Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1^CreERT2 and Glast^CreERT2 Murine Lines

Spiking properties of adult-born GCs in Ascl1^CreERT2 mice. A, Representative whole-cell current-clamp recordings in GCs at different ages. Spiking was elicited by depolarizing current steps of increasing amplitude (500 ms, 0–130 pA, 10 pA steps). Panels represent example traces at the indicated current steps. Calibration: left, 100 mV; right, 20 mV; 100 ms. B, Repetitive firing quantified as the number of spikes elicited by increasing current steps. C, Current threshold to elicit the first spike for the experiments shown in B. D, E, AP amplitude, AHP amplitude, and AP half-width were measured for the first spike (D) and all identified spikes (E). ***p < 0.001 compared with mature GCs. **p < 0.01 compared with mature GCs. *p < 0.05 compared with mature GCs. ns, Not significant. Sample sizes (presented as neurons/mice) were 34/6 (22 dpi), 39/5 (25 dpi), 42/6 (28 dpi), 35/5 (35 dpi), 31/5 (49 dpi), and 40/23 (mature). Red lines indicate mean ± SEM. Statistical comparisons were done using Kruskal-Wallis test followed by a post hoc Dunn's test.

Excitatory input connectivity of adult-born GCs in Ascl1^CreERT2 mice. A, Representative traces of sEPSC recorded at −70 mV in GCs at different ages. Top, Time-compressed traces allow the visualization of sEPSC frequency. Bottom, Expanded presentations show individual events. Calibration: top, 10 pA, 10 s; bottom, 2 pA, 100 ms. B, Frequency of sEPSC events, measured during 120 s. ***p < 0.001 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). **p < 0.01 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). *p < 0.05 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). C, sEPSC amplitude presented as mean value for each cell (Ci), and cumulative frequency for each cell age (Cii). Inset in Cii depicts amplitude histogram. Errors bars in Cii were omitted for clarity. Cumulative frequencies presented no differences with mature GCs by Kolmogorov–Smirnov test (minimum p > 0.24). D, Kinetic analysis of sEPSCs: rise time (20%–90%), decay time (90%–30%), and half-width. No significant differences were found for any of the parameters by Kruskal–Wallis test (amplitude, p = 0.06; rise time, p = 0.32; decay time, p = 0.10; width, p = 0.04). Sample sizes (presented as neurons/mice) were 29/6 (22 dpi), 34/5 (25 dpi), 34/6 (28 dpi), 17/3 (35 dpi), 31/5 (49 dpi), and 38/22 (mature). Red lines indicate mean ± SEM.

Morphology and neuronal markers for developing GCs generated in adult Ascl1^CreERT2 mice. A, Confocal images of Tom⁺ cells in the dentate gyrus of Ascl1^CreERT2;CAG^floxStopTom mice at different times after TAM induction. NeuN immunolabeling was performed to allow the identification of the GCL. Note the rise in the number of labeled cells from 3 to 14 dpi, characteristic of cell proliferation. White dashed lines were added to facilitate visualization of dendrites among densely packed tagged neurons. Imaging conditions were selected to render similar fluorescence intensity in all age groups. B, Examples of 22 and 35 dpi GCs obtained with identical imaging settings reveal a time-dependent increase in fluorescence intensity. Note the mixed population of high and low Tom intensities at 35 dpi. C, Representative images of single optical sections, and quantification of Sox2 (left) and NeuN (right) levels. Sample sizes: >50 Tom⁺ cells, with 2 mice for each time point (1 mouse for 28 dpi). D, Left, Representative images display single optical sections of DCX (top) and Cb (bottom) expression in Tom⁺ GCs at different times after induction. Inset, Higher magnification of Tom⁺ cells, to assess colocalization with DCX or Cb. Right, Quantification of DCX (top) and Cb (bottom) levels. Sample sizes: > 90 Tom⁺ GCs, with 2 or 3 mice for each time point. Error bars indicate mean ± SEM. All scale bars represent 20 μm.

Computational simulation of adult neurogenesis in Ascl1^CreERT2 mice. Neurogenesis results from the combination of a generation rate of newborn GCs (A, left; G(t)) and their survival probability (A, right; S(n)). Generation was represented by an exponential function starting at 1 dpi (T_inflexion) with a decay time of 5 d (τ_neurogenesis). T_inflexion and τ_neurogenesis were the only free parameters in the model. Survival probability was calculated based on experimental data (Brandt et al., 2003). The generation rate depends on the time after TAM induction, whereas the survival probability is a function of cell age. The number of newborn cells N(t, n) whose age is n at the time t was calculated as N(t, n) = G(t − n) × S(n). The population of GCs at different times after TAM induction has a substantial contribution of neurons whose age is close to the time after induction (B). To compute the expression of neuronal markers, we assumed that each labeled cell independently expresses DCX or Cb with a probability represented by respective sigmoid functions (C), according to previous experimental data (Espósito et al., 2005; Piatti et al., 2011). The model predicts expression levels of DCX (D, left, violet line) and Cb (D, right, red line) tightly close to experimental data (green bars, extracted from Fig. 4). For details on the model, see also Materials and Methods.

Intrinsic properties of adult-born GCs in Glast^CreERT2 mice. A, Confocal image of a 60-μm-thick hippocampal section depicting adult-born GCs (red) at 49 dpi. NeuN immunofluorescence (blue) allows visualizing the GCL, CA1, CA2, and CA3 pyramidal layers. Dendrites extend through the molecular layer (ML), and mossy fibers project across the hilus (H) to CA3, via the stratum lucidum. There are scattered Tom⁺ astrocytes throughout the section. Scale bar, 100 μm. B, Tom indelible expression was induced by TAM administration, and animals were killed at different ages to perform electrophysiological recordings in acute slices. C–E, Resting potential (C), input resistance (D), and capacitance (E) were measured in fluorescent adult-born neurons and unlabeled mature GCs. Sample sizes (presented as neurons/mice) were 38/5 (22 dpi), 37/6 (25 dpi), 37/4 (28 dpi), 33/4 (49 dpi), and 28/19 (mature). ***p < 0.001 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). **p < 0.01 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). *p < 0.05 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). ns, Not significant. Red lines indicate mean ± SEM.

Spiking properties of adult-born GCs in Glast^CreERT2 mice. A, Representative whole-cell current-clamp recordings in GCs at different ages, as indicated. Spiking was elicited by depolarizing current steps of increasing amplitude (500 ms, 0–130 pA, 10 pA steps). Panels represent example traces at the indicated current steps. Calibration: left, 100 mV; right, 20 mV; 100 ms. B, Repetitive firing quantified as the number of spikes elicited by increasing current steps. C, Current threshold to elicit the first spike for the experiments shown in B. D, E, AP amplitude, AHP amplitude, and AP half-width were measured for the first spike (D) and all identified spikes (E). ***p < 0.001 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). **p < 0.01 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). *p < 0.05 compared with mature GCs (Kruskal–Wallis test followed by a post hoc Dunn's test). ns, Not significant. Sample sizes (presented as neurons/mice) were 36/5 (22 dpi), 37/6 (25 dpi), 35/4 (28 dpi), 32/4 (49 dpi), and 27/16 (mature). Red lines indicate mean ± SEM.