Endogenous signaling through alpha7-containing nicotinic receptors promotes maturation and integration of adult-born neurons in the hippocampus

Abstract

Neurogenesis in the dentate gyrus occurs throughout adult mammalian life and is essential for proper hippocampal function. Early in their development, adult-born neurons express homomeric alpha7-containing nicotinic acetylcholine receptors (alpha7-nAChRs) and receive direct cholinergic innervation. We show here that functional alpha7-nAChRs are necessary for normal survival, maturation, and integration of adult-born neurons in the dentate gyrus. Stereotaxic retroviral injection into the dentate gyrus of wild-type and alpha7-knock-out (alpha7KO) male and female mice was used to label and birthdate adult-born neurons for morphological and electrophysiological measures; BrdU (5-bromo-2-deoxyuridine) injections were used to quantify cell survival. In alpha7KO mice, we find that adult-born neurons develop with truncated, less complex dendritic arbors and display GABAergic postsynaptic currents with immature kinetics. The neurons also have a prolonged period of GABAergic depolarization characteristic of an immature state. In this condition, they receive fewer spontaneous synaptic currents and are more prone to die during the critical period when adult-born neurons are normally integrated into behaviorally relevant networks. Even those adult-born neurons that survive the critical period retain long-term dendritic abnormalities in alpha7KO mice. Interestingly, local infection with retroviral constructs to knockdown alpha7-mRNA mimics the alpha7KO phenotype, demonstrating that the relevant alpha7-nAChR signaling is cell autonomous. The results indicate a profound role for alpha7-nAChRs in adult neurogenesis and predict that alpha7-nAChR loss will cause progressive impairment in hippocampal circuitry and function over time as fewer neurons are added to the dentate gyrus and those that are added integrate less well.

Figure 1.

Absence of α7-nAChRs decreases the chance of survival for adult-born neurons during the critical period. A, Immunofluorescent images of the dentate gyrus showing BrdU (red) and DAPI (blue) staining 4 weeks after injection of BrdU to WT (top) and α7KO (bottom) mice. B, Quantification of BrdU-immunopositive cells at 2 weeks (left; 2 WPI) and 4 weeks (right; 4 WPI) post-BrdU injection (mean ± SEM; n = 4 mice per condition). *p < 0.05, Student's t test. Scale bar, 40 μm.

Figure 2.

Adult-born neurons lacking α7-nAChRs receive less synaptic activity than normal. A, PSCs recorded in 3-week-old adult-born neurons at −80 mV holding potential, identified by GFP labeling from MMLV-GFP injection in vivo 3 weeks earlier: WT (top), α7KO (bottom). B, PSC frequency (left) and amplitude (right) for WT and α7KO neurons (mean ± SEM; n = 6 WT and 5 α7KO neurons). *p < 0.05, ***p < 0.001, Student's t test.

Figure 3.

Reduced dendritic arbors are found for 3-week-old adult-born neurons in mice lacking α7-nAChRs when analyzed by MMLV-GFP labeling in vivo. A, Deconvolved z-stack image of an adult-born granule neuron 3 weeks after the mouse was stereotaxically injected with MMLV-GFP in the dentate gyrus. Digitally magnified dendritic segments (inset) were used for measurement of spine numbers. B, Traces of dendrites from granule neurons in WT (left) and α7KO (right) mice 3 weeks after labeling with MMLV-GFP. C, Quantification of dendritic properties in terms of dendritic branch points (left) and dendritic length (right) of cells as in B (mean ± SEM; n = 3 mice per condition with >4 cells per animal). *p < 0.05, one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. Scale bars, 10 μm.

Figure 4.

Absence of α7-nAChRs causes adult-born neurons to have decreased dendritic complexity as visualized with GAD67 reporter mice. A, GFP expression in adult-born neurons in the dentate gyrus of GAD67-GFP reporter mice viewed at 10× (left) and 63× (right) magnification. B, Dendritic arbor traces of the most complex neurons observed in WT (left) and α7KO (right) mice. C, Dendritic branch point histograms (mean ± SEM; n = 3 mice per condition; 40 neurons per mouse). D, Cumulative frequency plot of dendritic branch points (n = 120 granule neurons each for WT and α7KO mice). ***p < 0.001, KS test.

Figure 5.

Delayed maturation of the chloride gradient in adult-born α7KO neurons extends the period of depolarizing GABAergic responses. A, Superimposed perforated patch-clamp recordings of GABAergic PSCs evoked in 3-week-old adult-born WT (left) and α7KO (right) neurons at the indicated holding potentials. The neurons were labeled in vivo with MMLV-GFP and visualized in freshly prepared slices at the time of recording. B, Peak amplitude of the evoked GABAergic PSC as a function of voltage in a WT (black) and an α7KO (red) neuron as in A. C, Interpolated reversal potentials (left; E_Cl; n = 6 WT and 5 α7KO) and resting membrane potentials (right; V_REST; n = 6 WT and 8 α7KO) for WT and α7KO neurons. D, NKCC1 immunostaining (green) of BrdU-labeled (red) 3-week-old adult-born neurons from a WT (top) and α7KO (bottom) dentate gyrus, mounted in DAPI-containing media to reveal nuclei (blue). E, Quantification of NKCC1 levels in neurons as in D (3 weeks of age) or from neurons in the outer third of the granule cell layer (mature) from the same mice (mean ± SEM; n = 3 animals per condition; ≥10 neurons per mouse). *p < 0.05, **p < 0.01, Student's t test.

Figure 6.

In the absence of α7-nAChRs, GABAergic PSCs display immature kinetics. A, Averaged and normalized evoked GABAergic PSCs from a WT (gray) and an α7KO (black) 3-week-old adult-born neuron. B, Quantification of rise time (left) and weighted decay (τ; right) of evoked GABAergic PSCs as in A (mean ± SEM; n = 5 WT and 4 α7KO). **p < 0.01, ***p < 0.001, Student's t test.

Figure 7.

The dendritic deficits found in adult-born α7KO neurons persist long after normal dendritic development is complete in WT. A, Deconvolved z-stack image of a 6-week-old adult-born neuron from an α7KO mouse labeled in vivo by injection of MMLV-GFP at the time of cell division. B, Dendritic arbor traces of 6-week-old adult-born neurons from WT (left) and α7KO (right) mice. C, Dendritic branch points (left) and dendritic length (right) of 6-week-old adult-born neurons visualized by MMLV-GFP labeling (mean ± SEM; n = 4 mice per condition with >4 cells per animal). *p < 0.05, Student's t test. Scale bars, 10 μm.

Figure 8.

Cell-autonomous signaling through α7-nAChRs supports dendritic maturation of adult-born granule neurons. A, Stereotaxic coinjection of lenti-α7RNAi (green) and MMLV-mcherry (red) yields both adult-born neurons expressing α7RNAi (top row; yellow cell) and adult-born neurons lacking RNAi expression (bottom row; red cells) in the same animal. Images are shown at 10× (left) and magnified at the region of interest (white box) to 63× (middle and right). B, Dendritic arbor traces of 3-week-old adult-born neurons expressing α7RNAi (top), scrambled RNAi (middle), or lacking RNAi expression in animals injected with lenti-α7RNAi (bottom). C, Dendritic branch points (left) and dendritic length (right) of 3-week-old adult-born neurons infected as in A and B (mean ± SEM; n = 3 mice per condition with >4 cells per animal). D, Dendritic traces of 3-week-old α7KO adult-born neurons expressing α7RNAi (top) or lacking RNAi expression in animals injected with lenti-α7RNAi (bottom). E, Dendritic branch points (left) and dendritic length (right) of 3-week-old α7KO adult-born neurons (mean ± SEM; n = 4 mice per condition). *p < 0.05, one-way ANOVA with Bonferroni's post hoc test for multiple comparisons.