Activation of adult-born neurons facilitates learning and memory

Journal name:
Nature Neuroscience
Volume:
15,
Pages:
897–904
Year published:
DOI:
doi:10.1038/nn.3108
Received
Accepted
Published online

Abstract

Thousand of local interneurons reach the olfactory bulb of adult rodents every day, but the functional effect of this process remains elusive. By selectively expressing channelrhodopsin in postnatal-born mouse neurons, we found that their activation accelerated difficult odor discrimination learning and improved memory. This amelioration was seen when photoactivation occurred simultaneously with odor presentation, but not when odor delivery lagged by 500 ms. In addition, learning was facilitated when light flashes were delivered at 40 Hz, but not at 10 Hz. Both in vitro and in vivo electrophysiological recordings of mitral cells revealed that 40-Hz stimuli produced enhanced GABAergic inhibition compared with 10-Hz stimulation. Facilitation of learning occurred specifically when photoactivated neurons were generated during adulthood. Taken together, our results demonstrate an immediate causal relationship between the activity of adult-born neurons and the function of the olfactory bulb circuit.

At a glance

Figures

  1. Light-induced activation of adult-born neurons.
    Figure 1: Light-induced activation of adult-born neurons.

    (a) Left, lentiviral vectors encoding ChR2-YFP were injected in the RMS. Right, confocal images of an olfactory bulb (OB) slice showing the immunoreactivity of ChR2-YFP–positive neurons (green) and DAPI staining (blue). Arrows point to YFP-positive cell somas observed in the GCL (middle) and the glomerular layer (GL, right). Scale bars represent 15 μm. The white dashed line delineates a glomerular structure. SVZ, subventricular zone. (b) Patch-clamp recordings of an adult-born granule cell expressing ChR2-YFP (10 wpi). Brief flashes of light (5 ms) evoked inward currents at −70 mV (bottom). Action potentials were triggered by light flashes (8 mW mm−2; current clamp = −2 pA, Vm = −68 mV; top). (c) Left, schematic of the in vivo photostimulation design. The blue/black shading illustrates the penetration depth of the light (see Supplementary Fig. 2 for quantification). Right, density of YFP and c-Fos double-positive neurons in non-stimulated mice (no stim) and after photostimulation (three 90-s pulse trains at 40 Hz delivered every 2 min, light stim). A significant difference was found between groups only in the GCL (**P < 0.01 with a Bonferroni test after repeated measures two-way ANOVA, group P = 0.04; 13,226 cells, n = 4). Error bars represent s.e.m.

  2. Olfactory discrimination learning.
    Figure 2: Olfactory discrimination learning.

    (a) In our task, the mouse breaks a light beam (red dashed line) across the odor port, which initiates a trial. Licking in the water port (WP) in response to a positive odor stimulus (S+) triggers water delivery (that is, the go response, left). In response to a negative odor stimulus (S–), the trained mouse retracts its head (that is, the no-go response, right) and rejects licking. (b) Accuracy (mean percentage of correct responses for five training blocks) is shown for easy odor pair (gray squares) and difficult odor pair (black squares). The learning curves of difficult and easy tasks were significantly different (group: F1,28 = 7.2, P = 0.01, repeated measures two-way ANOVA followed by Fisher LSD post hoc test, *P < 0.05, **P < 0.01, n = 12–16). A score of 50% corresponds to the success rate at chance level. (c) Data are presented as in b for the percentage of correct responses for each training block (group F1,23 = 8.57, P = 0.007 with a repeated measures two-way ANOVA, n = 12–16). For odor pairs we used 1% anisole versus 1% cineole (E1) and 0.1% (+)-limonene versus 0.1% (−)-limonene (D2). (d) Mean number of blocks required to reach the criterion of 85% of correct responses. ***P < 0.001, indicates significant differences from an easy task (main effect: F2,38 = 16.61, P < 0.0001, one-way ANOVA followed by Bonferroni post hoc test, n = 12–16). For odor pairs, we used E1, D2 and 1% (+)-carvone versus 1% (−)-carvone (D1). We defined an easy pair of monomolecular odorants when mice learnt to recognize distinct molecules in less than 200 trials (that is, mean of ten blocks to reach the criterion in control mice), whereas a difficult pair of odorants were molecularly similar odors or mixtures that required at least 360 trials (mean of 18 blocks) to reach the criterion of correct responses. (e) Mean percentage of correct responses for the memory test 10, 30 and 50 d post-training in a difficult task. The initial memory was still above chance level at 50 d post-training for both tasks (P < 0.001, one sample t test versus theoretical mean of 50%, n = 10), and was significantly higher for an easy task (E1) than for a difficult one (D2). #P = 0.012 with paired Student's t test, n = 10. Error bars represent s.e.m.

  3. Stimulation of adult-born neurons accelerates learning.
    Figure 3: Stimulation of adult-born neurons accelerates learning.

    (a) Diagram of the timing of the task events. Photoactivation (5-ms pulses) was provided at 10 or 40 Hz during a stimulation period of 500 ms, occurring at odor delivery (paired) or 500 ms later (delayed). (b,c) Paired stimulation of adult-born neurons at 40 Hz did not change the learning rate of an easy task (group F1,18 = 0.26, P = 0.61, repeated measures two-way ANOVA, n = 8–13, b), whereas a difficult task was learned quicker (group F1,17 = 8.75, P = 0.0008, repeated measures two-way ANOVA, c; ***P < 0.001, **P < 0.01, *P < 0.05, Fisher LSD test, n = 8–13) (see Supplementary Video 1). For odor pairs, we used E1 (b) and D1 (c). (d) No effect was found when using 10-Hz light stimulation (group F1,14 = 4.25, P = 0.06 with a repeated measures two-way ANOVA, n = 8–10). We used 0.6% of butanol + 0.4% pentanol versus 0.4% of butanol + 0.6% pentanol (D4) as the odor pair. (e) Similar results were found when counting the number of blocks required to reach the criterion (left, ##P = 0.003, Mann-Whitney test, n = 8–13), and an inverse correlation was found between the density of ChR2-positive cells (cells per mm2) counted in the GCL and the number of blocks required to reach the criterion when delivering 40-Hz paired-light stimuli (right, Pearson's test, n = 19). For odor pairs, we used D1, 1% (+)-terpinene versus 1% (−)-terpinene (D3), and 0.6% of ethyl-butyrate + 0.4% amyl-acetate versus 0.4% of ethyl-butyrate + 0.6% amyl-acetate (D5). (f) To evaluate discrimination acuity, we trained mice to discriminate mixtures with increasing complexity. Mice were first trained to distinguish between 1% (+)-carvone and 1% (−)-carvone. Once mice reached criterion, they were asked to discriminate between mixtures of the same odors. Each day, the ratio of (+)-carvone and (−)-carvone changed in both positive stimulus and negative stimulus odor solution (odor pair D1). The percentage of (+)-carvone/(−)-carvone in the positive stimulus is indicated. Only the last five training blocks are shown for the initial training using pure odors (indicated as 100). Both groups showed identical performance (group effect F1,13 = 0.47, P = 0.47, repeated measures two-way ANOVA, n = 8–13). (g) To assess memory, we tested mice 50 d after the discrimination task. The photostimulation (10 or 40 Hz) was applied simultaneously with odor onset (paired using E1 and D1 for 40 Hz and D4 for 10 Hz), or 500 ms after (delayed using D2), at both training and test sessions. #P = 0.015, significant differences between control and ChR2 groups with a Mann-Whitney test (n = 7–13). Error bars represent s.e.m.

  4. Light-induced responses in granule and mitral cells in vitro.
    Figure 4: Light-induced responses in granule and mitral cells in vitro.

    (a) Patch-clamp recordings of an adult-born granule cell at 10 wpi. Brief flashes of light (5 ms, ≥2 mW mm−2, Vm = −68 mV) delivered at 10 or 40 Hz depolarized the membrane potential and triggered action potentials. (b) Patch-clamp recordings of a mitral cell recorded in olfactory bulb slices at 10 wpi. Brief flashes of light (5 ms, 8.4 mW mm−2) were delivered at 10 and 40 Hz (voltage clamp = 0 mV). (c) A plot of the charge of light-induced synaptic GABAergic current in response to different light intensities and frequencies. The total charge of currents evoked at 40 Hz was significantly higher than those evoked by 10 Hz (*P < 0.05 with a Wilcoxon paired test, n = 10). Charge of GABAergic current is expressed in pico-Coulombs. (d) Traces of current-clamp recordings showing the firing activity of a mitral cell, which was depolarized above spike threshold by injecting a steady-step current (+100 pA, Vm = −62 mV). Mitral cell firing was repeatedly inhibited by blue light activation of adult-born neurons. The specific GABAAR antagonist SR95531 totally blocked the light-evoked synaptic inhibition (n = 12). Error bars represent s.d.

  5. Light-activation of ChR2-positive adult-born neurons control spontaneous and odor-evoked mitral cell firing activity in the awake mouse.
    Figure 5: Light-activation of ChR2-positive adult-born neurons control spontaneous and odor-evoked mitral cell firing activity in the awake mouse.

    (a) Example recording of spontaneous spiking activity of a mitral cell in the olfactory bulb of an awake head-restrained mouse. (b) Confirmation of single-cell recording by the similar spike waveform and amplitude (n = 500 spikes, top) and spike train autocorrelogram showing a clear refractory period (bottom). (c) Example peristimulus time histogram (PSTH) of mitral cell spontaneous spiking activity showing clear light-evoked inhibition after local stimulation (5, 10, 15 and 25 ms) through an optical fiber aimed at the neighboring GCL of the recorded cell. (d) PSTH and raster plots (20 sweeps) of mitral cell spontaneous spiking activity after 10-Hz (5 ms, 10 pulses, top) and 40-Hz light stimulation (5 ms, 40 pulses, bottom). (e) Mean spontaneous mitral cell firing rate 1 s before (pre), during 1-s light stimulation at 10 Hz (red, n = 35) or 40 Hz (blue, n = 37), and 1 s after (post). Gray lines show results from individual cells. ***P < 10−9, t test. (f) Histograms summarizing the percentage change of spontaneous firing inhibited by light after 10-Hz (red) and 40-Hz (blue) stimulation. Gray lines show data from individual cells recorded for both light stimuli (n = 35, **P < 10−18, paired t test). (g) PSTH and raster plots (30 sweeps) showing the influence of 10-Hz (5 ms, 10 pulses, top) and 40-Hz (5 ms, 40 pulses, bottom) light stimulation on odor-evoked mitral cell activity (odor stimulation, 3 s; odor valve opening at t = 0). (h) Magnitude of light-evoked inhibition of mitral cell spontaneous (square, left) and odor-evoked (diamond, right) firing activity during 10-Hz (red, spontaneous firing, n = 35 cells; odor evoked, n = 16 cells) and 40-Hz (blue, spontaneous firing, n = 37 cells; odor evoked, n = 21 cells) stimulation as a function of individual mean firing activity (Pearson's correlation coefficient R and P values are indicated). Error bars represent s.e.m.

  6. Light stimulation of postnatal-born neurons does not modify odor discrimination learning.
    Figure 6: Light stimulation of postnatal-born neurons does not modify odor discrimination learning.

    (a) Paired stimulation at 40 Hz of postnatal-born neurons did not change the learning rate of an easy and a difficult task (group F1,16 = 0.0007, P = 0.97 with repeated measures two-way ANOVA, n = 7–11). (b) Similar results were found when counting the number of blocks required to reach the criterion (P > 0.05, Mann-Whitney test, n = 7–11). (c) No correlation was found between the density of postnatal-born ChR2-positive cells (cells mm−2) counted in the GCL and the number of blocks required to reach criterion when delivering 40-Hz paired light stimuli (Pearson's test, n = 11). For odor pairs, we used E1 and D4. Error bars represent s.e.m.

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Author information

  1. These authors contributed equally to this work.

    • Sebastien Wagner

Affiliations

  1. Institut Pasteur, Laboratory for Perception and Memory, Paris, France.

    • Mariana Alonso,
    • Gabriel Lepousez,
    • Sebastien Wagner,
    • Cedric Bardy,
    • Marie-Madeleine Gabellec,
    • Nicolas Torquet &
    • Pierre-Marie Lledo
  2. Centre National de la Recherche Scientifique, Unité de Recherche Associée 2182, Paris, France.

    • Mariana Alonso,
    • Gabriel Lepousez,
    • Sebastien Wagner,
    • Marie-Madeleine Gabellec,
    • Nicolas Torquet &
    • Pierre-Marie Lledo
  3. Present address: The Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, California, USA.

Contributions

M.A. and P.-M.L. designed the experiments, discussed the results and wrote the manuscript. M.A. performed surgery, behavioral experiments and analyzed data. G.L. designed, performed and analyzed in vivo electrophysiology recordings. S.W. design olfactomers and light-stimulation devices. C.B. performed and analyzed in vitro electrophysiology recordings. M.-M.G. performed immunohistochemistry and N.T. designed the database for behavioral analysis.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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    Photostimulation of adult-born neurons during olfactory discrimination learning.

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