Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Summary

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

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

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....

Protocol

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.......

Discussion

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.......

Materials

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