Reid H.J. Olsen1, Szu-Aun Lim2, Isaac Haniff2, Juan Song1,3

1Department of Pharmacology, UNC Chapel Hill, 2UNC Chapel Hill, 3Curriculum 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 Gq-coupled CCK2 receptor, and exhibit Ca2+ transients following

stimulation with the CCK2-receptor selective form of CCK (CCK8) which can be blocked by the

CCK2R antagonist YM022. Moreover, we observed no calcium-response in neural stem cells

following application of the selective CCK1R 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 neuroblasts1

• CCK2 KO mice exhibit reduced adult neurogenesis2

• CCK promotes dendritic development in vitro which can be blocked by co-incubation with a

CCK2 receptor antagonist3

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 CCK2 Receptors mRNA

NSCs Respond to Activation of CCK2, but Not CCK1 Receptors

SR

10

1

Ba

se

lin

e

CC

K8

gCAMP6m-Ca2+ signal SR101-labeled NSCs in the DG of NestinCreERT2 x gCAMP6f/f at 3 DPI Tamoxifen

Allen Brain Atlas CCK2R

RNA in situ Hybridization Custom CCK2R RNA in situ

Hybridization

Custom CCK2R RNA in situ

Hybridization and GFAP IHC

DAPI CCK2 mRNA

GFAP+ NSC Composite

CCK2 Receptor Antagonist Blocks

CCK8-Induced Ca2+ response

CCK1 Agonist Does Not

Produce Ca2+ Response

Average NSC Response Average Amplitude of Ca2+ signal

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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+ neuron4

Activation of VGLUT3+ /CCK+ cells

Produces Ca2+ Responses in NSCs

Peak Amplitude of VGLUT3+ /CCK+

Cell-Mediated Ca2+ 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 CCK2

receptor mRNA, and respond to CCK. This can be blocked by the CCK2- receptor

antagonist YM022. NSCs do not respond to the selective CCK1-receptor agonist A71623. Activation of the

VGLUT3+/CCK+ cell population induces Ca2+ 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 Ca2+ imaging.

500 µm

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* *

**** ****

100 µm

Gray: DAPI; Red: mCherry; Green: CCK

* ** **

Regulation of Adult Neurogenesis by the Hippocampal Cholecystokinin Network