight path. The spatial distribution of the dendritic processes from the centroid of the cell body was studied using Wedge analysis. For this purpose, the xy plane was divided into 12 equiangular wedges. The total dendritic length in each wedge was then quantified (taking into account the z information) and displayed as a round directional histogram. The total length over all wedges corresponds to the total length of the neuronal dendritic arborization. Finally, in order to quantify the orientation of the analyzed neurons, the total dendritic length contained in the 4 most horizontally oriented wedges was divided by the length enclosed in the 4 most vertically oriented wedges. This ratio, called here “equipolarity,” would be equal to 1 for a perfectly radially oriented dendritic arbor and decreases with more vertically oriented processes.
Electrophysiological Statistical Analysis
All data are presented as mean ± SD unless otherwise stated. Mann–Whitney U
test was employed to compare electrophysiological properties between cell types. P
values of ≤0.05 were considered statistically significant. To classify 5-HT3A
-expressing neocortical neurons sampled without a priori knowledge, unsupervised clustering was performed using 28 electrophysiological parameters (see Supplementary Methods S1
) and the laminar location determined by infrared videomicroscopy. For neurons located at the border of layers I–II and II–III, the laminar location was digitized by 1.5 and 2.5, respectively. After standardizing the data, cluster analysis was performed using squared Euclidean distances and Ward's method linkage rules (Ward 1963
). Ward's clustering method has been used successfully by previous studies to define neuronal classes based on multiple electrophysiological, molecular, and/or morphological features (Tamas et al. 1997
; Cauli et al. 2000
; Karube et al. 2004
; Dumitriu et al. 2006
; Gallopin et al. 2006
; Halabisky et al. 2006
; Dávid et al. 2007
; Andjelic et al. 2008
; Helmstaedter et al. 2009
; Karagiannis et al. 2009
). Thorndike analysis of the critical threshold was conducted to suggest the likely number of different clusters in the data set (Thorndike 1953
). Descriptive statistics and cluster analysis were calculated with Statistica v 6.0 (Statsoft, Tulsa, OK).
Birth Dating In Vivo
Pregnant females of the Swiss genetic background received a single 5-bromo-2-deoxyuridine (BrdU) injection (IP, 50 mg/kg; in 0.9% NaCl) at gestational days E11.5, E12.5, E13.5, E14.5, E15.5, or E16.5. Animals, aged P25, were anesthetized as described above and perfused transcardially with 4% PFA. Cryosections (17 μm thick) were first processed for in situ hybridization to reveal 5-HT3A
mRNA transcripts and then processed for immunocytochemistry to detect BrdU. Nonradioactive in situ hybridization was performed as described in Schaeren-Wiemers and Gerfin-Moser (1993)
(products were purchased from Roche Diagnostics). Sense probes were used as control and did not show any labeling. Subsequently, sections were treated with 2 N HCl for 45 min, rinsed in 0.1 M PBS, pH 7.4, incubated during 1 h in PBS supplemented by normal goat serum (10%) and incubated overnight with anti-BrdU (1:100, Progen, GmbH, Germany) in PBS. After washing, sections were incubated 2 h in Alexa Fluor 488 goat antimouse antibody (1:200, Invitrogen) and were mounted in Vectashield containing Dapi. To estimate the number of BrdU-labeled cells among the 5-HT3A
-expressing population, a least 3 litters were analyzed per time point, and at least 2 animals per litter were processed for histology. For each anatomical area selected, 5 adjacent sections were analyzed per case. On each section, the number of 5-HT3A
+ neurons heavily labeled for BrdU (defined as having >50% of the nucleus immunolabeled; Gillies and Price 1993
) was estimated using a ×40 objective. The number of double-labeled cells was expressed as percentage of cells double labeled over the 5-HT3A
+ population. In the primary somatosensory cortex, the total number of cells per radial sector of (700-μm width) was pulled to produce the graphs.
In Utero Cells Transplantation
Homochronic transplantations were performed using E13/E13.5 and E14/E14.5 donor and host embryos. Donor embryos were collected into cold PBS following cervical dislocation of the pregnant mouse. Embryonic heads were dissected into cold L15 medium and embedded in 3% low-melting-point agarose (Sigma) in L15 medium. From these blocks, 270–300-μm-thick coronal sections were obtained using a Leica vibroslicer (Leica VTS1000) and were collected into cold L15 medium. CGE and AEP/preoptic (AEP/Po) explants were dissected out of the sections, collected into L15 medium, and kept on ice until transplantation. CGE or AEP/Po were mechanically dissociated prior transplantation. In addition, some experiments were performed using 5-HT3A
:GFP+ cells sorted by flow cytometry (see Supplementary Method S2
). For transplantation, time mate pregnant OF1 mice (Charles River) were anaesthetized with Xylazine–Ketamine (1 mg/kg/IP; 10 mg/kg/IP, in sterile saline solution). Uterine horns were exposed, and each embryo was manipulated under the uterine wall until position of the lateral ventricle was discernable. A glass micropipette (50 μm) containing an average of 5 × 104
cells in L15 stained with blue trypan (in 1 μL of solution) was introduced through the placenta in the ventricle (lateral or third) of each embryo. Cell transfer was achieved using mouth-control tubing attached to the pipette. The procedure was repeated for each embryo except for the most proximal and distal embryos. Injections were performed using E13/E13.5 5-HT3A
:GFP (CGE: n
= 6 litters, n
= 18 hosts; AEP/Po: n
= 5 litters, n
= 20 hosts) or using E14/E14.5 5-HT3A
:GFP donors (for cell suspension: CGE: n
= 10 litters, n
= 43 hosts; AEP n
= 12 litters, n
= 40 hosts, and for FACsorted cells: n
= 2 litters, n
= 5 hosts). After surgical recovery, animals were returned to their cages, and pups were reared until postnatal days 16–25 (P16–P25). Animals were processed as described above.
The distribution of 5-HT3A
:GFP+ grafted cells was determined on coronal sections counterstained with bis-benzimide. Quantification of GFP+ cells in the somatosensory cortex was carried out under a fluorescent microscope (Leica, DMR) using a 250
area under a ×20 objective lens or a 66
area under a ×40 objective lens. The laminar distribution of GFP+ cells was quantified at the level of the primary somatosensory area, in 500-μm-wide cortical strips (data obtained from 3 adjacent sections of at least 7 animals and expressed as mean ± standard error of the mean). The proportion of GFP+ grafted cells labeled for Parv, SOM, CR, VIP, or NPY was estimated in a cortical strip (700-μm width) in the primary somatosensory area and was expressed as percentage of double-labeled cells over the GFP+ population (data obtained from 3 adjacent sections of at least 12 animals from 5 different experiments).