A replication-deficient retroviral vector based on the Moloney murine leukemia virus was used to express enhanced GFP or mRFP1 (RFP) [73
] driven by a CAG promoter [39
]. Retroviral particles were assembled using three separate plasmids containing the capside (CMV-vsvg), viral proteins (CMV-gag/pol), and transgene (CAG-GFP or CAG-RFP). Plasmids were transfected onto 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, California, United States). Virus-containing supernatant was harvested 48 h after transfection and concentrated by two rounds of ultracentrifugation.
Subjects and stereotaxic surgery.
C57Bl/6J P7 pups were anesthetized by placing them on ice for 4 min. CAG-GFP expressing retrovirus was infused (0.7 μl in 1 min) into the dorsal area of the right DG (coordinates from bregma: antero-posterior = −1.5 mm, lateral = 1.5 mm, ventral = 1.5 mm) using a microcapillary calibrated pipette (Drummond Scientific, Broomall, Pennsylvania, United States). Pups were placed back with their mother after recovery (30–60 min on a warm pad). After weaning (at P21), mice were segregated by sex. At P42–P49, mice were anesthetized (100 μg ketamine/10 μg xylazine in 10 μl saline/g) and a second surgery was carried out to infuse the CAG-RFP retrovirus (0.7 μl in 5 min) into the dorsal aspect of the right DG (coordinates: −2 mm, 1.5 mm, 1.9 mm). Three days before the second surgery and thereafter, mice were housed with a running wheel to increase the efficiency of viral transduction [1
After weaning (P21), mice received a daily intraperitoneal injection of BrdU 50 mg/kg over 5 d. Four weeks after the last injection, mice were anesthetized (ketamine/xylazine) and perfused intracardially with 4% paraformaldehyde (PFA). Brains were removed and sectioned (40 μm) in a cryostat (Leica, Wetzlar, Germany). One in six sections throughout the hippocampus were studied by immunohistochemistry and confocal microscopy, and all sections containing GFP+ neurons were included in the analysis. Neuronal phenotype of GFP+ cells was assessed by morphology. The phenotype of GFP+/BrdU+ cells was corroborated by NeuN labeling.
Immunofluorescence and confocal microscopy.
Immunostaining for BrdU was done on 40-μm free-floating coronal sections. Double labeling of GFP and RFP was analyzed in 400-μm sections cut with a vibratome and fixed with 4% PFA. Antibodies were applied in TBS with 3% donkey serum and 0.25% Triton X-100. Primary antibodies were: BrdU (rat; 1:200; Boehringer Manheim, Roche, Basel, Switzerland) and GFP (chicken; 1:500; Invitrogen). NeuN (mouse monoclonal; 1:50) was included in all sections to label the GCL and to assess the neuronal phenotype. Corresponding secondary antibodies were used (donkey anti-chicken Cy2, donkey anti-rat Cy3, donkey anti-mouse Cy5; 1:250; Jackson ImmunoResearch, West Grove, Pennsylvania, United States). Images were taken with an optical pinhole of 1 μm using a Zeiss Pascal confocal microscope (Zeiss, Oberkochem, Germany). Only GFP+ cells located in the subgranular zone (SGZ) or GCL were included in the analysis. Co-localization was analyzed in single optical planes taken through the entire z-axis of each cell.
Electrophysiology: Whole-cell recordings.
Experiments were carried out in 56 slices from 30 mice injected at P7 and P42 (12 male and 18 female). Experiments on perforant path afferents were carried out 13.6 ± 0.5 wk (range: 12.9 to 14.6) after the second (P42) surgery. Experiments on GABAergic inputs and intrinsic membrane properties were carried out 6.7 ± 0.4 wk (range: 6.0 to 7.6) after the second (P42) surgery. Mice were anesthetized and decapitated. Brains were removed into a chilled solution containing (mM): 110 choline-Cl−, 2.5 KCl, 2.0 NaH2PO4, 25.0 NaHCO3, 0.5 CaCl2, 7 MgCl2, 20 dextrose, 1.3 Na+-ascorbate, 0.6 Na+-pyruvate, 4 kyn. Right hemisphere slices (400-μm thick) were cut in a vibratome and transferred to a chamber containing artificial cerebrospinal fluid (ACSF; mM): 125.0 NaCl, 2.5 KCl, 2.0 NaH2PO4, 25.0 NaHCO3, 2 CaCl2, 1.3 MgCl2, 1.3 Na+-ascorbate, 3.1 Na+-pyruvate, and 10 dextrose. Our stereotaxic injections mostly labeled dorsal DGCs. Experiments on perforant path afferents were carried out in coronal sections to render slices transversal to the dorsal hippocampus (the number of transverse dorsal slices was optimized by including a subtle angle to coronal sectioning). Experiments on GABAergic inputs were performed in horizontal slices. Slices were bubbled with 95% O2/ 5% CO2 (315 mOsm) and maintained at 30 °C during recovery. Recordings were carried out in ACSF at 23 ± 1 °C using microelectrodes (5–8 MΩ) pulled from borosilicate glass (KG-33, Garner Glass, Claremont, California, United States). ACSF was supplemented with 20 μM BMI for EPSCs recordings and 4 mM kyn for IPSCs recordings. Four internal solutions were used; all of them included (mM) 10 Hepes, 4 Tris-ATP, 0.3 Tris-GTP, 10 phosphocreatine, and Alexa Fluor 488 or 594 (10 μg/ml; Invitrogen), (pH 7.3) and 290 mOsm. In addition, EPSCs were recorded using (mM): 130 Cs-gluconate, 10 tetraethylammonium-Cl, 2 NaCl, 2 MgCl2, 0.2 EGTA, 2 QX-222; evoked IPSCs and intrinsic membrane properties were recorded with (mM): 120.0 Kgluconate, 20 KCl, 5 NaCl, 4 MgCl2, 0.1 EGTA; for spontaneous IPSC at VHold = −80 mV we used (mM): 19 Kgluconate, 121 KCl, 5 NaCl, 4 MgCl2, 0.1 EGTA; and for spontaneous IPSC at VHold = 0 mV, we used (mM): 140 CsCl, 5 NaCl, 2 MgCl2, 0.1 EGTA. Unless otherwise noted, all chemicals were from Sigma (St. Louis, Missouri, United States).
Whole-cell recordings (Axopatch 200B; Molecular Devices, Sunnyvale, California, United States) were filtered at 2 kHz, digitized (Digidata 1322A, Molecular Devices) and acquired onto a PC using jClamp (SciSoft [http://www.scisoftco.com/jclamp.html
)]. Series resistance was typically 15–25 MΩ. Criteria to include cells in the analysis were (1) co-labeling with Alexa Fluor 594 (for GFP+
cells) or 488 (for RFP+
cells) or visual confirmation of GFP or RFP fluorescence in the pipette tip, and (2) absolute leak current less than 150 pA. Unless otherwise noted, voltage-clamp recordings were performed at VHold
= −80 mV. Extracellular stimulation (50 μs, 0.1 Hz) was done using concentric bipolar electrodes (50-μm diameter; FHC, Bowdoinham, Maine, United States), a Grass S88 stimulator and a stimulus isolation unit (SIU5, Grass Instruments, West Warwick, Rhode Island, USA). In paired experiments, a development-born DGC (GFP+
) and one neighboring adult-born DGC (RFP+
) were sequentially recorded while maintaining electrode position, stimulation protocols, and stimulus strength (mostly 10 V, 50 μs) invariable. The order in which GFP+
cells were recorded was alternated across experiments. For orthodromic stimulation of MPP and LPP afferents, two stimulation electrodes were positioned on the middle and outer third of the ML, respectively, towards the crest of the DG. This resulted in appropriate separation of MPP versus LPP, as evidenced by the paired-pulse ratio and EPSC kinetics (E and F). For stimulation of GABAergic interneurons, the stimulation electrode was positioned on the GCL or outer third of the ML, as stated in the text. Electrodes were positioned approximately 250 μm away from the recorded cell. When more than one DGC of the same group was recorded for the same paired experiment, average values were used for comparisons.
The localization of the rare P7-born DGCs that continued to divide 2 wk after labeling (E) was restricted to the inner half of the GCL, whereas P7 neurons of the outer half did not incorporate BrdU. To exclude those rare neurons from our analysis, only P7-born DGCs of the outer half were selected for electrophysiological recordings.
Electrophysiology: Cell-attached recordings.
In this set of experiments, we have labeled neurons born at P42 using the CAG-GFP retrovirus, and took unlabeled (GFP−
) DGCs of the middle third of the GCL as pup born (see A and B; E. A. Mathews, N. Morgenstern, V. Piatti, S. Jessberger, A. F. Schinder, F. H. Gage, unpublished data). In additional support of this criterion, it has been shown that adult-born neurons largely remain within the inner third of the GCL [28
]. Recordings were carried out in 11 slices from eight mice, approximately 9 wk after infection. Simultaneous cell-attached recordings were carried out under voltage clamp at 0 mV using pipettes with a high tip resistance (10–14 MΩ). Stimuli were delivered to the middle third of the ML. Stimulus intensity was gradually increased (0.3–3 mA, 50 μs) and spiking probability for each neuron was obtained after 15–30 stimuli repeated at 15-s intervals. For each pair of cells, the single stimulus intensity that rendered the largest difference in spiking probability was selected for statistical analysis (D). Only experiments in which spiking was detected in both neurons were considered for analysis. Recordings were discarded if the seal resistance reached values below 8 GΩ. The integrity of the cell-attached patch was further confirmed by the absence of fluorescent dye in the cytoplasm.
Analysis of electrophysiological data.
Passive properties were calculated from the current response to a 5-mV hyperpolarizing step. Series and input resistances were measured from the peak and steady state values of the negative deflection; membrane capacitance was obtained after measuring the area under the capacitive current. Evoked postsynaptic currents were analyzed from average traces using ad hoc MATLAB (The Mathworks [http://www.mathworks.com/products/matlab/
]) routines. For AMPA/NMDA ratios, AMPA was measured as the peak amplitude of the EPSC at Vhold
= −80 mV. NMDA was measured as the EPSC amplitude at Vhold
= +50 mV, 33 ms after the stimulus artifact (~25 ms after the AMPA peak; arrowheads in A). At this time point, AMPA currents decay to less than 5% of their peak amplitude, and their contribution to the NMDA component becomes negligible [42
]. The overall significance of the comparisons of AMPA/NMDA ratio for each group was not dependent on the time point chosen for measuring the NMDA component (unpublished data).
In paired-pulse recordings, a baseline value was measured prior to the second stimulation artifact and subtracted from the amplitude of the second EPSC. For inter-pulse intervals less than 100 ms, the baseline value was extrapolated from the monoexponential fitting of the first EPSC decay phase. Rise and decay times were calculated from 20% to 80% and 100% to 40% of the peak amplitude, respectively. For individual sIPSC recorded at 0 mV (), decay time represents time constant from single-exponential fits of the decay phase. Kinetics of IPSCs evoked on the GCL and ML were measured from inward and outward traces recorded at VHold = −80 and approximately −40 mV, respectively. For GCL-evoked IPSCs, EGABA and GGABA were calculated from I-V curves constructed at five time points: (1) the rising phase of the IPSC; (2) the peak of the IPSC at VHold = −80 mV; and at (3) 85 ms, (4) 145 ms, and (5) 185 ms after the stimulation artifact. For ML-evoked IPSCs, EGABA and GGABA were calculated from I-V curves constructed at the time of the outward IPSC peak. In some experiments, the current amplitude at VHold = −80 mV deviated from linearity and was not considered for the I-V fitting. All sample traces of evoked responses are averages of more than ten sweeps.
Spontaneous IPSC were sampled at 10 KHz with Axoscope 9 (Molecular Devices), digitally filtered at 1 KHz, and detected offline with Mini Analysis software (Synaptosoft [http://www.synaptosoft.com/MiniAnalysis/
]). Amplitude and area thresholds for detection were 10 pA and 50 pA.ms, and the average standard deviation of baseline noise was 2 pA. All events were individually validated, and artifacts were discarded by visual inspection. For analysis of sIPSC peak amplitude (G and G), a mean value was obtained for each DGC. Individual sIPSCs were then exported to MATLAB, where kinetics were analyzed and two-dimensional histograms were obtained using routines developed in our laboratory. Kinetics of sIPSCs (H and H) were analyzed from the scaled average sIPSC from each cell. Events with multiple peaks were discarded from the analysis. For the construction of sIPSC scaled averages, traces were aligned at 10% of the rising phase. All statistical tests used two-tailed analysis.