Olfactory learning promotes input-specific synaptic plasticity in adult-born neurons

Abstract

The production of new neurons in the olfactory bulb (OB) through adulthood is a major mechanism of structural and functional plasticity underlying learning-induced circuit remodeling. The recruitment of adult-born OB neurons depends not only on sensory input but also on the context in which the olfactory stimulus is received. Among the multiple steps of adult neurogenesis, the integration and survival of adult-born neurons are both strongly influenced by olfactory learning. Conversely, optogenetic stimulation of adult-born neurons has been shown to specifically improve olfactory learning and long-term memory. However, the nature of the circuit and the synaptic mechanisms underlying this reciprocal influence are not yet known. Here, we showed that olfactory learning increases the spine density in a region-restricted manner along the dendritic tree of adult-born granule cells (GCs). Anatomical and electrophysiological analysis of adult-born GCs showed that olfactory learning promotes a remodeling of both excitatory and inhibitory inputs selectively in the deep dendritic domain. Circuit mapping revealed that the malleable dendritic portion of adult-born neurons receives excitatory inputs mostly from the regions of the olfactory cortex that project back to the OB. Finally, selective optogenetic stimulation of olfactory cortical projections to the OB showed that learning strengthens these inputs onto adult-born GCs. We conclude that learning promotes input-specific synaptic plasticity in adult-born neurons, which reinforces the top-down influence from the olfactory cortex to early stages of olfactory information processing.

Keywords: cortico-bulbar projections; glutamate; inhibitory circuits; piriform cortex; sensory systems.

Fig. 1.

Olfactory learning induced compartment-specific increase in spine density of adult-born GCs. (A) Experimental timeline. (B) Experimental groups. The OL (red) performed a discrimination learning task in which one odor of a pair was rewarded with water. The OE (blue) was exposed to the same odor presentation procedure without any reward contingency and received water at the end of the session. The AE (gray) was put in the same conditions as the OE group with the odor replaced by pure filtered air. (C) Daily odor discrimination performance of OL mice (n = 6). Mice were first trained to discriminate anisole versus cineole (1% in mineral oil) for the first week and β-ionone versus linalool (1%) for the second week. (D) GFP⁺ adult-born neurons at 32 dpi. Most adult-born neurons are GCs lying in the GCL with dendrites in the EPL. A few periglomerular cells in the GL are also visible. (Scale bar, 100 µm.) (E) Schematic drawing of an adult-born GC and its different dendritic domains. (F) Spine-density histograms for each dendritic domain (n = 95–100 dendritic segments analyzed from n = 5–6 mice per condition). The somatic density is per 10 µm², all other domains are per micrometer. **Bonferroni post hoc test, P < 0.005. Data are mean ± SEM. (G) Adult-born GC spines in the somatic and proximal dendritic domains (i) and in the apical dendritic domain (ii), illustrating filipodia-like spines (arrowhead) and mushroom-like spines (arrow). (Scale bars, 5 µm.)

Fig. 2.

Balance between excitatory and inhibitory synaptic inputs in adult-born GC. (A) PSD95-GFP⁺ adult-born GC, with PSD95-GFP⁺ puncta in green and dendritic morphology in red (anti-GFP). (Scale bar, 10 µm.) (B) Density of PSD95-GFP⁺ spines in the different dendritic domains (n = 25–45 dendritic segments per condition, n = 9 mice). The somatic density is per 10 µm², all other domains are per micrometer. **Bonferroni post hoc test, P < 0.01. (C) Gephyrin-positive clusters (red) in GFP⁺ adult-born GC spines. (Scale bar, 5 µm.) (Right, Inset) Higher 3D magnification of the boxed region (Scale bar, 1 µm). (D) Density of gephyrin-positive spines in the different dendritic domains of GFP⁺ cells (n = 45–50 dendritic segments per condition, n = 17 mice). The somatic density is per 10 µm², all other domains are per micrometer. **Bonferroni post hoc test, P < 0.016. Data are mean ± SEM.

Fig. 3.

Olfactory learning-induced remodeling of excitatory top-down inputs from the OC to adult-born GCs. (A) Transduction of the APC/AON with a ChR2-mCherry and the labeling of axonal projections to the OB. (Scale bar, 0.5 mm.) (B) Higher magnification showing the presence of cortico-bulbar axonal projections targeting the GCL and to a lesser extent the IPL and MCL. (Scale bar, 100 µm.) (C) Colocalization of VGlut1⁺ clusters (cyan) with mCherry⁺ fibers (red) in the GCL. (Scale bar, 1 µm.) (D) Conditional expression of ChR2-EYFP (green) in Cre-expressing M/T cells (Tbet-cre mice, n = 3 mice), ChrR2-mCherry expression in cortico-bulbar projections (red), doublecortin⁺ immature adult-born neurons (white). (Scale bar, 100 µm.) (E and F) ChR2-mCherry⁺ cortico-bulbar projections (magenta) and GFP⁺ adult-born neurons (green) (Scale bars: 500 µm in E; 100 µm in F.) (G) Electron micrographs showing DAB-labeled (asterisks) mCherry+ cortical axon terminals in the GCL establishing typical type-1 asymmetric synapses onto a presumed spine (S). Arrow, postsynaptic density. (Scale bar, 0.5 µm.) (H) PSD95-GFP⁺ adult-born GC making putative synapses with mCherry⁺ (magenta) cortical axonal boutons. (Scale bar, 10 µm.) (Right) High 3D magnification showing a putative synapse between a cortical presynaptic terminal (magenta) colocalized and wrapped around a postsynaptic PSD95-GFP⁺ spine. Arrows indicate PSD95-GFP⁺ puncta empty of any labeled presynaptic boutons. (Scale bars, 2 µm.) (I) Density of PSD95-GFP⁺ cortical synapses in the different dendritic domains. (n = 25–45 dendritic segments analyzed per condition, n = 9 mice). The somatic density is per 10 µm², all other domains are per micrometer. Bonferroni post hoc test, **P < 0.01; ***P < 0.001. Data are mean ± SEM. See also Fig. S1.

Fig. 4.

Spontaneous inhibitory and excitatory synaptic events onto adult-born GCs. (A, Upper) Traces showing spontaneous IPSCs (V_c = 0 mV). (Lower) sIPSC frequency (Hz, Left) and sIPSC amplitude (pA, Right). (B, Upper) Traces showing spontaneous EPSCs (V_c = −70 mV). (Lower) sEPSC frequency (Hz, Left) and sEPSC amplitude (pA, Right). *P < 0.05 with Mann–Whitney test. Data are mean ± SEM. See also Fig. S2.

Fig. 5.

Learning-induced enhancement of excitatory cortical top-down inputs onto adult-born GCs. (A) Evoked EPSC recording configuration during light stimulation of ChR2-expressing cortical top-down inputs from the APC/AON. (B) Amplitude of light evoked EPSCs (recorded at V_c = −70 mV) with respect to the light pulse duration (in milliseconds). (Inset) Superimposed traces in the 0.2-ms minimal stimulation condition (Left) and 2-ms stimulation condition (Right), illustrating the high failure rate and the similar amplitude of success responses during minimal stimulation. (C) Ratio between maximal and minimal evoked EPSCs. (D) Amplitude of minimal evoked EPSCs (pA). (E, Left) Minimal evoked EPSCs with paired-light stimulation (averaged from >30 individual EPSC; including failures; light-pulse duration, 0.2 ms; interspike interval, 50 ms). (Right) PPR (PPR = Amp2/Amp1). *P < 0,05 with Mann–Whitney test. Data are mean ± SEM. See also Fig. S2.