Methods
Retroviral vectors. Retroviral vectors, CAG-GFP/cre and CAG-mRFP1, were constructed
by replacing GFP cDNA in a previously described vector, CAG-GFP (Ref. 16), with
GFP/cre (Ref. 17) and mRFP1 cDNA (Ref. S1), respectively. mRFP1 cDNA was kindly
provided by Dr. Roger Y. Tsien at UCSD.
Subject. C57BL/6 mice were purchased from Harlan (Indianapolis, IN). Floxed NR1 mice
were originally produced by Dr. Susumu Tonegawa’s laboratory at MIT (Ref.18), provided
from the colony in Dr. Stephen Heinemann’s laboratory and maintained in our own colony
at the Salk Institute. ROSA26-GFP reporter mice (B6;129-Gt(ROSA)26Sortm2Sho/J) were
purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in our own colony.
Retroviral vectors (~1 x 107 particles/ml; 1.5 µl) were stereotaxically injected into the
dentate gyrus (1.5 mm lateral and 2 mm posterior to bregma and 2.3 mm ventral to brain
surface) in one hemisphere of the mice at the age of 6-8 weeks under anesthesia with
ketamine/xylazine solution. To inject a mixture of CAG-GFP/cre and CAG-mRFP1, two
viral preparations were mixed (1:1 in volume) and 1.5 µl was injected. CPP (Tocris,
Ellisville, MO; 10 mg/kg body weight, i.p.) was injected into fNR1 mice daily from 14 to
20 days after the injection of a mixture of CAG-GFP/cre and CAG-mRFP1.
Histology. The procedure for fixed sample preparation was previously described (Ref, 16).
Primary antibodies used were: rebbit anti-GFP (1:400; Molecular Probes, Eugene, OR),
rabbit anti-Prox1 (1:400; Chemicon, Temecula, CA), goat anti-DCX (1:400; Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti-cre (1:100; Novagen, Madison, WI), mouse
anti-NeuN (1:400; clone A60 provided by Dr. R. Mullen, Salt Lake City, UT), and rabbit
anti-activated-caspase-3 (1:400; Cell Signalling Technology, Beverly, MA). Secondary
antibodies used were: FITC-, Cy5- or Cy3-conjugated donkey anti-rabbit IgG, Cy5 or Cy3conjugated donkey anti-goat IgG, and Cy5-conjugated donkey anti-mouse IgG (1:250;
Jackson Immuno, West Grove, PA).
Fluorescence microscopy. To examine the colocalization of fluorescence signals in fixed
samples, we used a confocal microscope (Radiance 2100, Biorad) with a 40x oil-immersion
objective lens (NA 1.30). For quantitative analyses, to examine a large number of cells
precisely in minimal time, we set a focal plane with a maximal cross-sectional area of cell
body/nucleus for GFP or mRFP1 in each cell. When colocalization was not clear, one or
two additional focal planes were examined for colocalization. When colocalization was still
difficult to determine, z-series were taken with a 1-µm step size. To visualize the
morphology, z-series covering the whole neuronal structure were taken with a 1-µm step
size. For live imaging, we used the same confocal microscope with a 60x water-immersion
objective lens (NA 0.90). Brain slices were prepared from fNR1 mice injected with CAGGFP/cre and –RFP or only CAG-RFP, using Vibratome (Ted Pella, Redding, CA). Live
slices were placed on an imaging chamber that was perfused at room temperature with
magnesium-free artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3 KCl,
2 CaCl2, 1.1 NaH3PO4, 26 NaHCO3, and 10 dextrose and saturated with 95% O2 and 5%
CO2. Based on their position and morphology, we found mRFP1-positive or mRFP1,
GFP/cre-double-positive new neurons in the dentate gyrus. Then, to prevent indirect
stimulation of the new neurons through synaptic activity from other neurons, 1 µM TTX
and 20 µM DNQX were applied with bath application. After acquiring Z-series of these
new neurons, 500 µM NMDA was applied with bath application. After 30 min, the second
z-series were acquired. In addition to NMDA, a co-agonist of NMDAR, glycine (5 µM),
was applied to maximize NMDAR activation.
Density analysis of new neurons. To measure the density of fluorescently labelled new
neurons, sections were selected evenly from anterior to posterior regions of the dentate
gyrus. The numbers of mRFP1-positive only, GFP/cre-positive only and double-positive
new neurons (Prox1-positive) in the granule cell layer and the subgranular zone were
counted for each section. To calculate the density of new neurons, the number of new
neurons was divided by the number of examined sections containing any fluorescently
labelled cells. The examined sections without any fluorescent cells were not included in this
calculation to avoid variability caused by the difference in diffusion of injected virus in the
dentate gyrus. The densities of new neurons (mRFP1-positive only, GFP/cre-positive only,
and double-positive) were affected by the precise titer of virus. Therefore, the absolute
numbers of fluorescently labelled neurons were generally highly variable when different
viral preparations were injected, even if we tried to measure and match virus titer precisely.
However, with the same viral preparation, the densities of fluorescently labelled new
neurons were reliable between mice. Therefore, we always used the same viral preparations
for each experiment. For the same reason, for developmental analysis (Fig. 2a,b), the
density at each time point was normalized by the density at 7 days to directly compare the
decreasing rate of the density of new neurons transduced by different viruses (CAGGFP/cre and -mRFP1).
Electrophysyology. 13-15 days after injected with CAG-GFP or GFP/cre, 8-10 week-old
fNR1 mice were anaesthetized with halothane (Ayerst Laboratories) and rapidly
decapitated. The brains were immediately harvested and placed into ice-cold cutting
solution that consisted of (in mM): 124 NaCl, 3 KCl, 4 MgCl2, 0.2 CaCl2, 1.25 NaH2PO4,
26 NaHCO3, and 10-20 glucose. Vibratome (Redding, Ted Pella, CA) was used to cut 300
m thick slices. Slices were kept at room temperature (~25 0C) in ACSF composed of (in
mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10-20
glucose, bubbled with a mixture of 95% O2/5% CO2, making the final pH 7.4, 300-315
mOsm/kg. Experiments were performed at room temperature. GFP+ or GFP/cre+ cells in
acute brain slices were identified using wide field dual peak illumination at 488nm/595nm
and dual peak emissions at 520nm/615 nm. Pipettes were sealed on cell bodies using videoenhanced DIC optics. Intracellular solution contained (in mM) 130 K-methane sulfonate, 4
NaCl, 2 Mg-ATP, 0.3 Na-GTP and 10 HEPES; pH was adjusted to 7.3 with K-methane
sulfonate; 300 mOsm/kg. Pipette resistance was 6-8 M. Recordings were made in
voltage-clamp mode using Axopatch 200B amplifier (Axon Instruments, Foster City, CA).
Signals were filtered at 10 kHz. No correction was made for the junction potential between
the bath and the pipette. Recorded cells were filled with 5 M of Alexa Fluor 594
(Molecular Probes) through the recording pipette to ascertain that the recorded cell is
indeed EGFP positive. Presence of Alexa Fluor 594 in the recording pipette also helped to
position electrode close to the targeted cell using dual excitation/emission filter cube. To
check the neuronal identity and viability of recorded cells, we examined whether recorded
cells generated action potential in response to membrane depolarization. 
DAM) was applied in the presence of TTX (1 M), DNQX (20 M) and
Glycine (5 M) and in the absence of Mg2+. We recorded from 4 GFP-positive and 4
GFP/cre-positive neurons. Fluorescent images of recorded cells were taken after the
completion of recording using dual excitation/dual emission cube and Hamamatsu C-4742
CCD camera.
Electron microscopy. 4 weeks after injected with CAG-GFP/cre, 2-3 month-old mice were
perfused with saline, followed by a solution of phosphate buffer saline (PBS) containing
4% paraformaldehyde + 0.2% glutaraldehyde and sections were cut at a thickness of 100
μm. Cells were injected under a fluorescence microscope with 5% aqueous Lucifer yellow
(Sigma). Slices were then incubated with 2.8 mM 3,3'-diaminobenzidine tetrahydrochloride
(DAB) and 6 mM potassium cyanide and then irradiated under conventional
epifluorescence using a 75-W Hg lamp and a fluorescein filter set to induce
photoconversion of DAB into an electron-dense residue. Slices were then postfixed
overnight in a solution of 3% glutaraldehyde and processed conventionally for electron
microscopy. Serial sections were cut at a thickness of 35 nm and analyzed with a JEOL
100CXII electron microscope at a 48,000x magnification.
Reference
S1. Campbell, R. E. et al. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A
99, 7877-82 (2002).