Name: assaying circuit specific regulation of adult hippocampal neural precursor cells
Uploaded: 2019-07-24T12:30:00
Description: Watch this Scientific Journal Video about Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells at JoVE.com

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).

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
<ol>
	<li>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...

<ol>
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D., 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=A+functional+role+for+adult+hippocampal+neurogenesis+in+spatial+pattern+separation.">A functional role for adult hippocampal neurogenesis in spatial pattern separation.</a> Science. 325, (2009).</li><li>Sahay, A., 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=Increasing+adult+hippocampal+neurogenesis+is+sufficient+to+improve+pattern+separation.">Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation.</a> Nature. 472 (7344), 466-470 (2011).</li><li>Anacker, C., 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=Hippocampal+neurogenesis+confers+stress+resilience+by+inhibiting+the+ventral+dentate+gyrus.">Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus.</a> Nature. , 1 (2018).</li><li>Kuhn, H. 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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M., Warden, M. R., Diesseroth, K., Mcclung, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Optogenetic+Stimulation+of+the+Rodent+Central+Nervous+System.">In vivo Optogenetic Stimulation of the Rodent Central Nervous System.</a> J Vis Exp. , (2015).</li><li>Yizhar, O., Fenno, L. E., Davidson, T. 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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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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&#39;-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 .5mm</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 5uL 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>
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			<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&#38;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 [.1M] 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 ...

This protocol can answer how various circuits in the brain regulate adult neurogenesis and specifically how stimulating or inhibiting neural circuits affect the proliferation of adult neural stem cells. The advantage of this technique is that it is able to specifically target desired neural circuits, as well as reduce the amount of stress introduced to the animals. This method can provide insight into how different brain circuits regulate adult neurogenesis, in particular, how specific cell types that release certain neurotransmitters regulate adult neural stem cell proliferation.To begin this procedure, place the tissue sections in PBS and mount five to eight sections on a positively charged slide in serial order from anterior to posterior. Let the tissue sections dry at room temperature for two to five minutes and completely adhere to the slides. Next, prepare citrate buffer in a container.Heat the citrate buffer in a microwave oven for five minutes until the solution is boiling. In the meantime, place the mounted sections in a glass slide holder. After five minutes, carefully place the slide holder with the sections into a pipette box.Set the microwave oven power to 50%and the cook time to seven minutes. Start a timer for seven minutes, and monitor the solution. Stop the microwave oven when the solution starts to boil, and continue the microwave after boiling stops.Stop after the timer runs out, even if the cook time on the microwave has not finished. The goal of this step is to keep the water right below boiling temperature without letting the water overly boil. Too much boiling will remove tissue sections from the slide.Then, transfer the warm box with citrate buffer and tissue slides to an ice bucket for cooling. Cover the box, and wait for about 30 minutes or until the solution is cool to the touch, before proceeding to thymidine analog staining. To stain the tissue sections with thymidine analog, remove the tissue sections from the citrate buffer.Let the tissue sections dry and completely adhere to the slides, before drawing a border with a hydrophobic pen. Next, permeabilize the sections with permeabilization buffer for 20 to 30 minutes. Wash the sections twice using TBS-triton for five minutes each time.Then, prepare an Edu reaction solution. Incubate the sections in Edu reaction solution for 30 minutes to an hour. Subsequently, wash them three times in TBS-triton for five minutes each time.Cover the slides in aluminum foil, or place them in a light-protected chamber to protect from light after this step. At this stage, check if the Edu reaction works by using a fluorescent microscope. Edu-labeled cells should be observed if the reaction works.Now, block the mounted tissue sections using blocking buffer raised in the same animal as the secondary antibody for 30 minutes to an hour. Then, wash them twice in TBS-triton for five minutes each time. During the blocking step, prepare primary antibody solution by mixing the primary antibody in blocking buffer.Subsequently, add 250 microliters of primary antibody solution per slide to ensure the tissue sections are completely submerged, and incubate them overnight at room temperature. The next day, wash the tissue sections three times in TBS-triton for five minutes each to remove excess primary antibody. Then, incubate the tissue sections in fluorophore-conjugated secondary antibody prepared in blocking buffer solution for two hours at room temperature.Wash the tissue sections three times in TBS-triton for five minutes each to remove excess secondary antibody. Next, apply 300-micromolar DAPI solution at one to 100 in PBS for 15 minutes at room temperature. Afterward, wash the tissue sections three times in PBS for five minutes each to remove excess DAPI, and remove the PAP pen circle around the tissue using a cotton swab.Let the sections dry, before applying mounting media and covering them with coverslips. Using the imaging software, open the image of each dentate gyrus section as a composite image with the channels merged in distinct colors to easily visualize the colocalization. Then, measure the area of dentate gyrus in each section using the polygon selection tool, and record all the sections of each mouse.This will be the area of dentate gyrus used to calculate the density. Using the software plugin cell counter found under Plugins, Analyze, Cell Counter, Cell Counter, record the number of cells in the dentate gyrus that have colocalizing primary antibody and thymidine analog Edu from the composite image. Additionally, record the total number of Edu-positive and nestin-positive cells with a radial process.In the case of nestin, it is very important to pay attention to the cells'morphology. If quantifying neural stem cells, ensure that only cells with a radial process are quantified. Enter the cell counts in a spreadsheet software to compile all the data for analysis later.For example, to obtain the stem cell density, divide the sum of nestin-positive and Edu-positive cells by the sum of dentate gyrus volume in each animal. Calculate the volume of each section by multiplying the area with total Z-step increments, assuming that each step is one micrometer. In this protocol, total steps should be close to 40 since tissue is sectioned at 40 micrometers.Subsequently, calculate the overall number of proliferating cells, percent of proliferating neural stem cells, and total proliferating cells after stimulating contralateral mossy cells. By using a thymidine analog Edu and antigen retrieval for the nestin staining, the proliferating neural stem cells were successfully labeled. Additionally, by omitting the antigen retrieval step, Tbr2-positive neural progenitor and neuroblast and DCX-positive neuroblast and immature neurons were labeled.Here is an example of the area quantified and used to calculate cell density, and here is an example of the mounted tissue on a slide. Both successful and sub-par experiments are shown for comparison. Lastly, there are several different quantifications that can be obtained from a successful experiment.The quantifications include the density of proliferating neural stem cells, the percent of proliferating neural stem cells, total proliferating cells, and the total stem cell pool. Upon contralateral stimulation of mossy cells, a decrease in neural stem cell proliferation was observed. The most important step in this procedure is to control tissue boiling in the microwave oven.The tissue sections will be damaged if they are boiling for too long. After following this procedure, one could inject different viral vectors to target the same circuit. For example, if one were stimulating a neural circuit, one could inhibit it using an inhibitory DREADD virus.The techniques presented in this protocol aren't novel. However, the strength of this protocol is that it comprehensively covers all the steps required for answering questions about how circuit impacts adult neurogenesis.

Research

JoVE Journal

Neuroscience

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