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).

<ol>
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G., Eisch, A. J., Spalding, K., Peterson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+Phenotypic+Characterization+of+Adult+Neurogenesis.">Detection and Phenotypic Characterization of Adult Neurogenesis.</a> Cold Spring Harb Perspect Biol. , (2016).</li><li>Ansorg, A., Bornkessel, K., Witte, O. W., Urbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+Multiple+Labeling+with+Antibodies+from+the+Same+Host+Species+to+Study+Adult+Hippocampal+Neurogenesis.">Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis.</a> Journal of Visualized Experiments. (98), 1-13 (2015).</li><li>Podgorny, O., Peunova, N., Park, J. 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J., Mogri, M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+Optogenetics+in+Neural+Systems.">Primer Optogenetics in Neural Systems.</a> Neuron. 71 (1), 9-34 (2011).</li><li>Yeh, C., Asrican, B., Moss, J., Lu, W., Toni, N., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mossy+Cells+Control+Adult+Neural+Stem+Cell+Quiescence+and+Maintenance+through+a+Dynamic+Balance+between+Direct+and+Indirect+Pathways.">Mossy Cells Control Adult Neural Stem Cell Quiescence and Maintenance through a Dynamic Balance between Direct and Indirect Pathways.</a> Neuron. , 1-18 (2018).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+Stereotaxic+Surgery+in+Rodents.">Survivable Stereotaxic Surgery in Rodents.</a> Journal of Visualized Experiments. (20), 20-22 (2008).</li><li>Roth, B. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+the+subdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.">Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator.</a> The Anatomical Record. 231 (4), 482-497 (1991).</li><li>Kuhn, H., Dickinson-Anson, H., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+dentate+gyrus+of+the+adult+rat:+age-related+decrease+of+neuronal+progenitor+proliferation.">Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.</a> The Journal of Neuroscience. 16 (6), 2027-2033 (1996).</li><li>Gomez, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemogenetics+revealed:+DREADD+occupancy+and+activation+via+converted+clozapine.">Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.</a> Science. 357 (6350), 503-507 (2017).</li><li>Thompson, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dreadd+Agonist+21+(C21)+Is+an+Effective+Agonist+for+Muscarnic-Based+Dreadds+in+Vitro+and+in+Vivo.">Dreadd Agonist 21 (C21) Is an Effective Agonist for Muscarnic-Based Dreadds in Vitro and in Vivo.</a> ACS Pharmacology &amp; Translational Science. 72 (3), (2018).</li></ol>

The authors have nothing to disclose.

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...

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 response3,4,5,6. To influence behaviors, adult NSCs are highly regulated by various external stimuli in an activity dependent manner by responding to an array of local and distal chemical cues. These chemical cues include neurotransmitters and neuromodulators and act in a circuit specific manner from various brain regions. Importantly, circuit wide convergence of these chemical cues on NSCs allows for unique and precise regulation of stem cell activation, differentiation, and fate decisions.
One of the most effective ways to interrogate circuit regulation of adult NSCs in vivo is by pairing immunofluorescence analysis with circuit wide manipulations. Immunofluorescence analysis of adult NSCs is a commonly utilized technique, where antibodies against specific molecular markers are used to indicate the developmental stage of adult NSCs. These markers include: nestin as a radial glia cell and early neural progenitor marker, Tbr2 as an intermediate progenitor marker, and dcx as a neuroblast and immature neuron marker7. Additionally, by administering thymidine analogs such as BrdU, CidU, Idu, and Edu, cell populations undergoing S phase can be individually labeled and visualized8,9,10. By combining these two approaches, a wide range of questions can be investigated ranging from how proliferation is regulated at specific developmental stages, to how various cues affect NSC differentiation and neurogenesis.
Several options exist to effectively manipulate neural circuits including electrical stimulation, optogenetics, and chemogenetics, each with their own advantages and disadvantages. Electrical stimulation involves an extensive surgery where electrodes are implanted to a specific brain region which are later used to transmit electrical signals to modulate a targeted brain region. However, this approach lacks both cellular and circuit specificity. Optogenetics involves the delivery of viral particles that encode a light activated receptor that is stimulated by a laser emitted through an implanted optical fiber, but requires extensive manipulations, large cost, and complex surgeries11. Chemogenetics involves the delivery of viral particles that encode a designer receptor exclusively activated by designer drugs or DREADDs, which are subsequently activated by a specific and biologically inert ligand known as clozapine N-oxide (CNO)12. The advantage of utilizing DREADDs to manipulate local neural circuits that regulate adult NSCs lies in the ease and various routes of CNO administration. This allows for a less time-consuming approach with reduced animal handling, which is easily adaptable for long term studies to modulate neural circuits.
The approach described in this protocol is a comprehensive collection of various protocols required to successfully interrogate circuit regulation of adult hippocampal neurogenesis that combines both immunofluorescence techniques and circuit manipulations using chemogenetics. The method described in the following protocol is appropriate for stimulating or inhibiting one or multiple circuits simultaneously in vivo to determine their regulatory function on adult neurogenesis. This approach is best used if the question does not need a high degree of temporal resolution. Questions requiring precise temporal control of stimulation/inhibition at a certain frequency, can be better addressed using optogenetics13,14. The approach described here is easily adapted for long term studies with minimal animal handling especially where stress is a major concern.

<table><tbody><tr><td>24 Well Plate</td><td>Thermo Fisher Scientific</td><td>07-200-84</td><td></td></tr><tr><td>48 Well Plate</td><td>Denville Scientific</td><td>T1049</td><td></td></tr><tr><td>5-Ethynyl-2&#039;-deoxyuridine (Edu)</td><td>Carbosynth</td><td>NE08701</td><td></td></tr><tr><td>Alcohol 70% Isopropyl</td><td>Thermo Fisher Scientific</td><td>64-17-5</td><td></td></tr><tr><td>Alcohol Prep Pads</td><td>Thermo Fisher Scientific</td><td>13-680-63</td><td></td></tr><tr><td>Alexa-488 Azide</td><td>Thermo Fisher Scientific</td><td>A10266</td><td></td></tr><tr><td>Anti-Chicken Nestin</td><td>Aves</td><td>NES; RRID: AB_2314882</td><td></td></tr><tr><td>Anti-Goat DCX</td><td>Santa Cruz</td><td>Cat# SC_8066; RRID: AB_2088494</td><td></td></tr><tr><td>Anti-Mouse Tbr2</td><td>Thermo Fisher Scientific</td><td>14-4875-82; RRID: AB_11042577</td><td></td></tr><tr><td>Betadine Solution (povidone-iodine)</td><td>Amazon</td><td></td><td></td></tr><tr><td>Citiric Acid Stock [.1M] Citric Acid (21g/L citric acid)</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>Clozapine N- Oxide</td><td>Sigma-Aldrich</td><td>C08352-5MG</td><td></td></tr><tr><td>Confocal Software (Zen Black)</td><td>Zeiss Microscopy</td><td>Zen 2.3 SP1 FP1 (black)</td><td></td></tr><tr><td>Copper (II) Sulfate Pentahydrate</td><td>Thermo Fisher Scientific</td><td>AC197722500</td><td></td></tr><tr><td>Cotton Swabs</td><td>Amazon</td><td></td><td></td></tr><tr><td>Coverslip</td><td>Denville Scientific</td><td>M1100-02</td><td></td></tr><tr><td>Delicate Task Wipe Kimwipes</td><td>Kimtech Science</td><td>7557</td><td></td></tr><tr><td>Drill Bit 0.5 mm</td><td>Fine Science Tools</td><td>19007-05</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Thermo Fisher Scientific</td><td>E178-1</td><td></td></tr><tr><td>Hamilton Needle 2 inch</td><td>Hmailton Company</td><td>7803-05</td><td></td></tr><tr><td>Hamilton Syringe 5 &amp;mu;L Model 75 RN</td><td>Hmailton Company</td><td>Ref: 87931</td><td></td></tr><tr><td>High Speed Drill</td><td>Foredom</td><td>1474</td><td></td></tr><tr><td>Infusion Pump</td><td>Harvard Apparatus</td><td>70-4511</td><td></td></tr><tr><td>Injectable Saline Solution</td><td>Mountainside Health Care</td><td>NDC 0409-4888-20</td><td></td></tr><tr><td>Insulin Syringe</td><td>BD Ultra-Fine Insulin Syringes</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>Stereotax For Small Animal</td><td>KOPF Instruments</td><td>Model 942</td><td></td></tr><tr><td>Leica M80</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica Microtome</td><td>Leica</td><td>SM2010 R</td><td></td></tr><tr><td>LSM 780</td><td>Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Nair (Hair Removal Product)</td><td>Nair</td><td></td><td></td></tr><tr><td>Paraformaldahyde 4%</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Plus Charged Slide</td><td>Denville Scientific</td><td>M1021</td><td></td></tr><tr><td>Phosphate Buffered Solution (PBS)</td><td>Thermo Fisher Scientific</td><td>10010031</td><td></td></tr><tr><td>Puralube Vet Ointment</td><td>Puralube</td><td></td><td></td></tr><tr><td>Slide Rack 20 slide unit</td><td>Electron Microscopy Science</td><td>70312-24</td><td></td></tr><tr><td>Slide Rack holder</td><td>Electron Microscopy Science</td><td>70312-25</td><td></td></tr><tr><td>Small Animal Heating Pad</td><td>K&amp;amp;H</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Super PAP Pen 4 mm tip</td><td>PolySciences</td><td>24230</td><td></td></tr><tr><td>Surgical Scalpel</td><td>MedPride</td><td>47121</td><td></td></tr><tr><td>Tris Buffered Solution (TBS)</td><td>Sigma-Aldrich</td><td>T5912</td><td></td></tr><tr><td>Tri-sodium citrate Stock [0.1 M] Tri-sodium Citrate (29.4g/L tri-sodium citrate)</td><td>Sigma-Aldrich</td><td>C8532</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>93443</td><td></td></tr><tr><td>Tweezers</td><td>Amazon</td><td></td><td></td></tr><tr><td>Vet Bond Tissue Adhesive</td><td>3M</td><td>1469SB</td><td></td></tr></tbody></table>

assaying circuit specific regulation of adult hippocampal neural precursor cells

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.

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...

Watch this Scientific Journal Video about Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells at JoVE.com

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

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 ...

assaying-circuit-specific-regulation-adult-hippocampal-neural

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

6.4K Views.  University of North Carolina Chapel Hill. 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.

Video: Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Developmental Biology

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

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Chapters in this video

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders