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伯爵8685木虫 (正式写手)
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[求助]
求神经系统实验方法翻译
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ONLINE METHODS Mouse strains. CD1 mice (Charles River Laboratory) were iontoporated at P1 unless indicated otherwise. The Scnn1a-cre mouse line was obtained from Jackson Laboratories (stock number 009613). Where indicated, tamoxifen was administered intraperitoneally (100 μl, 10 mg ml−1) at P10. In utero electroporation of ChR2 was performed at E15.5 as previously described27,34. All of the experiments were carried out in accordance with the Institutional Animal Care and Use Committee of the University of Geneva and with permission of the Geneva cantonal authorities. Postnatal iontoporation. We anesthetized P1 pups on ice for 5 min and placed them on a stereotactic frame. A small skin incision was performed on the skull and 50–200 nl of DNA (5 μg μl−1) was injected using a 30-μm-tip glass micropipette mounted on a nanoject II nanoinjector (Drummond). Prior to injection,the plasmid solution was mixed with 1 μl of TCHD (100 mg ml−1) to enhance nuclear membrane permeability23,51. We placed 5-mm electrode paddles on each side of the skull 4 min after injection and gave two trains of ten pulses (1 Hz,50-ms duration) using a 50-mA constant-current protocol with an electroporator.After surgery, we kept the pups on a warm plate and placed them back with their mother. Mouse handling was performed in compliance with protocols approved by the Veterinary Office of the Canton of Geneva. Plasmids. We generated plasmids using a standard endotoxin-free Qiagen kit(12362). Fezf2 cDNA (Open Biosystems clone 6415359) was subcloned into a pCAGIG_IRES_GFP (Addgene number 11159) plasmid. The CCAT_loxp_RFP_loxp_GFP was a gift from G. Elder (Mount Sinai School of Medicine). The pCAGIG_STOP_Fezf2_IRES_GFP plasmid was made by subcloning the STOP sequence from Addgene plasmid 11925 into CAGIG_fezf2_IRES_GFP. The pCAG_ERT2_CreERT2 plasmid was from Addgene (number 13777). The Dlx5/6 plasmid was a gift from G. Fishell. The Chr2 T159C52 plasmid was subcloned into the pCAGIG_IRES_GFP vector. In situ hybridization and immunocytochemistry. We performed in situ hybridization for Rorb and Fezf2 and immunohistochemistry on 50-μm tissue sections as previously described27. Thicker sections (up to 400 μm) were used for Camera lucida reconstructions and detection of subcortical axons. For primary antibodies,we used mouse and rabbit antibodies to GFP (1:500, Invitrogene,a11120 and a11122), mouse antibody to SATB2 (1:200, Abcam, ab51502), rabbit antibody to CUX1 (1:500, Santa Cruz, sc-13024), guinea pig antibody to VGLUT2 (1:2,000, Millipore, ab2251), rabbit antibody to ER81 (1:500 Abcam, ab81086),rabbit antibody to Ankyrin-G (1:750, Santa Cruz, sc-28561) and rabbit antibody to Casp3 (1:200, Cell Signaling, 9661). Image acquisition, quantification and statistical analysis. Images were obtained with a Zeiss (LSM 700) confocal microscope and a Nikon 90i microscope and analyzed with Zen software, and reconstructed with Neurolucida software for neurite analysis. Analyses were performed between P6 and P20. The molecular identity and barrelcortex cytoarchitecture were assessed at P6 and confirmed at later time points. Electrophysiological analyses were performed between P10–18.Morphological analyses were performed at P18–20.L4 of the somatosensory cortex was identified by the presence of barrels or the presence of red neurons in L4Tom mice. The percentage of L4 neurons that were positive for CUX1, SATB2 or ER81 immunoreactivity was determined in all sections containing L4 GFP+ cells and DAPI was used to identify individualcells to count expression frequency in L4 and L5B.Colabeling of VGLUT2 and GFP was normalized using the equalize function of the Adobe Photoshop CS6 software and quantified using the ImageJ 1.45s histogram/list tool (US National Institutes of Health). Electron microscopy. The samples were processed as previously described53.Briefly, mice were perfused transcardially with phosphate-buffered saline, followed by fixative (4% paraformaldehyde (wt/vol), 1% glutaraldehyde (vol/vol), in 0.1 M phosphate buffer) and 50-μm-thick brain sections were immunostained for GFP (rabbit antibody to GFP, 1:1,000, Invitrogen, cat #A11122). Labeled cells were then revealed using a biotinylated secondary antibody (goat antibody to rabbit (F)ab fragment, 1:200, Jackson Laboratories, cat #111-066-047), an avidin- biotin-peroxydase complex (ABC Elite, Vector Labs) and a reaction with 3,3′-diaminobenzidine (Vector Labs) for 4 min. After postfixation in 2.5% glutaraldehyde followed by 1% osmium tetroxide (vol/vol) in 0.1 M phosphate buffer, the sections were dehydrated, embedded in durcupan resin (Sigma),and cells and dendrites of interest were identified. We imaged 60-nm ultrathin serial sections of the selected dendrites at a final magnification of 25,000× and performed three-dimensional rendering with Fiji software. Electrophysiology. We prepared thalamocortical slices from P10–18 mouse pups as described previously54,55. Slices were cut in cooled artificial cerebrospinal fluid containing119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl, 2.5 mM CaCl2, 1.0 mM Na2HPO4, 26.2 mM NaHCO3 and 11 mM glucose, bubbled with 95% O2 and 5% CO2. Slices were kept at room temperature and were allowed to recover for at least 1 h before recording. Under low magnification, the barrels in L4 could be readily identified, and high-power magnification was used to guide the recording electrode onto visually identified neurons. The internal solution contained 140 mM potassium gluconate, 5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 2 mM MgCl2, 4 mM Na2ATP, 0.3 mM Na3GTP and 10 mM sodium creatine-phosphate.Currents were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and digitized at 20 kHz (National Instruments Board PCI-MIO-16E4, Igor,WaveMetrics). The liquid junction potential was +12 mV.Voltage sag was measured in current clamp from resting membrane potential by presenting a hyperpolarizing current step (−100 pA, 500 ms) and was calculated by the difference between peak voltage and steady-state voltage. Ih was measured in voltage clamp using a −40-mV step (500 ms) and was calculated by the difference between the peak current and steady-state current. No synaptic blockers were present during these recordings. Cells were voltage clamped at−60 mV and thalamocortical EPSCs were evoked at a frequency of 0.1 Hz by a bipolar stimulating electrode placed in the VB nucleus. The lowest intensity evoking a response was used to avoid activation of corticothalamic fibers and other passing fibers. For intracortical stimulation, the stimulating electrode was placed in superficial layer 2 and the lowest intensity evoking a response was used.Photostimulation was performed using 10-ms blue laser light pulses every 30 s in L2/3 ChR2-expressing sections42, and photo-induced EPSCs were recorded from L4, L5B or Fezf2+ L4 neurons. mEPSCs were recorded in the presence of tetrodotoxin (0.5 μM). The frequency and amplitude properties of these currents were then analyzed using the Mini Analysis software package (v.4.3, Synaptosoft).Changes in cumulative miniature EPSC amplitude and inter-event interval distribution were analyzed for statistical significance using the nonparametric twosample Kolmogorov-Smirnov test (KyPlot) with a conservative critical probability level of P < 0.05. Statistical analysis. We used Student’s two-sided t test for parametric data and Fisher’s exact test for non-parametric data, and Kolmogorov-Smirnov test forcomparison of distributions, unless stated otherwise. |
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