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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.
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.
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....
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
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
24 Well Plate | Thermo Fisher Scientific | 07-200-84 | |
48 Well Plate | Denville Scientific | T1049 | |
5-Ethynyl-2'-deoxyuridine (Edu) | Carbosynth | NE08701 | |
Alcohol 70% Isopropyl | Thermo Fisher Scientific | 64-17-5 | |
Alcohol Prep Pads | Thermo Fisher Scientific | 13-680-63 | |
Alexa-488 Azide | Thermo Fisher Scientific | A10266 | |
Anti-Chicken Nestin | Aves | NES; RRID: AB_2314882 | |
Anti-Goat DCX | Santa Cruz | Cat# SC_8066; RRID: AB_2088494 | |
Anti-Mouse Tbr2 | Thermo Fisher Scientific | 14-4875-82; RRID: AB_11042577 | |
Betadine Solution (povidone-iodine) | Amazon | ||
Citiric Acid Stock [.1M] Citric Acid (21g/L citric acid) | Sigma-Aldrich | 251275 | |
Clozapine N- Oxide | Sigma-Aldrich | C08352-5MG | |
Confocal Software (Zen Black) | Zeiss Microscopy | Zen 2.3 SP1 FP1 (black) | |
Copper (II) Sulfate Pentahydrate | Thermo Fisher Scientific | AC197722500 | |
Cotton Swabs | Amazon | ||
Coverslip | Denville Scientific | M1100-02 | |
Delicate Task Wipe Kimwipes | Kimtech Science | 7557 | |
Drill Bit .5mm | Fine Science Tools | 19007-05 | |
Ethylene Glycol | Thermo Fisher Scientific | E178-1 | |
Hamilton Needle 2 inch | Hmailton Company | 7803-05 | |
Hamilton Syringe 5uL Model 75 RN | Hmailton Company | Ref: 87931 | |
High Speed Drill | Foredom | 1474 | |
Infusion Pump | Harvard Apparatus | 70-4511 | |
Injectable Saline Solution | Mountainside Health Care | NDC 0409-4888-20 | |
Insulin Syringe | BD Ultra-Fine Insulin Syringes | ||
Isoflurane | Henry Schein | 29405 | |
Stereotax For Small Animal | KOPF Instruments | Model 942 | |
Leica M80 | Leica | ||
Leica Microtome | Leica | SM2010 R | |
LSM 780 | Zeiss Microscopy | ||
Nair (Hair Removal Product) | Nair | ||
Paraformaldahyde 4% | Sigma-Aldrich | 158127 | |
Plus Charged Slide | Denville Scientific | M1021 | |
Phosphate Buffered Solution (PBS) | Thermo Fisher Scientific | 10010031 | |
Puralube Vet Ointment | Puralube | ||
Slide Rack 20 slide unit | Electron Microscopy Science | 70312-24 | |
Slide Rack holder | Electron Microscopy Science | 70312-25 | |
Small Animal Heating Pad | K&H | ||
Sucrose | Sigma-Aldrich | S0389 | |
Super PAP Pen 4 mm tip | PolySciences | 24230 | |
Surgical Scalpel | MedPride | 47121 | |
Tris Buffered Solution (TBS) | Sigma-Aldrich | T5912 | |
Tri-sodium citrate Stock [.1M] Tri-sodium Citrate (29.4g/L tri-sodium citrate) | Sigma-Aldrich | C8532 | |
Triton X-100 | Sigma-Aldrich | 93443 | |
Tweezers | Amazon | ||
Vet Bond Tissue Adhesive | 3M | 1469SB |
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