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Hypothalamic modulation of adult hippocampal neurogenesis in mice confers activity-dependent regulation of memory and anxiety-like behavior

Nature Neuroscience volume 25, pages 630–645 (2022)Cite this article

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Abstract

Adult hippocampal neurogenesis plays a critical role in memory and emotion processing, and this process is dynamically regulated by neural circuit activity. However, it remains unknown whether manipulation of neural circuit activity can achieve sufficient neurogenic effects to modulate behavior. Here we report that chronic patterned optogenetic stimulation of supramammillary nucleus (SuM) neurons in the mouse hypothalamus robustly promotes neurogenesis at multiple stages, leading to increased production of neural stem cells and behaviorally relevant adult-born neurons (ABNs) with enhanced maturity. Functionally, selective manipulation of the activity of these SuM-promoted ABNs modulates memory retrieval and anxiety-like behaviors. Furthermore, we show that SuM neurons are highly responsive to environmental novelty (EN) and are required for EN-induced enhancement of neurogenesis. Moreover, SuM is required for ABN activity-dependent behavioral modulation under a novel environment. Our study identifies a key hypothalamic circuit that couples novelty signals to the production and maturation of ABNs, and highlights the activity-dependent contribution of circuit-modified ABNs in behavioral regulation.

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Fig. 1: Activation of SuM-DG projections excites rNSCs through glutamate transmission.
Fig. 2: Stimulation of SuM neurons promotes production of rNSCs and neural progenitors.
Fig. 3: SuM GABA transmission regulates neural progenitors and early-stage immature neurons.
Fig. 4: SuM neurons promote maturation and dendritic spine development of late-stage adult-born immature neurons.
Fig. 5: Activation of SuM circuit-modified ABNs further improves memory performance.
Fig. 6: Bidirectional manipulation of SuM circuit-modified ABNs further modulates anxiety-like behavior.
Fig. 7: DG-projecting SuM neurons exhibit increased activity in EE.
Fig. 8: Ablation of SuM neurons abolishes EE-induced neurogenic effects and behavioral improvement mediated by ABNs.

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The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Song laboratory for comments and discussions, with special thanks to B. Asrican for making the final edits of the manuscript. We also thank B. Roth for providing floxed hM3Dq and hM4Di lines for this study. This work was supported by grants awarded to J.S. from NIH (nos. R01MH111773-01, R01MH122692-01, RF1AG058160-01 and R01NS104530-01) and the Alzheimer’s Association. Y.-D.L. was partially supported by a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation (no. 29600). Z.-K.C. was partially supported by grants-in-aid for scientific research from the National Natural Science Foundation of China (no. 8202010801). Z.-L.H. was supporred by the National Key Research and Development Program of China (no. 2020YFC2005300). Confocal microscopy was performed at the UNC Neuroscience Microscopy Core Facility (RRID: SCR_019060) with technical assistance from M. S. Itano. The UNC Neuroscience Microscopy Core was supported in part by funding from NIH-NINDS Neuroscience Center Support Grant no. P30 NS045892 and NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant no. U54 HD079124.

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Author notes

  1. These authors contributed equally: Ya-Dong Li, Yan-Jia Luo.

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Ya-Dong Li, Yan-Jia Luo, Ze-Ka Chen, Luis Quintanilla, Libo Zhang & Juan Song

  2. Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Ya-Dong Li, Yan-Jia Luo, Ze-Ka Chen, Luis Quintanilla & Juan Song

  3. Department of Pharmacology, School of Basic Medical Sciences; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, and Institutes of Brain Science, Fudan University, Shanghai, China

    Ze-Ka Chen & Zhi-Li Huang

  4. Neuroscience Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Luis Quintanilla

  5. International Institute for Integrative Sleep Medicine (WPI-IIIS) and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Yoan Cherasse & Michael Lazarus

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Contributions

J.S. supervised the project, designed the experiments and wrote the paper. Y.-D.L. designed the experiments, wrote the paper and carried out all aspects of in vivo experiments and data analysis. Y.-J.L. carried out all aspects of in vitro slice electrophysiology and helped with data analysis and preparation of the manuscript. Z.-K.C. and Z.-L.H. performed in vivo multichannel recording and related data analysis. L.Q. and L.Z. assisted with experiments and analysis. Y.C. and M.L. prepared the AAVs with genetic knockdown of Vgat or Vglut2. All authors discussed the manuscript.

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Correspondence to Juan Song.

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Extended data

Extended Data Fig. 1 SuM glutamate promotes proliferation of rNSCs and newborn progeny.

(a–b) Nestin-GFP-expressing cells in the SGZ show distinct electrophysiological properties. Membrane currents from the nestin-GFP positive cells shown in the left column were evoked by 50-ms voltage steps ranging from −135 mV to +25 mV from a holding potential of −65 mV (the recording protocol see the inset). From these recordings, corresponding current-voltage curves were obtained at the time points indicated by the black circle above the traces (right columns). (a) Example for a type-1 cell expressing passive, non-inactivating currents with a linear current–voltage relationship. (b) Examples for two type-2 cells expressing outwardly rectifying currents, but with different reversal potentials (around −40 mV in the left, and −80mV in the right). (c) Diagram and stimulation paradigm for optogenetic manipulation of SuMVgat-DG projections. (d) Sample images of EdU/Nestin and EdU/DCX staining in the DG. Scale bar = 20 µm. (e–f) Sample images of EdU/Nestin (e) and EdU/DCX (f) staining after optogenetic activation of SuMVgat-DG projections. Scale bar = 100 µm. g–k) Density of EdU+/Nestin+ (g), EdU+ (h), EdU+/DCX+ (i), total Nestin+ (j) and total DCX+ (l) cells in the whole DG after optogenetic activation of SuMVgat-DG projections. n = 6 mice in each group, two-sided unpaired t-test, g: P = 0.0099, h: P = 0.0012, i: P = 0.0029, j: P = 0.5730, i: P = 0.3101, respectively. (l) Sample images of EdU/Nestin staining upon optogenetic stimulation of SuMVgat-DG projections with expressing shVgat or shVglut2. Scale bar = 50 µm. (m–n) The density of total Nestin+ (m) and total DCX+ (n) cells in the whole DG after optogenetic activation of SuMVgat-DG projections with expressing shVgat or shVglut2 in the SuM. n = 7, 8, 6 mice in ChR2, shVglut2/ChR2 and shVgat/ChR2 group, respectively. P > 0.05 by one-way ANOVA.

Source data

Extended Data Fig. 2 Short-term optogenetic activation of SuM neurons in Ascl1-Ai9 mice.

(a–b) Optogenetic activation increased c-Fos expressions in the SuM. n = 3 mice for each group, P = 0.0309 by two-sided unpaired t-test. (c) Sample images of tdTomato+, Ki67+ and GFAP+/Sox2+ staining in the DG after optogenetic stimulation of SuM. The yellow arrow head indicated a Ki67+/tdTomato+/GFAP+ cell; the pink arrow head indicated a Ki67+/tdTomato+/Sox2+ cell. Scale bar = 100 µm. (d) Diagram of optogenetic stimulation of SuM neurons in Ascl1-Ai9 mice. Tamoxifen was i.p. administered at 80 mg/kg following 8 hours optogenetic stimulation of SuM neurons for 3 days. Mice were perfused on day 42 after the first tamoxifen injection. (e) Sample images of tdTomato+ cells in the DG at day 42 after tamoxifen injection. Scale bar = 100 µm. (f) The density of tdTomato+ cells in the DG. n = 4 mice for YFP group, n = 5 mice for ChR2 group, P = 0.9558 by two-sided unpaired t-test.

Source data

Extended Data Fig. 3 SuM neurons are essential for the proliferation of rNSCs and newborn progeny.

(a) Diagram of chemogenetic manipulation of SuM neurons in Vgat-Cre mice. AAV5-DIO-hM3Dq-mCherry or AAV5-DIO-hM4Di-mCherry and their control mCherry construct were bilaterally injected into SuM in the Vgat-Cre mice. After 3 weeks of virus expression, CNO 1 mg/kg was given by i.p. injection for 5 days. Mice were perfused on day 5 after 4 shoots of EdU at 40 mg/kg. (b) Sample image of hM3Dq-mCherry expression. Scale bar = 100 µm. (c, i) Sample images of EdU/Nestin (left) and EdU/DCX (right) staining after chemogenetic activation (c) or inhibition (i) of SuMVgat neurons. Scale bar = 100 µm. (d–f) Density of EdU+ (d), EdU+/Nestin+ (e), EdU+/DCX+ (f) in the DG after chemogenetic activation of SuMVgat neurons. n = 7 mice for each group, two-sided unpaired t-test, d: P = 0.0225, e: P = 0.0298, f: P = 0.0012, respectively. (g–h) Density of total Nestin+ (g) and total DCX+ (h) cells in the DG after chemogenetic activation of SuMVgat neurons. n = 7 mice for each group, two-sided unpaired t-test, g: P = 0.6761, h: P = 0.8048, respectively. (j–l) Density of EdU+ (j), EdU+/Nestin+ (k), EdU+/DCX+ (l) in the DG after chemogenetic inhibition of SuMVgat neurons. n = 7 mice for each group, two-sided unpaired t-test, j: P = 0.0483, k: P = 0.0287, l: P = 0.0023, respectively (m–n) Density of total Nestin+ (m) and total DCX+ (n) cells in the DG after chemogenetic inhibition of SuMVgat neurons. n = 7 mice for each group, two-sided unpaired t-test, m: P = 0.3325, n: P = 0.1046, respectively.

Source data

Extended Data Fig. 4 Electrophysiological recordings of adult-born neural progenitors or neuroblasts and immature neurons.

(a) Diagram of in vitro electrophysiological recording of progenitors/neuroblasts upon optogenetic stimulation of SuM-DG projections in Ascl1-Ai9 mice. (b) Confocal images of a biocytin-labeled tdTomato+ cell at 9 dpi after whole-cell patch clamp recording. Scale bar = 20 µm. (c) Electrophysiological characteristics of a neuroblast cell. Membrane currents from a non-responding cell were evoked by 50-ms voltage steps ranging from −135 mV to +25 mV at a holding potential of −65 mV. (d–e) Light stimulation failed to induce any currents in 9 dpi tdTomato+ cells, with bathing 4-AP or vigabatrin (0 of 5 cells; Vh = −65 mV; KCl-based pipette solution). (f) Diagram of in vitro electrophysiological recording of immature neurons at 9 or 12 dpi upon optogenetic stimulation of SuM-DG projections in Ascl1-Ai9 mice. (g) Confocal images of a biocytin-labeled tdTomato+ cell at 12 dpi after whole-cell patch clamp recording. Scale bar = 20 µm. (h) Electrophysiological characteristics of an immature cell at 12 dpi. Membrane currents from a non-responding cell were evoked by 50-ms voltage steps ranging from −135 mV to +25 mV at a holding potential of −65 mV. (i) Light stimulation failed to induce any currents in 12 dpi tdTomato+ cells (0 of 14 cells; Vh = −65 mV; GK-based pipette solution). (j) Proportion of connected and unconnected cells with the use of GK or KCl internal solution following blue light stimulation of SuM-DG projections. Numbers of cells are shown in parentheses.

Extended Data Fig. 5 Adult-born neurons at 32 dpi exhibit distinct electrophysiological properties.

(a–c) Comparison of intrinsic and active membrane properties in 32 dpi newborn neurons and mature GCs. Resting membrane potential (a), membrane capacitance (b), and input resistance (c) were measured in tdTomato+ newborn neurons and unlabeled mature GCs. n = 21 from 32 dpi newborn cells, n = 20 cells from mature GCs from 5 mice, P < 0.0001 by two-sided unpaired t-test. (d) The number of spikes elicited by increasing current steps in 32 dpi tdTomato+ adult-born neurons and unlabeled mature GCs. n = 20 cells for each group from 5 mice, P < 0.0001 by two-way ANOVA. (e, f) Sample traces showed a granule cell received both glutamate and GABA (e) or sole glutamate inputs (f) upon optogenetic activation of SuM-DG projections. (Cs-based pipette solutions). (g–h) Latency of light-evoked EPSCs (g) and IPSCs (h) in 32 dpi adult-born neurons and mature GCs. For EPSCs, n = 5 from 32 dpi newborn cells, n = 8 cells from mature GCs from 3 mice, P = 0.0057 by two-tailed unpaired t-test. For IPSCs, n = 3 from 32 dpi newborn cells, n = 7 cells for mature GCs from 3 mice, P = 0.0117 by two-tailed unpaired t-test. (i–j) Amplitude of light-evoked EPSCs (i) and IPSCs (j) in 32 dpi adult-born neurons and mature GCs. For EPSCs, n = 5 from 32 dpi newborn cells, n = 8 cells from mature GCs from 3 mice, P = 0.05 by two-tailed unpaired t-test. For IPSCs, n = 3 from 32 dpi newborn cells, n = 7 cells for mature GCs from 3 mice, P = 0.0737 by two-tailed unpaired t-test. (k) Proportion of glutamate and GABA inputs to GCs or tdTomato+ cells at 32 dpi following blue light stimulation of SuM-DG projections. The numbers of cells are shown in parentheses. (l–m) Newborn cells at 22 dpi (l) and 26 dpi (m) only received GABAergic inputs (Cs-based pipette solutions; Vh=+5 mV) from SuM neurons. (n) Sample images and quantification of iba1 and GFAP in the SuM of YFP control mice or laser stimulated (sti) mice for 32 days. n = 3 mice for each group. P = 5939 for (GFAP), P = 8139 (iba1) by two-tailed unpaired t-test. Values represent mean ± SEM. ∗P < 0.05; ∗∗P < 0.01 by unpaired t-test.

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Extended Data Fig. 6 Activity of adult-born neurons is critical for memory retrieval.

(a) Experimental protocol for acute chemogenetic activation of adult-born neurons during behavioral tests. (b) Representative confocal images of mCitrine+ hM3Dq+ cells in the DG of the Ascl1-hM3Dq or hM3Dq mice. mCitrine+ cells, indicating hM3Dq+ newborn neurons, were found in Ascl1-hM3Dq mice, but not hM3Dq mice. Scale bar = 100 µm. (c–d) Diagram of NPR (C) and CFC (D) tests. (e) Chemogenetic activation of adult-born neurons during memory retrieval increased the discrimination ratio in the NPR test. n = 14 mice for each group, P = 0.0484 by two-sided unpaired t-test. (f–i) Freezing time in the context-A at 2 h (f), 24 h (g), in the context-B at 24 h (h), and in the context-A at 7 days (i) after chemogenetic activation of adult-born neurons. CNO 0.5 mg/kg was administrated by i.p. injection 30 mins before memory retrieval tests. n = 13 mice for hM3 group, n = 12 mice for Ascl1-hM3 group, two-sided unpaired t-test, f: P = 0.5764, g: P = 0.0494, h: P = 0.6726, i: P = 0.0470, respectively. (j) Experimental protocol for acute chemogenetic inhibition of adult-born neurons during behavioral tests. (k) Representative confocal images of HA+ hM4Di+ cells in the DG of Ascl1-hM4Di or hM4Di mice. Scale bar = 100 µm. (l) Chemogenetic inhibition of adult-born neurons decreased the discrimination ratio in the NPR test. n = 7 mice for each group, P = 0.0202 by two-sided unpaired t-test. (m–p) Chemogenetic inhibition of adult-born neurons did not change the freezing time in the CFC test. CNO 1 mg/kg was administrated by i.p. injection 30 mins before memory retrieval tests. m–o: n = 13 mice for hM4 group, n = 9 mice for Ascl1-hM4 group. p: n = 6 mice for hM4 group, n = 7 mice for Ascl1-hM4 group, two-sided unpaired t-test, m: P = 0.9992, n: P = 0.7913, o: P = 0.8472, p: P = 0.9567, respectively.

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Extended Data Fig. 7 Chemogenetic manipulation of adult-born neurons in Ascl1-hM3Dq and Ascl1-hM4Di mice.

(a) Representative confocal images of mCitrine and c-Fos expression in the DG of Ascl1-hM3Dq or hM3Dq mice. Scale bar = 50 µm. (b) Percent of c-Fos expression in mCitrine+ cells and density of mCitrine+ cells in the DG of Ascl1-hM3Dq mice after i.p. injection of CNO at 0.5 mg/kg. n = 7 mice. (c) Sample trace showing that bath application of CNO at 10 µM induced depolarization of membrane potential and increased firing rates in an hM3Dq+ adult-born neuron. (d) Representative confocal images of HA-tag and c-Fos expression in the DG of Ascl1-hM4Di or hM4Di mice after administration of CNO at 1 mg/kg. (e) Density of HA-tag+ cells in Ascl1-hM4Di mice. n = 5 mice. (f) Sample traces showing that action potentials in response to current injections in an hM4Di+ cell before, after and wash out application of 10 µM CNO. (g) Representative heat map of locomotion tracing for an hM3Dq control and an Ascl1-hM3Dq mouse in the NPR test (left). Quantification of total locomotion in the NPR test (right). n = 10 mice for each group, P = 0.8410 by two-sided unpaired t-test. (h) Representative heat map of locomotion tracing for an hM4Di control and an Ascl1-hM4Di mouse in the NPR test (left). Quantification of total locomotion in the NPR test (right). n = 7 mice for hM4 group, n = 8 mice for Ascl1-hM4 group, P = 0.9189 by two-sided unpaired t-test. (i–j) Ascl1 labeled ABNs were found in the OB (i) and DG (j) 32 days after tamoxifen injection. Scale bar = 100 in i and j, scale bar = 10 µm in i1 and j1.

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Extended Data Fig. 8 Summary model for how circuit-modified hippocampal neurogenesis modulates hippocampal dependent behavior.

By combining circuit manipulation with lineage tracing of adult-born neural precursors, we demonstrate that SuM glutamatergic inputs act on the initial rNSC stage to promote self-renewal and neurogenic proliferation of rNSCs, leading to increased production of rNSCs and neural progenitors. Then, SuM GABAergic inputs indirectly acts on the neural progenitors potentially through dentate interneurons and directly acts on early-stage immature neurons to promote differentiation of neural progenitors and dendritic development of immature neurons, respectively, leading to increased number of immature neurons with longer and more elaborate dendrites. Finally, SuM GABAergic and glutamatergic inputs collectively act on late-stage immature neurons, leading to increased number of ABNs with enhanced maturity and increased dendritic spines. Therefore, stimulating SuM neurons leads to not only increased number of ABNs, but also enhanced developmental features of ABNs. Importantly, selectively manipulating the activity of circuit-modified ABNs further modulates memory performance and anxiety-like behavior as compared to activity-manipulation of control ABNs: activation of these circuit-modified ABNs further improves memory retrieval and reduces anxiety; while inhibition of these neurons exacerbates anxiety without affecting memory performance.

Extended Data Fig. 9 Circuit-modified adult-born neurons do not further modulate behavior without activity manipulation.

(a) Experimental protocol of optogenetic stimulation of SuM neurons for 32 days in wild-type mice. (b) Optogenetic stimulation of SuM neurons for 32 days did not change the discrimination ratio during the NPR test. n = 7 mice for mCherry group, n = 9 mice for ChR2 group, P = 0.8550 by two-sided unpaired t-test. (c–f) Optogenetic stimulation of SuM neurons for 32 days did not change freezing time during the CFC test. n = 7 mice for mCherry group, n = 9 mice for ChR2 group, two-sided unpaired t-test, c: P = 0.2851, d: P = 0.5718, e: P = 0.1534, f: P = 0.2468, respectively. (g–j) Optogenetic stimulation of SuM neurons for 32 days did not change behaviors in the open field, zero maze, and forced swimming tests. n = 7 mice for mCherry group, n = 9 mice for ChR2 group, two-sided unpaired t-test, g: P = 0.6731, h: P = 0.1251, i: P = 0.9092, j: P = 0.1179, respectively. (k) Sample image of DCX and c-Fos staining in HC or EE mice. The arrowhead indicates a c-Fos/DCX double positive cell. Scale bar: 20 µM. (l) Quantification of percent of c-Fos+DCX+/DCX+ cells. n = 4 mice in each group, P = 0.0478 by two-sided unpaired t-test. Values represent mean ± SEM. * P < 0.05 by unpaired t-test.

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Extended Data Fig. 10 Chronic inhibition of SuM reduced adult-born neurons and impaired hippocampal function.

(a) Experimental protocol of chemogenetic inhibition of SuM neurons for 32 days in Ascl1CreER::Ai9 mice. Behavior tests were performed later without CNO drinking. (b–c) Chemogenetic inhibition of SuM neurons for 32 days decreased density of ABNs. n = 4 mice, P = 0.0022 by two-sided unpaired t-test. (d–e) Chemogenetic inhibition of SuM neurons for 32 days decreased density of tdTomato/NeuN+ and tdTomato/DCX+ cells. n = 4 mice, P = 0.0234 (d), P = 0.0039 (e) by two-sided unpaired t-test. (f) The discrimination ratio in the NPR test after chronic inhibition of SuM for 32 days. n = 11 mice for mCherry group, n = 7 mice for hM4Di group., P = 0.0496 by two-sided unpaired t-test. (g–h) Freezing time in the context-A and context-B at 24 hours after encoding in the CFC test after chronic inhibition of SuM for 32 days. n = 11 mice for mCherry group, n = 7 mice for hM4Di group, P = 0.9313 (g), P = 0.5348 (h) by two-sided unpaired t-test. (i–j) Locomotion and time spent in the central area in the open field test after chronic inhibition of SuM for 32 days. n = 11 mice for mCherry group, n = 7 mice for hM4Di group, P = 0.3032 (i), P = 0.7419 (j) by two-sided unpaired t-test. (k) Time spent in the open arms in the zero-maze test after chronic inhibition of SuM for 32 days. n = 11 mice for mCherry group, n = 7 mice for hM4Di group, P = 0.0479 by two-sided unpaired t-test. (l) Time of immobility in the forced swimming test after chronic inhibition of SuM for 32 days. n = 11 mice for mCherry group, n = 7 mice for hM4Di group, P = 0.4929 by two-sided unpaired t-test.

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Li, YD., Luo, YJ., Chen, ZK. et al. Hypothalamic modulation of adult hippocampal neurogenesis in mice confers activity-dependent regulation of memory and anxiety-like behavior. Nat Neurosci 25, 630–645 (2022). https://doi.org/10.1038/s41593-022-01065-x

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