regulation of adult neurogenesis by the hippocampal...
TRANSCRIPT
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
** ** *
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