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Neurogenesis in the dentate gyrus occurs throughout adult mammalian life and is essential for proper hippocampal function. Early in their development, adultborn neurons express homomeric α7-containing nicotinic acetylcholine receptors (α7-nAChRs) and receive direct cholinergic innervation. We show here that functional α7-nAChRs are necessary for normal survival, maturation, and integration of adultborn neurons in the dentate gyrus. Stereotaxic retroviral injection into the dentate gyrus of wildtype and α7-knockout (α7KO) male and female mice was used to label and birthdate adultborn neurons for morphological and electrophysiological measures; BrdU injections were used to quantify cell survival. In α7KO mice, we find that adultborn 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 adultborn neurons are normally integrated into behaviorally relevant networks. Even those adultborn neurons that survive the critical period retain long-term dendritic abnormalities in α7KO mice. Interestingly, local infection with retroviral constructs to knockdown α7-mRNA mimics the α7KO phenotype, demonstrating that the relevant α7-nAChR signaling is cell-autonomous. The results indicate a profound role for α7-nAChRs in adult neurogenesis and predict that α7-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.
Continuing neurogenesis in the adult dentate gyrus is necessary for hippocampal function (Shors et al., 2001, 2002; Rola et al., 2004; Snyder et al., 2005; Winocur et al., 2006). Abnormalities in adult neurogenesis are likely to exacerbate major neurological disorders, including Alzheimer’s disease and depression (Gough, 2007; Sahay and Hen, 2007; Verret et al., 2007; Kotani et al., 2008; Perera et al., 2008) and contribute to addiction and relapse behavior (Noonan et al., 2009). Understanding how adultborn neurons develop and integrate into preformed neural networks may suggest targets for therapeutic intervention and assist in developing stem cell therapies for repairing damaged neuronal tissue (Sohur et al., 2006; Okano and Sawamoto, 2008).
Pioneering work on adult neurogenesis indicates that ionotropic receptor signaling plays important roles. Depolarizing GABAergic activity alters precursor proliferation and is necessary for dendritic growth of adultborn neurons (Liu et al., 2005; Ge et al., 2006). Glutamatergic activity through NMDA receptors encourages survival of adultborn neurons during a critical period when the neurons are first assimilated into behaviorally relevant networks (Tashiro et al., 2006; Tashiro et al., 2007). Nicotinic cholinergic input is also positioned well to influence adult neurogenesis. Early on, the neurons receive cholinergic innervation and express two major types of ionotropic nicotinic acetylcholine receptors (nAChRs): homopentameric α7-containing receptors (α7-nAChRs) and heteropentameric β2-containing receptors (β2*-nAChRs; Kaneko et al., 2006; Ide et al. 2008).
Substantial evidence indicates that cholinergic signaling regulates adult neurogenesis. Cholinergic forebrain lesion decreases adultborn neuron survival, and enhancing cholinergic activity increases survival (Cooper-Kuhn et al., 2004; Kaneko et al., 2006). Chronic nicotine exposure in vivo reduces adultborn neuron proliferation (Abrous et al., 2002; Scerri et al., 2005; Shingo and Kito, 2005), while β2-nAChR knockout (KO) mice show decreased proliferation, but normal survival, of hippocampal adultborn neurons (Harrist et al., 2004; Mechawar et al., 2004). Contributions of α7-nAChRs to adult neurogenesis are unknown.
In early postnates, α7-nAChRs contribute importantly to hippocampal development. Young hippocampal neurons in α7KO mice have a prolonged period of GABAergic excitation due to delayed appearance of the mature chloride transporter KCC2 and extended presence of the immature chloride transporter NKCC1 (Liu et al., 2006). Further, α7-nAChR signaling helps drive giant depolarizing potentials that shape network development and contribute to synaptic plasticity (Maggi et al., 2001, 2003; Le Magueresse et al., 2006). Subsequent α7-nAChR activity enhances both GABA and glutamate release (Gray et al., 1996; Alkondon et al., 1997A; Radcliff and Dani, 1998; Alkondon and Albuquerque, 2001).
Here we show that adultborn neurons in α7KO mice receive fewer spontaneous synaptic currents and are disadvantaged for survival during the critical period in vivo. The neurons develop with severely truncated dendritic arbors, a prolonged depolarizing chloride gradient, and GABAergic currents with immature kinetics. These results indicate that α7-nAChR signaling modulates the tempo of adultborn neuron physiological and morphological development. We further demonstrate that this function results from cell-autonomous α7-nAChR signaling and is necessary to prevent persistent morphological abnormalities of granule cells in the dentate gyrus.
All mice were C57Bl/6 background and used at 1–3 months of age. Control and experimental mice were age-matched in all experiments. Heterozygous α7KO mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Their offspring were genotyped by PCR, and homozygous α7KO mice were used for experiments. GAD67-GFP mice, generated and provided by Guoping Feng (Duke University), were crossed with the α7KO mouse line. To ensure that only GAD67-GFP heterozygous mice were used in experiments, homozygous GAD67-GFP mice were bred with nulls to produce test animals.
Lentiviral vectors were constructed to reduce α7-mRNA levels in vivo by RNA interference (α7RNAi). A Genscript algorithm was used to design sequences uniquely homologous to mouse α7-mRNA. A control scrambled sequence was obtained with Genscript sequence scrambler and shown not to be homologous with any sequence in the mouse genome. The sequences were inserted into lentiviral vectors (FG12; Addgene plasmid 14884) under an H1 promoter along with GFP under a ubiquitin promoter and linked to their reverse complement by the loop sequence TCTCTTGAA to form short-hairpin RNAs. Their compositions are: 5’-AGGCAGATATCAGCAGCTATA-3’, 5’-ACCACCAACATTTGGCTACAA-3’, and 5’-GAGAGTACGCTAAGATCCTAA-3’ for α7RNAi-1, α7RNAi-2, and scrambled RNAi, respectively. The effectiveness of the lentiviral α7RNAi constructs was tested by infecting 16-day-old mouse (E18 C57B1/6) hippocampal 3.5 cm cultures (plated at 62,500 cells/cm2) with 5 µl virus stock (1×108 PFU/ml), and collecting the cells 1 week later (Supplementary Fig. 1). Solubilized α7-nAChRs were immunoprecipitated with monoclonal antibody (mAb) 319 and radiolabeled with I125-α-bungarotoxin (I125-αBgt; Conroy et al., 2003). Total protein was measured using the Bradford assay (Bio-Rad Protein Assay). The α7RNAi-1 construct was used for all experiments reported here.
Mice were anesthetized by intraperitoneal injection of 10 mg/ml ketamine and 1 mg/ml xylazine in sterile 0.9% NaCl at a volume of 0.01 ml/g body weight. Toe pinch was used to determine effectiveness of anesthesia. Animals were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed and post-fixed in 4% paraformaldehyde in PBS overnight, then equilibrated in 30% sucrose. Next, the tissue was frozen, and coronal slices (40 µm) were cut using a cryostat microtome. Sections were dried on superfrost plus slides (Fisher) and processed immediately for immunostaining.
Slide mounted slices were blocked in 5% normal donkey serum in PBS with 0.5% triton X-100 (PBS-TX) for 30 minutes. Primary antibodies were diluted in the same blocking solution and applied overnight at 4°C. Primary antibodies included anti-NKCC1 mAb (1:1000 dilution, Developmental Studies Hybridoma Bank, University of Iowa), anti-5-Bromo-2-deoxyuridine (BrdU) mAb BU1/75 (1:200 dilution, Abcam), and anti-GFP mAb 3E6 (1:1000 dilution, Molecular Probes). Slides were then washed three times in PBS-TX for 10 minutes and incubated with secondary antibodies at 1:500 in PBS-TX for 2–4 hours at room temperature. After washing three times in PBS-TX for 10 minutes, slides were mounted with coverslips using Vectashield mounting medium containing dapi and stored in the dark at 4°C until imaged.
Imaging was performed within 48 hours of immunostaining using a Zeiss axiovert microscope with 3I deconvolution software for image analysis. For morphological measurements neurons were imaged at 63x magnification, and a z-stack was compiled by acquiring images every 0.5 µm through the section. Dendritic measurements were made in ImageJ using the NeuronJ tracing application. Spine counts were taken over a 20 µm segment of dendrite located within 100 µm of the granule cell layer boundary. NKCC1 fluorescence measurements were made in a 2 µm-thick ring surrounding the adultborn granule cell nucleus, defined by dapi and BrdU staining. Cell selection and quantification were performed blind to genotype.
A Moloney’s Murine Leukemia Virus construct expressing green fluorescent protein (MMLV-GFP) was provided by Fred Gage (Salk Institute) and modified to express mcherry (MMLV-cherry). Lentiviral constructs were purchased (FG12; Addgene plasmid 14884) and modified to express α7RNAi and scrambled sequences as described (see RNAi Constructs). Viruses were generated by transfecting the constructs into HEK293T cells. Harvest and concentration by ultra-centrifugation generated viral titers of 108 pfu/ml in sterile PBS. The viral suspensions were stereotaxically delivered as described (Van Praag et al., 2002) at the following position from Bregma: anteroposterior, −2mm; lateral, 1.7mm; ventral, −2mm. For electrophysiological experiments a second injection was made from Bregma: anteroposterior, −2.5mm; lateral, 2mm; ventral −2.2mm. Animal body temperature was maintained throughout the surgery until anesthesia wore off. After surgery, animals were housed singly and monitored to ensure no signs of infection, pain, or distress.
BrdU was injected intraperitoneally at 10 mg/ml in sterile 0.9% NaCl to yield a single dose of 50 µg/g body weight on each of four consecutive days. Mice were singly housed for 2 or 4 weeks following the initial injection until tissue preparation. Following perfusion all steps were performed blind to genotype. After cryostat sectioning, slices were dipped in 2N HCl for 30 minutes at 37°C, followed by 0.1 M borate buffer for 10 minutes at room temperature. After immunostaining, counts were made of BrdU-positive cells in the first third of the granule cell layer in every fourth section through the entire hippocampus. Adultborn neurons are largely confined to the first third of the granule cell layer (Zhao et al., 2006).
Following anesthesia (see tissue preparation), mice were decapitated. Brains were quickly removed from the skull and placed in ice-cold solution containing (in mM): sucrose 75, NaCl 87, KCl 2.5, CaCl2 0.5, MgCl2 7, NaHCO3 25, NaH2PO4 1.25, glucose 20, and bubbled with 95% O2/5% CO2, pH 7.4. Transverse, 250 µm thick hippocampal slices were cut using a vibratome (series 1000 Plus, Technical Products International Inc., St Louis, USA) and initially stored at 30°C in artificial cerebrospinal fluid containing (ACSF, in mM): NaCl 119, KCl 2.5; NaH2PO4 1, NaHCO3 26, MgCl2 1.3, CaCl2 2.5, glucose 10, pH 7.4, and equilibrated with 95% O2/5% CO2. Slices were allowed to recover at least 1 hour in oxygenated ACSF at 24°C prior to recording and then continuously perfused with the same solution at a rate of 2–3 ml/min while recording.
The whole-cell patch-clamp configuration was employed both in voltage-clamp and current-clamp modes. Microelectrodes (5–8 MΩ) were pulled from borosilicate glass capillaries (Sutter Instruments, Novato, CA, USA) with a P-97 pipette puller (Sutter Instruments).
To record spontaneous postsynaptic currents (PSCs), the electrodes were filled with an internal solution containing (in mM): CsCl 135, MgCl2 4, EGTA 0.1, HEPES 10, MgATP 2, NaGTP 0.3 and Na2phosphocreatine 10, pH 7.4, adjusted with CsOH to 280–290 mOsm. Cells were held at a membrane potential of −80 mV, and currents were recorded for five continuous minutes. The resting membrane potential was determined in current-clamp mode immediately after establishing the whole-cell configuration. The internal solution consisted of (in mM): K-gluconate 125, KCl 15, NaCl 8, EGTA 2, HEPES 10, MgATP 4, NaGTP 0.3, and Na2phosphocreatine 10, pH 7.3, adjusted with KOH to 280–290 mOsm. Cells with absolute leak current >100 pA at the holding potential (VHold) were discarded.
For perforated-patch recordings, gramicidin was diluted in the pipette solution (in mM: 135 CsCl, 4 MgCl2, 0.1 EGTA, 10 HEPES, pH 7.4, 300 mOsm) to a final concentration of 50 µg/ml immediately prior to use. Pipettes had resistances of 5–8 MΩ. Small voltage-steps (10 mV, 50 ms) were evoked prior to extracellular stimulation to monitor membrane and access resistance. If the holding current or either resistance changed significantly, the experiment was discarded. Extracellular stimulation (80–240 µA and 0.2 ms duration, 0.1 Hz) was performed using a concentric bipolar electrode (125 µm diameter; Frederick Haer Company, Bowdoin, ME), with a pulse generator (Master-8, A.M.P.I., Jerusalem, Israel) coupled through a stimulus isolator (S.I.U. 90; Neuro Data Instruments, New York, NY). The stimulation electrode was placed on the granule cell layer 300 µm away from the recorded cell. During the recording of evoked GABAergic PSCs, the cells were perfused with oxygenated ACSF containing CNQX (20 µM) and APV (20 µM) to block glutamatergic PSCs and isolate monosynaptic GABAergic PSCs. Recordings were performed under voltage-clamp at multiple holding potentials. Peak current amplitude and holding potential were plotted to yield the chloride equilibrium potential (ECl) from a linear fit of the I–V curve for each cell. As a control for perforated-patch integrity, in some cells ECl was redetermined after establishing a whole-cell configuration and adjusting the intracellular chloride concentration to yield a slightly positive ECl (2.7 mV). The measured values in these cases were in reasonable agreement with predicted values (Supplementary Fig. 2A). To ensure PSCs were GABAergic, gabazine was used to block GABAA receptors after ECl acquisition and demonstrate loss of the evoked events (Supplementary Fig. 2B). Current kinetic measurements were made from ≥ 5 averaged traces acquired at −80 mV holding potential. Current rise time was determined between 40% and 100% of peak amplitude using a single exponential best fit. Current decay was determined between 10% and 90% of peak amplitude. Since the best fit for decay was either one or two exponentials, weighted decay was calculated using the equation A1τ1 + A2τ2 where A is the relative amplitude and τ is the decay constant for each component.
All recordings were collected using a MultiClamp 700A amplifier (Molecular Devices, Sunnyvale, CA, USA), filtered at 2 kHz, and digitized at 5 kHz with pCLAMP 9 software (Molecular Devices); analysis was performed with Clampfit 9.2 software.
Student’s t test (t test) was used for comparing means if single pairs were involved. One-way ANOVA with Bonferroni’s post hoc test was used for comparing means for ≥ 3 groups. Kolmogorov-Smirnov (KS) statistical analysis was used to compare cumulative frequency distributions. *p<0.05, **p<0.01, ***p<0.001.
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
To determine if α7-nAChRs contribute to cholinergic regulation of adultborn neuron survival (Cooper-Kuhn et al., 2004; Kaneko et al., 2006), we birthdated adultborn neurons in WT and α7KO young adult mice by injecting BrdU and following their fate. Hippocampal slices were taken for BrdU immunostaining 2 and 4 weeks later, to allow quantification before and after the critical period for activity-dependent survival (Tashiro et al., 2006). No difference was seen in the number of BrdU-labeled cells in the dentate gyrus of WT and α7KO mice at 2 weeks (Fig. 1). A clear difference was found, however, at 4 weeks. At this time α7KO mice had significantly fewer BrdU-labeled granule neurons than did WT mice (Fig. 1). The results indicate that α7-nAChR signaling is necessary for optimal adultborn neuron survival through the critical period occurring between 2 and 4 weeks post neuronal birth.
Because survival through the critical period depends on glutamatergic signaling (Tashiro et al., 2006), we tested whether adultborn α7KO neurons receive reduced synaptic input. This was done by first labeling adultborn neurons in vivo using stereotaxic injection of MMLV-GFP, which can express only in dividing cells. Labeling is largely confined to neurons born within a 3-day window following virus injection (Zhao et al., 2006). Three weeks later we prepared fresh hippocampal slices, identified GFP-expressing neurons in the dentate gyrus region, and performed patch-clamp recording in voltage-clamp mode (Fig. 2A). Adultborn α7KO neurons had significantly fewer spontaneous postsynaptic currents (PSCs) than did WT neurons, and their PSCs were smaller in size (Fig. 2B). (Pharmacological blockade of glutamatergic and GABAergic transmission eliminated both spontaneous and evoked PSCs – see below and Ge et al., 2006). The results indicate that in the absence of α7-nAChRs, adultborn neurons receive less synaptic activity, offering a possible explanation for their reduced chances of surviving through the critical period.
To determine whether the reduced synaptic input to adultborn α7KO neurons possibly reflected fewer synapses being present on the cells, we carried out a morphological analysis. Young adult WT and α7KO mice were injected with MMLV-GFP stereotaxically into the dentate gyrus, and 3 weeks later taken for dendritic measurements of GFP-expressing neurons. Reductions in both the total dendritic length and in the number of dendritic branch points were found for adultborn α7KO neurons compared to their age-matched WT counterparts (Fig. 3). Because no differences were found in the number of spines per unit length of dendrite (15 ± 2 and 13 ± 2 spines/20 µm for WT and α7KO dendrites, respectively), we calculate that adultborn α7KO neurons are likely to have substantially fewer spines in aggregate than do WTs and therefore may have proportionately fewer synapses (though this would require other techniques to confirm). A reduction in synapses would account for the reduced number of PSCs, and, further, would suggest that α7-nAChR signaling may be necessary for normal dendritic growth on adultborn neurons.
To corroborate an α7-nAChR effect on dendritic growth in larger populations of adultborn neurons, we crossed α7KO mice with a GFP-reporter mouse line that expresses GFP under a partial GAD67 promoter. In the mature dentate gyrus of this line, GFP is expressed only in adultborn neurons 1–3 weeks post-neurogenesis (G. Feng, personal communication), prior to their assuming a glutamatergic fate (Toni et al., 2008). Examining 40 individual neurons from each of 3 WT/GFP and 3 α7KO/GFP mice revealed clear differences (Fig. 4A). Comparing either the most complex neurons (Fig. 4B) or the population histograms of dendritic branch points (Fig. 4C) showed that adultborn neurons in α7KO mice have a reduced dendritic complexity compared to WTs. KS statistical analysis of the dendritic branch point cumulative frequency histogram confirmed that the morphological differences were highly significant (Fig. 4D). BrdU-birthdating of adultborn neurons demonstrated that GFP expression occurred at comparable neuronal ages in the two mouse lines (data not shown). These results confirm that α7-nAChRs are required for normal dendritic growth in adultborn neurons of the dentate gyrus.
The finding of reduced dendritic arbors on adultborn α7KO neurons raised the question of whether the deficit was specific or might be part of a more global failure of the neurons to mature on schedule. One indicator of maturation is the time during development when the chloride gradient acquires an equilibrium potential (ECl) sufficiently negative as to support inhibitory GABAA-mediated currents. Prior to this, GABA is more depolarizing (and often excitatory) due to a reversed chloride gradient caused by early expression of the chloride transporter (importer) NKCC1. Subsequently, NKCC1 expression decreases and the chloride transporter (exporter) KCC2 appears. This inverts the chloride gradient, lowering ECl below threshold for action potentials and thereby rendering GABA inhibitory as required for adult function. Importantly, the initial period of depolarizing/excitatory GABAergic signaling is necessary both for early postnatal and adultborn neurons to develop properly and integrate into circuits (Rivera, et al., 1999; Ben-Ari Y 2002; Payne et al., 2003; Ge et al., 2006).
To determine if maturation of the chloride gradient is perturbed in adultborn neurons of α7KO mice, we performed gramicidin-induced perforated-patch-clamp recording on 3-week-old adultborn neurons. Stereotaxic injection of MMLV-GFP was used to label the neurons in vivo 3 weeks before preparing hippocampal slices. Recordings were then obtained from GFP-expressing cells. By measuring the amplitude of the evoked GABAergic PSC as a function of holding potential, we were able to construct I–V plots and calculate the mean reversal potential, this being ECl. Pharmacological blockers eliminated contributions from glutamatergic PSCs, and the GABAA receptor blocker gabazine confirmed that the PSCs being measured under these conditions were GABAergic (Supplementary Fig. 2B). The results reveal that 3-week-old adultborn α7KO neurons retain an ECl that is significantly more positive than that of age-matched WT neurons (Fig. 5A–C). No change was found in the mean resting membrane potential of adultborn α7KO and WT neurons at 3 weeks of age (Fig. 5C). Comparing ECl to the resting membrane potential reveals that GABAA receptor activation remains depolarizing in adultborn neurons from α7KO mice after it has switched to hyperpolarizing in WT mice.
Maturation of the chloride gradient usually involves loss of NKCC1 and appearance of KCC2 as noted above. To compare NKCC1 levels in α7KO and WT neurons, we birthdated the neurons by BrdU injection in vivo, and then prepared slices 3 weeks later for BrdU and NKCC1 immunostaining. Three-week-old adultborn α7KO neurons in the dentate gyrus displayed substantially higher levels of NKCC1 than did equivalent neurons in WT mice (Fig. 5D,E). NKCC1 immunostaining in mature neurons in the outer third of the granule cell layer, which contains few adultborn neurons, revealed no significant differences between WT and α7KOs. Loss of α7-nAChR signaling, therefore, delays the reduction in NKCC1 levels in adultborn neurons but does not permanently prevent the reduction from occurring in the broader population of mature granule cells. Taken together, the results indicate that endogenous signaling through α7-nAChRs is required to generate the normal maturation rate of the chloride gradient in adultborn neurons.
Another marker for maturation in adultborn neurons is the appearance of GABAA receptors containing α1 subunits. Early appearing forms of the receptor lack the α1 subunit and therefore lack the fast rise and decay kinetics associated with the mature form (Overstreet-Wadiche et al., 2005; Markwardt et al., 2009). We recorded evoked GABAergic PSCs using the perforated patch-clamp configuration on 3-week-old adultborn neurons in fresh slices previously labeled in vivo with MMLV-GFP as described above (Fig. 6A). Measuring the rise and decay kinetics of the events in adultborn neurons revealed significantly longer times in α7KO neurons compared to WTs (Fig. 6B). This suggests that adultborn α7KO neurons retain expression of immature GABAA receptor subunits over a longer developmental period than do WTs. Delayed appearance of α1-containing GABAA receptors, a prolonged presence of NKCC1 and depolarizing chloride gradients, and a retarded dendritic arborization in adultborn α7KO neurons indicate that α7-nAChR signaling normally helps drive neuronal maturation following neurogenesis in the adult dentate gyrus.
Adultborn α7KO neurons develop behind schedule and have a greater chance of dying during the critical period, but some do survive. The question remains as to whether adultborn α7KO neurons that survive the critical period continue to display deficits after the normal period of development. To examine this, we stereotaxically injected MMLV-GFP in vivo, birthdating adultborn neurons and filling them with fluor for subsequent visualization. After 6 weeks, hippocampal slices were prepared and examined for GFP-expressing cells. The images revealed that adultborn α7KO neurons retain deficits in dendritic length and complexity even after 6 weeks (Fig. 7). This is long after dendritic development has been completed in adultborn WT neurons (Zhao et al., 2006). The results indicate that even those adultborn α7KO neurons that survive the critical period retain morphological defects.
To gain insight into the mechanism by which α7-nAChR signaling regulates adultborn neuron maturation, we tested the hypothesis that the relevant α7-nAChRs act in a cell-autonomous manner, i.e. must be present on the adultborn neurons themselves. To address this we generated lentiviral constructs encoding α7RNAi with GFP, then stereotaxically co-injected this virus with MMLV expressing the red fluorescent protein mcherry into WT mice. This dual viral approach allowed us to compare the dendritic growth of α7RNAi-expressing (red and green = yellow) and α7RNAi-lacking (red) adultborn neurons in the same animal (Fig. 8A). Other WT mice were co-injected with lentivirus expressing a scrambled RNAi construct and MMLV-mcherry as an additional control. Adultborn neurons expressing α7RNAi showed significantly reduced dendritic complexity and length compared to either RNAi-lacking adultborn neurons in the same animal or scrambled RNAi-expressing adultborn neuron controls (Fig. 8B,C). In fact, the values obtained for adultborn neurons expressing α7RNAi were indistinguishable from those obtained for adultborn α7KO neurons of the same age, and both clearly reduced compared to adultborn WT (see Fig. 3C).
Lenti-α7RNAi often infected granule neurons in the outer third of the granule cell layer, which were likely to be fully mature prior to infection. Quantification of the dendritic arbors of such neurons showed no deficits (Supplementary Fig. 3), suggesting that the ability of α7RNAi to affect dendritic morphology is developmentally constrained. To control for potential off-target effects of the α7RNAi, we repeated this experiment in α7KO mice. Adultborn neurons in α7KO mice that received α7RNAi were not significantly different from RNAi-lacking adultborn neurons in the same animals (Fig. 8D,E). This indicates that the dendritic arbor defects caused by α7RNAi are dependent on the presence of α7-nAChRs in those neurons. Taken together these results demonstrate that α7-nAChR signaling acts in a cell-autonomous manner to regulate dendritic arborization of adultborn neurons, a key feature of their maturation.
We show here for the first time that endogenous nicotinic cholinergic signaling through α7-nAChRs in vivo enhances the survival and integration of adultborn neurons in the dentate gyrus. In the absence of functional α7-nAChRs, adultborn neurons have truncated, less complex dendritic arbors, receive fewer PSCs, display GABAergic PSCs with slower kinetics, and maintain an extended period of GABAergic depolarization due to an immature chloride gradient. Together this indicates that the neurons are in an immature state. Fewer neurons survive the critical period under these conditions, and the survivors have persistent deficiencies in dendritic arborization. As a result, fewer neurons are added to the adult dentate gyrus, thereby over time compromising renewal of the mossy fiber pathway relaying entorhinal input to the hippocampus.
Our results demonstrate that dendritic maturation of adultborn neurons is regulated by cell-autonomous α7-nAChR signaling. This excludes the possibility that the α7-nAChR effects are indirect, e.g. resulting from α7-nAChR-mediated increases in network activity impinging on the neurons. In other systems neurotransmitter-mediated regulation of dendritic morphology involves calcium signaling and subsequent changes in gene expression (Buttery et al., 2006; Aizawa et al., 2004; Gaudilliere et al., 2004; Borodinsky et al., 2003). Since α7-nAChRs have a high relative permeability to calcium (Bertrand et al., 1993; Seguela et al., 1993), can generate calcium events in hippocampal neurons even in the absence of detectable currents (Szabo et al., 2008; Fayuk and Yakel, 2007), and drive calcium-dependent gene transcription (Hu et al., 2002), this is the most plausible mechanism at present. Transcriptional regulation may also explain α7-nAChR-dependent changes in physiological maturation (Liu et al., 2006).
The deficits reported here resulted from loss of α7-nAChRs. None involved β2*-nAChRs, the other major class of nicotinic receptors in the hippocampus. Both are expressed at early stages by adultborn neurons, and the neurons receive direct cholinergic innervation (Kaneko et al., 2006; Ide et al. 2008). In contrast to α7-nAChRs, however, β2*-nAChRs appear to be essential primarily for initial generation of the cells, and only during middle age (Harrist et al., 2004; Mechawar et al., 2004). Differences in activation kinetics, ion permeabilities, surface locations, and tethering to intracellular signaling cascades provide candidate mechanisms enabling α7-nAChRs and β2*-nAChRs to exert distinct regulatory controls (Vernino et al., 1992; Seguela et al., 1993; Alkondon et al. 1997B; Zoli et al., 1998; Fenster et al., 1999; Chang and Berg, 2001; Wooltorton et al., 2003; Alkondon and Albuquerque 2005; Xu et al., 2006). Interestingly, the α7-nAChR dependence is also different from that of NMDA receptors. The latter are required by adultborn neurons for optimal survival but also appear to mediate competitive interactions when functional on other neurons (Tashiro et la., 2006). In contrast, α7-nAChRs are required whether or not other neurons receive input via α7-nAChRs.
Adultborn neurons examined here underwent their final mitosis in mice that were at least one month old. At this time the granule cell layer is fully formed, and neurogenesis is confined to the subgranular layer as it is throughout adulthood (Altman and Bayer, 1990; Esposito et al., 2005). Adultborn neurons generated in young and old animals appear to have similar fates with respect to differentiation and morphological end state (Morgenstern et al., 2008; Ahlenius et al., 2009). This suggests that the results obtained here are likely to apply broadly across the population of adultborn neurons in the dentate gyrus, though it should be noted that aged adults may differ from young adults in some respects. Importantly, results from adultborn α7KO neurons here were always compared to age-matched adultborn WT neurons.
The α7-nAChR requirement for timely maturation of the chloride gradient in adultborn neurons is similar to that reported previously for developing neurons in early postnatal mouse hippocampus and embryonic chick spinal cord (Liu et al., 2006). The mechanisms, however, may differ. Adult neurogenesis proceeds more slowly than in the embryo (Espositio et al., 2005; Overstreet-Wadiche et al., 2006), and adultborn neurons replace pre-existing synapses rather than increase the total as seen in the early postnate (Toni et al., 2007). The biggest difference, however, is likely to be the presence of spontaneous waves of excitation seen in much of the developing nervous system, including the dentate gyrus (Ben Ari et al., 1989; Kasyanov et al., 2004; Overstreet-Wadiche et al., 2006). In embryonic spinal cord and early postnatal hippocampus, nicotinic activity initiates or enhances these waves (Hanson and Landmesser, 2003; Le Magueresse et al., 2006). The waves may enable nicotinic activity to act ubiquitously (though indirectly) to excite large populations and coordinate their maturation. The adult dentate gyrus has no comparable waves of excitation (Overstreet-Wadiche et al., 2006), and may instead rely on direct cholinergic input to guide the development and integration of adultborn neurons. Nonetheless, α7-nAChRs appear to have some common effects in the early postnatal and adult dentate gyrus on neuronal development: adultborn and early postnatal neurons rely on α7-nAChR signaling to terminate the initial period of GABAergic excitation/depolarization.
A period in which GABA currents are depolarizing (and likely excitatory) is widely thought essential for proper neuronal development and integration for both early postnatal and adultborn neurons (Ben-Ari, 2002; Represa and Ben-Ari, 2005; Tozuka et al., 2005; Ge et al., 2006; Cancedda et al., 2007). Extended periods of depolarizing/excitatory GABA signaling, as found in α7KOs, might therefore be expected to correlate with increased dendritic arborization and innervation. The opposite was found: adultborn α7KO neurons have (1) shorter, less complex dendritic arbors, (2) reduced synaptic input, (3) an immature form of GABAA receptors apparently lacking α1 subunits (Overstreet-Wadiche et al., 2005), and 4) increased likelihood of dying during the critical 2–4 weeks post-neurogenesis (Tashiro et al., 2006). The results indicate that adultborn neurons require α7-nAChR signaling for normal development and integration, advancing them to some trigger point that enables the chloride gradient to mature and render GABA currents inhibitory.
Adult neurogenesis is essential for proper hippocampal function. Adultborn neurons integrate into functional hippocampal networks and are activated by hippocampal-dependent learning tasks (Ramirez-Amaya et al., 2006; Tashiro et al., 2007). Ablation of the precursor cells that normally generate adultborn neurons impairs certain types of hippocampal-dependent learning and memory (Shors et al., 2001, 2002; Rola et al., 2004; Snyder et al., 2005; Winocur et al., 2006). Key functions for adultborn neurons include coding time and place integration (Aimone et al., 2006), spatial pattern separation (Clelland et al., 2009), reinforcement of preexisting memories (Trouche et al., 2009), and transition of memories from hippocampal to cortical circuits (Kitamura et al., 2009). Recently adultborn neurons have been shown to play a role in diminishing addictive behavior and reducing the incidence of relapse (Noonan et al., 2009). Loss of α7-nAChR signaling will cause cumulative deficits to accrue over time in hippocampal circuitry as fewer adultborn neurons survive and those that do are less likely to be appropriately integrated into functioning circuits. The expected outcome would be a pronounced decline in hippocampal function, perhaps most acute for memories that are space- and time-dependent (Aimone et al., 2006; Clelland et al., 2009; Trouche et al., 2009).
Early deficits in Alzheimer’s disease involve loss of cholinergic neurons and a diminution of cholinergic signaling (Whitehouse et al., 1982; Francis et al., 1999; Nordberg, 2001; Lyness et al., 2003; O’Neil et al., 2007). The β-amyloid peptide, which accumulates during the disease (Selkoe, 1994), impairs choline uptake and acetylcholine release, further compromising cholinergic signaling (Auld et al., 1998; Kar et al., 1998). Moreover, β-amyloid peptide has been reported to inhibit α7-nAChR function either directly or indirectly (Wang et al., 2000a,b; Liu et al., 2001; Pettit et al., 2001; Dougherty et al., 2003; Grassi et al., 2003; Lee and Wang, 2003; Pym et al., 2005), though it has also been reported to have α7-nAChR agonist activity at low concentrations (Dineley et al., 2001, 2002; Dougherty et al., 2003; Grassi et al., 2003; Wang et al., 2003). Several studies have reported specific decrements in α7-nAChRs associated with Alzheimer’s disease (Hellstrom-Lindahl et al., 1999; Guan et al., 2000; Lee et al., 2000; but see Reid et al., 2000). The present studies predict that the loss or blockade of α7-nAChRs in Alzheimer’s disease would exacerbate the symptoms by decreasing the incorporation of adultborn neurons. Supporting this idea is the observation that donepezil, an acetylcholinesterase inhibitor approved as a drug for treatment of Alzheimer’s disease, has been shown to promote adultborn neuron survival during the critical period (Kaneko et al., 2006).
Our results identify α7-nAChRs as potential pharmacological targets for amplifying adultborn neuron integration and survival. An impediment to prescribing nicotinic agonists, however, is the observation that prolonged nicotine exposure at concentrations encountered by smokers can have detrimental effects on the survival of adultborn neurons (Abrous et al., 2002; Scerri et al., 2005; Shingo and Kito, 2005). The nicotine-mediated death of adultborn neurons occurs early in their development whereas the beneficial effects of endogenous α7-nAChR signaling seen here become apparent 2–4 weeks after neurogenesis. Either additional nAChR subtypes are involved or the manner of receptor activation is critical. This motivates further examination of mechanisms controlling nicotinic regulation of adult neurogenesis.
Grant support was provided by the NIH (NS012601 and N0S35469) and the Tobacco-Related Disease Research Program (16RT–0167). We thank Gouping Feng (Duke University) for the GFP-reporter mouse line, and Xiao-Yun Wang for expert technical assistance. N.R.C. is a TRDRP predoctoral fellow. C.C.F. is a Fundação Calouste Gulbenkian Graduate Fellow. A.W.F. is an NRSA predoctoral fellow.