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Reid H.J. Olsen 1 , Szu-Aun Lim 2 , Isaac Haniff 2 , Juan Song 1,3 1 Department of Pharmacology, UNC Chapel Hill, 2 UNC Chapel Hill, 3 Curriculum in Neurobiology, UNC Chapel Hill Neurogenesis Occurs in the Adult Hippocampus and is Regulated by Neuronal Activity Adult neurogenesis is a unique and poorly understood form of neuroplasticity that in humans is essentially restricted to the dentate gyrus (DG) of the hippocampus. This process is tightly regulated by the activity of local neuronal populations and afferent projecting systems. The identity of specific cell types, neurotransmitters, and receptors that facilitate this regulation remains critically understudied. Studies utilizing animal and cell culture models suggest that the neuropeptide cholecystokinin (CCK) serves as proliferative and survival signals for neural stem cells (NSCs) in the adult brain. Utilizing DREADD technology, we have tested the hypothesis that in vivo activation of CCK-cells promotes proliferation of NSCs. We have found that NSCs express mRNA for the G q -coupled CCK 2 receptor, and exhibit Ca 2+ transients following stimulation with the CCK 2 -receptor selective form of CCK (CCK8) which can be blocked by the CCK 2 R antagonist YM022. Moreover, we observed no calcium-response in neural stem cells following application of the selective CCK 1 R agonist A71623. Finally, we found that in vivo chemogenetic stimulation of CCK-releasing neurons in the DG produces an increase in stem cell proliferation. CCK-cells are a heterogeneous population and can co-release GABA, we stimulated the Vglut3 + CCK-basket cell population in vivo, and separately knocked down CCK synthesis via shRNA. Stimulation of the CCK-expressing basket cell population (Vglut3 + ) in ex vivo slice produced calcium transients in neural stem cells, and an increase in proliferating neural stem cells. Knockdown of CCK in the dentate produced a striking decrease in neural stem cell proliferation. These data strongly implicate CCK as a positive regulator of adult neurogenesis. Moving forward, we will assess effects at later stages of development in adult born neurons, and characterize behavioral consequences related to neurogenesis. ABSTRACT Introduction • CCK promotes proliferation and survival of cultured neuroblasts 1 • CCK 2 KO mice exhibit reduced adult neurogenesis 2 • CCK promotes dendritic development in vitro which can be blocked by co-incubation with a CCK 2 receptor antagonist 3 Dentate CCK-Cell Fibers Ramify in the Granule Cell Layer Hypothesis In Vivo Activation of Hippocampal CCK-Cells Promotes Proliferation of Adult Neural Stem Cells DAPI CCK8 Composite CCK-Cre x AAV-DIO-mCherry The Neuropeptide CCK is Implicated in Adult Neurogenesis 20 μm 100 μm Hippocampal NSCs Express CCK 2 Receptors mRNA NSCs Respond to Activation of CCK 2, but Not CCK 1 Receptors SR101 Baseline CCK8 gCAMP6m-Ca 2+ signal SR101-labeled NSCs in the DG of NestinCre ERT2 x gCAMP6 f/f at 3 DPI Tamoxifen Allen Brain Atlas CCK 2 R RNA in situ Hybridization Custom CCK 2 R RNA in situ Hybridization Custom CCK 2 R RNA in situ Hybridization and GFAP IHC DAPI CCK 2 mRNA GFAP + NSC Composite CCK 2 Receptor Antagonist Blocks CCK8-Induced Ca 2+ response CCK 1 Agonist Does Not Produce Ca 2+ Response Average NSC Response Average Amplitude of Ca 2+ signal ** ** * Results CCK-shRNA Scramble shRNA CCK8 GAPDH * AAV-Mediated shRNA Knockdown of CCK is Effective In Vivo Knockdown of CCK Reduces NSC Proliferation CCK Knockdown Reduces NSC Proliferation CCK Knockdown Reduces Total Proliferating in the DG Chemogenetic Activation of DG CCK-Cells induces NSC Proliferation Stereotaxic AAV Injection 4-weeks recovery 4 days CNO in drinking water (5 mg/200 ml) EdU Pulse EdU incorporation into dividing cells (green); Nestin + NSCs with radial process Experimental Timeline CCK-Cell Stimulation Increases Percentage of Proliferating NSCs CCK-Cell Stimulation Increases Density of Proliferating NSCs No Change in Total NSC Density CCK-Cell Stimulation Increases Total Proliferation in the DG VGLUT3 + /CCK + Basket Cells Activates NSCs in Ex Vivo Slice and Increase NSC Proliferation In Vivo hM3D-mCherry (red) and ProCCK (green) in VGLUT3 + neuron 4 Activation of VGLUT3 + /CCK + cells Produces Ca 2+ Responses in NSCs Peak Amplitude of VGLUT3 + /CCK + Cell-Mediated Ca 2+ response hM3D-mCherry in VGLUT3 + Basket Cells (green) MCM2 + / (red) Nestin + NSCs Activation of VGLUT3 + /CCK + cells Increases NSC Proliferation Conclusions and Future Directions NSCs express CCK2 receptors Adult neural stem cells express CCK 2 receptor mRNA, and respond to CCK. This can be blocked by the CCK 2 - receptor antagonist YM022. NSCs do not respond to the selective CCK 1 -receptor agonist A71623. Activation of the VGLUT3 + /CCK + cell population induces Ca 2+ responses in NSCs. In Vivo Activation of CCK-Cells Increases NSC Proliferation Stimulation of CCK + cells in the DG in vivo produces an increase in NSC proliferation in two separate orthologous mouse-lines. Total proliferating cells also increase, but with no commensurate increase in the density of total NSCs. This suggests that CCK may stimulate NSCs to proliferate asymmetrically. Loss of CCK reduces NSC proliferation and Total Proliferation in the DG In Vivo. The shRNA-mediated knockdown of CCK provides further support for the role of CCK in regulating adult neurogenesis. Future Goals We will further probe the significance of these findings using behavioral assays to investigate the importance of this system in specific neurogenesis-associated tasks. We will also investigate later time-points such as cell-differentiation and fate-choice, survival, and dendritic and synaptic integration. 1. Langmesser, S. et al. Journal of cellular biochemistry (2007) 2. Stanic, D. et al. PNAS (2008) 3. Zhang, L.l. et al. Neuroscience Letters (2013) 4. Somogyi et al., Eur J Neuroscience (2004) References RHJO: NINDS NRSA 1F31NS093917 – 01 SAL: UNC SURF Fellowship IH & SAL: UNC Office of Undergraduate Research Travel Grant Funding Acknowledgements We acknowledge RHJO’s thesis committee (Drs. Bryan Roth, Tom Kash, Eva Anton, & Ken McCarthy. Brad Lowell gifted VGLUT3-Cre mice. shRNA plasmid was a gift from Dr. Ralph DiLeone. CCK and Pro-CCK antibodies were a gift from Drs. Andrea Varro and Margery Beinfeld, respectively. Hyojin Kim assisted with Ca 2+ imaging. 500 μm *** * * **** **** 100 μm Gray: DAPI; Red: mCherry; Green: CCK * ** ** Regulation of Adult Neurogenesis by the Hippocampal Cholecystokinin Network
