Conceived and designed the experiments: LAM MSE AFS. Performed the experiments: LAM MSE. Analyzed the data: LAM MSE AFS. Wrote the paper: LAM AFS. Prepared retroviral stocks: GL.
The authors have declared that no competing interests exist.
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.
The dentate gyrus is the main gateway to the hippocampus and it constitutes a primary neurogenic niche of the adult brain. Neural progenitor cells of the subrgranular zone give rise to dentate granule cells (DGCs) that develop and mature over several weeks. A substantial fraction of those newly generated neurons become integrated in the hippocampal network and are then maintained throughout adulthood
The participation of newly generated neurons in hippocampal processing has been highlighted by recent reports whereby reduced or blocked neurogenesis impair performance in hippocampus-dependent learning tasks
To put forward the hypothesis that young neurons behave as a distinct neuronal population within the active hippocampal networks it is crucial to determine when they become engaged in firing activity, and how their activity compares to the remaining units of the network. We have used retroviral labeling and electrophysiological tools to study intrinsic membrane properties and input-output conversion in developing DGCs of the adult mouse hippocampus. We found that three- to four-week-old neurons display weak glutamatergic inputs, yet spike reliably in response to perforant path stimulation. In these immature neurons ionic currents are efficiently converted into membrane depolarization due to their high input resistance. As neurons mature the membrane resistance decreases, with a consequent reduction in excitability. In addition, we found a developmental increase in the inward rectifier K+ conductance (Kir) that highlights Kir channels as regulators of the excitability in newborn DGCs in the adult hippocampus.
The activity of the hippocampal network can only be modified by neurons that can integrate incoming signals to produce a firing behavior and alter the state of postsynaptic targets. Firing is ultimately shaped by the concerted action of synaptic integration and excitability. To investigate the impact of developing DGCs in the adult hippocampal network, a retrovirus encoding GFP driven by a strong promoter was used to label adult-born DGCs and acute brain slices were prepared 18 to 29 days after retroviral injection (dpi), at which time neurons are still immature, yet afferent excitatory connections become established
The excitability of developing neurons in the adult dentate gyrus was investigated by monitoring passive and active membrane properties in whole-cell recordings. Injection of current steps of small amplitude readily generated action potentials in young DGCs, whereas increasingly larger currents were required to reach the membrane threshold for action potential in more mature neurons (
(A) Whole-cell current clamp recordings in neurons of different ages, as indicated on top of each panel. Spiking was elicited by depolarizing current steps of increasing amplitude (step = 10 pA). Each panel shows a subset of five representative traces with steps 10, 30, 50, 70 and 90 pA (from bottom to top). Scale bars: 100 mV, 100 ms. (B) Repetitive firing quantified as the number of spikes elicited by increasing current steps. Sample sizes are N = 10 (19 dpi), 20 (22 dpi), 29 (25 dpi), 13 (28 dpi), 24 (49 dpi) and 51 (mature). (C) Current threshold to elicit the first spike for the experiments shown in (B). (
young | mature | ||
519±30 (68) | 224±7 (89) | ||
30.6±1.0 (68) | 56.9±2.1 (89) | ||
32.5±2.4 (25) | 34.0±2.0 (25) | ||
−75.6±0.5 (42) | −80.6±0.5 (60) | ||
29.0±1.7 (42) | 58.8±2.5 (60) | ||
−42.8±0.9 (38) | −38.4±0.9 (55) | ||
−57.7±0.5 (23) | −59.8±0.4 (23) | ||
113.8±0.8 (23) | 118.8±0.4 (23) | ||
266±8 (23) | 309±6 (23) | ||
307±20 (26) | 423±6 (29) | ||
189±13 (26) | 288±16 (29) | ||
1.73±0.32 (16) | 5.18±0.34 (20) | ||
3.64±0.13 (40) | 3.37±0.13 (40) | ||
21.8±0.9 (40) | 25.1±0.9 (40) | ||
7.01±0.18 (42) | 5.83±0.18 (39) | ||
34.0±1.8 (43) | 37.3±2.0 (42) |
Tau measured by fitting a single exponential equation to the membrane potential response to a current pulse (10 pA)
gNa+ measured at Vh = −20 mV
gK+ measured at steady state at Vh = 70 mV
gKir+ measured as described in
Welch's correction for differences in variances was applied.
Mean±SEM are shown, with cell numbers in parentheses. Statistical analyses were performed by two-tailed t-tests.
The input resistance of a neuron (Rin) reflects the cell size and the density of ion channels open at resting. High values of Rin are associated with an enhanced excitability, as small inward currents can elicit large membrane depolarizations. Young neurons displayed high values of Rin (in the GΩ range) that decreased as they reached mature developmental stages (
To determine whether an excitatory drive can trigger action potentials in immature DGCs, postsynaptic responses to afferent glutamatergic stimulation were recorded in the presence of GABA receptor antagonists (picrotoxin 100 µM and CGP 55845 100 nM). Excitatory postsynaptic currents (EPSCs) were evoked by increasing stimulus intensities delivered to the medial perforant path (
(A) Illustration depicting electrophysiological recordings of postsynaptic responses in acute hippocampal slices. Stimulation was performed in the medial perforant path (pp), and whole-cell recordings were obtained in young (green) or mature (blue) granule cells. GCL, granule cell layer; ML, molecular layer; H, hilus. (B) Example traces of EPSCs evoked by increasing stimulus strength (0.2–1 mA, 50 µs) obtained from DGCs of different developmental stages (shown on the top). Each trace is an average of five epochs. Scale bars: 100 pA, 50 ms. (C) Maximal peak EPSC amplitude for young and mature DGCs. (#) denotes
Stimuli that evoked the larger EPSC amplitudes were selected to assess spiking by an excitatory drive. DGCs were practically unable to spike by 19 dpi most likely due to their weak excitatory input combined with their limited ability to generate action potentials (
Since young and mature DGCs display distinct excitability, spike properties and input strength, it would be predictable that they are differentially recruited into an active network. To address this question spiking probability of young (21–29 dpi) and mature DGCs were measured in current clamp, while repetitive stimuli of increasing strength were delivered at low frequency to the medial perforant path (
(A) Example of a recording performed to measure spiking probability in response to presynaptic stimuli of different intensities in the same neuron. Each column shows typical membrane potential traces in response to stimuli repeated at low frequency (0.07 Hz). Probabilities are shown on the top. Scale bars: 50 mV, 100 ms. (B) Quantitative analysis of spiking probability. Each experiment involved consecutive recordings of a young and a mature neuron at all stimulus intensities in the same slice. Two-way ANOVA revealed no differences between young and mature cells (
It is surprising that young and mature DGCs have different functional properties but display similar firing behavior. To better understand this phenomenon we investigated how synaptic currents are transduced into membrane depolarization. Current- and voltage clamp recordings were combined to monitor subthreshold postsynaptic responses evoked by afferent stimulation with increasing strength. Thus, each stimulus generated an EPSP / EPSC pair whereby a given postsynaptic depolarization was associated to a particular synaptic current (
(A) Typical traces recorded in a young and a mature neuron displaying evoked EPSPs (upper panels) and EPSCs (lower panels) for the same series of increasing stimuli (0.1–1 mA) delivered to the medial perforant path. Scale bars: 5 mV/50 pA, 50 ms. (B) Data from a representative experiment showing EPSP / EPSC pairs and their linear regression curves. (C) Average slope of linear regressions. Statistical difference was analyzed by a two-tailed
To better understand the observed differences in firing behavior we characterized action potentials, voltage-gated Na+ and K+ currents, and inward rectifier K+ currents (Kir), which contribute to neuronal excitability in subthreshold conditions
(A) Characterization of action potentials recorded in young and mature neurons. Spiking threshold, N = 38 (young) and 55 (mature); (*) denotes
The observations described above suggest that the activity of DGCs might be regulated by the level of Kir expression. To address this question neuronal excitability was studied in the presence of extracellular Ba2+ (200 µM), a well known blocker of Kir channels
(A) Kir conductance in the absence or presence of BaCl2. (**) denote
DGCs generated in the adult hippocampus receive functional afferents, spike in response to an excitatory drive, and release glutamate onto postsynaptic target cells (this work,
Kir channels are involved in the resting membrane potential and conductance, exerting a role in the regulation of cellular excitability
Voltage-gated Na+ and K+ channels displayed a slight age-dependent increase, consistent with the observed changes in action potential threshold and shape. This minor increment indicates that voltage-gated currents were close to plateau levels in young neurons. Consistent with this notion we observed only a minor increase in the rising slope of the action potential, a parameter that reflects the density of Na+ channels
A replication-deficient retroviral vector based on the Moloney murine leukemia virus was used to express enhanced GFP (or Kir-IRES-GFP in experiments shown in
Female C57Bl/6J mice, 6–7 weeks of age, were anesthetized (100 µg ketamine/10 µg xylazine in 10 µl saline/g). CAG-GFP expressing retrovirus was infused (0.9 µl in 7 min) into the dorsal area of the right dentate gyrus (coordinates from bregma: antero-posterior = −2 mm, lateral = 1.5 mm, ventral = 1.9 mm) using a microcapillary calibrated pipette (Drummond Scientific, Broomall, PA) as described previously
Experiments were carried out in 352 neurons from 92 mice. Mice were anesthetized and decapitated at 18 to 56 days post injection (dpi), as described below. Brains were removed into a chilled solution containing (in 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, and 4.0 kynurenic acid. Horizontal slices (400-µm thick) were cut in a vibratome (Leica VT1200 S, Nussloch, Germany) and transferred to a chamber containing (in 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 (315 mOsm). Slices were bubbled with 95% O2/5% CO2 and maintained at 30°C for at least 1 hour before experiments started. Adjacent sections to the injection site were discarded to avoid effects of inflammation. Recordings were performed at 23 ± 2°C using microelectrodes (4–6 MΩ) pulled from borosilicate glass (KG-33; King Glass, Claremont, CA) and filled with (in mM): 120 K-gluconate, 20 KCl, 5 NaCl, 4 MgCl2, 0.1 EGTA, 10.0 HEPES, 4.0 Tris-ATP, 0.3 Tris-GTP, 10 phosphocreatine, Alexa Fluor 594 (5 µg/ml; Invitrogen), pH 7.3, and 290 mOsm. Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), digitized (Digidata 1322A; Molecular Devices), and acquired at 20 KHz onto a personal computer using the p-Clamp 9 software (Axon CNS, Molecular Devices).
All experiments were carried out in neurons that showed action potentials in response to current injection through the patch pipette. Developing neurons expressing GFP were binned in the following age groups: 18–20 dpi (“19 dpi” group), 21–23 dpi (“22 dpi”), 24–26 dpi (“25 dpi”), 27–29 dpi (“28 dpi”), and 42–56 dpi (“49 dpi”). GFP+ cells of different ages were identified in the granule cell layer using FITC fluorescence optics (DMLFS; Leica). In previous works we have compared mature neurons born in 15-day-old embryos, 7-day-old pups and adult mice and found no significant functional differences
Whole-cell voltage-clamp recordings were performed at a holding potential (Vh) of −70 mV, unless otherwise noted. Criteria to include cells in the analysis were co-labeling with Alexa Fluor 594 or visual confirmation of GFP in the pipette tip and absolute leak current <100 pA at Vh. Series resistance was typically 10–20 MΩ, and experiments were discarded if higher than 25 MΩ. Membrane capacitance and input resistance were obtained from current traces evoked by a hyperpolarizing step of 10 mV. In current-clamp recordings the resting membrane potential was kept at −70 mV by passing a holding current. The threshold current for spiking was assessed by successive depolarizing current steps (5 or 10 pA; 500 ms) to drive the membrane potential (Vm) from resting to 0 mV. Action potential threshold was defined as the point at which the derivative of the membrane potential dV/dt deviated from the mean baseline value by >2 standard deviations
Extracellular stimulation of the medial perforant path was performed using concentric bipolar electrodes (50 µm diameter; Frederick Haer Company, Bowdoinham, ME) and an Iso-Flex stimulator (A.M.P.I.; Jerusalem, Israel). The stimulation electrode was placed orthodromically on the middle third of the molecular layer at >250 µm from the recorded cell. Functional inputs were assessed at stimulus strengths of 0.1–1.3 mA (50 µs) repeated at 15-s intervals. Excitatory postsynaptic currents (EPSCs) and potentials (EPSPs) were recorded in the presence of picrotoxin (100 µM) and CGP 55845 (100 nM), antagonists of GABAA and GABAB receptors. Peak amplitudes were calculated from the average of 5 traces (EPSCs) and 10–20 traces (EPSPs). Simultaneous cell-attached recordings were carried out under voltage clamp at 0 mV using pipettes with a high tip resistance (10–14 MΩ). Stimulus intensity was gradually increased (0.3–2 mA, 50 µs) and, for each cell, spiking probability was obtained after 10–20 stimuli. For each pair of cells, the lower stimulus intensity at which both (young and mature) neurons fired was selected for statistical analysis of spiking probability. Whole-cell recordings were carried out at the end of all experiments to verify granule cell phenotype by spiking properties and morphology after filling with a fluorescent dye. 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.
Spiking probability vs. input strength. Spiking probability vs. EPSC amplitude measured in young and mature DGCs. Input strength was binned into three categories according to the EPSC amplitude. (*) and (**) denote
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Graphic analysis used to determine spiking threshold. (A) Representative action potentials from young (green) and mature neurons (blue). Scale bars: 50 mV, 5 ms. (B) The plot depicts the derivative of the membrane potential (dV/dt) in relation to the membrane potential (Vm). Arrows indicate spiking thresholds for a young and a mature DGCs.
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Ontogeny of Kir currents in adult-born DGCs. (A) Average I–V curves, with N = 13 (8 dpi), N = 14 (17 dpi), N = 18 (24 dpi), N = 12 (29 dpi) and N = 15 (35 dpi). (B) Kir conductance calculated for the experiments shown in (A).
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Kir blockade by extracellular Ba2+. (A) Example current traces of young (25 dpi) and mature neurons recorded in the absence (left) or presence (right) of BaCl2 (200 µM). Voltage steps from −45 to −130 mV (step 5 mV, 100 ms) for a 25 dpi and a mature neuron. Scale bars: 200 pA, 30 ms. (B) Mean I–V plots obtained from N = 24 (young), 8 (young+Ba2+), 28 (mature) and 23 (mature+Ba2+).
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We thank Guillermo Lanuza for the mouse Kir 2.1 cDNA, and Benedikt Berninger, Josef Bischofberger, and Antonia Marín-Burgin for critical comments to improve this manuscript.