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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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 desired manipulation, proper delivery of a thymidine analog, animal age, immunostaining efficiency, successful transcardial perfusions, and unbiased quantification of images. For example, inaccurate viral delivery may cause off target effects that result in a phenotype unrelated to the circuit in question. Additionally, low quality immunofluorescence techniques may hide the true number of present cells and therefore produce a phenotype that is not biologically relevant. Another very important factor to control is the age of the mice when performing experiments, considering that adult neurogenesis is age dependent22. Lastly, it is important that each section is unbiasedly scored. To reduce bias, take a methodical approach and ensure that the person scoring is proficient at identifying the stages of adult NSC development using morphological information. Additionally, blind both control and treatment groups and reveal their identities after image quantifications. As an additional measure to reduce bias, two separate individuals can quantify the same data set to validate observed results.
There are several limitations associated with this approach to study circuit activity dependent regulation of adult NSCs and newborn progeny. The first limitation is that this approach does not provide information about the specific cell types within a circuit that mediate the overall effect on NSCs from manipulating the circuit in question. This means that although there might be a phenotypic effect on adult NSCs, the effect may be acting through one or several intermediate cell types. An efficient way to address this concern is to pair these studies with electrophysiology to pin down the intermediaries. An additional limitation of this protocol is the need to have either a specific Cre mouse (5-HTR2A) line or a viral construct (AAV5-camKII-hM3d-mcherry) that can target the desired circuit. If an effective cell specific Cre mouse line is not readily available for a question of interest, the ability to study this circuit becomes increasingly difficult. However, many cell types in the brain have Cre specific mouse lines. A lesser limitation of this protocol is related to CNO as an effective inert ligand. Recently, studies demonstrated that CNO, the inert chemical used to activate DREADDs, metabolizes to clozapine, which may cause behavioral phenotypes23. However, an efficient way to address is to include proper controls in each experiment. An example of proper controls includes both a CNO and DREADD control, where CNO is administered in combination with a control reporter virus (AAV5-DIO-mCherry), and a saline only control where no CNO is administered to a reporter virus group. By including these controls, the effects of only CNO can be isolated. Alternatively, a secondary inert ligand known as C21, has been recently demonstrated to have similar efficacy and potency with no demonstrated behavioral effects24. Lastly, a final limitation of this protocol is controlling the amount of CNO that each animal consumes during the experiment. Different animals drink CNO-containing water at varying degrees and may therefore have a range of effects on adult neurogenesis. In general, a mouse tends to drink about 4 mL of CNO water in a 24 h period. This means that at the concentration of 1 mg/200 mL animals receive a total of 0.02 mg of CNO per day which is comparable to the amount of a single CNO dose injected intraperitoneally. If timely coupled administration of CNO is a concern, switching to intraperitoneal injections may be a better alternative.
The advantage of using this protocol is the degree of specificity achieved when modulating adult neurogenesis. Past neurogenesis studies have utilized systemically administered pharmacological agonist or antagonist to modulate circuit components. These non-specific manipulations may produce phenotypic differences but provide little insight about mechanisms involved in adult neural stem cell regulation. Additionally, this protocol can be easily modified to investigate various circuit wide effects on adult neurogenesis. For example, by switching to an inhibitory DREADD, or by targeting one or multiple brain regions at once, one can ask an array of questions to understand circuit specific regulation of adult neurogenesis. Another advantage of using this protocol over previous approaches is that, the use of a nestin antibody eliminates transgenic animal breeding of fluorescently encoded neural stem cell reporters such as nestin:GFP, increasing efficiency and reducing time per experiment8. Furthermore, this technique limits rodent handling when administering CNO, which reduces rodent stress during experiments. It is important to mitigate stress when studying stress-sensitive processes. Lastly, this approach is easily amendable to include a behavioral assay, for example, if one were interested in asking if the contralateral mossy cell circuit that modulates NSCs also plays a role in spatial learning or stress resilience.
The main technical difficulty when using this approach is accurate viral delivery. Becoming a proficient rodent surgeon takes practice and can take significant troubleshooting. It is therefore advisable to perform a series of pilot experiments to test viral titer, labeling efficiency, and viral spread. We have found that certain serotypes have different spreading patterns and that the AAV2 serotype spreads less than AAV5 or AAV8. Additionally, it is best to have a trusted viral packaging provider for each of these experiments. By performing pilot surgeries, many of these concerns can be addressed and one can save time. It is also recommended that one test different CNO concentrations to stimulate or inhibit the desired circuits. In general, 1 mg/kg will sufficiently activate tested circuits, but certain cell types may require more or less CNO. It is important to note that the dose of CNO administration can differentially affect certain circuits specifically when looking at something such as mossy cells15.
Alternative applications of this protocol include simultaneous behavioral testing, modulation of alternative circuits, and additional analysis of neurogenesis features. To perform behavioral testing, one could follow the protocol described and after administering CNO, perform a specific behavioral task, such as a novel location assay, or a spatial navigation task. The benefit of this approach is that a single experiment would yield both behavioral and circuit specific information that could lead to a circuit specific behavioral phenotype. To modulate alternative circuits, one can use a combination of different Cre lines and viral vectors. For example, if one were interested in understanding how inhibiting dopaminergic neurons from the ventral tegmental area (VTA) or VTA modulates adult neurogenesis, one could use a tyrosine hydroxylase Cre mouse line and inject a Cre dependent hM4D (inhibitory) DREADD virus in to the VTA to determine dopaminergic specific regulation of adult neurogenesis. The possibilities to target alternative brain regions using this approach are vast and can be strategically used to interrogate compelling neural circuits. Lastly, this approach allows one to investigate additional stages of adult neurogenesis. If for example one wanted to understand how stimulating mossy cells affects arborization or dendritic length of immature neurons, one would follow a similar protocol but perform alternative analysis such as sholl analysis.
In summary, this protocol provides a detailed step-by-step process to assay circuit activity dependent regulation of adult NSCs and neurogenesis via DREADD technology. The strength of this protocol lies in its ability to be easily modified to address a vast range of questions regarding circuit specific adult neural stem cell regulation. With the advancement of the clustered regularly interspaced short palindromic repeats (CRISPR) technology, it is now easier to generate cell specific Cre mouse lines to pair with sophisticated viral constructs to address increasingly complex questions expanding the applicability of this protocol.

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 promoted stem cell quiescence (Figure 1C). We verified accurate viral delivery before the analysis of tissue (Figure 2A,B). Additionally, we verified activation of mossy cells via c-fos immunohistochemistry experiments (data not shown). In the case of improper viral injection, exclude animal from further analysis. An improper injection is one that fails to target the desired coordinates, has most of the expression outside the desired region, or has little to no viral delivery. For this experiment, mossy cells in the hilus of the DG were the intended target, and if injections were outside of the hilus, they were excluded. By using a thymidine analog, Edu, and antigen retrieval for the nestin staining outlined in sections 5.2 and 5.3, we were able to successfully label proliferating neural stem cells (Figure 3A). Additionally, by omitting the antigen retrieval step, section 5.2, we were able to label Tbr2 positive neural progenitor and neuroblast, and DCX positive neuroblast and immature neurons (Figure 3A). We demonstrate an example of the area quantified and used to calculate density and provide an example of mounted tissue on a slide (Figure 3B and Figure 4A). Moreover, both successful and sub-par experiments are provided as references for experimental approaches (Figure 4B). Lastly, there are several different quantifications that can be obtained from a successful experiment (Figure 5A-D)15. The quantifications include the density of proliferating neural stem cells (Nestin+/Edu+/volume), the percent of proliferating neural stem cells (Nestin+/Edu+/total nestin), total proliferating cells (Edu+/volume) and total stem cell pool (Nestin+/volume). Upon contralateral stimulation of a mossy cells, a decrease in neural stem cell proliferation was observed. Similar quantifications can be obtained for neural progenitor and immature neurons by using the appropriate antibody.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59237/59237fig1.jpg" /> 
Figure 1: Experimental approach to assay circuit regulation of adult neural stem cells. (A) Schematic representing the different steps outlined in the protocol. (B) Timeline of experimental approach used to stimulate mossy cells in rodents. (C) Injection schematic targeting contralateral mossy cells for stimulation. (D) Schematic of the developmental lineage of adult neural stem cells in the subgranular zone (SGZ), granule cell layer (GCL), and molecular layer (ML) with corresponding antibodies used at different developmental stages. The different developmental stages include either quiescent or activated radial neural stem cells (NSC), neural progenitors (NP), neuroblast (NB), immature granule cells (GC), and mature GC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59237/59237fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59237/59237fig2.jpg" /> 
Figure 2: Demonstration of effective viral delivery. (A) Immunofluorescence images of accurate viral delivery. Viral particles express an mCherry fluorescent label which target mossy cells in the hilus (white boxed region). DAPI labels cell nuclei. The image side of accurate viral delivery demonstrates clear mossy fiber projections from the contralateral injection side. (B) Immunofluorescence images of off target viral injections. Note that in this case contralateral mossy fibers are absent in the image side. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59237/59237fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59237/59237fig3.jpg" /> 
Figure 3: Analysis of proliferating neural stem cells and progeny. (A) Immunohistochemistry images of thymidine analog Edu colocalizing with specific cell stage markers nestin (neural stem cells and progenitors), Tbr2 (neural stem cells, neural progenitors), and DCX (neuroblast, immature neurons) indicated by white arrowheads. Scale bar = 10 &#181;m. (B) Representative measurement of the area within the dentate gyrus used to calculate density. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59237/59237fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59237/59237fig4.jpg" /> 
Figure 4: Demonstration of immunofluorescence preparation. (A) Schematic demonstrating stereological separation of mounted tissue sections from anterior to posterior axis. Red box denotes the side imaged. (B) Demonstration of successful and sub-par immunofluorescence experiments for neuronal lineage markers. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59237/59237fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59237/59237fig5.jpg" /> 
Figure 5: Contralateral activation of mossy cells decreases neural stem cell proliferation. (A) Immunohistochemistry quantifications of Nestin+/Edu+ cells in the dentate gyrus demonstrate a decrease in proliferation after stimulation of contralateral mossy cells. (B) Decrease in the percent of proliferating neural stem cells in the group with activated contralateral mossy cells. (C) There was no significant change in neural stem cell density in either group. (D) There was no change in overall levels of proliferating cells in the dentate gyrus. Values represent means &#177; standard error of measurement. p &#60; 0.05 (n = 3 for control, n = 5 for the hM3D group). This figure has been adapted from Yeh et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59237/59237fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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 ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

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. ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

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.