Method Article

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

DOI:

10.3791/59237

⸱

July 24th, 2019

 ,  ,  ,  , 
1Department of Pharmacology, University of North Carolina Chapel Hill, 2Neuroscience Center, University of North Carolina Chapel Hill, 3Neuroscience Curriculum, University of North Carolina Chapel Hill

In This Article

Summary

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The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Abstract

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Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

Introduction

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Adult neurogenesis is a biological process by which new neurons are born in an adult and integrated into the existing neural networks1. In humans, this process occurs in the dentate gyrus (DG) of the hippocampus, where about 1,400 new cells are born each day2. These cells reside in the inner part of the DG, which harbors a neurogenic niche, termed the subgranular zone (SGZ). Here, hippocampal adult neural stem cells (NSCs) undergo a complex developmental process to become fully functional neurons that contribute to the regulation of specific brain functions, including learning and memory, mood regulation, and stress resp....

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Protocol

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All procedures including animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina Chapel Hill.

1. Stereotaxic Injection of Viral Particles

  1. Determine the neural circuits in question. This will determine the virus and the mouse line utilized for the following procedure.
    NOTE: For this example, contralateral mossy cell projections are stimulated to analyze its effects on adult neurogenesis. Viral particles encoding AAV5-hSyn-DIO-hM3Dq-mCherry are delivered to the DG of 5ht2A-Cre mice15.
  2. Administer melox....

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Results

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Following the experimental procedures described above (Figure 1A,B), we were able to determine the effects of stimulating contralateral mossy cell projections on the neurogenic niche within the hippocampus. By utilizing a Cre-dependent Gq-coupled stimulating DREADD virus paired with a mossy cell labeling 5-HT2A Cre-line, we were able to selectively activate excitatory projections from mossy cells onto the contralateral DG and determined that strong mossy cell stimulation pro.......

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Discussion

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The goal of this protocol is to assess how manipulating specific neural circuits regulates adult hippocampal neurogenesis in vivo using a series of immunohistochemistry techniques. Assaying activity dependent regulation of adult neurogenesis mediated by specific neural circuits is a valuable technique with great potential for modifications to study a diverse range of neural circuits. The success of these types of experiments depends on multiple factors including accurate viral delivery, proper viral selection for the des.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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L.J.Q. was supported by the National Institute of Mental Health of the National Institutes of Health under Diversity Supplement R01MH111773 as well as a T32 training grant T32NS007431-20. This project was supported from grants awarded to J.S. from NIH (MH111773, AG058160, and NS104530).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
24 Well PlateThermo Fisher Scientific07-200-84
48 Well PlateDenville ScientificT1049
5-Ethynyl-2'-deoxyuridine (Edu)CarbosynthNE08701
Alcohol 70% IsopropylThermo Fisher Scientific64-17-5
Alcohol Prep PadsThermo Fisher Scientific13-680-63
Alexa-488 AzideThermo Fisher ScientificA10266
Anti-Chicken NestinAvesNES; RRID: AB_2314882
Anti-Goat DCXSanta CruzCat# SC_8066; RRID: AB_2088494
Anti-Mouse Tbr2Thermo Fisher Scientific14-4875-82; RRID: AB_11042577
Betadine Solution (povidone-iodine)Amazon
Citiric Acid Stock [.1M] Citric Acid (21g/L citric acid)Sigma-Aldrich251275
Clozapine N- OxideSigma-AldrichC08352-5MG
Confocal Software (Zen Black)Zeiss MicroscopyZen 2.3 SP1 FP1 (black)
Copper (II) Sulfate PentahydrateThermo Fisher ScientificAC197722500
Cotton SwabsAmazon
CoverslipDenville ScientificM1100-02
Delicate Task Wipe KimwipesKimtech Science7557
Drill Bit 0.5 mmFine Science Tools19007-05
Ethylene GlycolThermo Fisher ScientificE178-1
Hamilton Needle 2 inchHmailton Company7803-05
Hamilton Syringe 5 μL Model 75 RNHmailton CompanyRef: 87931
High Speed DrillForedom1474
Infusion PumpHarvard Apparatus70-4511
Injectable Saline SolutionMountainside Health CareNDC 0409-4888-20
Insulin SyringeBD Ultra-Fine Insulin Syringes
IsofluraneHenry Schein29405
Stereotax For Small AnimalKOPF InstrumentsModel 942
Leica M80Leica
Leica MicrotomeLeicaSM2010 R
LSM 780Zeiss Microscopy
Nair (Hair Removal Product)Nair
Paraformaldahyde 4%Sigma-Aldrich158127
Plus Charged SlideDenville ScientificM1021
Phosphate Buffered Solution (PBS)Thermo Fisher Scientific10010031
Puralube Vet OintmentPuralube
Slide Rack 20 slide unitElectron Microscopy Science70312-24
Slide Rack holderElectron Microscopy Science70312-25
Small Animal Heating PadK&H
SucroseSigma-AldrichS0389
Super PAP Pen 4 mm tipPolySciences24230
Surgical ScalpelMedPride47121
Tris Buffered Solution (TBS)Sigma-AldrichT5912
Tri-sodium citrate Stock [0.1 M] Tri-sodium Citrate (29.4g/L tri-sodium citrate)Sigma-AldrichC8532
Triton X-100Sigma-Aldrich93443
TweezersAmazon
Vet Bond Tissue Adhesive3M1469SB

References

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  1. Zhao, C., Deng, W., Gage, F. H. Mechanisms and Functional Implications of Adult Neurogenesis. Cell. 132 (4), 645-660 (2008).
  2. Spalding, K. L., et al. Dynamics of hippocampal neurogenesis in adult humans. Cell. 153 (6), 1219-1227 (2013).
  3. Hill, A. S., Sahay, A., Hen, R.

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Tags

Adult NeurogenesisNeural Stem CellsDREADDs TechnologyStereotaxic InjectionChemogenetic StimulationThymidine AnalogImmunofluorescence LabelingConfocal ImagingAntigen RetrievalMossy Cells

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