Postnatal and adult subventricular zone (SVZ) neurogenesis is believed to be primarily controlled by neural stem cell (NSC)-intrinsic mechanisms, interacting with extracellular and niche-driven cues. Although behavioral experiments and disease states have suggested possibilities for higher level inputs, it is unknown whether neural activity patterns from discrete circuits can directly regulate SVZ neurogenesis. We identified a previously unknown population of choline acetyltransferase (ChAT)+ neurons residing in the rodent SVZ neurogenic niche. These neurons showed morphological and functional differences from neighboring striatal counterparts and released acetylcholine locally in an activity-dependent fashion. Optogenetic inhibition and stimulation of subependymal ChAT+ neurons in vivo indicated that they were necessary and sufficient to control neurogenic proliferation. Furthermore, whole-cell recordings and biochemical experiments revealed direct SVZ NSC responses to local acetylcholine release, synergizing with fibroblast growth factor receptor activation to increase neuroblast production. These results reveal an unknown gateway connecting SVZ neurogenesis to neuronal activity-dependent control and suggest possibilities for modulating neuroregenerative capacities in health and disease.
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We thank A. Gittis (Carnegie Mellon), J. Yakel (National Institute of Environmental Health Sciences), J. Ting (Massachusetts Institute of Technology), M. Prado (Robarts Research Institute), N. Calakos, M. Caron and V. Bennett for helpful discussions, D. Kleinfeld (University of California, San Diego) for M1-CNiFER cells, N. Kessaris (University College London) for Gsx2-Cre driver, Duke Pathology EM Facility for help with sample preparation, Cold Spring Harbor Laboratory Ion Channel Physiology Course instructors N. Golding, A. Lee and M. Nolan for inspiration, K. Abdi, G. Lyons, Q. Xiao, M. Rinehart and D. Fromme for project assistance, and J. Grandl and G. Pitt for manuscript comments. This work was supported by the David & Lucile Packard Foundation, US National Institutes of Health grant R01NS078192, and a George & Jean Brumley Endowment (C.T.K.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Representative DCX staining of differentiated SVZ adherent cultures treated pharmacologically. (b) Western blot analyses of protein lysates from individually treated culture plate wells, showing titratable increase in DCX protein levels following acetylcholine (ACh) treatment. Ser: serotonin; Dopa: dopamine; Glut: glutamate; Muscimol: GABA agonist. (c) Western blot analyses of control or ACh-treated (10 μm) cultures in the presence of muscarinic antagonist atropine (Atrop) or nicotinic antagonist mecamylamine (Mec). Western blot analyses for each condition were repeated 5 times with consistent results. Scale bar: 50 μm.
(a) Genomic map of ank3 floxed-allele showing insertion of LoxP sites around exons 22 and 23, within Spectrin binding domain of Ank3. (b) Western blot analysis of whole brain lysates from P0 animals, showing successful deletion of higher molecular weight isoforms of Ank3 in ank3KO/KO (KO) mutants (left panel). Western blot analysis of FACS-sorted ChAT+ neurons from ChATIRES-Cre/+; R26R-tdTomato mice, showing expression of higher molecular weight isoforms of Ank3 protein (right panel). (c) Ank3 IHC staining in tdTomato-labeled ChAT+ neurons in control (Ctrl) and Ank3-cKO (cKO) mice, showing proximal axonal segments (arrows). Imaris software was used to create 3D image reconstructions (lower panels). (d) Representative whole-cell, current-clamp recordings from striatal ChAT+ neurons in response to 100 ms of 250 or 500 pA current pulses in Ctrl, Het, and Ank3-cKO animals. (Right) Quantification of spike numbers to 100 ms of current pulse, 250 or 500 pA. * P < 0.002, t28 = 3.661, unpaired Student's t test, n = 15 in each group (5 animals). Box plots show mean, median, quartiles, range. Scale bar: 2 μm (c).
(a) Representative views of ventricular whole-mount DCX staining from P30 Nkx2.1-Cre; ChATflox/+ (Ctrl) and Nkx2.1-Cre; ChATflox/flox (cKO) mice. (b) Representative Ki67 and Mash1 IHC staining of SVZ niche from P30 Nkx2.1-Cre; ChATflox/+ (Ctrl) and Nkx2.1-Cre; ChATflox/flox (cKO) mice. Fluorescence signals inverted to black on white for clarity in both (a) and (b). (c) Quantifications of SVZ Ki67+, Mash1+, DCX+ cell numbers. n = 5. Box plots show mean, median, quartiles, range. Scale bars: 50 μm (a,b).
(a) IHC staining for tdTomato, ChAT, VAChT in ChATIRES-Cre/+; R26R-tdTomato transgenic mice, showing co-localization in subep-ChAT+ neurons. (b) Additional example traces for subependymal (Subep) or striatal ChAT+ neurons in ventricular and coronal views. (c) Quantifications of subependymal and striatal ChAT+ neuron dendritic branch point numbers. Box plots show mean, median, quartiles, range, n = 10. (d) 3D Sholl analyses of subependymal (Subep) and striatal (Str) ChAT+ neuron dendritic morphology. Box plots show mean, median, quartiles, range, n = 10. Scale bars: 20 μm (a), 50 μm (b).
Supplementary Figure 5 Comparisons of striatal and subependymal ChAT+ neuron electrophysiological properties.
(a) Representative whole-cell, current-clamp recordings of striatal and subep-ChAT+ neurons in response to 100 (red), 250 (green), or 500 pA (black) current injections for 100 ms. (Right) Number of evoked spikes during 100 ms current pulses of 100, 250, or 500 pA. Box plots show mean, median, quartiles, range, n = 15 in each group (5 animals). (b) Representative whole-cell current-clamp recordings of striatal and subep-ChAT+ neurons in response to 500 ms negative current injection (following 100 ms 20 pA positive current pulse). Dotted-lines indicate amplitude of sag potential. (c) Analyses of striatal and subep-ChAT+ neuron resting membrane potential, spike initiation threshold, and depolarization to spike, acquired by increasing depolarizing current pulses (100 ms duration) until spike initialization. Box plots show mean, median, quartiles, range, n = 15 (5 animals).
Schematic representations of (a) M1-CNiFER cell detection of ACh; (b) M1-CNiFER cells transplanted into SVZ niche in acute slice preparation; (c) M1-CNiFER cells transplanted into SVZ niche in acute slice preparation from ChATIRES-Cre/+; R26R-ChR2EYFP mice.
IHC staining of P30 SVZ coronal sections, with antibodies against α3-nicotinic (α3-nAChR), α4-nicotinic (α4-nAChR), or pan-muscarinic (mAChR) receptors (red); GFAP (blue); GFP (from ChAT-GFP transgene, green); and DAPI (red, in left panels). GFAP+ SVZ NSCs (white dashed-lines) touching the lateral ventricle (LV) are positive for nicotinic and muscarinic receptors (Imaris 3D). They are adjacent to GFP+ processes from ChAT+ neurons (arrows). Antibodies against nicotinic and muscarinic receptors that showed IHC specificity were made in rabbit, and were stained individually. Scale bar: 10 μm.
(a) Schematic representation of experimental setup in P30 SVZ acute slices from nestin-CreERtm4 (N4); R26R-tdTomato; ChAT-ChR2EYFP mice. (b) Representative images of tdTomato+ subependymal NSCs in P30 brain slices, lineage-traced via nestin-CreERtm4; R26R-tdTomato following tamoxifen injection, filled with DiO (green) through glass micropipette. (c) Representative images showing DiO injections and subsequent IHC staining with Nestin, S100β, Mash1, or DCX antibodies. (d) Representative voltage-clamp recordings from tdTomato+ ependymal niche cells, Mash1+ “C” cells, DCX+ neuroblasts, following 10 ms 473 nm light pulses @ 30 Hz for 1 second (red traces are baselines without light). Inward current in DCX+ neuroblast is sensitive to cholinergic blockers mecamylamine (Mec., 40 μM), atropine (Atrop., 5 μM). Blue bar = duration of light-stimulation train. (e) Quantifications of light-evoked current responses in (d). * P < 0.0002, F2,8 = 34.49, one-way ANOVA, n = 5. Box plots show mean, median, quartiles, range. Scale bars: 5 μm (b,c).
(a,c) Schematic representations of in vivo light-stimulation. LV = lateral ventricle, Ctx = cortex.(b,d) Representative Ki67, Mash1, DCX, Nestin/Ki67 IHC staining of SVZ niche from P30 ChATIRES-Cre/+; R26R-ChR2EYFP (b) or ChATIRES-Cre/+; R26R-ArchGFP (d) mice, with (+) or without (–) 48 hrs of light-stimulation in vivo (473 nm for ChR2 or 556 nm for Arch). Fluorescence signals inverted to black on white for clarity. Representative subependymal Nestin+ cells (*) co-localizing with Ki67 (arrows) in right panels. (e,f) Representative p-rpS6 IHC staining of striatal ChAT+ neurons (arrows) adjacent to the SVZ, from P30 ChATIRES-Cre/+; R26R-ChR2EYFP mice following 48 hrs of 473 nm light-stimulation (e), or from P30 ChATIRES-Cre/+; R26R-ArchGFP mice following 48 hrs of 556 nm light-stimulation (f), comparing ipsilateral to uninduced contralateral striatum after ventricular stimulation, imaged at identical settings from same section. Cy5 channel used for p-rpS6 secondary antibody staining, for clarity represented in green channel for co-localization with tdTomato. Scale bars: 20 μm (b,d), 10 μm (e,f).
(a) Representative Iba1, CD11b, NG2 IHC staining of SVZ niche from P30 ChATIRES-Cre/+; R26R-ChR2EYFP mice, with (+) or without (–) 48 hrs of 473 nm light-stimulation in vivo. Dashed-lines = proliferating SVZ regions. (b) Quantifications of SVZ Iba1, CD11b, NG2 IHC staining data from (a). P = 0.169, F2,12 = 2.07 (NG2); P = 0.175, F2,12 = 2.02 (NG2/Ki67). One-way ANOVA, n = 5. Box plots show mean, median, quartiles, range. (c) Representative Thbs4 IHC staining on SVZ sections, 48 and 84 hrs post cortical optical fiber implantation to target the lateral ventricle (LV). Note the delayed Thbs4 upregulation in SVZ niche ipsilateral (ipsil) to fiber implantation (arrows). contra = contralateral hemisphere. Scale bars: 20 μm (a), 50 μm (c).
(a) Western blot analyses on effects of ACh or FGF (12 ng/ml) in SVZ NSC FGFR phosphorylation. Anti-FGF (α-FGF) was used at 13 μg/ml, except for “low” condition in lane 8 (6.5 μg/ml). (b) Western blot analysis of FGFR phosphorylation following application of either 100 μM ACh, or 12 ng/ml FGF to SVZ NSC adherent cultures. Note the lack of ACh-mediated increase in FGFR phosphorylation 90 minutes after treatment. (c) ELISA analyses of FGF concentrations in culture media during SVZ NSC differentiation in vitro, with ACh added (100 μM once at time zero) compared to control (no ACh). * P < 0.008, z = 2.739 (24 hrs), z = 2.611 (48 hrs), Wilcoxon two-sample test, n = 5. Box plots show mean, median, quartiles, range. (d) Western blot analysis of EGFR phosphorylation in SVZ NSC adherent cultures after treatment with 100 μM ACh, 20 ng/ml EGF (EGF Cond1), or 40 ng/ml EGF (EGF Cond2). (e) Western blot analyses on the effects of ACh and FGF on DCX+ neuroblast production in SVZ NSC adherent culture. All Western analyses (a,b,d,e) were repeated 5 times with consistent results. (f) Representative IHC staining and quantifications of phosphorylated FGFR (pFGFR) co-localization with GFAP+ SVZ NSCs in P30 niche using Imaris, showing decreased expression in Ank3-cKO animals compared to littermate controls. pFGFR fluorescence signal inverted to black on white for clarity. * P < 0.008, z = 2.611 Wilcoxon two-sample test, n = 5. Box plots show mean, median, quartiles, range. Scale bar: 10 μm (f).
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Paez-Gonzalez, P., Asrican, B., Rodriguez, E. et al. Identification of distinct ChAT+ neurons and activity-dependent control of postnatal SVZ neurogenesis. Nat Neurosci 17, 934–942 (2014). https://doi.org/10.1038/nn.3734
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