All animal procedures were in compliance with animal care guidelines and were approved by Stanford University's Administrative Panels on Laboratory Care (A-PLAC). Approval number is 14007.
To create a Cre-dependent tTA construct (pCA-ZtTA) we performed a 3-way ligation using the following fragments: 1) The tTA 
open reading frame with a Kozak sequence (CCGCCACC) was generated by PCR with a pair of primers (5′-AAACTCGAGCCG CCACCATGTCTAGATT-3′
and 5′-TAGAATTCGGGGATCCTC G-3′
), followed by XhoI and EcoRI restriction digestion; 2) The loxP-flanked βgeo (a lacZ-neomycin phosphotransferase fusion) coding sequence was obtained from the Z/EG plasmid 
by restriction digestion with XbaI, subsequent treatment with Klenow to fill-in the 5′ overhangs, and then digestion with XhoI; 3) The expression vector pCA-HZ2 
containing a chicken β-actin promoter with CMV enhancer 
and an SV40 T-antigen poly(A) signal was digested with SmaI and EcoRI. After ligation, the resulting pCA-ZtTA vector was sequenced, cut with PacI and AscI, and subcloned into the pROSA26-PA targeting vector 
to generate the final targeting construct pROSA26-ZtTA. Before ES cell injection, the functionality of this vector was tested by Western blotting of lysates of COS cells transiently transfected with pROSA26-ZtTA, a TRE-GFP reporter and a Cre plasmid. Only when all three plasmids were co-transfected, we observed high GFP expression (data not shown).
pTRE-Bi-SG-T and piiTRE-Bi-SG-Tii
We generated pTRE-Bi-SG-T by a 3-way ligation using the following fragments: 1) A GFP-tagged mouse Synaptophysin (Syp) containing plasmid (gift from Shasta Sabo, Case Western Reserve University) was digested with NheI and BssSI, generating a fragment containing full-length Syp fused to a portion of GFP; 2) An unpublished vector pBT255.32, containing mut4EGFP 
, was cut by EcoRI and BssSI and yielded a fragment containing a portion of mut4EGFP to reconstitute Syp-mut4EGFP in the final ligation; 3) An unpublished vector pBT248.31 containing the TRE-Bi promoter and tdTomato 
fused to 3 Myc epitopes (tdT-3Myc) was cut by NheI and EcoRV to place Syp-mut4EGFP under the control of a bidirectional TRE (pBi; Clontech, Mountain View, California; Catalog No. 631006; GenBank Accession No.: U89932). The final ligation product was verified by restriction digestion and sequencing. To generate piiTRE-Bi-SG-Tii, a PmeI/PacI fragment from pTRE-Bi-SG-T was subcloned into pBT226.1. pBT226.1 is a derivative of pJC13-1 
and contains a PacI/AscI/PmeI polylinker flanked by two copies of the 1.2 kb-long chicken β-globin HS4 insulator on each side. It was created from pJC13-1 by removal of EcoRI-flanked HS2 enhancer and by replacement of BamHI-flanked Neo reporter by a PacI/AscI/PmeI polylinker. We tested the functionality of pTRE-Bi-SG-T and piiTRE-Bi-SG-Tii constructs by co-transfecting them with a tTA-expressing plasmid into dissociated hippocampal culture (data not shown).
was linearized by AgeI digestion and purified by phenol/chloroform extraction. Gene targeting in mouse R1 ES cells 
was performed by the Stanford Transgenic Facility. Following G418 selection, clones that contained the knock-in were identified by long-range genomic PCR as previously described 
. To detect correct targeting at the 5′ end of ROSA26, we amplified ~1.5 kb genomic DNA fragment using primers: Rosa3 (CCACTGACCGCACGGGGATTC
) and Rosa4 (TCAATGGGCGGGGGTCGTT
), and LA Taq with GC buffer I (Takara, Cat No. RR02AG). To detect the correct targeting at the 3′ end of ROSA26, we amplified ~6 kb genomic DNA fragment using primers Rosa8 (GGATCCCCGAATTCTAGATAACTGATCATAATCAGCC
) and Rosa9 (GGGGAAAATTTTTAATATAAC
), and LA Taq with LA PCR Buffer II (Takara, Cat No. RR002M). Two correctly targeted ES cell clones were expanded and injected into C57BL/6J blastocysts to generate chimeric mice. Germline transmission was verified by PCR as previously described 
. F1 ZtTA mice were either intercrossed to generate homozygous mice or crossed to Nestin-Cre 
, Nestin-CreER.line1 and Nestin-CreER.line5 
, or β-actin-CreER 
The bacterial plasmid backbone was removed from pTRE-Bi-SG-T using PacI and PmeI, and piiTRE-Bi-SG-Tii using AccI and NdeI, and separated from the inserts using gel purification. The two constructs were independently introduced into mice to generate randomly integrated transgenes via standard pronuclear injection into FVB zygotes at the Stanford Transgenic Facility. The transgenic mouse founders were identified by PCR genotyping using four primers: (CTTCAAGTCCG CCATGCCCGA) and (TCCAGCAGGACCATGTGATCGC), which detect GFP as a band of 420 bp, and (CAAATGTTGCTTGTCTGGTG) and (GTCAGTCGAGTGCACAGTTT), which detect the control band of 200 bp.
For phenotypic screening, the PCR-positive founders were crossed to mice containing Foxg1-tTA, which is expressed in the mouse forebrain 
Tissue Preparation and Immunohistochemistry
All animal procedures were in compliance with animal care guidelines and were approved by Stanford University's Administrative Panels on Laboratory Care (A-PLAC). Approval number is 14007. For histology, mice were anesthetized with an intraperitoneal injection of 2.5% Avertin (400–600 mg/kg body weight; Sigma, Cat. No. T48402) and perfused intracardially with 0.1 M phosphate buffer saline (PBS; Sigma, Cat. No. P3813) to remove the blood and then with 4% paraformaldehyde (PFA; Sigma, Cat. No. 441244) in 0.1 M PBS. Tissues were isolated and fixed in 4% PFA in 0.1 M PBS at 4°C overnight, washed three times in PBS, cryoprotected for >24 hours in 30% sucrose in PBS, and embedded in Tissue-Tek OCT (VWR, Cat. No 25608-930) prior to cryostat sectioning. Tissues were sectioned at 25 µm or 50 µm thickness unless otherwise specified. 25 µm sections were collected on microscope slides (VWR, Cat. No. 48311-703) and immunostained if needed as described below. 50 µm sections that were to be immunostained were usually collected in PBS and further processed in a “floating” mode. The floating sections were immunostained in 24-well plates following the procedure below and subsequently mounted on slides.
For immunofluorescence, cryosections were washed three times for 10 min in PBS, blocked with 10% normal donkey serum (NDS) in PBS +0.3% Triton X-100 (Sigma, Cat. No. 234729) (PBT) for 1 hour at room temperature, and stained at 4°C overnight with primary antibodies in 5% NDS in PBT. Following four washes for 10 min in PBT, the sections were stained at 4°C overnight with secondary antibodies. The sections were washed four times for 10 minutes with PBT, treated with DAPI (Sigma, Cat. No. D8417), rinsed for 10 minute in PBS, and were mounted in Fluoro-Gel mounting medium (EMS, Hatfield, PA, Cat. No.17985-10). Images were taken using an LSM510 confocal system (Carl Zeiss Inc., Oberkochen, Germany) or a Zeiss fluorescence microscope with a CCD camera and processed using LSM software (Zeiss) and Image J (http://rsbweb.nih.gov/ij/
Primary antibodies against the following proteins were used: GFP (chicken, 1
500; Cat. No. GFP-1020; Aves Labs, Tigard, OR), Myc (goat, 1
200; Cat. No. 600-338; Novus, Littleton, Colorado). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. West Grove, Pennsylvania and included: donkey anti-chicken FITC (1
200; Cat. No. 703-095-155), donkey anti-goat Cy3 (1
500, Cat. No. 705-165-147). Pre-absorption of goat anti-Myc antibody (see above) was conducted by cutting three wild-type brains into small pieces after fixation in 4% PFA overnight at 4°C, then blocking the brain pieces at 4°C overnight with 10% NDS in PBT, and finally incubating the fixed brain pieces with shaking in 30 ml of PBT with anti-Myc antibody at 1
200 dilution for >2 days at 4°C.
Hippocampal Neuron Culture and FM Dye Staining
Dissociated hippocampal neurons in culture were prepared from P0 mouse pups (genotype: ZtTA/wt; TRE-Bi-SG-T/wt; Nestin-Cre/wt) according to procedures described previously 
and used between 14 and 43 days in vitro
(DIV). Briefly, the hippocampal regions were dissected out of brains of triple trangenic mice, cut into small pieces, and incubated in a digestion solution consisting of 10 mg/ml trypsin (Sigma, Cat No. T1005) and 0.5 mg/ml DNAase type IV (Sigma, Cat. No. D5025-15KU). The pieces were then mechanically dissociated in Hank's solution supplemented with 12 mM MgSO4
and 0.5 mg/ml DNAase type IV. The cells were recovered by centrifugation and plated onto Matrigel (BD Biosciences -Collaborative biomedical, Cat. No. 35-4234) -coated (6 mg/cm2
) coverslips (Carolina Biological Supply Company, Cat. No. ER-63-3009) at a density of 25,000–50,000 cells/cm2
. Culture medium consisted of minimal essential medium (Invitrogen, Cat. No. 51200-038), 0.6% D-glucose, 1 mM NaHCO3
, 2.4 g/l bovine transferrin (Calbiochem, Cat. No.: 616420), 0.25 g/l insulin (Sigma, Cat No. I6634), 0.3 g/l L-glutamine, 5%–10% fetal calf serum (Invitrogen, Cat. No. 16010-159), and 8 µM Cytosine β-D-arabinofuranoside hydrochloride (Sigma, Cat. No. C6645) Cultures were maintained at 37°C in a 95% air, 5% CO2
humidified incubator, and culture medium was replaced every 3 days.
Neurons were loaded with 8 µM FM 4-64 (Invitrogen, Cat. No. F34653) dye solution by field stimulation at room temperature. With field stimulation, neurons were exposed to FM 4-64 during and up to 60 s after stimulation with plantium electrodes (2–120 s, 10 or 20 Hz). After loading, neurons were washed with Tyrode's solution for 5–10 min. Images were taken under a fluorescent microscope (Nikon TE2000) and puncta counting analysis was performed with Metamorph software.
One triple transgenic mouse (genotype: ZtTA/wt; BI-TRE-Syp-GFP/wt; Nestin-CreER.line5/wt, with 2 mg tamoxifen injected at E9.5) was perfused at P35 intracardially with 0.1M PBS and 4% PFA in 0.1 M PBS, and the tissue was processed for array tomography 
. The cerebellum was dissected out, further fixed in the same fixative using microwave irradiation (PELCO 3451 laboratory microwave system; Ted Pella), then dehydrated up to 70% ethanol and embedded in LRWhite resin (medium grade, SPI). Serial ultrathin sections (70 nm) were cut on an ultramicrotome (Leica), mounted on coverslips and immunostained with antibodies against Synapsin I (rabbit, Millipore AB1543P, 1
100) and MAGUK (mouse, NeuroMabs, 75-029, 1
100). As secondary antibodies we used Alexa 594 goat anti-mouse, and Alexa 647 goat anti-rabbit (A11032 and A21245, Invitrogen, 1
150). Sections were mounted using SlowFade Gold antifade reagent with DAPI (Invitrogen). The immunostained serial sections were imaged using a Zeiss AxioImager.Z1 fluorescence microscope with AxioCam HRm CCD camera and a Zeiss 63×/1.4 NA Plan Apochromat objective. Images from serial sections were aligned using ImageJ and the Multistack Reg plugin.
To quantify the colocalization of GFP with endogenous Synapsin and MAGUK, 4 volumes (20×20×2 mm each) from the molecular layer of the cerebellum were analyzed. All GFP puncta that were present on more than one 70 nm section were chosen from a stack of images from the GFP channel. Using the ImageJ ROI function, the same region was then located on the Synapsin channel stack and on the MAGUK channel stack and evaluated for the presence of immunolabeling.
Quantification of Dox Regulation
Confocal microscopy was used to take a single 8 µm thick optical section of the cerebellum from mice with the genotype of ZtTA/wt; TRE-Bi-SG-T/wt; β-actin-CreER/wt that were raised under different Dox conditions. Three images per animal were taken at random within the granular layer of the cerebellum. Two animals per Dox condition were used for analysis. Average fluorescent intensity within a cell body was measured for every GC body within a given confocal image by using ImageJ after manually positioning the same circle that is smaller than any of the GC bodies within each GC body. For each image, 10 background measurements were applied to normalize the background noise. For each Dox condition, 6 images from 2 animals were used for analysis.
Quantitative Analysis of Cerebellar Granule Cells
Serial 50 µm coronal sections of cerebellum were immunostained and imaged through a 63× (1.3 NA) oil objective by 0.4 µm optical sectioning using a Zeiss 710 confocal microscope (Carl Zeiss Inc., Oberkochen, Germany). The ascending segments and the parallel fibers were selected that met the following criteria: (1) the entire ascending segment can be followed until the bifurcation point of parallel fiber, (2) the entire cell body and dendritic processes are present, (3) the parallel fibers can be followed within the section (not broken). Given the fact that a single entire parallel fiber (several millimeters long) is always cut into pieces in cryostat sections, it is not practical to trace the entire parallel fiber. Thus for parallel fibers, we traced the axon from the bifurcation points for as long as possible (). Processes (filaments) and synaptic puncta within each section were traced in 3D using the Imaris 6.2 software (Bitplane). Following 3D tracing, the number of synaptic puncta, the filament length between puncta, the total length of the filaments, and the fluorescence intensity of puncta were quantified by using Imaris or ImageJ. Numerical analysis and statistics were performed in Microsoft Excel, Matlab or Graphpad Prism. We calculated the density of synapses (per 100 µm) in a particular region of a GC by taking the total number of puncta in a region/total length of region (in µm) * 100.
For measuring the percentage of Syp-GFP fluorescent signal in a given region of a GC, we processed the confocal stack image of each labeled GC with the “maximal intensity projection” function of ImageJ. Then we measured the total intensity of Syp-GFP for axonal region (X) and the dendritic region (Y), respectively, while excluding the cell body region. We calculated the percentage of Syp-GFP signal for a given GC in the two regions: Syp-GFPaxon
Y/(X+Y)*100. We applied this type of measurement to both mature and immature GCs (10 cells for each).