mice (Kaeser et al., 2008
) were crossed to two different Cre mouse lines to obtain recombination in the thalamus and in the cerebral cortex. Breeding was performed on a mixed background 129Sv × C57BL/6. To minimize potential variation due to background issues, littermate controls of either sex were used for all the experiments and processed simultaneously with the recombined samples.
To obtain thalamic-specific recombination, the serotonin transporter (Sert) Cre mouse line was used. This is a knock-in of nls-Cre in the 5′UTR region of Sert
; Zhuang et al., 2005
). In these mice, Cre is active in the ventrobasal thalamus (VB) from E15.5 (Narboux-Nême et al., 2008
). Unexpectedly, some offspring of RIM1flox/flox
breeding with SertCre
strain gave rise to null alleles (RIM−
allele), indicating occasional germline expression of Cre that needed to be controlled in all littermates. In our breeding we usually obtained: Rim1f/−
, which are referred to as RIM-DKOSert
, and Rim1f/−
, which are referred to as Controls (Ctrl). In their home cages, RIM-DKOSert
could not be distinguished from their control littermates and showed normal survival rates.
For cortex-specific recombination, we used the Emx1-Cre mouse line, a knock-in of Cre in 3′ from the stop codon (Iwasato et al., 2000
). In these mice, Cre is active in the glutamatergic cortical neurons and in glia but not in GABAergic neurons at E12.5 (Iwasato et al., 2000
). Rim recombination with Emx1-Cre strain is referred to as RIM-DKOEmx1
, and their littermates were used as controls. The RIM-DKOEmx1
mice were smaller than their control littermates and all died around weaning.
We used the TaumGFP-nls-LacZ
reporter mouse line to check recombination efficiency. This is a knock-in in the Tau gene of a construction containing a stop cassette flanked by loxP sites, a MARCKS sequence fused to the green fluorescent protein (GFP) and an IRES-NLS-LacZ cassette (Hippenmeyer et al., 2005
). In the presence of Cre, neurons express a membrane-bound GFP and β-Galactosidase (β-Gal) in the nucleus.
Experiments were conducted in compliance with the standard ethical guidelines (European Community guidelines on the care and use of laboratory animals and French Agriculture and Forestry Ministry guidelines for handling animals).
Genotyping was done on tail lysates prepared by immersing tissue in 50 mm
NaOH, at 95°C for 1 h, and then neutralizing with 1 m
Tris-HCl. PCRs were conducted as previously described (Kaeser et al., 2008
RIM1 and RIM2 quantitative PCR
Brains from postnatal day 7 (P7, P0 being the day of birth) RIM-DKOSert
and controls were dissected out and directly frozen on liquid nitrogen for 10 s and transferred to dry ice. The VB and somatosensory cortex were microdissected on 120 µm cryostat section. RNAs were extracted from samples and genomic DNA was removed with “RNAqueous-Micro Kit” (Applied Biosystems). First-strand cDNA was synthesized by reverse transcription of 50 ng of total RNA with Superscript-II reverse transcriptase (Invitrogen) according to standard protocols. Reverse transcriptase was omitted in some samples as negative control. Relative expression levels of RIM1 and RIM2 mRNA were determined by real time RT-PCR using Absolute SYBR Green Mix (ABgene) and a set of primers specific for the RIM1 and RIM2 floxed sequences. RIM expression was normalized to mouse CyclophilinB mRNA expression. Data were analyzed with the 2–DCt method (Livak and Schmittgen, 2001
). Values are expressed as the mean of 3–5 separate experiments, each comprising triplicates.
Mice were killed by an overdose of xylazine (1.92 mg/kg body weight, i.p.) and pentobarbital (100 mg/kg i.p.). They were perfused with 4% paraformaldehyde in phosphate buffer. Brains were dissected and either rinsed in PBS for cytochrome oxidase staining, or postfixed overnight at 4°C. Brains were cryoprotected overnight in 30% sucrose in PBS at 4°C before sectioning at 60 µm with a freezing microtome. To obtain tangential sections, cortices were dissected and flattened between two glass slides with spacers before postfixation.
The cytochrome oxidase (CO) staining of brain sections was conducted on free-floating sections. They were placed in the CO reaction solution (5 mg diaminobenzidine, 5 mg cytochrome C, 0.4 g sucrose in 0.1 mm Tris, pH 7.6) for 12–24 h at 37°C.
Immunocytochemistry was performed on free-floating sections. They were incubated overnight at 4°C in appropriate primary antibodies including: rabbit polyclonal antibody (Ab) c-Fos (1:1000, Santa-Cruz Biotechnology), rabbit polyclonal Ab CDP/Cux1 (1:500, Santa-Cruz), rabbit polyclonal Ab β-Gal (1:5000, Rockland), mouse monoclonal Ab NeuN (1:1000, Millipore), rabbit polyclonal Sert (1:1000, Calbiochem), Guinea pig polyclonal Ab vGlut2 (1:2000, Millipore Bioscience Research Reagents). After several rinses, species-specific fluorescent secondary Ab (1:500 Invitrogen) were incubated for 2 h, before mounting with Mowiol (Calbiochem). vGlut2 immunohistochemistry was preceded by a 10 min incubation in 50 mm sodium citrate at 97°C. For peroxidase revelation of Sert, biotinylated anti-rabbit (1:300, Vector) followed by avidin-biotin-peroxidase complex (1:400, GE Healthcare) was used before the diaminobenzidine reaction.
hybridization was performed on free-floating frozen sections as described previously (Narboux-Nême et al., 2008
). NBT/BCIP was used as blue substrate for in situ
revelation. The following plasmids containing full-length cDNA were used: RIM1 (IMAGE:40047877) and RIM2 (IMAGE:4505661).
Measures and cell counts
Quantification of areas
vGlut2 immunostained sections were photographed at a ×1.2 magnification using a stereomicroscope (Olympus). The total cortical area was measured on the tangential sections. The posteromedial barrel subfield (PMBSF) was outlined with ImageJ software and its area measured. Within this area, the vGlut2-positive regions correspond to the barrel hollows containing the dense TC axon terminals. The vGlut2-positive patches were outlined and their areas measured. The total PMBSF area minus the sum of vGlut2-positive areas was used as an estimate of the total interbarrel area. We refer to this value as the septal area in the rest of the manuscript, although strictly speaking, it comprises two different elements, the cellular wall surrounding the hollows and a septal region between the barrel walls. Interestingly double immunostainings for vGlut2 and Tenascin, an extracellular glycoprotein that surrounds structural and functional developing barrels in S1 (Crossin et al., 1989
; Steindler et al., 1990
) showed that tenascin-positive regions corresponded precisely to the vGlut2-negative regions.
Quantification of neuronal density
Photomicrographs of tangential sections immunostained for vGlut2 and Cux1 were acquired with a 40× objective using a Leica SP2 confocal microscope. The C2 barrel was placed in the center of the field and a single confocal section was acquired. The vGlut2-positive area was delimited after automatic thresholding using a fixed threshold value (MetaMorph Software). This area was defined as the barrel hollow. A 20 µm thick belt surrounding the barrel hollow was computed using MetaMorph imaging software. This area was defined as the barrel wall (Ballester-Rosado et al., 2010
). The Cux1-positive cell nuclei were counted with the cell counter plug-in (ImageJ software) within the barrel hollow and the wall compartments. Stereological methods were applied to avoid multiple counting. The cell counts were normalized per surface area for each compartment, providing cell density estimates for the hollow and wall compartments.
Quantification of colocalization
Pictures of coronal sections from P10 SertCre/+ TaumGFP-ires-LacZ and Emx1Cre/+ TaumGFP-ires-LacZ, immunostained for β-Gal together with NeuN, were acquired with the confocal microscope. Using the cell counter plug-in of ImageJ, the number of β-galactosidase-positive neurons within the NeuN population was measured in the VB and posterior medial (POm) nuclei of the thalamus and in the cerebral cortex layers.
All measures were done blind to genotype. For statistical analysis, the distributions of the results were tested with a Shapiro-Wilk test, followed by an f test. We found a normal distribution in the experiments analyzed. In most cases, the variance between groups was similar and a nondirectional Student’s t test was applied. When variance was different, a nondirectional homoscedastic t test was used. To compare small populations (n < 6), Mann–Whitney tests were applied.
TC slice preparation
Animals (P5–P7) were anesthetized by intraperitoneal injection of pentobarbital (15 mg/kg) and decapitated. The brain was quickly removed and placed in ice-cold (2–4°C) oxygenated (5% O2
, 95% CO2
) standard artificial CSF (ACSF). TC slices were cut (thickness, 400 µm) using a vibratome (VT100S; Leica), as previously described (Laurent et al., 2002
). The slices were first maintained 1 h at 33°C and later at room temperature (22–24°C) in oxygenated standard ACSF.
For recordings, the slices were glued on a polylysine-coated small (1.2 mm diameter) glass coverslip, which was then placed in a small (~1 ml) chamber, perfused at 3 ml/min with ACSF at near physiological temperature (33–34°C). Recordings were obtained from layer IV neurons identified under visual control using an upright fixed-stage microscope (Axioskop FS, Zeiss) equipped with infrared Nomarski optics and a video camera (Cascade 512B, Roper Scientific). Somatic whole-cell recordings were performed using borosilicate glass pipettes with a tip resistance of 2–3 MΩ and an Axopatch 200B amplifier (Molecular Devices). The recorded neurons were maintained in voltage-clamp mode using whole-cell patch-clamp techniques. Membrane capacitance and serial resistance were not compensated. Voltage and current signals were filtered at 5 kHz, digitized at 100 kHz using a digital board (Digidata 1322A, Molecular Devices), and stored on computer. The protocols were generated using the program pClamp10 (Molecular Devices). The series resistance (R
s) was estimated using a short-duration (20 ms) negative voltage step (3 mV) preceding by 200 ms the electrical stimulation of the afferent fibers. Typical R
s, calculated at the beginning of the current step using Ohm’s law, were of the order of 5–20 MΩ, and recordings were discarded when R
s changed by >20% during recording. Afferent TC fibers were stimulated by applying short-duration (20 µs) current steps with an isolated voltage stimulator (DS2A, Digitimer), through bipolar tungsten electrodes (SNEX200x50, Rhodes Medical Instruments) placed on the ascending TC pathway in the internal capsule (IC). As shown previously (Laurent et al., 2002
), low-frequency (0.03 Hz) extracellular IC stimulation evoked stable glutamatergic EPSC in the layer IV spiny stellate neurons. The unitary EPSC due to single afferent fiber stimulation (Laurent et al., 2002
) was found by progressively increasing the stimulation intensity until a plateau unitary response was obtained (see ; Takahashi, 1992
; Silver et al., 1996
). To unambiguously define the single fiber unitary EPSC, we plotted the amplitude of the responses in relation with the stimulation intensity. A shown in our previous work at cortical (Laurent et al., 2002
), and thalamic (Evrard and Ropert, 2009
) synapses, it is sometimes possible to distinguish axonal failure from release failure. In most cases, there was no evidence for axonal failures and the response changes from complete failures at smaller intensity (<2.3 mA, ), to a stable unitary response at higher intensities. In such cases the stimulation intensity was maintained above threshold.
Figure 2 Reduction of the TC synaptic transmission in the RIM-DKOSert mice. The TC synapses were analyzed in P5 to P7 brain slices preserving the TC pathway. TC axons were stimulated at low frequency (0.03 Hz) in the internal capsule and the TC EPSCs were recorded (more ...)
The data were analyzed offline using Clampex (Molecular Devices), Excel (Microsoft), and Igor Pro 4.1 (WaveMetrics). The amplitude of the AMPA receptor (AMPAR)-mediated component of the EPSC at −70 mV was measured between the baseline and the peak of the response by averaging the signal during a short time window (40 µs). The amplitude of the NMDAR component of the EPSC was measured at +30 mV by averaging the current recorded during an identical time window (40 µs) placed 15 ms after the stimulation. The EPSC failure was measured at −70 mV and was defined as a response that was smaller than twice the SD of the baseline current. The EPSC latency was defined as the time when the EPSC reached 10% of its peak amplitude. Paired-pulse ratios (PPRs) were obtained by applying two stimulations at various time intervals and calculated at −70 mV as the second EPSC amplitude divided by the first EPSC amplitude (EPSC2/EPSC1) including failures. The EPSC kinetics was quantified by fitting their decay with a biexponential function y0 + Afast e−1/τfast + Aslow e−1/τslow, Afast and Aslow being the amplitude of the fast and slow components respectively, τfast and τslow their time constant, y0 the offset.
Solutions and drugs
All chemicals were purchased from Sigma unless otherwise specified. Standard ACSF contained the following (in mm): 126 NaCl, 2.85 KCl, 1.25 KH2PO4, 1.5 MgSO4, 2 CaCl2, 26 NaHCO3, 5 Na-pyruvate and 10 glucose. The following compounds were bath applied: d-(−)-2-amino-5-phosphonopentanoic acid (d-AP5, 50 µm; Ascent Scientific), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX, 10 µm; Ascent Scientific), [6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide] (gabazine, 10 µm; Ascent Scientific).
The intracellular pipette solution contained the following (in mm): 120 Cs methylsulfonate, 10 CsCl, 10 HEPES, 4 K-ATP, 2 MgCl2, 0.4 Na-GTP, and 0.2 EGTA, pH adjusted to 7.35 using CsOH. All voltage values were corrected for a −9 mV liquid junction potential.
Pre-embedding immunolabeling electron microscopy
Adult mice (2 Ctrl and 2 RIM-DKOSert) were anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde + 0.5% glutaraldehyde in cold 0.1 m phosphate buffer. Brains were further postfixed in 4% paraformaldehyde + 15% sucrose for 2 h at 4° and then cut in 80 µm thick coronal sections with a vibratome. Thorough rinses were followed by a blocking step in 5% normal goat serum + 5% BSA and then an overnight incubation at room temperature in guinea-pig polyclonal Ab vGlut2 (1:8000 Millipore Bioscience Research Reagents) diluted in PBS + 2% Normal Goat Serum.
For immunoperoxidase procedures, a biotinylated anti-guinea pig IgG (Vector, CA) was applied as secondary Ab (1/200 in PBS, 2 h), an ABC peroxidase complex (Vectastain Elite) diluted 1/400 was used for amplification and revelation was performed with 0.05% diaminobenzidine as chromogen.
For immunogold labeling, a 4 h incubation in ultra-small gold conjugate of goat anti-guinea pig IgG (1/100; Aurion) was followed by extensive washings, 10 min postfixation in 2% glutaraldehyde, and the 0.7 nm gold beads were then silver enhanced (HQ silver; Nanoprobes).
After OsO4 postfixation (2% for immunoperoxidase labeling, 1% for silver-gold revelation), sections were dehydrated in graded acetone with a 2% uranyl acetate en bloc staining step in acetone 70%, and finally embedded in Epon resin. Ultrathin sections were examined with a Philips CM100 electron microscope, operated at 80 kV and imaged with a Gatan digital camera.
To locate vGlut2-positive synapses within layer IV, the borders of the layer were outlined on a micrograph of the Nissl-stained semithin section immediately preceding the ultrathin sections. Boundary lines were then transferred on a low-magnification electron micrograph to identify the zone in which the positive synapses were examined.
Mice were killed and perfused with 0.9% NaCl. Brains were dissected and soaked in the Golgi-Cox filtrated solution (K2
) for 2 d, rinsed in 30% sucrose for 1 week and sectioned at 200 µm with a vibratome (Leica VT1000). Sections were mounted on slides, color reacted in 30% NH4
OH for 30 min, and fixed in 30% AL4 Kodak photo fixative for 30 min. They were counterstained with cresyl violet, dehydrated and coverslipped with Eukitt (Electron Microscopy Sciences). Neurons were drawn using a camera lucida with a 100× oil-immersion objective for dendritic analyses. A total of 50 Ctrl and 41 RIM-DKOSert
layer IV spiny stellate cells were reconstructed from 4 mice of each genotype. Morphometric analysis of dendrites was done with NeuronJ software. Asymmetry of dendrites was measured as described previously (Datwani et al., 2002
), briefly, a line was drawn between the tip of each dendrite and the center of the soma. Neurons were considered as asymmetric when 50% or more of these lines representing dendrites were oriented in a 90° angle. Otherwise, they were counted as symmetric. The proportions between genotypes were tested with a χ2
test. A two dimensional Sholl analysis (Sholl, 1953
) was used to analyze dendritic branching patterns. Concentric circles of 10 µm intervals were brought over each cell with the center of the circles positioned in the middle of the soma. Intersections of different dendritic orders and circles were counted.
The cell count plug-in of the ImageJ program was used to count the number of branches and bifurcation points. The same program was used to count spines on 20 µm portions of the secondary and tertiary dendrites from stacks of photomicrographs captured using a 100× oil objective on a Leica microscope. Statistical comparisons used ANOVA.
Voltage-sensitive dye imaging
Voltage-sensitive dye (VSD) imaging of the cortical activity evoked by tactile stimulation was performed on 6- to 8-week-old mice under urethane anesthesia (1.7 mg/g), as previously described (Ferezou et al., 2007
). Briefly, a large fraction of mouse sensorimotor cortex was exposed unilaterally and stained for 1 h with the VSD RH1691 (Optical imaging, 1 mg/ml, in Ringer’s solution containing [in mm
]: 135 NaCl, 5 KCl, 5 HEPES, 1.8 CaCl2, 1 MgCl2). After removing the unbound dye, the cortex was covered with agarose (0.5%) and a coverslip. Cortical imaging was done through a tandem-lens fluorescence microscope (Scimedia), equipped with 2 Leica PlanApo 1× objectives, a 630 nm excitation filter, a 650 nm dichroic, and a long pass 665 nm emission filter. Alternate sequences of images were acquired every 20 s (at 500 frames/s), using a CMOS-based camera (MiCam Ultima, SciMedia), with or without piezzo electric stimulus (2 ms) delivered to the right C2 whisker. Data were analyzed using custom-written routines in IgorPro (Wavemetrics). Subtraction of the averaged unstimulated sequences was used to correct for photobleaching.
Sensory-mediated c-Fos expression
All whiskers of adult mice were clipped on the right side, under light anesthesia (ketamine, 100 mg/kg IP). The following day, mice were placed in a cage containing new objects (typically a cardboard tube, two plastic goblets and two balls of paper for each cage). Mice explored this new environment for 1 h, after which they were perfused. For c-Fos immunostaining, brains were sectioned at 50 µm with a vibratome (Leica VT1000). Immunohistochemistry was performed as described above.
Training and testing were conducted in Plexiglas boxes (white floor, gray walls) of 17 × 32 × 40 cm, in the dark. Before the test, animals were habituated to the empty boxes in 10 min sessions for two consecutive days. For the test, two objects were placed in the boxes and animals were allowed to freely explore for 10 min. Sessions were recorded and analyzed using Viewpoint software. The software determined both the position of the nose and the center of mass of the mouse. Center of mass position over time determined the speed of the mouse during exploration, and object exploration was defined when the nose was within a 2 cm large circle around the object. Eight control and 9 RIM-DKOSert mice were used for this test.
Beam walking test
The test was conducted as described previously (Carter et al., 2001
). Briefly, mice were trained for 4 d to walk on an elevated 22 mm section beam to reach a platform. On the trial day, each mouse was given three trials to cross the beam and the time used to do so was noted. When mice did not succeed to reach the platform within 2 min, the trial was considered as a failure. The two best times over the three trials were averaged to determine each mouse’s crossing time. All failures per genotype were summed and averaged to estimate the probability of failure per trial. Seven Ctrl and 9 RIM-DKOSert
mice were used for this test.