Functional Convergence of Neurons Generated in the Developing and Adult Hippocampus

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function

Reliable Activation of Immature Neurons in the Adult Hippocampus

Neurons born in the adult dentate gyrus develop, mature, and connect over a long interval that can last from six to eight weeks. It has been proposed that, during this period, developing neurons play a relevant role in hippocampal signal processing owing to their distinctive electrical properties. However, it has remained unknown whether immature neurons can be recruited into a network before synaptic and functional maturity have been achieved. To address this question, we used retroviral expression of green fluorescent protein to identify developing granule cells of the adult mouse hippocampus and investigate the balance of afferent excitation, intrinsic excitability, and firing behavior by patch clamp recordings in acute slices. We found that glutamatergic inputs onto young neurons are significantly weaker than those of mature cells, yet stimulation of cortical excitatory axons elicits a similar spiking probability in neurons at either developmental stage. Young neurons are highly efficient in transducing ionic currents into membrane depolarization due to their high input resistance, which decreases substantially in mature neurons as the inward rectifier potassium (Kir) conductance increases. Pharmacological blockade of Kir channels in mature neurons mimics the high excitability characteristic of young neurons. Conversely, Kir overexpression induces mature-like firing properties in young neurons. Therefore, the differences in excitatory drive of young and mature neurons are compensated by changes in membrane excitability that render an equalized firing activity. These observations demonstrate that the adult hippocampus continuously generates a population of highly excitable young neurons capable of information processing

Slow-Dendritic sIPSCs

<div><p>(A) and (B) Example of traces of outward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scales indicate 0.5 s/50 ms (top/bottom), 10 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 695 events) and adult DGCs ([D] <i>n</i> = 1,160 events). Color scale indicates the relative frequency for each bin.</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult DGCs (red). Same data as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 10 neurons; adult, <i>n</i> = 16; <i>p</i> = 0.94; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (pup, <i>n</i> = 10; adult, <i>n</i> = 14; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 50 ms. All experiments conducted in the presence of kyn at V_hold = 0 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.37; decay time, <i>p</i> = 0.41).</p></div

Short-Term Plasticity of Entorhinal Glutamatergic Afferents

<div><p>(A) Average EPSCs recorded at V_Hold = −80 mV (downward deflections) and +50 mV (upward deflections) from pup and adult DGCs (<i>n</i> = 8 to 11) upon stimulation of MPP or LPP. Dashed line indicates zero level. Arrowheads denote time points for quantification of AMPA (open triangles) and NMDA (filled triangles) currents shown in (B). Criteria for AMPA/NMDA quantification are detailed in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a> section. Scale bars indicate 20 ms, 100 pA.</p> <p>(B) AMPA/NMDA ratio from pup and adult DGCs (<i>n</i> = 9 to 13) in response to MPP or LPP stimulation. Two-way ANOVA revealed a significant effect of MPP versus LPP (<i>p</i> = 0.006), but no significant effect of pup versus adult (<i>p</i> = 0.63).</p> <p>(C) Averages of EPSCs in response to paired-pulse stimulation of the MPP or LPP delivered at increasing interpulse intervals (20, 50, 100, and 500 ms). Traces are averages of 7–14 cells aligned and normalized to the first EPSC. Stimulation artifacts and late decay phases of the second EPSC were removed for clarity. Scale bar indicates 100 ms.</p> <p>(D) Paired-pulse ratio as a function of interpulse interval for the experiments shown in (C). Two-way ANOVAs revealed a significant effect of interpulse interval for MPP (dashed lines, <i>p</i> < 0.0001) and LPP (solid lines, <i>p</i> < 0.0001), but no significant effect of pup (green lines, solid circles) versus adult (red lines, open circles) for either MPP (<i>p</i> = 0.073) or LPP (<i>p</i> = 0.72) stimulation (<i>n</i> = 9 to 14)</p> <p>(E) Example of EPSCs from a pup (green) and an adult DGC (red) in response to MPP stimulation (ten pulses, 50 Hz) Traces are normalized to the first EPSC amplitude. Scale bar indicates 40 ms.</p> <p>(F) Relative EPSC amplitudes measured in response to 50-Hz stimulation evoked as shown in (E). No difference was found between pup and adult responses (two-way ANOVA, <i>p</i> = 0.49, <i>n</i> = 10 pups [solid circles], <i>n</i> = 4 adults [open circles]). All recordings were carried out in the presence of 20-μm BMI. Neurons were approximately 18 wk old (pup) and approximately 13 wk old (adult). All plots depict mean ± SEM.</p></div

Fast-Perisomatic sIPSCs

<div><p>(A) and (B) Example of traces of inward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scale bars indicate 1 s/50 ms (top/bottom), 40 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 5,871 events) and adult DGCs ([D] <i>n</i> = 8,183 events). Color scale indicates the relative frequency for each bin (square areas in the graph).</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult (red) DGCs. Data are the same as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 12 neurons; adult, <i>n</i> = 15; <i>p</i> = 0.99; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (<i>n</i>, same as in [F]; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 10 ms. All experiments conducted in the presence of kyn at V_hold = −80 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.96; decay time, <i>p</i> = 0.72).</p></div

Firing Behavior Elicited by Excitatory Inputs

<div><p>(A) Action currents in cell-attached configuration recorded from an adult-born DGC in response to MPP stimulation at increasing stimulus strengths (0.5–1.5 mA, 50 μs). Six representative epochs are shown. Spiking probability (p) is shown below the traces. The asterisk (*) marks the stimulation artifact. Scale indicates 10 ms, 50 pA.</p> <p>(B) Sample experiment of simultaneous cell-attached recordings of DGCs born in pup and adult brain in response to MPP stimulation (0.4 mA, 50 μs). Action currents indicate a higher spiking probability in the pup DGC. Scales indicate 10 ms, 50 pA (left) and 20 pA (right).</p> <p>(C) Sample experiment in which the spiking probability is higher in the adult-born DGC (1.5 mA, 50 μs). Scale indicates 10 ms, 30 pA.</p> <p>(D) Firing behavior of DGCs born in pup and adult brain during simultaneous paired experiments. No significant difference was found (<i>n</i> = 14 pairs, <i>p</i> = 0.8, Wilcoxon signed rank test). All recordings were carried out in the presence of BMI (20 μM). In this set of experiments, adult-born neurons were retrovirally labeled with GFP, whereas unlabeled DGCs of the middle third of the GCL were considered postnatally born (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a>). Repetitive (>15 episodes) slow frequency stimulation was used to measure the spiking probability for each neuron at the given stimulus.</p></div

GABAergic Inputs Elicited by GCL Stimulation

<div><p>(A) Example of evoked IPSCs recorded from a pup-born DGC in response to extracellular stimulation of the GCL before (control), during (BMI), and after (wash) application of 20 μM BMI. Scale bars indicate 25 ms, 20 pA. Inset: schematic diagram of the DG showing the position of the bipolar electrode for the stimulation of GCL interneurons. Experiments were carried out with internal solution containing low [Cl⁻].</p> <p>(B) Typical traces of IPSCs evoked on a pup (green) and adult (red) DGC recorded from the same slice in a paired experiment. Scales indicate 25 ms, 40 pA.</p> <p>(C) Paired analysis of peak IPSC amplitude recorded from a pup versus adult DGCs (<i>n</i> = 19 pairs, <i>p</i> = 0.97, paired <i>t</i>-test). Mean ± SEM are shown on the sides.</p> <p>(D) Kinetics of evoked IPSCs (<i>n</i> = 40 adults, <i>n</i> = 22 pups; rise time, <i>p</i> = 0.31; decay time, <i>p</i> = 0.19; unpaired <i>t</i>-tests). Inset: scaled average traces of evoked IPSCs. Scale bar indicates 50 ms.</p> <p>(E) and (F) Examples of I-V curves of evoked IPSCs recorded from pup (E) and adult DGCs (F). Top: sample traces of IPSCs recorded at the holding potentials shown on the right (mV). Scale bars indicate 50 ms, 100 pA (E) and 50 pA (F). Bottom: I-V plots measured at time points indicated with arrowheads (early: filled triangles/dashed lines, late: open triangles/solid lines).</p> <p>(G) E_GABA measured at early and late time points. Two-way ANOVA (<i>n</i> = 11, pup; <i>n</i> = 16, adult) revealed a significant effect of early versus late points (<i>p</i> < 0.0001), but not of pup versus adult (Adu) neurons (<i>p</i> = 0.56).</p> <p>(H) Conductance profile of IPSCs. Each value denotes the slope conductance measured from the I-V curve at the time points indicated on the sample traces (inset). No significant differences were found between pup (green) and adult (red; 11 ≤ n ≤ 16, <i>p</i> = 0.62, two-way ANOVA). Scale indicates 50 ms. All experiments were conducted in the presence of kyn (4 mM) in slices obtained from mice aged 13–14 wk. Neurons were approximately 12 wk old (P7) and approximately 7 wk old (P42).</p></div

Fluorescent Labeling of DGCs Born during Early Postnatal and Adult Neurogenesis

<div><p>(A) and (B) Retroviral delivery of GFP into DGCs generated at P7 (A) and P42 (B), analyzed 7 wk after each injection. The GCL was labeled by immunohistochemistry for the neuronal marker NeuN (blue). Images are merges of 27 (A) and 21 (B) confocal planes taken from coronal sections (40-μm thick). H, hilus; ML, molecular layer.</p> <p>(C) and (D) Double retroviral labeling of DGCs generated at P7 (GFP⁺, green) and P42 (RFP⁺, red). Images are merges of nine (C) and 20 (D) confocal planes taken from fixed transverse sections of the DG (400-μm thick) from 13-wk-old mice.</p> <p>(E) Double labeling of DGCs with GFP (green) and BrdU (red): intrahippocampal injections of CAG-GFP retrovirus in P7 were followed by daily injections of BrdU carried out from P21 to P25; brains were analyzed at P53. The image is a merge of 16 confocal planes.</p> <p>(F) Example of a sporadic event of co-localization of GFP, BrdU, and NeuN shown by a single optical section for the green, red, and blue channels. Their overlay is shown together with the orthogonal projections onto the <i>x-z</i> (top) and <i>y-z</i> (right) planes.</p> <p>(G) Number of GFP⁺ or BrdU⁺ cells per mouse (left) and the percentage of GFP⁺ cells showing BrdU label (right). Data are mean ± standard error of the mean (SEM) (<i>n</i> = 3 mice). Scale bars indicate 50 μm (A–E) or 10 μm (F).</p></div

GABAergic Inputs Evoked by ML Stimulation

<div><p>(A) Example of evoked IPSCs recorded from a pup-born DGC in response to extracellular stimulation of the ML before (control), during (BMI), and after (wash) application of 20 μM BMI. Scale bars indicate 25 ms, 20 pA. Inset: schematic diagram of the DG showing the position of the bipolar electrode for the stimulation of ML interneurons. Experiments were carried out with internal solution containing low [Cl⁻].</p> <p>(B) Kinetics of IPSCs evoked on pup versus adult DGCs (pup, <i>n</i> = 6; adult, <i>n</i> = 14; rise time, <i>p</i> = 0.85; decay time, <i>p</i> = 0.90, paired <i>t</i>-tests). Inset: scaled averages of IPSCs (pup, green; adult, red). Scale bar indicates 50 ms.</p> <p>(C) and (D) Examples of I-V curves of evoked IPSCs recorded from pup (C) or adult (D) DGCs. Top: sample traces of IPSCs recorded at the holding potentials shown on the right (mV). Scale bar indicates 50 ms, 10 pA. Bottom: I-V plots measured at the time indicated by the arrowheads.</p> <p>(E) E_GABA of IPSCs (pup, <i>n</i> = 7; adult, <i>n</i> = 11; <i>p</i> = 0.67, <i>t</i>-test).</p> <p>(F) Slope conductance measured at the IPSC peak (<i>n,</i> same as in [E]; <i>p</i> = 0.23; <i>t</i>-test). All experiments conducted in the presence of kyn (4 mM) in neurons aged 12 wk (P7) and 7 wk (P42).</p></div